SOP Gentri Medical

Document Name: LABORATORY POLICIES
AND PROCEDURES
GENTRI MEDICAL CENTER
AND HOSPITAL INC. Document No.: LAB – 13-01
Doc. Property: LABORATORY DEPARTMENT
Santusan st., Manggahan, Gen Trias Cavite

Tel. (046)-424-08-88 Page No.: Page 1 of 225
Effective Date: JUNE 2013
LABORATORY DEPARTMENT
GENERAL POLICIES AND

PROCEDURES
Prepared by: Rolan Dela Rosa, M.D. Reviewed by: Divina Rojas, M.D. Approved by: Jerrimo Genuino, M.D.
Date: June 2013 Date:June 2013 Date:June 2013
AND PROCEDURES

REVISION HISTORY
REASON
REV REV FOR PREPARED REVIEWED APPROVED EFFECTIVE
DCN
NO. DATE REVISION BY BY BY DATE
AND PROCEDURES

Table Of Contents
Department Organizational Chart 6
Hospital Mission and Vision 7
Laboratory Policy Statement 8
Purpose 8
Scope 8
Department Mission 9
Department Vision 9
Department Objectives 9
Materials 9
Definition of Terms 9
Department Organization 12
Job Responsibilities 12
Pathologist 12
Chief Medical Technologist 13
Medical Technologist 14
Phlebotomist/Laboratory Technician 14
Histopath Technician 15
Laboratory Aide 15
Schedules and Shifting 15
Accounts and External Affairs 17
Departmental Meetings 17
Departmental Reports 18
Licensing and Accreditation 19
Complaints and Incident Reports 20
Release of Results 21
Requisition Of Reagents And Supplies 23
Personnel Policies 23
Hours Of Work/ Rest Periods 24
Change Of Shift Schedules 24
Overtime 24
Tardiness 25
AND PROCEDURES

Sick Leave 25
Day-Off 26
Vacation Leave 26
Leave Of Absence/ Leave Without Pay 27
Time Keeping 27
Compliance, Licensure, and Testing 27
Computer Use and Security 28
Public Image 29
MAINTENANCE OF PHYSICAL PLANT & EQUIPMENT 29
Contingency Procedures 35
Equipment Breakdown (Section Machines) 35
Microhematocrit and Clinical Centrifuge 35
Hot Air Oven and Incubator 35
Microscope 35
No Reagents Available 35
Power Failure 36
Puncture Wounds, Cuts and Abrasions 36
Ingestion Of Potentially Infectious Material 36
Potentially Infectious Aerosol Release 36
Broken Containers and Spilled Infectious Substances 36
Breakage of Tubes Inside Sealable Buckets (Safety Cups) 37
Fire and Natural Disasters 37
Emergency Services: Whom to Contact 38
Emergency Equipment 38
Total Quality Management 39
Policy on Continuing Program on Staff Development 41
Development of Training Program 43
Annual Training Program 43
Personal Training Requisition 44
Laboratory Guidelines 45
General Guidelines 45
Pre-Analytical Phase 45
Pre-Collection Variables 45
Common Interferences 48
The Request Form 50
Timing of Collection 52
AND PROCEDURES

Timed Specimens/ Multiple Specimens 52

Volume Requirements 53
Criteria for Rejection of Specimen 56
Fasting Specimens 56
Specimen Processing 57
Blood Storage and Preservation 58
Stat (Emergency) Request for Laboratory Services 58
Bio-safety 58
Standard Protocols 69
Test Requisition 62
Receipt of Request Form: 62
Receipt of Specimen for Laboratory Test 63
Collection and Handling of Specimens 64
Charging Of Laboratory Services 64
Releasing Of Results 67
Standard Operating Procedures 71
Blood Collection 72
Hematology 80
Microscopy 98
Serology 175
Chemistry 187
Microbiology 197
Result Forms 217
Flow Charts 230
AND PROCEDURES

ORGANIZATIONAL CHART
AND PROCEDURES

GENTRI MEDICAL CENTER AND HOSPITAL

MISSION
To provide the best quality health care enabled by highly specialized and
competent medical practitioners and staff operating in an eco-friendly and
globally attuned environment.
VISION
To become a Premiere Referral Medical Center in Cavite and the Southern
Tagalog Region.
AND PROCEDURES

LABORATORY POLICIES
The pathology laboratory provides and interprets analytical and morphological

information to assist in the diagnosis of clinical problems and the monitoring of disease progress
and treatment. The provision of a legible and appropriate clinical history on the request form,
together with a properly collected specimen, allows the pathology laboratory to issue relevant
and accurate results, and to assist the clinician in the interpretation of these results in the clinical
context.
PURPOSE
To document the general laboratory procedures and policies in order to guide medical,
paramedical, and non-medical hospital personnel.It is the purpose of this document to
establish policy, provide instruction, and set forth the basic principles to be followed in the
laboratory. The policy is written to comply with DOH standards and regulations.
SCOPE
This covers the basic clinical laboratory procedures from patient test requisition, receipt of
request, specimen collection and handling, specimen processing and analysis to reporting of
results.
AND PROCEDURES

1.0.VISION:
The department envisions on becoming the premier tertiary referral
laboratory in General Trias Cavite.
2.0.MISSION:
Rendering genuine and proper Patient care, providing aid and assistance to
Doctors in disease diagnosis through fast, accurate and reliable results
3.0 OBJECTIVES:
 Be responsible for the logical process from the acquisition of the specimen
to the production of data and the final report of test results.
 Strive to improve professional skills and knowledge and adopt scientific
advances that benefit the patients and improve the delivery of test results.
 Safeguard the dignity and privacy of patients.
 Actively seek to establish cooperative and specific working relationship
with other health professionals,
 Exercise professional judgment, skill and care while meeting established
standards.
 Provide expertise to advise and counsel other health professionals with
regards to laboratory examinations and technology.
 Render appropriate and efficient laboratory services provided by the
hospital.
4.0 MATERIALS
Please refer to master list of equipment, materials and supplies.
5.0 DEFINITION OF TERMS:

5.1. Abstinence: Term that denotes cessation of sexual contact for 3-5 days
for semen analysis.
5.2. Accuracy: the nearness or closeness of the assayed value to the true
or target value.
5.3. Contaminated: A term used to describe a specimen that is either
adulterated, exposed or mixed with unnecessary
substances that may obscure or affect examination result.
AND PROCEDURES

5.4. Department: term used to denote the Laboratory Department.

5.5. Diagnostic sensitivity: is the ability of the test to detect the proportion of
individuals with that disease who test positively with the
test; and it indicates the ability of the test to generate more
true-positive results and few false negative
5.6. Diagnostic Specificity: is the ability of the test to detect the proportion of
individuals without the disease who test negatively for
the disease; and it reflects the ability of the method to
detect true-negatives with very few false-positives.
5.7. External Quality Control : concludes all statistical quality control methods which are
performed periodically (i.e. Every month, every two
months, twice a year) by the laboratory personnel with
the contribution of an external center (referral laboratory,
scientific associations, diagnostic industry etc.). It checks primarily the accuracy of the
laboratory’s analytical methods.
5.8. Fasting: abstinence from food and liquids for required period of
time.
5.9. Head: Term used to denote the Pathologist
5.10. Hemoconcentration: The change in interstitial components due to fluid shift
from inside to outside the cells; this may be due to
changes in body state prior to blood collection.
5.11. Hemodilution: The change in interstitial components due to fluid shift
from outside to inside of cells; this may be due to changes in
body state prior to blood collection
5.12. Hemolysis: destruction of red cell corpuscles with liberation of
hemoglobin into the surrounding fluid; can be seen as the
reddish pigment in serum/plasma after centrifugation.
5.13. Internal Quality Control: concludes all statistical quality control methods, which are
performed every day by the laboratory personnel with the
laboratory’s materials and equipment. It checks primarily
the precision (repeatability or reproducibility) of the
method.
AND PROCEDURES

5.14. Lipemia: presence of fat globules in the blood; can be seen as the
yellowish cloudy portion in serum/plasma after
centrifugation.
5.15. NEQAS: “National External Quality Assurance”; a monthly program
instilled by the Department of Health to monitor efficiency
and reliability of national laboratory participants.
5.16. Over Fasting: abstinence from food and liquids more than the required
period of time.
5.17.Personnel: Term used to denote all Laboratory employees.
5.18.Practicability: the degree by which a method is easily repeated.
5.19. Precision or reproducibility: The ability of an analytical method to give repeated results
on the same sample that agree with one another.
5.20. Quality Assessment: (also known as proficiency testing) is a means to determine
the quality of the results generated by the laboratory.
5.21. Quality control: in the medical laboratory is a statistical process used to
monitor and evaluate the analytical processes that produce
patient results.
5.22. Quantity insufficient: A term used to describe an inadequate sample collected for
the required examinations; a repeat collection must always
be made to handle this problem.
5.23. Reliability: the ability of an analytical method to maintain accuracy and
precision over an extended period of time during which
equipment, reagents, and personnel may change.
5.24. Repeat Collection: Repeat collection of specimen either due to quantity
insufficiency, hemolysis, lipemia, hemoconcentration,
hemodilution, over fasting, under fasting, etc.
5.25. Routine request: test which follows the regular schedule for receiving of
specimens and releasing of results.
5.26. Sensitivity: is the ability of an analytical method to measure the
smallest concentration of the analyte of interest.
5.27. Serial Examination: a series of exams done at regular intervals; usually
requested for monitoring purposes.
5.28. Specificity: is the ability of an analytical method to measure only the
analyte of interest.
AND PROCEDURES

5.29. STAT request: test in which result is released within one hour or less for
immediate diagnosis and management; always given
priority.
5.30.Supervisor: Term used to denote the Chief Medical Technologist
5.31. Under Fasting: abstinence from food and liquids less than the required
period of time.
6.0 DEPARTMENT ORGANIZATION
The administrative responsibility of the laboratory lies with the Chief of Clinics, Medical
Director, and Pathologist as head. The head of the laboratory, the Pathologist, regulates all
clinical laboratory services. The Chief Medical Technologist assumes responsibility for
coordinating everyday activities of the laboratory and communication with the rest of the
staff.
7.0 JOB RESPONSIBILITIES:

7.1 CHIEF PATHOLOGIST
Pathologist has the ultimate responsibility for laboratory test results, the quality and
safety standards of the laboratory and for advising clinicians on the interpretation of test
results and further investigation of the patient.
7.1.1 The primary role of a pathologist is to perform or supervise tests on blood, other
body fluids, body secretions and samples of tissue taken at surgery or as a part of
a medical examination or autopsy.
7.1.2 Overall coordinator for the services, and training programs of the Department.
7.1.3 Responsible for the preparation of budget, laboratory rates, fees and cost analysis
of the different laboratory procedures in consultation with the staff and
Laboratory Administrator
7.1.4 Coordinates with the staff regarding the organizational set-up, work flow and job
description
7.1.5 Recommends to the Laboratory administrator acquisition of new equipment,
setting-up of new laboratory tests and hiring of new laboratory personnel.
7.1.6 Acts as overall head of each laboratory section and institutes quality control
measures for each section
7.1.7 Guides the Medical Technologists in the performance of their duties.
7.1.8 Attends to any problems that may arise in the Department.
7.1.9 Such other duties as may arise from time to time are prescribed by proper
authorities.
AND PROCEDURES

7.2 CHIEF MEDICAL TECHNOLOGIST

A Chief Medical Technologist functions to effectively assist the department head in the
supervision of the activities of the laboratory in accordance with the national standards
and administrative policies of the hospital and to assist in planning, organizing and
conducting program for the department.
7.2.1. Directs and monitors all activities of the unit
7.2.2. Guides the staff in the performance of their duties
7.2.3.Makes requisition and maintains an inventory of laboratory supplies and other
necessary materials for laboratory use
7.2.3.1. Performs monthly and annual census
7.2.4. Formulates standard operating procedures, policies, guidelines, rules and
regulations in the laboratory in consultation with the staff and Pathologist.
7.2.5. Prepares schedule of duties of laboratory staff and schedule of rotation of all
rotatingMedical Technologists in the various sections of the laboratory; a copy of
which shall be posted in the Laboratory bulletin board.
7.2.6. Performs necessary communications affecting the Laboratory:
7.2.6.1. Informs the Department Head about any problems or complaints
encountered in the Laboratory and discusses with them possible
solutions
7.2.6.2. Goes over current literature for latest laboratory procedures and submits it
to the Laboratory Administrator and Department Head for discussions and
approval of demonstrations and lectures for all Medical Technologists.
7.2.6.3. Accomplishes all the necessary documents for renewal of the Department’s
license to operate
7.2.7. Assists the Department Head in preparing and updating the costing of all
laboratoryexaminations
7.2.8. Assumes full responsibility of the Department Head in case of his/ her absence.
7.2.9. Such other duties as may arise from time to time be prescribed by proper
authorities.
7.3 MEDICAL TECHNOLOGIST:
AND PROCEDURES

7.3.1. Responsible for the proper collection, preservation and storage of specimens, and
analysis of specimen.
7.3.2.Advise patients on preparations and procedures to be observed prior to extraction.
7.3.3. Screens and rejects unsatisfactory specimens receive
7.3.4. Processes the completion of routine and special analysis
7.3.5. Responsible for the maintenance and orderliness of the section and Machine/s
7.3.5.1 Performs routine machine start-up and quality control to be used for the
day and record results
7.3.5.2. Establishes and interprets quality control charts, trends and shifts.
7.3.5.3. Perform machine calibration as needed.
7.3.5.4. Performs equipment and reagent evaluation
7.3.6. Double checks specimens with panic and abnormal results and refers them to Chief
Medical Technologist or Department Head for second opinion before releasing
7.3.7. Record-keeping and Documentation
7.3.7.1. Records all results in the proper logbook and signs all official results
before release
7.3.7.2. Keeps records of patients’ results, quality control, and maintenance log
sheet and service report of equipment in the section
7.3.7.3. Checks the reagents and materials needed for the weeks and ensures the
availability of supplies
7.3.7.4. Responsible for the monthly and annual census of the section
7.3.8. Supervises and extends assistance to Junior Medical Technologists and gives
proper endorsement to the next shift.
7.4. PHLEBOTOMIST / LABORATORY TECHNICIAN:
7.4.1. Responsible for the proper collection, preservation and storage of specimen
7.4.2. Observes proper decorum when greeting and accommodating patients and
inquiries
7.4.3. Responsible for receiving specimens from in- and out- patients and releasing
results
7.4.4. Responsible for charging patients.
7.4.5. Responsible for typing communications
7.4.6. In-charge of all forms, receipts and supplies needed by the different sections
AND PROCEDURES

7.4.8. Files examination results, receiving and releasing logbooks

7.4.9. Performs such duties that may be assigned by proper authorities.
7.5. HISTOPATH TECHNICIAN
7.5.1. Responsible for the maintenance and orderliness of the section
7.5.2. Ensures that request forms have been properly filled-out and that specimen/s is /
are properly labelled prior to charging and processing
7.5.3. Properly enters all data of specimen received in the logbook and assigns
histopath /cytology numbers
7.5.4. Stores and files all slides and paraffin blocks after the case has been read and
signed-out
7.5.5. Performs all staining procedures for Histopathology
7.5.6. Performs inventory of all reagents for Histopathology and makes necessary
requests
7.6. LABORATORY AIDE:
7.6.1. Responsible for the cleanliness and orderliness of the department
7.6.2. Washes test tubes, slides and vials
7.6.3. Delivers results of in-patients to the different nurses station
7.6.4. Obtains supplies for the Department.
7.6.5. Performs other duties as from time to time as prescribed by proper authorities
8.0. SCHEDULES AND SHIFTING

8.1. The department shall operate on a 24-hour basis having applying a 12-hour
shifting schedule among Medical Technologist staff andan 8-hour shifting
schedule among Phlebotomists and other personnel.
8.1.1. MEDICAL TECHNOLOGIST STAFF:
8.1.1.1. There shall be two (2) shifting schedule per day (7:00am-7:00pm
and 7:00pm-7:00am).
8.1.1.2 Three (3) Med.Tech. shall function in the AM Shift, this shall
comprise two (2) Med.tech. staffs and one (1) Supervisor staff.
Additional staff shall be dependent on the need and workload and
shall further be subjected to the decision of the supervisor.
AND PROCEDURES

8.1.1.3. One (1) Med.Tech. staff shall function during the PM shift.
Additional staff shall be dependent on the need and workload and
shall further be subjected to the decision of the supervisor.
8.1.1.4. In cases wherein full-time staff is not available, reliever Med.Tech.
may be requested to fill-in the schedule, provided that this
endorsement and changes has been brought upon the attention of
the HR Department, under the permission of the Head of the
department or Supervisor.
8.1.1.5. Supervisor and/or senior staffs should always be available in the
AM shifts for management and supervision of the daily laboratory
processes.
8.1.2. PHLEBOTOMIST/LABORATORY TECHNOLOGIST STAFF:
8.1.2.1. There shall be one (1) staff available every 8-hour shifting starting
from 6:00am onwards.
8.1.2.2. In cases wherein there is a need for additional staff, a phlebotomist
staff may be called upon to extend duty, in which these hours may
accrue as additional day-off or over-time.
8.2. The OPD and Industrial laboratory shall only function on Mondays- Saturdays
starting 7:00am-5:00pm with a cut-off time of 4:30pm to allow enough
processing time and release of results prior to the 5:00pm end. This policy shall
not be absolute, and is dependent on the need of the situation and the decision
of the head or supervisor of the department.
8.3. Other considerations:
8.3.1 Although the OPD laboratory starts at 7:00am, patients who are on fasting
(NPO) state are exempted from this policy and shall be given priority. Patients
may be extracted for examinations requiring fasting starting from 6:00am up to
9:00am ONLY. This is to allow and assure proper collection on a basal body
state.
8.3.2 Over-fasting or under-fasting patients should be advised. In case the patient
insists on blood collection, accomplish a patient consent form.
8.3.3. Time extension shall ONLY be applicable in the Industrial laboratory when five
(5) or more patients arrived for Medical Examination before 5:00pm.
8.3.4. Early cut-off time may ONLY be applied in the Industrial laboratory when:
AND PROCEDURES

8.3.4.1. There is an on-going remote annual medical and the situation requires
additional staff to do field work.
8.3.4.2. The Med. Tech. staff was pulled-out of his assigned department without
possible staff to fill-in in his post.
8.3.5. Laboratory personnel who performed home service extraction is exempted
from daily biometrix time keeping (automated time recording) and is subjected
to use manual daily time records upon approval of the department supervisor.
9.0 ACCOUNTS AND EXTERNAL AFFAIRS
The department supervisor should be the one responsible in assisting and managing
external affairs (Sales representatives, Suppliers, etc.) ONLY to the extent of his duties.
All activities and dealings shall be endorsed to the Department head for approval and to
the Purchasing Department for recommendation. The department shall not transact with
these parties at all cost, it is ONLY the Purchasing department who is allowed and
authorized to conduct purchases and transact business with external parties.
10.0 DEPARTMENTAL MEETINGS
The department shall have scheduled meetings to maintain and monitor service provision
and processes of the laboratory. The department supervisor shall spearhead the meetings
while other laboratory personnel shall be delegated to deliberate/discuss specific agenda.
AGENDA SCHEDULE PERSON-IN-CHARGE

Every first Monday of the
Service Provision Concerns Chief Med. Tech.
month
Industrial Laboratory Every first Tuesday of the Industrial Laboratory
Concerns month Section Head
Every first Wednesday of OPD Laboratory Section
Out-Patient Concerns
the month Head
Pathologists and Chief
Staff Development Program Annually
Med. Tech.
Inter-departmental Pathologists and/or
Whenever deemed needed
Concerns Chief RMT
Inventory and Monthly, every last
Staff Med. Tech.
Consumption Monday of the month
Staff Meeting Weekly, every Friday Staff Med. Tech.
AND PROCEDURES

11.0 DEPARTMENTAL REPORTS

The department should prepare daily, monthly, and annual reports in order to fully assess
and justify its needs and department performance.
REPORT SCHEDULE OF DEADLINE PERSON-IN-CHARGE

Statement of
Every after the eight (8) hour Shift Phlebotomist-on-duty
procedure charges
Inventory of supplies Daily Section Head
Workload/Census Daily Chief Med. Tech.
Over Time forms Daily Chief Med. Tech.
Schedule Per Shift Chief Med. Tech.
Net and Gross Income Daily Chief Med. Tech.
Supply Requisition Monthly Chief Med. Tech.
11.1 Laboratory Result Reports
11.1.1. The reports of laboratory exams are recorded on their respective logbooks,
and (2) copies of results of examinations will be distributed to their
respective nursing unit.
11.1.2. The reports of routine requests will be distributed to nurse on duty of the
different department twice per eight (8) hour shift. Results for STAT or
emergency cases will be forwarded once the result is available or within 1
hour whichever is faster
11.1.3. Upon receiving the laboratory results, the nurse on duty will sign in the
releasing logbook, provided for that purpose wherein reports are attached
to the chart of the patient.
11.2 Recording/Documentation Of Laboratory Results
11.2.1 Record all results in their respective logbooks.
11.2.2. Any calculations, dilutions, conversions, used in obtaining the final results
must be recorded in the working logbook.
11.2.3. Double check all results before releasing and recording.
11.2.4. All final reports are checked and signed by the Medical Technologist who
performed the test.
11.3 Procedure For Reporting Work Load
11.3.1. Completed analysis/test ought to be appropriately transcribed in the
proper result form and specific section logbook.
AND PROCEDURES

11.3.2. Properly accomplished result form must be meticulously reviewed before

releasing to the pertinent ward or department.
11.3.3. Individual person receiving the result is required to print his/her name on
the receiving logbook of the laboratory.
11.3.4. At the end of the day, all analysis tests performed within the day are
recorded and tallied in a census.
11.3.5. The census incorporated all records on a daily, weekly, monthly and
annually tallies.
Guidelines For Record And Specimen Retention
PARTICULAR RECORD TYPE RETENTION

RECORDS Test Records/Logbooks 5 years
Quality Control Chart 2 years
Procedure Manual Indefinite
Instrument Maintenance Indefinite
REPORTS Laboratory Reports 1 year
Pathology Reports 10 years
Cytopathology Reports 10 years
SPECIMEN Serum/Body Fluids 48 hours
Blood Smear 1 week
Cytology Slides 5 years
FNAB Slides 5 years
Paraffin Block Indefinite
Pathology Slides 10 years
Microbiology Smears 1 week
12.0 LICENSING AND ACCREDITATION
As per DOH requirement, the department shall renew its license and comply with the
prerequisites of issuance of license to operate and accreditation. The following stated
below should annually be prepared and accomplished by the department in preparation
for the renewal application:
12.1. Accomplished Renewal Application Form.
12.2. Adequate number on laboratory staff along with their proof of qualification and
proof of employment.
12.3. Records of machine calibration and quality control.
12.4. Reagent Certificate of Registrations and Material Safety Data Sheets.
AND PROCEDURES

12.5. Copy of Laboratory Standard Operating Procedures.

12.6. External Quality Assurance Program Certificates for the required sections of the
laboratory.
13.0 COMPLAINTS AND INCIDENT REPORTS
13.1 Complaints are commonly encountered in the laboratory and laboratory personnel
are prone to patient criticisms. It is the department’s responsibility to employ
maximum tolerance and courtesy at all times in order to gain patrons.
13.2 In the event of any complaints or incidents involving patient concerns, it is every
personnel’s responsibility to inform the department supervisor of the situation as
soon as the problem is encountered. In case the supervisor is not present, the senior
staff may take charge of the situation.
13.3 Whenever complaints or incidents have been encountered a corresponding written
report regarding the case should be accomplished and submitted to the department
supervisor. All complaints reported will be investigated and the personnel involve
shall be subjected to disciplinary actions.
13.4 At any rate a personnel submits complaints against a co-employee a written report
should be filed to the department supervisor. Any complaints involving employees
intrinsic to the work environment shall be referred to the HR department by the
supervisor.
13.5 Reporting And Analysis Of Adverse Events And Incidents
13.5.1. Proper and thorough investigation after gathering the necessary

information directed to the involved staff/personnel. (Name of complainant
and personnel involved, date and time and the details)
13.5.2. Advise the person involved to do the IR (Incident Report).It will be
addressed to the head of the laboratory or her/his associate(s) as soon as
possible, for proper management.
13.5.3. Any inputs from the identified situational analysis should be relayed to the
rest of the staff to convey and enhance proper handling and management of
any similar incidents.
14.0 RELEASE OF RESULTS

14.1. The department shall employ scheduled release of results per type of examination
or section of the laboratory. This policy shall not be absolute and shall be
AND PROCEDURES

subjected to reassessment depending of the department protocols and

status of laboratory processes.
IMMUNOLOGY, SEROLOGY, and CHEMISTRY examinations:
TIME OF TIME OF RELEASE

COLLECTION
6:00 AM- 9:00 AM 11:00 AM
9:00 AM–12:00 PM 2:00 PM
12:00PM- 2:00PM 4:00 PM
3:00PM- 5:00PM 7:00 AM the next working day
14.2. All blood chemistry examination results will be released on the scheduled time,
unless:
14.2.1. Patient requested for the result to be released due to scheduled check-up or
with just reason.
14.2.2. Request is a STAT procedure or the patient is currently in the Emergency
Room and is under observation.
14.2.3. Result is a prerequisite for the patient to be discharged in the hospital.
14.3. HEMATOLOGY examinations:
All hematology examinations will be released within one (1) hour. Stat procedures
shall be given priority and will be released less that one (1) hour or once it is
available, whichever is faster. Unless the request is a special procedure,
results shall be released once the result is available.
14.4. MICROSCOPY examinations:
All microscopy examinations will be released within one (1) hour. Stat procedures
shall be given priority and will be released less that one (1) hour or once it is
available, whichever is faster. Unless the request is a special procedure,
results shall be released once the result is available.
14.5. HIV testing
HIV testing is a special procedure to be performed exclusively by a certified HIV
proficient Medical Technologist. Results should ONLY be released to the
individual itself or the patient’s guardian by the laboratory personnel who
conducted the examination. Strict compliance of patient confidentiality should
always be observed.
AND PROCEDURES

14.6. Patient Confidentiality

Strict implementation of this policy shall be practiced. Results or any other patient
information shall not be divulged unless prior formal endorsement or
authorization letter has been provided by the patient himself, allowing access
to his information. All necessary actions should be endorsed to the
Department Supervisor. In case of current admission, patient results shall not
be relayed thru phone or verbally. All results shall be formally given via its
designated result forms duly signed and verified by the Medical
Technologist.
14.6.1. Require Endorsement or Authorization Letter
14.6.2. Inform Supervisor of the requested information
14.6.3. Log the released copies of results in the Releasing Logbook for
documentation.
14.6.4. File the letter submitted by the requester.
14.7. Delay in Release of Results
In case of any circumstance causing delay of release of results, it is imperative to:
14.7.1. Inform the Patient or Department of possible delay and estimated time
of release of results.
14.7.2. Inform the cause of delay as well as continuously monitor patient
(OPD) and department (IPD) if in case the result is badly or
urgently needed. OPD shall have the right to cancel their request in
case results have been delayed one hour from the scheduled
time of release. In case of urgent tests for admitted patients, the
laboratory department shall have the discretion to refer the test to
another diagnostic laboratory.
14.7.3. To monitor proper time of release, Turn-around time should be noted
in the Patient’s requests and end of processing time should be
recorded in the receiving logbook. The patient’s arrival and end of
procedure shall indicate if target Turn-around time is
accomplished.
14.7.4. Any incidence of delay without just cause shall be discussed in the
weekly staff meeting.
15.0 REQUISITION OF REAGENTS AND SUPPLIES
AND PROCEDURES

15.1. Medical Technologist on duty sees to it that reagents, supplies and others are
adequate for a month’s consumptions.
15.2. All reagents, supplies and others are requested by the Chief Medical Technologist
thru Purchase Requisition Form, indicating the volume, unit cost per kit and the
total cost of the items to be purchased, countersigned by the Purchasing department
for approval.
15.3. Reagents and supplies are being purchased from reputable suppliers.
15.4. Inventory of reagents, supplies is usually done before the end of each month, and the
yearly inventory submitted by the department supervisor to the Accounting
Department for records and checking.
16.0 PERSONNEL POLICIES
Employees are important members of the GMCH family; accordingly, each employee has
certain rights and privileges. In addition, each has responsibilities to the Hospital, to its
Department, and to its fellow employees. The Laboratory Department has prepared this
copy of its Department Personnel Policies, which has been reviewed accordingly with the
Medical and Labor Relations Department Codes.
16.1. HOURS OF WORK/ REST PERIODS
16.1.1.
The standard work shift is twelve (12) hours for Medical Technologists
and eight (8) hours for other personnel.
16.1.2. Meal periods of one (1) hour shall be on a decking schedule to provide
maximum provision of services.
16.1.3. An employee is required to finish eight (8) hours of duty per day, six (6)
times a week; in that case, any additional hours beyond the required hours
of duty shall qualify as Overtime.
16.2. CHANGE OF SHIFT SCHEDULES
16.2.1. Employees shall not trade, change, or work additional shifts without
approval of the department supervisor.
16.2.2. Employees who wish to trade, change or work additional shifts should
formally apply with the required forms.
16.2.3. Employees shall be given two (2) preferred day-off per a 15-day shifting
schedule, provided that the chosen day-off does not affect the whole
AND PROCEDURES

working schedule and does not cause any inconvenience with the
Department.
16.3. OVERTIME
16.3.1. Premium overtime is defined as hours worked in excess of either:

16.3.1.1. Forty-eight (48) hours per week when specified holidays do not
occur and vacation or sick leave is not used.
16.3.1.2. Eight (8) hours per day or ninety-six (96) hours per biweekly
period when specified holidays do not occur and vacation or sick
leave is not used.
16.3.2.
Straight overtime is defined as:
16.3.2.1. Hours worked in excess of part-time appointment and no more
than a regularly scheduled full-time appointment (regularly
scheduled hours)
16.3.2.2. Hours worked on non-specified holidays, if eligible for holiday
pay.
16.3.2.3. Hours worked in excess of: (i) forty-eight (48) hours per week or
(ii)eight paid hours per day or ninety-six (96) hours per
biweekly and sick or vacation leave is used.
16.3.2.4. Straight hours are paid at the straight time rate and also require
prior approval.
16.3.3. Employees will be informed when hired that they may be required to work
for overtime with minimal notice, given the need for adequate staffing of a
hospital at all hours.
16.3.4. The HR Department/ Supervisor must approve all overtime in advance.
Only if a supervisor is unavailable and an emergency arises may an
employee work overtime without prior approval, provided, endorsement
to the necessary key persons be accomplished as soon as possible.
16.3.5. The department supervisor shall be accountable for all overtime and it is
their responsibility to minimize overtime and assign it in an equitable
manner in accordance with the Hospital Policies.
16.4. TARDINESS
16.3.1. It is the Employees responsibility to be at the workstation and ready to

work no later than fifteen (15) minutes after the start of every shift.
AND PROCEDURES

16.3.2.
If tardiness is anticipated, the employee must call the work section as
soon as possible and leave a message with the supervisor indicating
anticipated time of arrival. The employee shall report to the supervisor or
the person in-charge upon arrival.
16.3.3. Excessive or unexcused tardiness of one hundred (100) minutes will
result in a loss of pay equivalent to one (1) working day salary.
16.5. SICK LEAVE
16.5.1. Sick leave accrues according to the GMCH Policies.
16.5.2. Employees are responsible for notifying the department supervisor or the
person-in-charge of their absence due to illness and shall provide a
satisfactory proof of illness from a licensed physician.
16.5.3. The department supervisor or the person-in-charge is to be notified at

least two (2) hours (>2 hours) before the start of work.
16.5.4. The abuse of sick leave, or excessive use of sick leave cay result to
corrective actions up to and including dismissal.
16.5.5. When an employee completely uses all sick leave credits, the employee
may use compensatory time earned or vacation time accrued to cover
illness with prior approval from supervisor and authorizing individuals.
16.5.6. Request for sick leave due to medical or dental appointments for
employees and/or their family members shall be made to the Supervisor
at least one (>1) week in advance. Employees who require numerous
medical or dental appointments shall arrange an appointment schedule
with prior approval.
16.6. DAY-OFF
16.6.1. Day-off for personal reasons shall be requested to the Supervisor at least
one (>1) week in advance. For permanent employees, day-off may be
covered by vacation, compensatory time earned, or leave without pay.
16.7. VACATION LEAVE
AND PROCEDURES

16.7.1. Vacation leave accrues according to GMCH Policies.
16.7.2. The Supervisor shall schedule vacation at the convenience of the

Department. Vacation shall be requested fro the Supervisor at least one
(>1) month in advance.
16.7.3. The Supervisor shall approve vacation leave based on seniority. The
Supervisor is responsible for ensuring an equitable distribution of
preferred vacation assignments.
16.7.4. Request for change of vacation schedules may be approved if there is no

negative impact upon the Department.
16.7.5. At any rate, an employee may recommend a Reliever during vacation

leave to fill-in the vacant schedules, provided, that the reliever is well and
capable of all the procedures and services offered by the Department.
16.8. LEAVE OF ABSENCE/ LEAVE WITHOUT PAY
16.8.1. All leave must be requested and granted in writing. Any change of the
time or schedule must also be requested and granted in writing.
16.9. TIME KEEPING
16.9.1. It is the employee’s responsibility to enter the required payroll system

transactions to record the hours worked on each schedule day. If an
employee is absent, the Supervisor will enter the appropriate remarks for
that scheduled day. Should an employee notice a problem with the
recorded hours of work or fail to register in the automated log system,
notify the supervisor or the person-in-charge as soon as possible.
16.9.2. The Department Supervisor shall keep for each employee a current
record of sick and vacation leave, and compensatory time accrued and
taken. Overtime worked should also be kept in record and shall be paid
on a biweekly basis.
AND PROCEDURES

16.10. COMPLIANCE, LICENSURE, AND TESTING
16.10.1. Only appropriate licensed personnel are authorized to review and verify
analytic results, perform calibration procedures and clinical laboratory
tests, and examinations classified as high complexity under DOH, NCCLS,
etc. standards.
16.10.2. Unlicensed personnel are allowed to enter patient demographic data,

order and accession test procedures, verify specimen collection, accession
and transcribe patient results. Performing and verification of clinical
laboratory tests are not allowed.
16.10.3. The transcription and computer entry of analytical results by unlicensed
personnel are limited to results that have been previously reviewed and
verified with signature release, by appropriate licensed personnel.
16.11. COMPUTER USE AND SECURITY
16.11.1. General Policies:
16.11.1.1. Any patient data or records are confidential and may not be
revealed to unauthorized persons, agencies, or
institutions without written consent of the patient. Any
questions or concerns regarding authorization or release of
patient information should be brought to the attention of the
Department Supervisor.
16.11.1.2. GMCH computer system contains confidential medical record

and patient account information that is only accessible to
authorized users. It is the laboratory personnel’s responsibility
as an authorized user to protect the confidentiality of patient
information and data to which he/she has access.
16.11.1.3. The accessing of patient data is restricted to only what is

required to perform job duties.
16.11.1.4. Any record with patient information or data is to be disposed
of in the appropriate waste container.
AND PROCEDURES

16.11.1.5. There shall be no discussion of any patient information in

public areas.
16.11.1.6. Computer login and password codes are for personal use only
and are not to be given to others for use.
16.11.1.7. Every personnel is responsible for maintaining the integrity of
the computer system. Any knowledge of the altering, deleting,
damaging, or destroying of a computer’s data, program, system,
or network is to be brought to the attention of a supervisor or
computer staff.
16.11.1.8. The computer is not for personal use, nor for the conducting of
personal electronic mail (i.e., e-mail) messages, or accessing
the World Wide Web (e.g. "surfing the web") for information
that is not work related.
16.11.2. Laboratory Information System:
16.11.2.1. The LIS is considered a component of GMCH Laboratory
Information System and only accessible to authorized
employees only.
16.11.2.2. The LIS contains patient data and laboratory results that are
part of the patient's medical records. It is therefore laboratory
personnel’s responsibility as an authorized user of the LIS to
protect the confidentiality of patient information and data
including laboratory results.
16.11.2.3. Because LIVE patient data is in the TEST side of MYSIS, staff
should be careful when selecting patients for purposes of
testing. Use obvious ‘patients’ that are specifically created for
testing purposes (i.e. “TEST patient”). Avoid using LIVE
patients when testing.
16.11.2.4. The MYSIS Mail feature is NOT for personal use, nor for
conducting communications or messages that are personal in
nature. Employees are to restrict the use of Internet Mail for
work and communication of work and laboratory related
information only. Adherence to this policy will be enforced and
repeat offenders will be subject to disciplinary action.
16.11.2.5. Only laboratory supervisors or other individuals with specific
authorization are allowed to change a result once entered and
verified (Note: this does not alter the "paper trail" of a
corrected result), submit patient charges and credits, modify
AND PROCEDURES

laboratory system tables or programs, or perform system

administrative procedures.
16.11.2.6. The use of the laboratory computer related to the testing,
crossmatching, and issuing of blood or blood products is
restricted to the staff of the Blood Bank/Donor Center and
other specifically authorized licensed individuals (e.g., CLS).
16.12. PUBLIC IMAGE
16.12.1. Frequent or constant contact with patients, physicians, and non-laboratory
employees places an important obligation upon the Clinical Laboratories
to present the hospital and laboratories in a professional manner.
16.12.2. Employees are expected to conduct themselves in a professional manner
with patients, visitors, physicians, other hospital personnel, co-workers
and supervisors.
16.12.3. Employees are expected to be courteous when addressing staff, patients,
and visitors directly or on the phone. Telephones should be answered
“Clinical Laboratories (or section name). This is....speaking. May I help
you?”
16.12.4. An Employee’s personal appearance must reflect professionalism
16.12.5. Personnel with patient contact must wear a buttoned lab coat or
appropriate uniform.
16.12.6. GMCH photo identification I.D. MUST be worn above the waist and must be
clearly visible.
16.12.7. The dress code applies to all employees and must be neat, clean and
appropriate for the work place. Per hospital policy, unacceptable dress
includes items such as sheer garments, halter or tank tops, oversized or
baggy garments, garments designed to be worn as athletic gear, such as
leggings or spandex pants. Blue jeans, and apparel containing phrases or
pictures unrelated to the professional environment of the Medical Center
are also unacceptable. Shoes must be safe, clean, in good repair and
appropriate for the work to be performed. Thongs and bare feet are not
acceptable.
16.12.8. A professional environment must be maintained. Radios should not be
played at high volume and must not be audible to telephone callers. Radios
should not be played in any area open to the public or where direct
interaction with non-laboratory personnel such as physicians and nurses
takes place (e.g., at Central Processing). Personal headsets related to
Walkman and similar devices may not be worn on the job except during
AND PROCEDURES

break periods. TV’s may not be used in any laboratory area except as part
of an educational training activity sponsored by the laboratory.
17.0 MAINTENANCE OF PHYSICAL PLANT AND EQUIPMENT
17.1. Regular check-up of the physical plant of the laboratory such as

ventilation, lighting, electrical, water supply will be checked by the
hospital’s in house maintenance personnel to ensure continuous workflow
in the laboratory.
17.2. The department shall have a separate logbook for its preventive
maintenance records to be accomplished by the house maintenance
personnel.
Program For Calibration, Preventive Maintenance And Repair For The Equipment
Facility Monitoring Visit

Aircon Monthly
Light Source Semi-Annual
Electrical Wirings Annually
Sockets and Outlets Semi-Annually
Water Pipes Annually
Sink Annually
Faucets Semi-Annual or as needed
17.3. To make sure that the performance of all equipment are in accordance
with the required specifications defined by the facility. Schedule for
equipment monitoring, calibration and maintenance are established and
AND PROCEDURES

followed. Proper documentation, record maintenance of all repairs,

monitoring and calibration must be reserved and periodically reviewed.
17.4. Maintenance of equipment is performed by the Medical Technologists on a

daily basis, which includes cleaning and removing off of dirt on machines.
17.5. Running of normal and abnormal controls are also done to check
calibration of machine to ensure accurate results.
17.6 Quarterly and annual maintenance of equipment should be requested to

and performed by the company where the machine is bought.
17.7. All controls, status checking are recorded in appropriate logbooks for
record keeping.
17.8. All problems encountered with regards to performance of the machine

should be reported immediately to the immediate supervisor and is
recorded on a Preventive Maintenance Sheet.
17.9. Temperature Monitoring of the laboratory refrigerator must be recorded

to monitor proper preservation of reagents.
17.10. The Medical Technologist should monitor environment temperature to

ensure good working conditions of machines.
17.11. Safety precautions must be established, posted and imposed.

Schedule Of Maintenance Service And Repair
Equipment Description
COBAS C111 1. Monthly maintenance/service
2. Calibration every six months.
3. Records of maintenance, service and repair will be kept in a
folder specific for Cobas C111 only.
AND PROCEDURES

SYSMEX X5-800i 1. Monthly maintenance/service

folder specific for Sysmex X5-800i only.
SYSMEX KX-21 1. Monthly maintenance/service
folder specific for Sysmex KX-21 only.
SYSMEX CA-20 1. Monthly maintenance/service
folder specific for Sysmex CA-20only.
BLOOD BANK 1. Quarterly.
REFRIGARATOR 2. Calibration every 6 months.
3. Weekly cleaning.
folder specific for Blood bank refrigerator.
REFRIGARATORS 1. Quarterly.
2. Calibration every 6 months.
3. Weekly cleaning.
folder specific for Refrigerators only.
CLINICAL 1. Monthly maintenance/service
CENTRIFUGES 2. Annual calibration.
folder specific for clinical Centrifuge only.
UF- 500i 1. Monthly maintenance/service
folder specific for UF-100i only.
NIKON AND 1. Annual maintenance/service.
OLYMPUS 2. Daily cleaning.
MICROSCOPES 3. Annual calibration.
folder specific for microscopes.
AND PROCEDURES

INCUBATOR 1. Monthly maintenance/service

2. Annual calibration.
folder specific for Incubator only.
HOT AIR OVEN 1. Monthly maintenance/service
2. Annual calibration.
Laboratory Maintenance
3. Records of maintenance,Schedule
service and repair will be kept in a
folder specific for Hot Air Oven only.
Procedure/Task Schedule Personnel
HAIER BIOSAFETY 1. Monthly maintenance/service
CABINET 2.Slides
Washing of Test tubes and Calibration every six months. RMT/PHLEBOTOMIST
DAILY
Garbage disposal (Noninfectious) DAILYfor Biosafety Cabinet
folder specific RMT/PHLEBOTOMIST
only.
MICROHEMATOCRIT 1. Monthly maintenance/service
Garbage Disposal RMT c/o Disposal
CENTRIFUGES 2. Annual calibration.
(Infectious/Sharps) 3. Records ofDAILY
maintenance, serviceCompany
and repair will be kept in a
folder specific for Microhematocrit
RMTCentrifuge
c/o only.Disposal
Disposal of Specimens WEEKLY Company
Disposal of Reagents (Expired, RMT c/o Disposal
Contaminated, etc.) WHEN NEEDED Company
Cleaning f Trash cans/bins WEEKLY MAINTENANCE
Cleaning of Cabinets WHEN NEEDED RMT/MAINTENANCE
Cleaning of Sink DAILY RMT
Cleaning of Windows WEEKLY MAINTENANCE
Mapping of Floor DAILY MAINTENANCE
EVERY AFTER
Cleaning of Counter tops SHIFT RMT
Cleaning of Refrigerator MONTHLY MAINTENANCE
Monitoring Supplies DAILY RMT
EVERY END OF
Checking Reagent Expiration THE MONTH RMT
Prepared by:Temperature Monitoring Reviewed by: Divina
Rolan Dela Rosa, M.D. DAILY
Rojas, M.D. RMT
Approved by: Jerrimo Genuino, M.D.
Defrosting of refrigerator WHEN NEEDED RMT
AND PROCEDURES

18.0 CONTINGENCY PROCEDURES
18.1. EQUIPMENT BREAKDOWN (SECTION MACHINES)

Minor troubleshooting shall be made by the medical technologist on duty if
troubleshooting was unsuccessful refer to the service technician. If still
unsuccessful, tests are then sent out to the nearest hospital or laboratory with a
Memorandum of Agreement. (This is until a back-up machine is made available).
18.1.1. Information Dissemination
The laboratory shall be responsible for informing all concerned
departments (i.e. NSO) regarding the machine breakdown. As soon as the
machine operation has been restored, the laboratory should immediately
inform the concerned departments.
18.1.2. Back-up machine
In lieu of the machine breakdown, back-up machines are readily available so
as not to affect the flow of the specimen.
18.1.3. Record Keeping
RMT should write an incident report regarding the machine breakdown in
the Maintenance Logbook. The report should contain pertinent information
regarding the incident that happened. RMT should also compile the Service
Report given by the service engineers as part of the incident report.
18.2. MICROHEMATOCRIT AND CLINICAL CENTRIFUGE
If breakdown of primary Centrifuge happens, there is still one back-up centrifuge
unit available for use.
18.3. HOT AIR OVEN AND INCUBATOR
If breakdown of the Hot air oven happens, immediately refer the problem to the
service technician for repair of machine and report the need for back-up machine.
18.4. MICROSCOPE
Minor troubleshooting by the medical technologist on duty, if troubleshooting was
unsuccessful refer to the service technician. If the breakdown happens there is still
one back-up microscope unit available.
18.5. NO REAGENTS AVAILABLE
In cases wherein stocks and consumption of reagents are not properly monitored,
the Medical Technologist on duty calls the Reference laboratory to inform them of
Send-out orders. Emergency purchase order should be accomplished by the Medical
Technologist to the supplier.
18.6. POWER FAILURE
AND PROCEDURES

Every machine in the Laboratory should be equipped with UPS to provide enough
electricity to finish the test being performed and to allow proper shut down of the
machine. Daily checking of UPS should be done and charging of the battery should
be made regularly.
18.7. PUNCTURE WOUNDS, CUTS AND ABRASIONS
The affected individual should remove protective clothing, wash the hands and any
affected area(s), apply an appropriate skin disinfectant, and seek medical attention
as necessary. The cause of the wound and the organisms involved should be
reported, and appropriate and complete medical records kept.
18.8. INGESTION OF POTENTIALLY INFECTIOUS MATERIAL
Protective clothing should be removed and medical attention sought. Identification
of the material ingested and circumstances of the incident should be reported, and
appropriate and complete medical records kept.
18.9. POTENTIALLY INFECTIOUS AEROSOL RELEASE (OUTSIDE A BIOLOGICAL
SAFETY CABINET)
All persons should immediately vacate the affected area and any exposed persons
should be referred for medical advice. The laboratory supervisor and the biosafety
officer should be informed at once. No one should enter the room for an appropriate
amount of time (e.g. 1 h), to allow aerosols to be carried away and heavier particles
to settle. If the laboratory does not have a central air exhaust system, entrance
should be delayed (e.g. for 24 h). Signs should be posted indicating that entry is
forbidden. After the appropriate time, decontamination should proceed, supervised
by the biosafety officer. Appropriate protective clothing and respiratory protection
should be worn.
18.10. BROKEN CONTAINERS AND SPILLED INFECTIOUS SUBSTANCES
Broken containers contaminated with infectious substances and spilled infectious
substances should be covered with a cloth or paper towels. Disinfectant should then
be poured over these and left for the appropriate amount of time. The cloth or paper
towels and the broken material can then be cleared away; glass fragments should be
handled with forceps. The contaminated area should then be swabbed with
disinfectant. If dustpans are used to clear away the broken material, they should be
autoclaved or placed in an effective disinfectant. Cloths, paper towels and swabs
used for cleaning up should be placed in a contaminated-waste container. Gloves
should be worn for all these procedures. If laboratory forms or other printed or
written matter are contaminated, the information should be copied onto another
form and the original discarded into the contaminated-waste container.
AND PROCEDURES

Breakage of tubes containing potentially infectious material in centrifugesnot having

sealable bucketsIf a breakage occurs or is suspected while the machine is running,
the motor should be switched off and the machine left closed (e.g. for 30 min) to
allow settling. If a breakage is discovered after the machine has stopped, the lid
should be replaced immediately and left closed (e.g. for 30 min). In both instances,
the biosafety officer should be informed.
Strong (e.g. thick rubber) gloves, covered if necessary with suitable disposable
gloves, should be worn for all subsequent operations. Forceps, or cotton held in the
forceps, should be used to retrieve glass debris.
All broken tubes, glass fragments, buckets, trunnions and the rotor should be placed
in a noncorrosive disinfectant known to be active against the organisms concerned.
Unbroken, capped tubes may be placed in disinfectant in a separate container and
recovered.
The centrifuge bowl should be swabbed with the same disinfectant, at the
appropriate dilution, and then swabbed again, washed with water and dried. All
materials used in the clean-up should be treated as infectious waste.
18.11. BREAKAGE OF TUBES INSIDE SEALABLE BUCKETS (SAFETY CUPS)
All sealed centrifuge buckets should be loaded and unloaded in a biological safety
cabinet. If breakage is suspected within the safety cup, the safety cap should be
loosened and the bucket autoclaved. Alternatively, the safety cup may be chemically
disinfected.
18.12. FIRE AND NATURAL DISASTERS
Fire and other services should be involved in the development of emergency
preparedness plans. They should be told in advance which rooms contain potentially
infectious materials. It is beneficial to arrange for these services to visit the
laboratory to become acquainted with its layout and contents.
After a natural disaster, local or national emergency services should be warned of

the potential hazards within and/or near laboratory buildings. They should enter
only when accompanied by a trained laboratory worker. Infectious materials should
be collected in leak proof boxes or strong disposable bags. Biosafety staff on the
basis of local ordinances should determine salvage or final disposal.
18.13. EMERGENCY SERVICES: WHOM TO CONTACT
AND PROCEDURES

The telephone numbers and addresses of the following should be prominently

displayed in the facility:
18.13.1. The institution or laboratory itself (the address and location may not be
known in detail by the caller or the services called)
18.13.2. Director of the institution or laboratory
18.13.3. Laboratory supervisor
18.13.4. Biosafety officer
18.13.5. Fire services
18.13.6. Hospitals/ambulance services/medical staff (names of individual clinics,
departments, and/or medical staff, if possible)
18.13.7. Police
18.13.8. Medical officer
18.13.9. Responsible technician
18.13.10. Water, gas and electricity services.
18.14. EMERGENCY EQUIPMENT
The following emergency equipment must be available:
18.14.1. First-aid kit, including universal and special antidotes
18.14.2. Appropriate fire extinguishers, fire blankets
18.14.3. Full protective clothing
18.14.4. Full-face respirators with appropriate chemical and particulate filter
canisters
18.14.5. Room disinfection apparatus, e.g. sprays and formaldehyde vaporizers
18.14.6. Stretcher
18.14.7. Tools, e.g. hammers, axes, spanners, screwdrivers, ladders, ropes
18.14.8. Hazard area demarcation equipment and notices.
19.0 TOTAL QUALITY MANAGEMENT
19.1. On Quality Assurance Program
Quality assurance should be maintained in the laboratory thus internal and external
evaluation program must be established and performed.
AND PROCEDURES

Medical Technologist responsible for each section ought to keep all proper
documentation of internal quality assessment perform, likewise participation of the
institution’s laboratory for quality laboratory performance should be a continuous
program aside from the external quality assessment conducted by the National
Reference Laboratory, SACCL, EAMC, PNLC, NKTI and RITM.
19.2. Quality Control Measures Of Machines And Equipment
19.2.1. Automated daily maintenance of machines.

19.2.2. Calibration summary.
19.2.3. Strict quality control observance.
19.2.4. Regular visit of Professional Service Engineer to ensure that machines are
working properly.
19.2.5. Monitoring of machines and equipment by the users to detect any change
in the performance of machines.
19.2.6. Placing the procedural manuals in a conspicuous place for easy
consultation as the need arises.
19.2.7. Proper documentation of maintenance because a number of medical
technologist will be using machines and equipment. It is important to
document all maintenance in order to facilitates troubleshooting and to
have a clear idea of the problem areas for a particular machine or
equipment.
19.3. Indicators Of Quality Performance
19.3.1. Internal Quality Performance
19.3.1.1. Chemistry
AND PROCEDURES

19.3.1.1.1. Reagents are run with the inclusion of Level 1 and Level 2
controls to assist the medical technologist in monitoring
the accuracy and precision of clinical assay.1.1.
19.3.1.1.2. The values of Level 1 and Level 2 controls are listed in the
quality control files. They are then computed and graphed to
know the accuracy and reliability of the reagents as
well as the Medical Technologist running the Chemistry
examinations.
19.3.1.1.3. A quality control chart or graph for every Chemistry test is

performed on daily basis.
19.3.1.2. Hematology
19.3.1.2.1. Calibrators and controls are being run to check the working
status of the machine.
19.3.1.2.2. A quality control graph is stored in the machine and can be

printed to check the validity of the reagents and if the
machine is working properly.
19.3.1.2.3. For manual testing of CBC and other hematological

examinations, the reagents are checked upon delivery, lot no.
of the reagents and its expiration date to ensure reagents
stability.
19.3.1.3. Clinical Microscopy
Reagent strips are properly stored, when in use, they are capped
and covered to prevent possible contamination. Checking
of quality of reagents shall be made prior to testing.
AND PROCEDURES

19.3.1.4. Blood Bank
Strict monitoring of bloodstocks should be imposed and

properly documented. Temperature Monitoring should be
fully accomplished to ensure proper storage. Equipment
for storage should be calibrated and preventive
maintenance should be accomplished.
19.3.1.5. Bacteriology
Control measures for the bacteriology equipment should be

properly implemented. Proper sterilization and storage
should be practiced so as to maintain quality of testing and
prevent nosocomial infections.
19.3.2. External Quality Performance
19.3.2.1. Philippine Council for Quality Control Assurance in Clinical

Chemistry (NEQAS)
19.3.2.2. Department of Health – National Center for Health Facility –

External Quality Assessment in Clinical Chemistry (EQAs)
19.3.2.3. Philippine Council for Quality Assurance in Hematology (NEQAS)
20.0 POLICY ON CONTINUING PROGRAM ON STAFF DEVELOPMENT
Training programs are an important management resource for assisting Departments in

increasing staff competency, administering programs more effectively, and assuring high
quality of health provision. The overall objective of the training program is to assists in the
achievement of organizational goals and objectives.
In line with this, as a PAMET (Philippine Association of Medical Technologist) member, it is

a requirement to attend symposiums/seminars at least once a year. The management
should shoulder registration where appropriate fees and other sponsorship program are
under budgetary allocations. It is believed that through continuing education, professional
AND PROCEDURES

growth and development of laboratory medicine, professionalism and ethical conduct are
being served.
20.1 The Laboratory Department shall make provision for training activities designed to
meet employee needs including but not limited to the following:
20.1.1. Preparing for newly assigned job duties.

20.1.2. Expanding knowledge and understanding of their job’s subject field.
20.1.3. Providing knowledge and understanding of new and changing ideas.
20.1.4. Remaining current on program changes, new programs, and other
subject areasrelated to their duties and responsibilities
20.2. The Laboratory Department shall provide for an Annual Training Program for its
personnel. GMCH financial participation shall be available only for those training
costs specified by the training program.
20.3. The Pathologist and the associates are in charge for the approval of all the trainings
needed by the medical technologist/s to uphold quality health care services. The
request for the training needed by the Medical Technologist will be sent to the
Medical Director through the recommendation of the Pathologist/s. The Medical
Director is in charge to screen and approve the requested training needed. The
approval will be forwarded to the Finance Department to give necessary budget for
the seminar.
20.4. The training cost identified are subjected to reimbursement as Staff development
Costs provided that the following are met:
20.4.1.
Attendees of the training program has successfully finished the required
hours of training, provided further, that the attendees make available or
present training certificates, certificate of participation, or such, as proof
that they have indeed completed the training.
20.4.2. Declaration of costs and expenses should be applied for reimbursement
seven (7) working days after the said training attended.
21.0 DEVELOPMENT OF TRAINING PROGRAM:
21.1. Laboratory Department shall design, and utilize at least annually, processes to
assess training needs. The Laboratory department shall select Needs Assessment
processes that may include but are not limited to:
o Questionnaires to all personnel designed to cover all major training need areas
AND PROCEDURES

o Departmental Meetings structured to reveal personnel or other training needs

o Internal studies of persistent department problems
o Analyses of required skills d knowledge of job functions compared with analyses
of the skill and knowledge of staff currently holding such jobs
o Individual or unit employee performance report
o Measurement of program or service delivery effectiveness
o Surveys of service recipients or inter-department groups
21.2. Training needs assessment should be conducted to result in statements of training
needs which:
o Reflect the perception of more than one organizational level of the department.
o Are related to organizational goals and objectives.
o Are described in terms of expected impact on the organizations, the individual or
the program.
22.0 ANNUAL TRAINING PROGRAM
By January of each year, the Department shall file an Annual Training Program which shall
cover the fiscal year and which adheres to the definitions, guidelines, policies and direction
contained in these regulations.
The Chief and Head of the Department shall cooperate in preparation of the program,
which shall include at least the following information:
o A statement of training goals and objectives for the next fiscal year, which relates
to the goals and objectives of the various program components.
o A copy of the department’s organizational chart.
o A budget and staff description, which differentiates between full- and part- time
training employees and training assignments, which regard the following:
 Total number of personnel
 Itemized budget for all staff development and training costs
o Training needs assessment, including descriptions of the following:
o Processes used in the annual training needs assessment.
o Identified needs and the methods by which the department plans to meet them.
23.0 PERSONAL TRAINING REQUISITION
AND PROCEDURES

An employee may personally apply for Training Leave provided that the training/seminar
requested is relevant to their job description, provided further that the department shall
not suffer any difficulties or complications whatsoever in the absence of the employee. The
hospital shall have the right to decide whether or not to carry/pay for Training expenses of
its Employees, not unless both parties agreed upon certain terms of conditions.
23.1. A laboratory personnel is eligible/qualified for Training without pay when:
a. Holding a Probationary position in the company.

b. Willing to accept terms/ contract in exchange of any trainings paid for by the
company.
c. Request for Training Leave is properly accomplished, endorsed and is approved.
23.2. A laboratory personnel is eligible/qualified for Training with pay when:
a. Holding a permanent position in the company.

b. Willing to accept terms/ contract in exchange of any trainings paid for by the
company.
c. Request for Training Leave is properly accomplished, endorsed and is approved.
LABORATORY GUIDELINES
PURPOSE
This is to establish a documented procedure in the Department of Clinical Laboratory to

ensure that the processing of the laboratory examinations to the clients is done according to
the standards.
SCOPE
AND PROCEDURES

This procedure covers the processes involved in doing the laboratory examinations to the
clients.
1.0 GENERAL GUIDELINES

1.1. PRE-ANALYTICAL PHASE
Preanalysis refers to all the complex steps that must take place before a sample can
be analyzed. Preanalytic factors include patient-related variables (diet, age, sex, etc.),
specimen collection and labeling techniques, specimen preservatives and
anticoagulants,specimen transport, and processing and storage. Potential sources of
error or failure in this process include improperly ordered tests, sample
misidentification, improper timing, improper fasting, improper anticoagulant/blood
ratio, improper mixing, incorrect order of draw, and hemolyzed or lipemic
specimens. The most frequent preanalytic errors include improperly filling the
sample tube, placing specimens in the wrong containers or preservatives, and
selecting the incorrect test. The following are some of the variables that may be
encountered in the laboratory.
1.2. PRE-COLLECTION VARIABLES
In preparing a patient for phlebotomy, care should be taken to minimize physiologic

factors related to activities that might influence laboratory determinations. These
include diurnal variation, exercise, fasting, diet, ethanol consumption, tobacco
smoking, drug ingestion and posture.
Diurnal variation.This may be encountered when testing for hormones, iron, acid
phosphatase, and urinary excretion of most electrolytes such as sodium, potassium,
and phosphate.
Exercise.Physical activity has transient and long-term effects on laboratory

determinations. Transient changes may include an initial decrease followed by an
increase in free fatty acids, and lactate may increase by as much as 300%. Exercise
AND PROCEDURES

may elevate creatine phosphokinase (CK), aspartate aminotransferase (AST), and

lactate dehydrogenase (LD), and may activate coagulation, fibrinolysis, and platelets.
These changes are related to increased metabolic activities for energy purposes and
usually return to preexercise levels soon after exercise cessation. Long- term effects
of exercise may increase CK, aldolase, AST, and LD values. Chronic aerobic exercise is
associated with lesser increases in plasma concentration of muscle enzymes such
as CK, AST, alanine aminotransferase (ALT), and LD. Decreased levels of serum
gonadotropin and sex steroid concentrations are seen in long-distance athletes
while prolactin levels are elevated.
Diet.An individual’s diet can greatly affect laboratory test results. The effect is
transient and is easily controlled. Glucose and triglycerides, absorbed from food,
increase after eating. After 48 hours of fasting, serum bilirubin concentrations may
increase. Fasting for 72 hours decreases plasma glucose levels in healthy women
to 45 mg/dL (2.5 mmol/L), while men show an increase in plasma triglycerides,
glycerol, and free fatty acids, with no significant change in plasma cholesterol.
When determining blood constituents such as glucose, triglycerides, cholesterol, and
electrolytes, collection should be done in the basal state. Eating a meal, depending
on fat content, may elevate plasma potassium, triglycerides, alkaline
phosphatase, and 5-hydroxyindoleacetic acid (5-HIAA). Stool occult blood tests,
which detect heme, are affected by the intake of meat, fish, iron, and
horseradish, a source of peroxidase, causing a false-positive occult blood reaction.
Physiologic changes may include hyperchylomicronemia, thus increasing turbidity
of the serum or plasma and potentially interfering with instrument readings.
Stress.Mental and physical stresses induce the production of adrenocorticotropic

hormone (ACTH), cortisol, and catecholamines. Total cholesterol has been reported
to increase with mild stress, and HDL cholesterol to decrease by as much as 15%.
Hyperventilation affects acid-base balance and elevates leukocyte counts, serum
lactate, or free fatty acids.
AND PROCEDURES

Posture.Posture of the patient during phlebotomy can have an effect on various

laboratory results. An upright position increases hydrostatic pressure, causing a
reduction of plasma volume and increased concentration of proteins. Albumin and
calcium levels may become elevated as one changes position from supine to upright.
Elements that are affected by postural changes are albumin, total protein, enzymes,
calcium, bilirubin, cholesterol, triglycerides, and drugs bound to proteins. Incorrect
application of the tourniquet and fist exercise can result in erroneous test results.
Using a tourniquet to collect blood to determine lactate concentration may result in
falsely increased values. Prolonged tourniquet application may also increase serum
enzymes, proteins, and protein-bound substances, including cholesterol, calcium,
and triglycerides, as the result of hemoconcentration when plasma water leaves the
vein because of back pressure. After bed rest in the hospital, a patient’s hemoglobin
(Hb) can decrease from the original admitting value enough to falsely lead a
physician to suspect internal hemorrhage or hemolysis. This effect can be amplified
by intravenous fluid administration. Patients should be advised to avoid changes in
their diet, consumption of alcohol, and strenuous exercise 24 hours before having
their blood drawn for laboratory testing.
Age.Age of the patient has an effect on serum constituents. Young defines four age
groups: newborn, childhood to puberty, adult, and elderly adult (Young, 2001). In
the newborn, much of the Hb is Hb F, not Hb A, as seen in the adult. Bilirubin
concentration rises after birth and peaks at about 5 days. In cases of hemolytic
disease of the fetus and newborn (HDFN), bilirubin levels continue to rise. This often
causes difficulty in distinguishing between physiologic jaundice and HDFN. Infants
have a lower glucose level than adults because of their low glycogen reserve. With
skeletal growth and muscle development, serum alkaline phosphatase and
creatinine levels, respectively, also increase. The high uric acid level seen in a
newborn decreases for the first 10 years of life, then increases, especially in boys,
until the age of 16 (Young, 2001). Most serum constituents remain constant during
adult life until the onset of menopause in women and middle age in men. Increases
of about 2 mg/dL (0.05 mmol/L) per year in total cholesterol and 2 mg/dL (0.02
mmol/L) per year in triglycerides until midlife have been reported. The increase in
AND PROCEDURES

cholesterol seen in postmenopausal women has been attributed to a decrease in

estrogen levels. Uric acid levels peak in men in their 20s but do not peak in women
until middle age. The elderly secrete less triiodothyronine, parathyroid hormone,
aldosterone, and cortisol. After age 50, men experience a decrease in secretion rate
and concentration of testosterone and women have an increase in pituitary
gonadotropins, especially follicle-stimulating hormone (FSH).
Gender. After puberty, men generally have higher alkaline phosphatase,

aminotransferase, creatine kinase, and aldolase levels than women; this is due to the
larger muscle mass of men. Women have lower levels of magnesium, calcium,
albumin, Hb, serum iron, and ferritin. Menstrual blood loss contributes to the lower
iron.
1.3. COMMON INTERFERENCES
1.3.1. Tobacco Smoking
Tobacco smokers have high blood carboxyhemoglobin levels, plasma

catecholamines, and serum cortisol. Changes in these hormones often result in
decreased numbers of eosinophils, while neutrophils, monocytes, and plasma fatty
free acids increase. Chronic effects of smoking lead to increased Hb concentration,
erythrocyte (RBC) count, MCV, and leukocyte (WBC) count. Increased plasma levels
of lactate, insulin, epinephrine, and growth hormone and urinary secretion of 5-
HIAA are also seen. Vitamin B12 levels may be substantially decreased and have
been reported to be inversely proportional to serum thiocyanate levels. Smoking
also affects the body’s immune response. Immunoglobulin (Ig)A, IgG, and IgM are
lower in smokers, and IgE levels are higher. Decreased sperm counts and motility
and increased abnormal morphology have been reported in male smokers when
compared with nonsmokers.
1.3.2. Collection-Associated Variables
On occasion, when there is a problem finding a vein for phlebotomy, the specimen
may be hemolyzed as the result of sheer forces on the red blood cells. Hemolysis can
AND PROCEDURES

also be caused by using a needle that is too small, pulling a syringe plunger back too
fast, expelling the blood vigorously into a tube, shaking or mixing the tubes
vigorously, or performing blood collection before the alcohol has dried at the
collection site. Hemolysis is present when the serum or plasma layer is pink.
Hemolysis can falsely increase blood constituents such as potassium, magnesium,
iron, LD, phosphorus, ammonium, and total protein.
Because of the extremely important role of potassium in cardiac excitation,

elevations due to hemolysis can be problematic, especially for emergency room
patients who are at risk of hemolysis during frantic blood collection.Even with no
hemolysis, the range of potassium concentrations can be broad in a combination of
healthy and sick individuals. Low levels of hemolysis cause only minor elevations,
but very strong hemolysis can raise the potassium level by 2 to 3 mEq/L into a
critical range.
Another special case where pseudohyperkalemia can occur is in patients with

extremely high blast counts in acute or accelerated phase leukemias. Those blasts
can be fragile and may lyse during standard phlebotomy, releasing potassium. In
contrast, specimens with very high WBC counts that are collected gently can show
pseudohypokalemia when potassium is taken up by highly metabolically active
leukemic cells along with glucose; such specimens can be transported on ice to slow
this enzymatically mediated uptake.
Normally platelets release potassium during clotting, so serum has a slightly higher
value of potassium than plasma from the same individual; this difference is
accentuated when the platelet count is extremely elevated.
To avoid problems with hemoconcentration and hemodilution, the patient should be

seated in a supine position for 15 to 20 minutes before the blood is drawn. Extended
application of the tourniquet can cause hemoconcentration, which increases the
concentrations of analytes and cellular components. When blood collection tubes
that contain various anticoagulants/additives are used, it is important to follow the
proper order of draw and to thoroughly mix an anticoagulated tube of blood after it
AND PROCEDURES

has been filled. Failure to mix a tube containing an anticoagulant will result in failure
to anticoagulate the entire blood specimen, and small clots may be formed.
Erroneous cell counts can result. If a clot is present, it may also occlude or otherwise
interfere with an automated analyzer. It is very important that the proper
anticoagulant be used for the test ordered. Using the wrong anticoagulant will
greatly affect the test results.
Each collection tube containing an anticoagulant has a specific manufacturer’s color

code. Icteric or lipemic serum provides additional challenges in laboratory analysis.
When serum bilirubin approaches 430 mmol/L (25 mg/L), interference may be
observed in assays for albumin (4-hydroxyazobenzene-2-carboxylic acid [HABA]
procedure), cholesterol (using ferric chloride reagents), and total protein (Biuret
procedure). Artifactually induced values in some laboratory determinations result
when triglyceride levels are elevated (turbidity) on the basis of absorbance of light
of various lipid particles. Lipemia occurs when serum triglyceride levels exceed 4.6
mmol/L (400 mg/dL). Inhibition of assays for amylase, urate, urea, CK, bilirubin, and
total protein may be observed. To correct for artifactual absorbance readings,
“blanking” procedures (the blank contains serum, but lacks a crucial element to
complete the assay) or dual-wavelength methods may be used. A blanking process
may not be effective in some cases of turbidity, and ultracentrifugation may be
necessary to clear the serum or plasma of chylomicrons.
1.4. THE REQUEST FORM

The patient identification section of the request form must be completed with care,
and the information on the specimen must correspond to that on the request form.
The importance of correct identification is most clearly illustrated by the potentially
lethal consequences of misidentification in the area of blood transfusion, but serious
repercussions can occur in all areas of investigation. DNA genetic testing (molecular
genetics) is another situation where a clerical error can lead to a serious situation
since a genetic disorder may be excluded or falsely associated with an individual
and/or family members and this error may not be detected for many years.
Laboratories do (and should) reject samples where the identification data on the
form and/or specimen are incomplete or inconsistent.
AND PROCEDURES

The request form must include appropriate clinical information, including

medications. Any difficulty in obtaining the specimen should also be noted on the
request form. Request forms for microbiology should include comment on antibiotic
therapy. The quality of the information provided to the laboratory directly affects the
quality of the interpretation provided to the requesting clinicians.
LABORATORY SERVICES
Hematology CBC
Platelet Count
Clotting Time and Bleeding Time
Peripheral Blood Smear
PTT/PT
ESR
CT/BT
Peripheral Blood Smear
Clinical Microscopy Urinalysis
Fecalysis
Pregnancy Test
Fecal Occult Blood
Semenalysis
Blood Banking and Blood Typing
Immuno-Serology Cross Matching
Hepatitis Testing
RPR/VDRL
Clinical Chemistry FBS
BUA
BUN
Creatinine
Lipid Profile
Liver Profile
Cardiac Profile
Serum Electrolytes
Thyroid Function Test
HBA1c
Bacteriology Culture and Sensitivity
AND PROCEDURES

AFB
Gram Staining
Blood C/S
Blood ARD
Anatomic Pathology Histopath
Pap’s Smear
FNAB
1.5. TIMING OF COLLECTION
Sometimes, samples have to be collected at a specific time. Failure to follow the
planned time schedule can lead to erroneous results and misinterpretation of a
patient’s condition. Timed specimens are ordered for a variety of reasons, usually to
monitor changes in a patient’s condition, to determine the level of a medication, or
to measure how well a substance is metabolized. Specimens for some tests must be
collected with the patient fasting, or with knowledge of when food was last taken (eg
glucose). Some tests must be collected in the basal state or with due regard to
diurnal variations. Hormonal suppression or stimulation tests require accurately
timed collections. Blood for drug monitoring assays is usually collected as a “trough”
(pre-dose) sample, but in certain cases (eg flucytosine) a “peak” sample is required.
Some tests may be performed only after prior arrangement with the laboratory eg
lymphocyte function studies, platelet function tests, glucose tolerance tests.
1.6. TIMED SPECIMENS/ MULTIPLE SPECIMENS

If more than one timed specimen for the same is collected, the collection time MUST
be indicated on each tube (i.e. glucose tolerance testing, drug level analysis etc.)
1.7. VOLUME REQUIREMENTS

Volume requirements are listed for each test (see specimen/minimum volume
section for specific test). To determine the amount of whole blood needed to yield
the required volume of plasma or serum, multiply the specimen required by 2 to 2.5.
1.8. OTHER SPECIMEN COLLECTION & HANDLING
Collection of specimen involves its proper technique, transport of the specimen to
the laboratory and the proper identification of specimen. All specimens submitted
must be of sufficient quantity, fresh and free from hemolysis (blood serum sample)
AND PROCEDURES

and should be accompanied by a completely filled up laboratory request. Specimens

coming from outside should be transported in ice.
LABORATORY EXAMINATIONS AND THEIR COLLECTION REQUIREMENTS
Carbamazepene/Tegretol* Non-fasting Serum Red Top (3 cc. Whole blood

* / 0.5 cc serum)
Cannabinoids/Marijuana** Chain of Custody Urine 60ml Sterile container
Chlorine Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Cholesterol Fasting for 10 Serum Red Top (3 cc. Whole blood
hours / 0.5 cc serum)
Carbon Dioxide** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
CPK Total** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
CPK MB Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Creatinine Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Dilantin/Phenytoin** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Ferritin** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Glucose, Fasting Fasting for 8 Serum Red Top (3 cc. Whole blood
Glucose, Random Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Hepatitis Profile** Non-fasting Serum Red Top (5 cc. Whole blood
/ 2.0 cc serum)
HIV 1 & 2** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Iron** Fasting for 10-12 Serum Red Top (3 cc. Whole blood
LDH** Non-fasting Serum Red Top (3 cc. Whole blood
AND PROCEDURES

/ 0.5 cc serum)
Lipase** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Lipid Profile Fasting for 12 Serum Red Top (5 cc. Whole blood
Liver Profile (SGOT, SGPT, Non-fasting Serum Red Top (5 cc. Whole blood
Bilirubin T, Bilirubin D, / 2.0 cc serum)
Protein T, Albumin)
Magnesium Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Opiates** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Potassium Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Phenobarbital Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Protein, total Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
SGOT Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
SGPT Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Sodium Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Triglycerides** Fasting for 12 Serum Red Top (3 cc. Whole blood
TIBC** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Uric acid Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
VDRL** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
RA Factor** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
CRP** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
AND PROCEDURES

Thyroid Hormone (T3, T4, Non-fasting Serum Red Top (3 cc. Whole blood
TSH)** / 0.5 cc serum)
PSA** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
AFP** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
CEA** Non-fasting Serum Red Top (3 cc. Whole blood
/ 0.5 cc serum)
Complete Blood Count Non-fasting Whole Violet top (depending on
Blood tube volume required
Protime Non-fasting Whole Violet top (depending on
Activated Partial Non-fasting Whole Blue top (depending on
Thromboplastin Time Blood tube volume required
Erythrocyte Sedimentation Non-fasting Whole Blue top (depending on
Rate Blood tube volume required
Arterial Blood Gas Non-fasting Whole Green top (depending on
Urine Analysis Random/First Urine Sterile screw-cup
voided container
Stool Analysis Random Stool Sterile screw-cup
container
Semen Analysis Abstinence (3-5 Semen Sterile screw-cup
days) container
NOTE:
Liver Profile
ALT, (SGPT), AST (SGOT), Protein Total, Albumin, A/G Ratio, Bilirubin Total, Bilirubin, Direct,
Bilirubin Indirect.
Lipid Profile
Cholesterol, Triglycerides, HDL, LDL, VLDL
Cardiac Profile
AND PROCEDURES

CK-MB, AST (SGOT), Cholesterol, Triglycerides, HDL, LDL, VLDL, Glucose

Renal Profile
Creatinine, Urea Nitrogen, Uric acid, ALT (SGPT), Protein total, Albumin, Glucose, Sodium,
Potassium, Chloride
1.9. CRITERIA FOR REJECTION OF SPECIMEN

All specimens must be collected, labeled, transported, and processed according to
established laboratory procedures that include sample volume, special handling needs, and
container type. Failure to follow specific procedures can result in specimen rejection. A
specimen may be rejected based when:
1.9.1. Hemolysis/Lipemia
1.9.2. Clots Present In An Anticoagulated Specimen
1.9.3. Specimen When Test Requires Fasting
1.9.4. Improper Blood Collection Tube
1.9.5. Short Draws, Wrong Volume
1.9.6. Improper Transport Conditions (Ice For Blood Gases)
1.9.7. Discrepancies Between Requisition And Specimen Label
1.9.8. Unlabeled Or Mislabeled Specimen
1.9.9. Contaminated Specimen/Leaking Container
1.10. FASTING SPECIMENS

Prior to collection, no food or beverages are to be ingested, except water for 10-12 hours.
Water and medication are allowed unless specifically prohibited by test requirements or a
physician’s order.
1.11. SPECIMEN PROCESSING

The handling of specimens must be in compliance with the regulations in the Laboratory
Safety Manual. This includes, but is not limited to:
1.11.1 The use of gloves, protective lab coats, protective pipetting shields, protective
eyeware, and face shields, as appropriate for the task being performed.
AND PROCEDURES

1.11.2. Handwritten labels, or pre-printed labels are to be affixed to sample tubes

(vacutainers, capillary tubes or syringes) immediately after the specimen has
been obtained.
1.11.3. Tubes should be handled gently and kept in a stable, vertical position after
collection to promote better clot formation and to prevent hemolysis.
1.11.4. Red top are allowed to clot for 30 minutes at room temperature. Do not release
the clot by rimming the tube with a wooden applicator stick prior to
centrifugation.
1.11.5. Samples are to be centrifuged within 30 minutes of receipt unless constituent
stability is such that more rapid processing is indicated. Serum may be separated
from the cells by decantation.
1.11.6. If delays in testing are anticipated, the sample should be refrigerated or frozen,
depending on constituent stability. Tubes should be placed into the centrifuge in a
pattern designed to balance the centrifuge head. The same size tubes should be
placed exactly opposite each other.
1.11.7. If an odd number of specimens are to be processed, tube filled with water should
be used for balance.
1.11.8. Tubes are centrifuged with stoppers in place or with an adequate closure.
1.11.9. Serum tubes are spun for 10 minutes and plasma tubes are spun for 15 minutes.
1.11.10. Remove serum or plasma with a plastic transfer pipette within 2 hours of
collection (except for Glucose, must be removed immediately).
1.11.11. Do not re-centrifuge specimens except shortly after original spinning and before
any aliquot has been taken.
1.11.12. Aliquots and dilutions must be clearly labeled with marking pen.
1.12. BLOOD STORAGE AND PRESERVATION
During storage, the concentration of a blood constituent in the specimen may change as a
result of various processes, including adsorption to glass or plastic tubes, protein
denaturation, evaporation of volatile compounds, water movement into cells resulting in
hemoconcentration of serum and plasma, and continuing metabolic activities of leukocytes
AND PROCEDURES

and erythrocytes. These changes occur, although to varying degrees, at ambient

temperature and during refrigeration or freezing. Storage requirements vary widely by analyte.
SPECIMEN TEMPERATURE DURATION
Routine Hematology 2-4 oC 48 Hours
Coagulation Tests Room Temp. (24-27oC) Indefinite
Serology -2 - -4 oC 1 Week
Chemistry -2 - -4 oC 48 Hours
1.13. STAT (EMERGENCY) REQUEST FOR LABORATORY SERVICES

1.13.1. STAT request are given immediate priority.
1.13.2. After completion of the stat exam, the nursing unit shall be informed that
results are ready; the ward personnel may pick up the written report from the
laboratory.
1.13.3. Results should NOT be relayed through the phone to the attending
physician/consultant unless otherwise the circumstances require
immediate response (emergency cases).
1.14. LABORATORY BIOSAFETY
The clinical laboratory can potentially expose staff to a variety of hazards through contact
with patients, specimens, equipment, and routine daily tasks. These potential hazards
cannot be completely eliminated but they can be contained to avoid harm to staff. Extreme
care must be exercised to minimize spread of infection. This code is a listing of the most
essential laboratory practices and procedures.
1.14.1. Access
1.14.1.1. The international biohazard warning symbol and sign must be displayed
on the doors of the rooms.
1.14.1.2. Only authorized persons should be allowed to enter the laboratory
working areas.
AND PROCEDURES

1.14.1.3. Laboratory doors should be kept closed.

1.14.1.4. Children should not be authorized or allowed to enter laboratory-working
areas.
1.14.1.5. Access to animal houses should be specially authorized.
1.14.1.6. No animals should be admitted other than those involved in the work of
the laboratory.
1.14.2. Personal protection
1.14.2.1. Laboratory coveralls, gowns or uniforms must be worn at all times for
work in the laboratory.
1.14.2.2. Appropriate gloves must be worn for all procedures that may involve
direct or accidental contact with blood, body fluids and other potentially
infectious materials or infected animals. After use, gloves should be
removed aseptically and hands must then be washed.
1.14.2.3. Personnel must wash their hands after handling infectious materials and
animals, and before they leave the laboratory working areas.
1.14.2.4. Safety glasses, face shields (visors) or other protective devices must be
worn when it is necessary to protect the eyes and face from splashes,
impacting objects and sources of artificial ultraviolet radiation.
1.14.2.5. It is prohibited to wear protective laboratory clothing outside the
laboratory, e.g. in canteens, coffee rooms, offices, libraries, staff rooms and
toilets.
1.14.2.6. Open-toed footwear must not be worn in laboratories.
1.14.2.7. Eating, drinking, smoking, applying cosmetics and handling contact lenses
is prohibited in the laboratory working areas.
1.14.2.8. Storing human foods or drinks anywhere in the laboratory working areas
is prohibited.
1.14.2.9. Protective laboratory clothing that has been used in the laboratory must
not be stored in the same lockers or cupboards as street clothing.
1.14.3. Procedures
1.14.3.1. Pipetting by mouth must be strictly forbidden.

1.14.3.2. Materials must not be placed in the mouth. Labels must not be licked.
1.14.3.3. All technical procedures should be performed in a way that minimizes the
formation of aerosols and droplets.
AND PROCEDURES

1.14.3.4. The use of hypodermic needles and syringes should be limited. They must
not be used as substitutes for pipetting devices or for any purpose other
than parenteral injection or aspiration of fluids from laboratory animals.
1.14.3.5. All spills, accidents and overt or potential exposures to infectious
materials must be reported to the laboratory supervisor. A written record
of such accidents and incidents should be maintained.
1.14.3.6. A written procedure for the clean-up of all spills must be developed and
followed.
1.14.3.7. Contaminated liquids must be decontaminated (chemically or physically)
before discharge to the sanitary sewer. An effluent treatment system may
be required, depending on the risk assessment for the agent(s) being
handled.
1.14.3.8. Written documents, which are to be removed from the laboratory need to
be protected from contamination while in the laboratory.
1.14.4. Laboratory working areas
1.14.4.1. The laboratory should be kept neat, clean and free of materials that are
not pertinent to the work.
1.14.4.2. Work surfaces must be decontaminated after any spill of potentially
dangerous material and at the end of the working day.
1.14.4.3. All contaminated materials, specimens and cultures must be
decontaminated before disposal or cleaning for reuse.
1.14.4.4. Packing and transportation must follow applicable national and/or
international regulations.
AND PROCEDURES

STANDARD PROTOCOLS
2.0 STANDARD PROTOCOLS
The following are standard procedures to be employed in the laboratory that shall
serve as protocols for all laboratory personnel in performing examinations.
2.1 TEST REQUISITION

2.1.1 Nurse / ROD/ Attending Physician accomplishes Laboratory Request form
for the examination desired.
2.1.2 Request form includes the following data:
AND PROCEDURES

2.1.2.1. Patient’s full name, age, sex, date of birth

2.1.2.2. Date requested
2.1.2.3. Room number
2.1.2.4. Requesting physician
2.1.2.5. Type of specimen
2.1.2.6. Examination desired
2.1.2.7. Type of request (routine / stat)
2.1.2.8. Diagnosis / clinical impression
2.1.2.9. Date and time received
2.1.2.10. specimen number
2.1.3. Certain examinations require specific data; request forms for these are
provided by the laboratory
2.1.3.1. Surgical Pathology Request Form: available in the Operating
Room and Laboratory
2.1.3.2. Gynecologic / Cytology Request Form: available in the OPD and
Laboratory
2.2. RECEIPT OF REQUEST FORM:
2.2.1. Ensures to it that requests are properly categorized into routine
,STATprocedure, or serial collection.
NOTE: Exams which do not indicate STAT on the request form are considered as
Routine exam
2.2.2. Ensures that the requiredpatient demographics are written on the request.
2.2.3. CT Scan and Ultrasound Guided Biopsy
Note: Written requests with attached medical abstract are submitted to the
Laboratory at least 24 hours before the procedure.
2.3. RECEIPT OF SPECIMEN FOR LABORATORY TEST

2.3.1. In-Patients
2.3.1.1. Ensures that the request form is properly filled-out with the
required patient demographics and specimen is satisfactory
and acceptable.
AND PROCEDURES

NOTE: Request form may not be completely filled-out if it is

a STAT request
2.3.1.2. Receives the specimen from the nursing staff and signs on the
Receiving logbook of the nursing unit, otherwise, rejects specimen
and requests for repeat collection.
NOTE: Repeat blood collection shall be accomplished by laboratory
personnel(Phlebotomist or RMT) as part of the routine warding
procedure, unless request is a STAT or a serial collection procedure.
2.3.1.3. Upon receipt of request and specimen, the request shall be
entered in the laboratory Receiving logbook and a charge slip shall
be accomplished.
2.3.1.4. Labels the specimens with assigned laboratory number and
submit specimen to the designated section for examination.
2.3.2. Out-Patient
2.3.2.1. Checks the request for examinationrequirements (Fasting or non-
fasting; routine or special test), then, instructs patients if
necessary.
2.3.2.2. Issue a charge slip and instructs patient to proceed to specimen
collection.
2.3.2.3 Asks patient for official receipt, enters request into the Receiving
logbook, andproceed to collection procedure, otherwise, advices
patient to return to the reception area for instructions. The
collection procedure is as stated below:
2.3.2.3.1. Issues specimen cups to patient and advices proper
procedure for collection.
2.3.2.3.2. Performs phlebotomy procedure to patient.
2.3.2.4. Informs patient estimate schedule for release of results.
2.4. RECORDING OF REQUEST
2.4.1. Recording of request should always be accomplished prior to collection
procedures (for Out-patient) or testing procedures (for In-patient) into its
corresponding logbooks.
2.5. COLLECTION AND HANDLING OF SPECIMENS
AND PROCEDURES

2.5.1. Issues specimen cups and instructs patient for preparation and specimen
collection procedure.
2.5.1.1. For fasting specimens, patient is instructed for the required fasting
periods.
2.5.1.2. For Non-fasting specimen/Random samples, collection may be
accomplished anytime, provided that the patient is well instructed
of the proper collection procedure.
2.5.1.3. For other examinations requiring specific preparations, instruct
patient of the procedure and schedule the examination if possible.
2.5.2. Specimens should be properly handled and transported to the designated
section of the laboratory. The following are the necessary procedures to
employ.
2.5.2.1. Specimens collected should ALWAYS be properly collected in correct
containers, with enough volume, and labeled with the patient’s
name, age, gender, date and time of collection.
2.5.2.2. Avoid prolonged transport time if necessary.
2.5.2.3. Specimen for examinations should always have their corresponding
request forms otherwise, specimen will be rejected.
2.5.3. Urine Collection
2.5.3.1. Routine specimen should be collected in a sealable clean dry
container; pediatric urine bag may be used if necessary.
2.5.3.2. Approximatelyinstruct patient to collect 5-10ml of sample,
preferably, first morning, mid-stream clean catch urine.
2.5.3.3. For random collection, a mid-stream, clean catch urine may be
suitable.
2.5.3.4. For timed collection, instruct the patient or the nursing unit of the
procedure and preservation technique.
2.5.3.4.1 Discard the first voided urine then collect and record all
urine excreted for the next 24 hours starting from the time
of last urine void.
2.5.3.4.1. Upon collection, preservative should always be mixed with
the sample and refrigerated.
AND PROCEDURES

2.5.3.5. For urine culture, cleanse the urethra thoroughly and collect mid-
stream urine in a sterile bottle.
2.5.4. Stool Collection
2.5.4.1. Routine specimen should be collected in a sealable clean dry
container.
2.5.4.2. A pea-size sample may be submitted for formed stool, in case
patient is having diarrhea or is suffering from symptoms, instruct
patient to collect the watery/bloody part of the stool.
2.5.4.3. Upon collection, Instruct patient to avoid contamination and to
submit the sample one (1) hour after collection.
2.5.4.4. Forstool culture, instruct patient to collect specimen in a sterile
bottle.
2.5.5. Blood Collection
- Refer to Operation Manual
2.5.6. Sputum Collection
For culture, gram stain and acid-fast bacilli (AFB stain), instruct patient to
collect sputum upon promoting a deep cough. To ensure a suitable sample,
collect specimen first thing in the morning before eating or brushingand place
sample in a wide-mouthed sterile container provided by the laboratory and
submit immediately.
2.5.7. CSF And Other Body Fluids
2.5.7.1. Ensure specimen integrity and quantity enough and suitable for Cell
count, Chemistry, and Microbiology examinations.
2.5.7.2. Order of draw should always be considered as to where to submit
the specimen. Refer to Operation Manual.
2.5.8. Bacteriology specimens
2.5.8.3. For exudates and discharges, two (2) swab specimens must be
collected and placed in a sterile vial / thioglycollate tube.
2.5.8.4. Specimens collected should immediately be brought to the
laboratory for processing.
2.5.9. Surgical pathology specimens
The Laboratory provides the OR and OPD with various fixatives (10%
buffered formalin, 95% ethyl alcohol) for specimen preservation.
AND PROCEDURES

2.5.9.1. Preservation:
2.5.9.1. Fixes specimen in 10% formalin and submit immediately
to the laboratory.
2.5.9.2. For cytologic examination, submit unfixed specimen
immediately. If specimen cannot be processed within 12
hours, specimen must be fixed in an equal volume of 50%
ethyl alcohol and refrigerated until it can be processed.
2.5.9.3. For cytologic examination of smears, fix specimen
immediately in 95% ethyl alcohol.
NOTE: It is the prerogative and responsibility of the Laboratory to reject unsatisfactory

specimens and request for a repeat collection
2.6 CHARGING OF LABORATORY SERVICES

2.6.1. Prepare the charge ticket using the HIMS system
2.6.2. Instructs patient to submit the charge ticket to the billing section after
recording the charges in the Receiving logbook
2.6.3. Charges routine and STAT examinations according to categories: OPD, HMO,
Ward, Small Private, Suite, etc.
2.6.4. Charges professional fees aside from the processing and handling fees for the
following exams:
a) Histopathologic examinations
b) Cytologic examinations
c) Special procedures (ct and ultrasound guided biopsies)
d) Peripheral blood smear
e) Malarial smear
2.6.5. STAT charges
2.6.5.1. Add a surcharge of 50% to the regular rate to cover for:
a) Additional Reagents And Controls
b) Additional Cost For Repair And Maintenance Of Equipment and
Instruments
c) Additional Personnel
2.6.5.2. Considers all out-patient requests coming from the Emergency Room
(ER) as “stat” with surcharge
AND PROCEDURES

2.6.5.3. Considers as “STAT” without surcharge the following examinations:

a) CSF qualitative and quantitative analysis
b) Pregnancy test
c) Wet mounts
e) CPK-MB
f) Troponin I
g) Seminalysis
h) Creatinine from CT scan request
i) Stool Analysis
NOTE: Repeat examinations for unsatisfactory specimen or exams, which are done
twice for confirmation, are not charged.
The following stated above may vary depending on availability of tests.
2.6.6. Charge service fee and supplies that will be used for examinations done
outside the Laboratory (send-out) in which specimen is collected / processed
by the Laboratory
2.6.7. Senior citizens with a valid ID are given 20% discount.
2.8 RELEASING OF RESULTS
2.8.1. Results shall be validated by authorized personnel and should not be released
without the signatory.
2.8.2. Results will be released according to the established laboratory policies (refer
to General Policies).
2.8.3. Upon releasing, the Releasing logbook should be accomplished signed by the
NOD of the nursing unit along with the date and time of release.
2.8.4. Result forms are color-coded as to the following:
SECTION COLOR CODE

Hematology Pink
Clinical Chemistry White
Clinical Microscopy Yellow/ Green
Serology Blue
Miscellaneous White
AND PROCEDURES

2.8.5. Procedure for releasing:

2.8.5.1. IN-PATIENT
2.8.5.1.1 Laboratory aide delivers result to every nurse station
2.8.5.1.2. Nurse on duty / Nursing staff signs on the receiving
logbook his full-name, date and time of receipt and
attaches received results immediately to the patient’s
chart
2.8.5.2.OUT-PATIENT
2.8.5.1. Patient / relative signs on the releasing logbook upon
presenting official receipt of payment
2.8.5.2. Laboratory staff signs on the official receipt before
releasing result to the patient
2.8.5.3. Release results
2.8.6. REFERRAL OF RESULTS
2.8.6.1. Med. Tech refers questionable results to Chief Med. Tech
who in turn refers to Pathologist
NOTE: Releasing of results may be delayed for any referral
for confirmation to the Pathologist
2.8.6.2. Peripheral blood smears, Malarial smears and Lupus
Erythematosus (LE) smears are referred to the Pathologist
before releasing results
2.8.7. STAT requests are released within or less than 1 hour
except when there are multiple stat examinations being
done at the same time and when questionable results are
referred to the Pathologist.
2.8.8. Routine results are released within a minimum of 1 hour
after receipt of the specimen
2.8.9. The prescribed schedule of release of results should always
be followed (refer to Laboratory Policies).
2.8.10. Serial examinations are released within 2 hours
2.8.11. Bacteriology
2.8.11.1. AFB stain
AND PROCEDURES

2.8.11.1.1. Results are released within 24 hours after receipt of

the specimen for In-patients. For Out Patients,
results are released upon completion of the 3rd day
collection
2.8.11.2. Culture and Sensitivity
2.8.11.2.1. Release of results depends on the type of
request and the number of isolated
organisms
2.8.11.2.2. Follow-up of results can be done after
48 hours.
2.8.12. Surgical Pathology
2.8.12.1. Results are released within 1 week except when
longer fixation is required or additional studies are
needed (i.e. additional sections, special stains,
referred for second opinion)
2.8.12.2. For CT and Ultrasound Guided biopsies, a
preliminary result (adequacy of specimen and
tentative diagnosis) is issued to the Consultant
Radiologist. Final written report is released within
1 week.
2.8.12.3. Surgical Pathology and Gynecologic Cytology Report
(Modified Bethesda System)
Report forms are available in 3 color-coded copies:
2.8.12.3.1. White: patient’s copy (for out-patients) / chart
copy (for in-patients)
Blue: doctor’s copy
Pink: laboratory’s copy
6.8.13. Follow-up and Relaying of Results:
6.8.11.1. Follow-up and relaying of results is allowed only for
patients in critical units (i.e. ER, ICU, NICU) but is
not advised.
AND PROCEDURES

6.8.11.2. Follow-up and relaying of STAT results by phone is

allowed only if results have not yet been released
after the time limit of one hour has elapsed
6.8.11.3. Follow-up of results of routine requests is allowed
only if such results were not delivered on time.
Relaying of result by phone is highly discouraged.
When relaying results by phone, note the name of
the nurse who received the call and the time during
which the results were relayed.
STANDARD OPERATING PROCEDURES

Laboratory services have become an integral and inseparable component of modern medicine
and public health. Laboratories play a decisive role in the diagnosis, treatment, prognosis and
monitoring of both communicable and non-communicable diseases.
Quality assurance in laboratory services, aimed at improving reliability, efficiency and facilitating
inter-laboratory comparability in testing, is the backbone of quality health care delivery. The use
of standard operating procedures in laboratory testing is one of the most crucial factors in
achieving quality. This helps both in proper patient management and generates reliable disease
surveillance data. This document provides guidelines on standard operating procedures for
diagnosing diseases of public health importance at intermediate and peripheral levels.
AND PROCEDURES

The available Operations Manual designed for the specific needs of the Laboratory are the
following:
Blood Collection
Hematology
Microscopy
Serology
Microbiology
Chemistry
Blood Bank
STANDARD OPERATING
PROCEDURES
AND PROCEDURES

BLOOD COLLECTION
1.0. VENIPUNCTURE BLOOD COLLECTION
Venipuncture is accomplished using a needle/adapter assembly attached to an

evacuated glass/plastic test tube with a rubber/plastic stopper. Blood may also be
collected in a syringe and transferred to the appropriate specimen container
(evacuated tube system).
1.1. Advantages:
1.1.1 Multiple and repeated examinations can be performed on the same specimen.
1.1.2. Aliquots of the specimen may be frozen for future reference.
1.1.3. There is no variation in blood values if specimens are obtained from different
veins.
1.2. Disadvantages:
AND PROCEDURES

1.2.1. The venous method is somewhat lengthy procedure that requires more
preparation than the capillary method.
1.2.2. Prolonged stasis produced by the tourniquet must be avoided, because it
produces hemoconcentration.
1.3. Materials:
1.3.1. Collection Tubes

1.3.2. Cotton balls/ Gauze pad
1.3.3. Isopropyl Alcohol (70%)
1.3.4. Syringe
1.3.5. Tourniquet
1.3.6. Micropore/Transpore
1.4. Procedure:
1.4.1 Verify that computer-printed labels match requisitions. Check patient

identification band against labels and requisition forms. Ask the patient for
his or her full name.
1.4.2 If a fasting specimen or a dietary restriction is required, confirm patient
has fasted or eliminated foods from diet as ordered by physician.
1.4.3 Read the patient’s request form carefully. Decide how much blood is
needed and prepare the correct bottle or tube to be used for each test.
1.4.4 If blood is to be used for different laboratory investigations, plan the
sequence in which blood samples must be taken. (For example, the first 1
ml of blood must be discarded when blood is taken for coagulation assays.)
1.4.5 Before taking the blood, wash your hands with soap and water. Ask the
patient to sit alongside the table used for taking blood. Lay the patient’s
arm, palm upwards, and support it by placing a small cushion under the
elbow .
1.4.6 Apply a tourniquet and ask the patient to make a fist without vigorous
hand pumping.
1.4.7 Select a suitable vein for puncture.
1.4.8 Cleanse the venipuncture site with 70% isopropyl alcohol. Allow the area
to dry.
1.4.9 Anchor the vein firmly.
1.4.10 Enter the skin with the needle at approximately a 15-degree angle or less
AND PROCEDURES

to the arm, with the bevel of the needle up:

1.4.10.1. Follow the geography of the vein with the needle.
1.4.10.2. Insert the needle smoothly and fairly rapidly to minimize patient
discomfort.
1.4.10.3. If using a syringe, pull back on the barrel with a slow, even
tension as blood flows into the syringe. Do not pull back too
quickly to avoid hemolysis or collapsing the vein.
1.4.10.4. If using an evacuated system, as soon as the needle is in the vein,
ease the tube forward in the holder as far as it will go, firmly
securing the needle holder in place. When the tube has filled,
remove it by grasping the end of the tube and pulling gently to
withdraw, and gently invert tubes containing additives. Release
the tourniquet when blood begins to flow. Never withdraw the
needle without removing the tourniquet.
1.4.11. Withdraw the needle, and then apply pressure to the site. Apply adhesive
bandage strip over a cotton ball or gauze to adequately stop bleeding and
to avoid a hematoma.
1.4.12. Mix and invert tubes with anticoagulant; do not shake the tubes. Check
condition of the patient. Dispose of contaminated material in designated
containers (sharps container) using Universal Precautions.
1.4.13. Label the tubes before leaving patient’s side with: a. patient’s first and last
nameb. identification numberc. date of collection d. time of collection e.
identification of person collecting specimen
1.4.14. Deliver tubes of blood for testing to appropriate laboratory section or
central receiving and processing area.
1.5. Additional Considerations
1.5.1. Always check and prepare extraction materials.

1.5.2. Ensure that the patient has no foreign objects in their mouth.
1.5.3. Introduce yourself and entertain patient quarries regarding the procedure.
If the patient requests additional information, refer the patient to their
physician. Providing too much information to the patient can lead to
misinformation and possible legal action.
1.5.4. Never argue with a patient, exercise maximum tolerance; if the patient
refuses to have blood work drawn, write "Refuse to Extract" on the
requisition and let the patient sign the request, inform the nursing staff
AND PROCEDURES

and the doctor of the situation.

1.5.5. Specimens collected from a hematoma area may cause erroneous test
results.
1.5.6. Because of potential harm to the patient due to lymphostasis, do not draw
blood from the side on which a mastectomy was performed unless you
have a written order from her physician.
1.5.7. A-V Fistula/Shunts - when the patient has a fistula, do not use that arm for
venipuncture, the pressure from the tourniquet could cause a clot in the
shunt.
1.5.8. If the patient appears to be unruly, ask the nursing unit staff for assistance.
If the patient continues to be violent and poses a hazard to the staff
drawing the blood, do not proceed. Document and communicate this
information to the physician.
1.5.9. Carefully observe the patient for signs of reaction during and immediately
following the venipuncture. If complications arise call for assistance and
inform the physician about the situation.
1.5.10. Immediately report an accidental needle stick or contamination of a break
in the skin by blood or excreta to your supervisor.
2.0. CAPILLARY PUNCTURE
Capillary blood is obtained from the tip of a finger in adults and from the great toe or
the heel in infants. For routine assays requiring small amounts of blood, skin
puncture is a simple method by which to collect blood samples in pediatric
patients. In the neonate, skin puncture of the heel is the preferred site to collect a
blood sample; in older children, the finger is the preferred site.
2.1. Advantages:
2.1.1. Capillary blood can be obtained with ease.

2.1.2. Capillary blood is the preferred material for making peripheral blood smear.
2.1.3. Skin puncture is useful in adults with (1) extreme obesity, (2) severe burns,
and (3) thrombotic tendencies, with point-of-care testing or with patients
performing tests at home (blood glucose).
AND PROCEDURES

2.1.4. Skin puncture is often preferred in geriatric patients because the skin is
thinner and less elastic; thus a hematoma is more likely to occur from a
venipuncture.
2.2. Disadvantages:
2.2.1. Venipuncture of deep veins in pediatric patients may rarely cause (1) cardiac
arrest, (2) hemorrhage, (3) venous thrombosis, (4) reflex arteriospasm
followed by gangrene of an extremity, (5) damage to organs or tissues
accidentally punctured, (6) infection, and (7) injury caused by restraining an
infant or child during collection. Only a small specimen can be obtained, and
repeated examinations require new specimens.
2.2.2. Blood in microtubes frequently haemolyses, and haemolysis interferes with
most laboratory tests.
2.2.3. Test results on capillary blood cannot be compared with test results in venous
blood.
2.2.4. The finger is not only sensitive but difficult to adequately sterilize in the time
usually available. In patients with lowered resistance to infection, a specimen
taken from the finger is much likely to lead to infection than one taken from
the arm.
2.2.5. Red and white cell counts and enumeration of Platelets and Reticulocytes
should not be performed on capillary blood, because of the difficulty in
standardizing capillary blood flow.
2.3. Materials:
2.3.1. Capillary tube

2.3.4. Pricker
2.4. Procedure:
2.4.1. Select an appropriate puncture site.
2.4.1.1. For infants younger than 12 months old, this is most usually the
lateral or medial plantar heel surface.
2.4.1.2. For infants older than 12 months, children, and adults, the palmar
surface of the last digit of the second, third, or fourth finger may be
AND PROCEDURES

used.
2.4.1.3. The thumb and fifth finger must not be used, and the site of puncture
must not be edematous or a previous puncture site because of
accumulated tissue fluid.
2.4.2. Warm the puncture site with a warm, moist towel no hotter than 42° C; this
increases the blood flow through arterioles and capillaries and results in
arterial-enriched blood.
2.4.3. Cleanse the puncture site with 70% aqueous isopropanol solution. Allow the
area to dry. Do not touch the swabbed area with any nonsterile object.
2.4.4. Make the puncture with a sterile lancet or other skin-puncturing device, using
a single deliberate motion nearly perpendicular to the skin surface. For a heel
puncture, hold the heel with the forefinger at the arch and the thumb
proximal to the puncture site at the ankle. If using a lancet, the blade should
not be longer than 2 mm to avoid injury to the calca- neus (heel bone).
2.4.5. Discard the first drop of blood by wiping it away with a sterile pad. Regulate
further blood flow by gentle thumb pressure. Do not milk the site, as this may
cause hemolysis and introduce excess tissue fluid.
2.4.6. Collect the specimen in a suitable container by capillary action. Closed
systems are available for collection of nonanticoagulated blood and with
additives for whole blood analysis. Open-ended, narrow-bore disposable glass
micropipets are most often used up to volumes of 200 μL. Both heparinized
and nonheparinized micropipets are available. Use the appropriate
anticoagulant for the test ordered. Mix the specimen as necessary.
2.4.7. Apply pressure and dispose of the puncture device.
2.4.8. Label the specimen container with date and time of collection and patient
demographics.
2.4.9. Indicate in the report that test results are from skin puncture.
3.0 ARTERIAL PUNCTURE
The artery to be punctured is identified by its pulsations. A nonanesthetized arterial

puncture provides an accurate measurement of resting pH and partial pressure of carbon
dioxide (pCO2). In spite of possible theoretical error caused by patient hyperventilation
resulting from the pain of the arterial puncture. The use of butterfly infusion sets is not
recommended.
AND PROCEDURES

Arterial punctures are technically more difficult to perform than venous punctures.
Increased pressure in the arteries makes it more difficult to stop bleeding, with the
undesired development of a hematoma. In order of preference, the radial, brachial, and
femoral arteries can be selected. Before blood is collected from the radial artery in the
wrist, one should do a modified Allen test to determine whether the ulnar artery can
provide collateral circulation to the hand after the radial artery puncture. The radial artery
is more difficult to puncture, but complications occur less frequently. The major
complications of arterial puncture include thrombosis, hemorrhage, and possible infection.
When performed correctly, no significant complications are reported except for possible
hematomas.
3.1 Modified Allen Test
3.1.1. Have the patient make a fist and occlude both the ulnar (opposite the thumb
side) and the radial arteries (closest to the thumb) by compressing with two
fingers over each artery.
3.1.2. Have the patient open his or her fist, and observe if the patient’s palm has
become bleached of blood.
3.1.3. Release the pressure on the ulnar artery (farthest from the thumb) only, and
note if blood return is present. The palm should become perfused with blood.
Adequate perfusion is a positive test indicating that arterial blood may be
drawn from the radial artery. Blood should not be taken if the test is negative.
Serious consequences may occur if this procedure is not followed, which may
result in loss of the hand or its function.
3.2. Materials:
3.2.1. Collection Tubes

3.2.4. Syringe
3.2.5. Tourniquet
3.3. Procedure:
3.3.1. Prepare the arterial blood gas syringe according to established procedures. The needle
AND PROCEDURES

(18–20 gauge for brachial artery) should pierce the skin at an angle of approximately
45–60 degrees (90 degrees for femoral artery) in a slow and deliberate manner. Some
degree of dorsiflexion of the wrist is necessary with the radial artery, for which a 23–25
gauge needle is used. The pulsations of blood into the syringe confirm that it will fill by
arterial pressure alone.
3.3.2. After the required blood is collected, place dry gauze over the puncture site while
quickly withdrawing the needle and the collection device.
3.3.3. Compress the puncture site quickly, expels air from the syringe, and activate the needle
safety feature; discard into sharps container.
3.3.4. Mix specimen thoroughly by gently rotating or inverting the syringe to ensure
anticoagulation.
3.3.5. Place in ice water (or other coolant that will maintain a temperature of 1°–5° C) to
minimize leukocyte consumption of oxygen.
3.3.6. Continue compression with a sterile gauze pad for a minimum of 3 to 5 minutes
(timed). Apply an adhesive bandage.
STANDARD OPERATING
PROCEDURES
AND PROCEDURES

HEMATOLOGY
HEMATOLOGY
Hematology section involves the study of the cellular components of the blood. These
components are differentiated. This study may assist in the diagnosis of disease such as anemia
and leukemia. Procedures for hematology examinations vary depending on the type or procedure.
The laboratory department shall be using Automated Cell Counters (Sysmex) as its main
equipment in hematology determinations (Refer to Manufacturer’s Manual). Providedin this
operations manual are the different manual procedures that may be used as secondary
procedures in case of equipment breakdown.
Examination offered:
A. Routine Examinations:
a. Complete Blood Count
1. Hematocrit
2. Hemoglobin
3. RBC Count
4. WBC Count
5. Differential Count
b. Actual Platelet Count
B. Special Examinations:
AND PROCEDURES

a. Peripheral Blood Smear

b. Clotting Time
c. Bleeding Time
d. ESR
e. Sickle cell test
1.0. BLOOD SMEAR PREPARATION

A blood smear enables the medical technologist to view the cellular components of blood
in a natural state as possible. The morphology of the cellular components can be studied.
1.1. Specimen:
The best specimen for a blood smear is a capillary blood which has no anticoagulant
added. However, a satisfactory smear may be made from venous blood which has the
anticoagulant EDTA added to it, provided the smear is made within 2 hours of collections.
Other anticoagulants should not be used since they change the morphology of the cells.
1.2. Materials/Equipment:
1.2.1. Material for capillary puncture
1.2.2. Microscopic slides
1.3. Procedure: Wedge Method
1.2.1. Place a 1" x 3" glass microscope slide with a frosted end on a flat surface (usually the
counter top of a laboratory bench).
1.2.2. Attach a label on the slide or write the patient name, specimen identification
number, and date of preparation on the frosted surface.
1.2.3. Place a 2 - 3 mm drop of blood approximately 1/4" from the frosted slide, using a
wooden applicator stick or glass capillary tube.
1.2.4. Hold the slide by the narrow side between the thumb and forefinger of one hand at
the end farthest from the frosted end.
AND PROCEDURES

1.2.5. Grasp a second slide ("spreader slide") between the thumb and forefinger of the
other hand at the frosted end.
1.2.6. Place the edge of the spreader slide on the lower slide in front of the drop of blood
(side farthest from the frosted end).
1.2.7. Pull the spreader slide toward the frosted end until it touches the drop of blood.
Permit the blood to spread by capillary motion until it almost reaches the edges of
the spreader slide.
1.2.8. Push the spreader slide forward at a 30o angle with a rapid, even motion. Let
the weight of the slide do the work.
1.2.9. Air dry slide.
1.4. Factors Affecting The Smear:

1.4.1. The length and thickness of the smear is affected by the size of the drop of blood and
the angle at which the operator is held.
1.4.2. Thick smear occurs when the angle of the spreader is too high or the drop of blood is
too large. Thin smear occurs when the drop of blood is too small or the angle is too
low.
1.4.3. Thin uneven smears may also occur when too much pressure is applied to the
spreader.
1.4.4. The faster the spreading motion, the thinner the smear.
2.0. HEMATOCRIT DETERMINATION

Hematocrit determination may be ordered separately or as part of a complete blood count.
It is a simple procedure, which make it an ideal test to follow the progress of anemia or
bleeding patients.
2.1. Principle:
A small amount of whole blood is centrifuged to determine maximum packing of
erythrocytes, expressed as PCV or HCT.
2.2. Specimen: Whole blood anticoagulated with EDTA (2 mg/ mL of blood) or heparin
2.3. Reagents/ Materials/Equipment:
2.3.1. Capillary tube (with patient’s sample),
2.3.2. Microhematocrit Centrifuge
2.4. Procedure: Adam’s method (Microhematocrit method)
2.4.1. Draw blood into capillet (at least 2/3 full) by placing the end of the capillary tube
over the punctured finger and allowing it to fill by the force of gravity.
2.4.2. Seal the fine-polished (colored) end of the tube with sealing clay and then paraffin.
AND PROCEDURES

2.4.3. Place the capillary tubes into the radial grooves of the microhematocrit centrifuge
with the sealed end towards the periphery. Make sure to place a balancer to the
opposite side of the tube.
2.4.4. Tighten the head cover in a clockwise direction and close the centrifuge cover.
2.4.5. Centrifuge for 5 minutes at 10,000 rpm.
2.4.6. Allow the centrifuge to stop on its own.
2.4.7. Read the value in a microhematocrit reader.
2.5. Normal: Male= 0.42-0.48
Female= 0.37-0.42
3.0 HEMOGLOBIN DETERMINATION

Hemoglobin concentration provides an estimate of the oxygen-carrying capacity of blood.
3.1. Principle:
3.2. Specimen: Whole blood anticoagulaed with EDTA (2 mg/,L of blood) or heparin
3.3.1. Sahli’s haemoglobinometer (comparator)
3.3.2. Comparison tube 3.0.02ml pipette (Hemoglobin pipette with rubber tubing and
mouthpiece)
3.3.3. Two Pasteur pipettes
3.3.4. Glass rod to stir
3.3.5. Hydrochloric acid
3.3.6. Distilled Water
3.4. Procedure: Acid Hematin Method
3.4.1. Add hydrochloric acid ( 1: 10 diluted ) to the haemometer tube ( comparison tube )
up to lowest graduation ( 0.02 gram )
3.4.2. Sterilize the fingertip with Isopropyl alcohol surgical spirit and allow it to dry
3.4.3. Using sterile lancet prick the finger tip
3.4.4. Wipe away first few drops of blood
3.4.5. Suck blood into the hemoglobin pipette ( capillary pipette ) up to 20 cu.mm( avoid
air bubbles coming into a tube )
3.4.6. Wipe the outside tip of pipette clean with tissue
3.4.7. Immediately transfer the blood to the comparison tube
3.4.8. Suck blood back into the pipette several times and blow out again into the tube ( to
mix blood with HCl )
AND PROCEDURES

3.4.9. Place the haemometer tube in the stand and allow for 10 minutes (during this
period Hcl lysis red cell and released hemoglobin on reacting with Forms a dark
brown colored acid Hematin )
3.4.10. Now using a Pasteur pipette to add a few drops of distilled water and stir the
contents with a glass rod.
3.4.11. Continue to add water drop by drop and stir the contents each time until the
solution is just darker than the standard.
3.4.12. Carefully add one or two drops of water till the color exactly matches with that of
the Standard and note the reading while taking the reading hold the
haemoglobinometer against good daylight at arm’s length
3.4.13. The comparison tubes represent 100 percent haemoglobin with reference to a
standard, whichis 14. 8 g Hb / 100 ml of blood
4.0 ERYTHOCYTE COUNT
4.1. Principle:
The most commonly used method employs the Improved Neubauer countingchamber .
There are two Chambers per Haemocytometer, each consists ofnine large squares, each
measuring 1mm2.
4.2. Specimen: Whole blood anticoagulaed with EDTA (2 mg/,L of blood) or heparin
4.3.1. RBC pipette
4.3.2. Neubauer Counting Chamber
4.3.3. Hayem’s Fluid
4.3.4. Microscope
4.4. Procedure:
4.4.1. Select the diluting fluid pipet
4.4.2. Clean and dry the pipet
4.4.3. Prepare the sample by mixing it thoroughly and properly
4.4.4. Suck blood up to 0.5 mark; wipe the tip of the pipet.
4.4.5. Suck diluting fluid (hayems fluid) up to 101 marks
4.4.6. Mix the solution by rotating the pipet
4.4.7. Place pipet on a horizontal surface
4.4.8. Clean the chamber and the coverslip
4.4.9. Place the cover slip on the central platform of the chamber, mix contents of the bulb
thoroughly.
AND PROCEDURES

4.4.10. Discard the first 2 drops of the fluid from the pipet
4.4.11. Place the tip of the pipet on the surface of the chamber at 45 degrees and allow the
diluted blood to flow under the cover slip.
4.4.12. Place chamber on a flat surface.
4.4.13. Calculation:
Area of five medium- sized squares= 0.2 mm2
Volume of five medium sized squares= 0.02 mm3
Dilution factor= 1:200
Depth of the chamber= 10
RBC Count= total red cells counted (n) x DF x Depth factor / total area counted (0.2
mm3)
RBC Count= n x DF x VCF
RBC Count= n x 200 x 50
RBC Count= n x 10, 000
5.0. LEUCOCYTE COUNT

The leucocyte or white cells count is the number of white cells in one cubic millimeter of
blood. Blood is diluted with Acetic acide which the non-nucleated erythrocytes.
5.1. Principle:
A suitably diluted specimen is loaded onto hemocytometer and the cellular elements
are identified and counted microscopically and reported as number of cells per liter.
5.2. Specimen : Whole blood anticoagulated with EDTA
5.3.1. WBC pipette
5.3.2. Neubauer Counting Chamber
5.3.3. Acetic Acid
5.3.4. Microscope
5.4. Procedure:
5.4.1. Draw blood up to 0.5 mark of a WBC pipet (capillary or venous blood).
5.4.2. Draw diluting fluid (acetic acid) to 11 mark with accuracy.
5.4.3. Shake the pipet for 3 minutes horizontally by hand.
5.4.4. Discard the first 3 to 4 drops from the pipet.
5.4.5. Load both counting chambers of the hemacytometer.
5.4.6. Using the low power objective, count the number of white cells in each 4 secondary
squares. In order to avoid confusion in counting, cells that touch the upper and left
AND PROCEDURES

borderlines are counted as if they are within the squares and neglect those cells
thattouch the lower and right borderlines.
NOTE:
The white cells are round. If you see objects that are not round or if you see objects that do not hold
their round shape when you move the fine adjustment back and forth, do not count them. Those
objects are only artifacts in the fluid or counting chamber, if the number in each big square varies
nor than 10, the count must be discarded and the counting chamber must be cleaned and re-
charged.
5.5. Computation:
Number of WBCs per cubic mm= WBC counted / (Squares counted x Depth x Dilution)
If more than five nucleated RBCs are present, the leucocytes count should be corrected
using the following formula:
Corrected WBC count = uncorrected WBC count x 100 / ( 100 + Nucleated RBC seen in 100
WBC)]
5.6. Normal values: 5,000-10,000 per cubic millimeter
6.0. DIFFERENTIAL LEUCOCYTE COUNT

6.1. Principle:
An appropriately prepared and stained blood film is systematically scanned microcopically
to estimate leukocyte count, identify morphologic erythrocyte abnormalities, estimate
paltelet number, and classify leukocytes into group types. (Steininger)
6.2. Specimen : Whole blood anticoagulated with EDTA
6.3.1. Stained blood smear
6.3.2. Cedarwood oil
6.3.3. Shillings differential counter
6.3.4. Microscope
6.4. Procedure:
6.4.1. Check slide identification
6.4.2. Place a drop of cedarwood oil on the upper edge of the smear (feathery end).
6.4.3. Using OIO begin at the upper edge of the smear to move the slide down to the lower
edge, marking down the type of each leucocyte that appears on the field.
AND PROCEDURES

6.4.4. When the lower edge is reached, moves the slide sideways of a short distance,
classifies all leucocytes seen while moving the slide and then moves it up.
6.4.5. When you have identified and recorded 100 white cells, note down the total number
of each variety.
6.4.6. Determine the percentage of each type of leucocyte that you have counted.
7.0. ACTUAL PLATELET COUNT
7.1. Principle:
Whole blood is diluted with a 1% ammonium oxalate solution. The isotonic balance of the
diluent is such that all erythrocytes are lysed while the leukocytes, platelets, and
reticulocytes remain intact.The standard dilution for platelet counts is 1:100. This dilution
is prepared using the leukocyte/platelet Unopette system.The dilution is mixed well and
incubated to permit lysis of the erythrocytes. Following the incubation period, the dilution
is mounted on a hemacytometer. The cells are allowed to settle and then are counted in a
specific area of the hemacytometer chamber under the microscope. The number of
platelets is calculated per m L (x 109/L) of blood.
7.2. Specimen :Whole blood anticoagulated with EDTA, or free-flowing capillary blood may be
used.
7.3.1. Two leukocyte/platelet Unopette reservoirs; each containing 1.98 mL of the

following diluent:
Ammonium oxalate 11.45 g
Sorensen’sphosphate buffer 1.0 g
Thimerosal 0.1 g
QS with distilled water to 1 liter
7.3.2. Two Unopette capillary pipets, 20 μL

7.3.3. Hemacytometer with cover glass
7.3.4. Petri dish with filter paper
7.3.5. Hand counter
7.3.6. Microscope
AND PROCEDURES

7.4. Procedure
7.4.1. Prepare two leukocyte/platelet Unopettes as follows:

7.4.1.1. Using the protective shield on the capillary pipet, puncture the diaphragm
as follows:
7.4.1.1.1. Place reservoir on a flat surface. Grasping the reservoir in one

hand, take the pipet assembly in the other hand and push the tip
of the pipet shield firmly through the diaphragm in the neck of
the reservoir, then remove.
7.4.1.2. Remove the shield from the pipet assembly with a twist and fill the
capillary pipet with whole blood. Transfer the whole blood to reservoir as
follows:
7.4.1.2.1. Wipe excess blood from the outside of the capillary pipet, making
certain that no blood is removed from the capillary bore
7.4.1.2.2. Squeeze the reservoir slightly to force out some air. Maintain
pressure on the reservoir.
7.4.1.2.3. Cover opening of overflow chamber of the pipet with your index
finger and seat the pipet securely in the reservoir neck.
7.4.1.2.4. Release pressure on the reservoir. Then remove your finger from
the pipet opening. Negative pressure will draw the blood into the
diluent.
7.4.1.2.5. Squeeze the reservoir gently two or three times to rinse the
capillary bore, forcing diluent into, but not out of, the overflow
chamber, releasing pressure each time to return the mixture to
the reservoir.
7.4.1.2.6. Place your index finger over the pipet opening and gently invert
several times to thoroughly mix the blood with diluent.
7.4.1.2.7. Let stand for 10 minutes to allow erythrocytes to hemolyze.
7.4.2. Clean the hemacytometer and cover glass by flooding them with 70% alcohol. Dry
thoroughly with gauze or tissue; do not allow the alcohol to dry on the
hemacytometer. Be sure to remove all lint. Place the cover glass in position over the
ruled area.
AND PROCEDURES

7.4.3. Following incubation, mix diluted blood thoroughly by inverting reservoir to

resuspend cells. Charge hemacytometer as follows:
7.4.3.1. Convert to dropper assembly by withdrawing the pipet from the reservoir
and reseating it securely in its reverse position.
7.4.3.2. Clean the capillary bore by inverting the reservoir, gently squeeze the sides,
and discard the first three or four drops.
7.4.3.3. Place the pipet tip on the edge of the ruled area of the counting chamber.
Carefully charge the hemacytometer with diluted blood by gently squeezing
the sides of the reservoir to expel the contents until the chamber is
properly filled.
7.4.3.4. Repeat procedure to charge the other side of the hemacytometer with the
first Unopette reservoir.
7.4.3.5. Place the hemacytometer on moistened filter paper in a Petri dish, and
allow to stand 10 minutes to permit the cells to settle.
7.4.3.6. Using this same procedure, charge a second hemacytometer with the
second Unopette reservoir.
7.4.4. Carefully place the hemacytometer on the microscope stage. Perform cell count as
follows:
7.4.5. With the low-power (10x) objective, locate the ruled area and the center large
square (1 mm2). Examine the entire center square for even distribution of platelets,
then carefully switch to the high-dry-power (40x) phase objective for counting
platelets. With phase microscopy, platelets appear as round or oval bodies.
7.4.6. Platelets are counted in the entire center large square (1 mm2) as follows:
7.4.6.1. Count the platelets in the first row of squares going from left to right, then
from right to left in the second row; follow this pattern until all rows are
counted.
7.4.6.2. Within each square, count all platelets touching the top and left-hand
borders. Do not count any cells touching the bottom or right-hand borders.
7.4.6.3. Use the fine adjustment knob to focus up and down to identify the platelets.
7.4.7. Repeat this counting procedure for the other side of the hemacytometer
7.4.8. Record the counts for each center square. The difference between these two counts
should not exceed 10%.
7.4.9. Count the platelets on the second hemacytometer following the above counting
procedure.
7.5. Calculations
AND PROCEDURES

7.5.1. The calculation formula for hemacytometer cell counts determines the number of cells
within 1 mL (1 mm3) of blood. To make this determination, the total number of cells
counted must be corrected for the initial dilution of blood and the volume of diluted
blood used. The standard dilution of blood for platelet counts is 1:100; therefore the
dilution factor is 100. The volume of diluted blood used is based on the area and depth
of the counting area. The area counted is 2 mm 2 and the depth is 0.1 mm; therefore the
volume factor is 0.2 mm3.
cells/mm3= total number of cells counted x dilution factor x 1 / volume factor cell
cells x 109/L = cells /μL x 103 μL /L
8.0. ESTIMATED PLATELET COUNT

Platelets are seen as round or oval purple bodies. They vary in size normally from 1-5
micra. They are difficult to be counted accurately because of their small size and their
tendency to clump.
The platelet count is a important test to investigate bleeding disorders and to assess
clotting ability, or to monitor drug treatments.
8.1. Procedure: Indirect platelet count
8.1.1. Using a properly prepared and stained blood smear count the number of
platelets in ten fields.
8.1.2. The total number of platelets counted multiplied by two.
8.2. Normal values: 150-400 x 10^9/L
9.0. PERIPHERAL BLOOD SMEAR
A stained smear is evaluated in a procedure called differential count in which the relative
number of leucocytes could be done. Erythrocytes and platelets can also be evaluated. Also,
stained smears may be examined to identify blood parasites like malaria.
9.1. Principle:
Wright’s stain is a methyl alcohol solution of an acid dye and a basic dye. The acid is
known as eosin, it is red in color. The basic is known as methylene blue, it is blue in
color.In the staining process, a buffer solution is used to control the acid-base balance of
AND PROCEDURES

the stain.
used.
9.4. Procedure:
9.4.1. Make a blood smear
9.4.2. Air dry the smear.
9.4.3. Using the Wright’s stain, dip slides with fixative (ethyl alcohol). (5 dips)
9.4.4. Dip slides with primary stain (eosin). (5 dips)
9.4.5. Dip slides with secondary stain (methylene blue). (5 dips)
9.4.6. Wash gently with water.
9.4.7. Allow the slides to air dry. Examine under oil immersion.
10.0. CLOTTING TIME
The clotting time is used to screen for problems in the blood clotting or coagulation
mechanism. It measures the amount of time needed for the blood to form a fibrin clot. This
procedure tests the function of the coagulation factors rather than of the platelets in any of
one or more of the coagulation factors. This test however is not specific, though it may still
be used to screen pediatric patients before surgical procedures. A venous blood sample is
collected in a glass tube. The time it takes for the blood to coagulate (clot) is measured.
10.1. Principle:
The time required for blood to clot in a glass tube; a measure of the intrinsic system of
coagulation. In the Lee-White method, blood in test tubes is maintained at a constant
temperature and examined regularly until clotting occurs; the test can be also be
performed in capillary tubes
used.
10.3.1. Materials for capillary pncture
10.3.2. Glass slide
10.3.3. Timer
10.4. Procedure:Lee-White Method
10.4.1. Sterilize the site of puncture with 70% alcohol.
10.4.2. Allow the area to dry.
10.4.3. Make a firm quick stab on the site of puncture. Make sure that there will be a free
flowing blood.
AND PROCEDURES

10.4.4. Wipe off the first drop of blood with dry cotton.
10.4.5. Record the time of appearance of the second drop after 2 minutes.
10.4.6. Place a clean glass slide over the blood. Avoid touching and pressing the wound.
10.4.7. At half-minute intervals, draw the pointed part of the lancet across the drop of
blood.
10.4.8. When threads of fibrin cling to the end of the pointed part of the lancet stop and
record the time.
NOTE: The clotting time is the interval between the shedding of the second drop of blood until some
threads of fibrin cling to the pointed part of the lancet.
10.5. Normal Values: 5-10 minutes
11.0 BLEEDING TIME
The bleeding time measures the interaction between platelets and injured vascular
endothelium. It is simple but extremely useful to for studying the initial phase of
hemostasis formation of platelets plug. It is quite a sensitive test. Where a positive test
(prolonged bleeding time) nearly always indicates a clinical abnormality. Where clinical
suspicion is high but the initial test is negative. It should be repeated at least 3x at one
week interval before finally being reported as normal bleeding time.
11.1. Principle:
An incision 5 mm long x 1 mm deep is made on the lateral aspect of the volar surface of the
forearm and the time to cessation of bleeding is measured. Constant pressure supplied by a
sphygmomanometer) of 40 mm Hg. is applied and a disposable incision device is used to
standardize the procedure. Provided that fibrinogen levels and platelet count is normal,
this procedure will detect defective platelet function and is used as a screening test for
inherited and acquired platelet defects.
used.
11.3.1. sphygmomanometer
11.3.2. Pricker
11.3.3. Filter paper
AND PROCEDURES

11.3.4. Timer
11.3.5. Bandage
11.4. Procedure: IVY Method
11.4.1. Select a site on the patient's arm on the lateral aspect volar surface that is free of
veins, bruises, edematous areas, and scars and is approximately 5 cm below the
antecubital crease.
11.4.2. Clean the site with the alcohol prep.
11.4.3. Place the sphygmomanometer around the patient's arm approximately two inches
above the elbow and maintain 40 mm Hg.
11.4.4. Remove the "trigger" safety and place the incision device on the site with minimal
pressure so that both ends of the device touch the skin. Do not press hard.
11.4.5. Depress the "trigger" to make the incision then remove the device.
Discard the device in a "sharps" container.
11.4.6. Start the timing device and blot the edge of the incision at 30-second intervals
with the filter paper. Do not touch the incision with the filter paper.
11.4.7. Note the time that bleeding stops and report to the nearest 30 seconds.
11.4.8. To minimize scaring, bandage with a bandage is applied perpendicular to the
incision.
11.5. Normal Values: 2- 9 minutes.
12.0 ESR (ERYTHROCYTE SEDIMENTATION RATE)

12.1. Principle:
An ESR test measures the speed at which RBC falls to the bottom of an upright westergren
tube. This measurement is important because when abnormal protein are present in the
blood, typically due to inflammation or infection, they cause RBCs to clump together and
sink more quickly. It is useful in detecting inflammation in the body that may be caused by
infection, some cancers and certain autoimmune disease such as rheumatoid arthritis,
lupus and Kawasaki disease.
12.2. Specimen: Trisodium Citrate (0.4ml)
12.3.1. Winthrobe Tube
12.3.2. Timer
12.4. Procedure: Westergren Method
AND PROCEDURES

12.4.1. Mix Na Citrate for 4x. Label sedivial and remove sedivial cap.Using a transfer pipet,
obtain an aliquot of blood and fill the sedivial with blood to fill the line. Recap and
mix thoroughly.
12.4.2. Place sedivial in rack on a level surface. Insert the anti-zero tube into the sedivial
continue inserting until the tube test at the bottom of sedivial.
12.4.3. Verify the blood if at the zero mark and that there are no bubbles in the tube.
12.4.4. Allow the sample to stand undisturbed for exactly 1 hour and then read the results
of the sedimentation rate in mm/hr.
13.0 RETICULOCYTE COUNT

13.1. Principle:
The reticulocyte is a non-nucleated immature red cell containing residual RNA. A
supravital stain, new methylene blue, is used to precipitate the RNA into dark blue
filaments/granules to I dentify the reticulocyte.
13.2. Specimen: EDTA whole blood
13.3. Reagents/ Materials/Equipment: New methylene blue, 12x75 mm tubes, pipettes and glass
slides.
13.4. Procedures:
13.4.1 Put 2 drops of new methylene blue in the bottom of a 12x 75 mm tube using a
pipette and 2 drops of well mixed EDTA blood to the tube.
13.4.2 Mix blood / stain mixture.
13.4.3 Incubate mixture at least 5 minutes.
13.4.4 Mix solutions again and prepare 2 good smears, label and air dry.
13.4.5 Read the result using oil immersion field.
13.4.6 Examine at least 1000 red cells
13.4.7 Count the total number of red cells and the number of reticulocytes.
13.4.8 Calculate the percent of reticulocytes as follows:
Number of reticulocytes / 1000 x 100
Note: In order to decrease the microscopic field and thus make it easier to count the cells,
place a piece of paper containing a "Window" in the eyepiece of the microscope.
13.4.9. Reticulocytes are juvenile red cells that contain fine, deep violet granules
(remnants of the ribosomes and the ribonucleic acid present in the precursor cell)
arranged in a network.
13.5. Reference values
AND PROCEDURES

Adults & children = 0.2 - 2.0%

Infants = 2-6%
14.0 SICKLE CELL TEST:
The purpose of this test is to detect sickle cell disorder (Anaemia or Trait). Sickle cell
anaemia is caused by an abnormal form of haemoglobin known as haemoglobin-S, which
tends to precipitate in such a way that the red cell takes the sickling shape.
14.1. Principle:
One drop of blood is mixed with one drop of a sodium metabisulfite reagent on a slide. If
the red cells contain abnormal haemoglobin, they will become sickle-shaped (or half-moon
shape). The reagent sodium metabisulfite removes oxygen from the cells, allowing sickling
to take place.
used.
14.3.1. Sodium metabisulfite 2% Aqueous Solution:

14.3.2. Sodium metabisulfite (Na2S2O5) 0.2g
14.3.3. Distilled water 10ml
14.3.4. Always should be freshly prepared before use
14.4. Method:
14.4.1. Place a small drop of capillary blood in the centre of a slide.

14.4.2. Add an equal drop of the fresh sodium metabisulfite solution.
14.4.3. Mix carefully and cover with a cover slide, make sure that no air bubbles form. Seal
with Vaseline or DPX.
14.4.4. Place the slide in a wet chamber.
14.4.5. Examine under the microscope after 15 minutes using the X 40 objective. If the
result is negative, re-examine after one hour, then after a further hour and after 24
hours.
14.4.6. The test is negative if the red cells remain round.
AND PROCEDURES

14.4.7. The test is positive if the cells become sickle-shaped, or banana-shaped, often with
spikes.
STANDARD OPERATING
PROCEDURES
MICROSCOPY
AND PROCEDURES

MICROSCOPY
1.0. ROUTINE URINALYSIS
A urinalysis is a group of tests that detect and semi-quantitatively measure various

compounds that are eliminated in the urine, including the byproducts of normal and
abnormal metabolism as well as cells, including bacteria, and cellular fragments.
Urine is produced by the kidneys, located on either side of the spine at the bottom of the
ribcage. The kidneys filter wastes and metabolic byproducts out of the blood, help regulate
the amount of water in the body, and conserve proteins, electrolytes, and other compounds
that the body can reuse. Anything that is not needed is excreted in the urine and travels
from the kidneys to the bladder, through the urethra, and out of the body. Urine is
generally yellow and relativelyclear, but every time someone urinates, the color, quantity,
concentration, and content of the urine will be slightly different because of varying
constituents. Many disorders can be diagnosed in their early stages by detecting
abnormalities in the urine. These include increased concentrations of constituents that are
not usually found in significant quantities in the urine, such as: glucose, protein, bilirubin,
red blood cells, white blood cells, crystals, and bacteria. They may be present because there
are elevated concentrations of the substance in the blood and the body is trying to
AND PROCEDURES

decrease blood levels by “dumping” them in the urine, because kidney disease has made
the kidneys less effective at filtering, or in the case of bacteria, due to an infection.
1.1. Collection of Urine Specimen
Containers for the collection of urine should be wide-mouthed, clean and dry. If the urine
specimen has to be transported for any length of time it should contain an appropriate
preservative to prevent bacterial overgrowth or hatching of viable ova.
1.1.1. Early morning urine specimen - Early morning urine provides the most
concentrated sample.
1.1.2. Random urine specimen - A random urine sample, taken at any time of the day,
will enable the laboratory toscreen for substances which are indicators of kidney
infection.
1.1.3. 24-Hour urine specimen - The 24-hour urine specimen is collected in a clear 2-
litre bottle with a stopper. Onthe first morning the patient gets up and urinates; this
urine is not collected. All theurine passed during the rest of the day and night is
collected in the bottle. The nextmorning the patient gets up and collects the first
urine of the morning in the bottle.The bottle should then be taken immediately to
the laboratory.Measure the volume of urine with a measuring cylinder and record it.
1.1.4. Terminal urine specimen - The patient urinates the last portion of urine into an
open container.
1.1.5. Urine specimens collected using a catheter - Collection of urine using a catheter
must be carried out by a qualified physician ornurse. The procedure is used for
certain bacteriological tests, mainly in women.Usually, however, a specimen
collected in the normal way following thorough cleansingis acceptable for this
purpose.
1.1.6. Urine specimens from infants - Urine can be collected into a plastic bag with an
adhesive mouth. The bag is fixedaround the genitalia and left in place for 1–3 hours,
depending on the examinationrequested. Colostomy bags can be used.
1.2. Preservation of Urine Specimens
1.2.1. Urine passed at a clinic and examined immediately does not require preservation.
AND PROCEDURES

1.2.2. Urine that is not processed immediately should be put in a refrigerator

temeperature.
1.2.3. If urine has been collected to check for the presence of Schistosoma haematobium
ova but it may not be examined for several hours, it should be acidified with a few
drops of 10% acetic acid (reagent no. 2).
1.3. Macroscopic Examination Of The Urine Sample
1.3.1. Physical Examination

1.3.1.1. Urine Color
The color of urine varies from person to person and from time to time.
Normal urine is straw color or pale yellow. Deviations of almost colorless
to black may be due to normal metabolic functions, physical activity,
ingested materials, or pathologic conditions.
1.3.1.2. Transparency
This is a general term that refers to the clarity of a urine specimen. This is
done by visually examining the mixed specimen in a clear container
against a light source.
1.3.2. Chemical Examination
The chemical phase of urine analysis utilized a well-mixed urine specimen. The
urine specimen is usually collected during the time at which excretion or
presence of the analyte is highest.
1.3.2.1. pH - Determination of the pH of urine is useful for the identification of

crystalline deposits. Some crystals are deposited only in acid urine, others
only in alkaline urine. Urine pH becomes alkaline on standing, because of
the action of urea – splitting bacteria, which produce ammonia. The urine
pH of an uncovered specimen will become alkaline because carbon dioxide
vaporizes from the urine. Dietary factors affect urine pH.
Ingestion of large quantities of citrus fruits, dairy products, and
vegetables produces alkaline urine, whereas a diet high in meat
and certain foods produces acidic urine.
1.3.2.2. Specific Gravity - Specific gravity is a measurement of the kidney’s ability

to concentrate urine. Specific gravityis used to evaluate the concentrating
and excretory power of the kidneys. High specific gravity indicates
concentrated urine. Low specific gravity indicates dilute urine. Specific
AND PROCEDURES

gravity refers to the weight of the urine compared with that of distilled
water. Particles in the urine give it weight or specific gravity
1.3.2.3. Protein/albumin - Protein is a sensitive indicator of kidney

function. Normally, protein is not present in the urine because
the spaces in the normal glomerular filtrate membrane are too
small to allow its passage. If glomerular membrane is injured, the
spaces in the filtrate become larger, and the protein seeps into the filtrate,
then into the urine. If this persists at a significant rate, hypoproteinemia
can develop as a result of severe protein loss through the kidneys.
This decreases the normal capillary oncotic pressure that holds
fluid within the vasculature and causes severe interstitial edema.
1.3.2.4.
Glucose - Glucose is the most commonly found sugar substance in urine,
particularly in diabetic patients and patients suffering from chronic renal
failure. Glucose is a reducing substance. It reduces the blue copper sulfate
in Benedict solution to red copper oxide, which is insoluble. Lactose is also
a reducing sugar and is occasionally seen in the urine of pregnant women.
1.3.3. Reagents/ Materials
1.3.3.1. Test Tubes (5 Ml To 10 Ml Capacity)

1.3.3.2. Reagent Strips
1.3.3.3. Thermometer
1.3.3.4. Pipettes And Medicine Droppers
1.3.3.5. Alcohol Lamp
1.3.3.6. Evaporating Dish
1.3.3.7. Graduated Cylinder
1.3.3.8. Indirect Heating Set-Up: Hot Plate, 250 Ml Beaker, Boiling Chips And Test Tube
Floaters
1.3.3.9. Benedict’s Reagent
1.3.3.10. 10% Acetic Acid
1.3.3.11. Phenolphthalein
1.3.3.12. Benzidine Reagent
1.3.3.13. 3% Hydrogen Peroxide
AND PROCEDURES

1.3.4. Procedure:
1.3.4.1. Dry Chemistry: Reagent Strip Method

1.3.4.1.1. Briefly but completely dip the reagent strip into a well-mixed specimen.
1.3.4.1.2. Remove the excess urine as you withdraw the strip from the specimen
container.
1.3.4.1.3. Allow color development to occur according to the manufacturer’s
instructions before comparing the color reactions with the comparator
chart provided with the kit. Wait for the specified length of time for
reaction to take place.
CHEMICAL TESTS READING TIME
Glucose 30 seconds
pH 60 seconds
Sugar 45 seconds
Protein 60 seconds
1.3.4.1.4. Read the results against a light source
1.3.4.1.5. Record the results.
1.3.4.1.6. Care of reagent strips:
1.3.4.1.6.1. Store with a desiccant in an opaque, tightly closed
container.Store below 30 degrees; do not freeze.
1.3.4.1.6.2. Do not expose to volatile fumes.
1.3.4.1.6.3. Always check for expiration date.
1.3.4.1.6.4. Do not use if chemical pads become discolored.
1.3.4.1.6.5. Remove strips immediately prior to use.
1.3.4.2. Wet Chemistry

1.3.4.2.1. Screening For Bence-Jones Proteinuria By Heat Precipitation
1.3.4.2.1.1. Using water bath, heat an aliquot of a well-mixed

specimen(around 2/3 of a 10 ml capacity tube).
1.3.4.2.1.2. Monitor the temperature of the sample and observe for
coagulation at temperatures between 40 to 60 degrees
celsius. If no coagulation appears, report the specimen as
negative.
1.3.4.2.1.3. If coagulation occurs, continue heating the sample up to 100
degree celsius. If the coagulation disappears at this
AND PROCEDURES

temperature, report the specimen as positive for bence-jones

proteinuria screening test.
1.3.4.2.1.4. If the coagulation persists, report the specimen as negative.
1.3.4.2.2. Qualitative Test For Proteinuria By Heat And Acid Method
1.3.4.2.2.1. Using direct heating, heat an aliquot of a well-mixed

specimen(around 2/3 of a 10 ml capacity tube) making sure
that the only upper portion of the test tube is exposed to the
flame.
1.3.4.2.2.2. Continue heating the upper portion of the tube until either
boiling or cloudiness is observed.
1.3.4.2.2.3. If only boiling of the sample occurs, terminate the test and
report the specimen as negative.
1.3.4.2.2.4. If the cloudiness is observed, stratify the specimen with a
maximum of 1 ml 10% acetic acid while the specimen is still
hot.
1.3.4.2.2.5. A persistent cloudiness indicates proteinuria whereas the
disappearance of the cloudiness after the addition of the
acetic acid indicates a negative result.
1.3.4.2.3. Semi-Quantitative Test For Glucosuria By Copper Reduction Test
1.3.4.2.3.1. Mix 5ml of benedict’s reagent with ml aliquot of urine
sample gently by inversion.
1.3.4.2.3.2. Heat preparation using a water bath for a maximum of 5
minutes. (Note: start the timer while the solution starts to
boi).
1.3.4.2.3.3. Read the result immediately:
No change in color – negative(-)
Green solution without precipitate – trace(T)
Green solution with yellow precipitate – plus one (+)
Yellow solution with yellow precipitate – plus two (++)
Yellow-orange solution with brick red precipitate – (+++)
1.5.2.4. Qualitative Test For Ketonuria By Legal’s Test
1.5.2.4.1. Mix 1drop of phenolphthalein and 5 ml of aliquot of urine
sample in an evaporating dish.
1.5.2.4.2. Add naoh or koh drop by drop until permanent pale pink-
AND PROCEDURES

colored solution is achieved.

1.5.2.4.3. Add 10 drops of fresh sodium nitopusside solution and mix by
swirling or stirring.
1.5.2.4.4. A purple or violet color indicates the presence of acetone.
1.5.2.5. Semi-Quantitative Test For Blood, Hemoglobin, And Myoglobin For

Benzidine Test
1.5.2.6. Mix 1 ml of benzidine reagent with 1 ml of urine in an
evaporating dish.
1.5.2.7. Add 0.5 ml of 3% hydrogen peroxide and mix by swirling or
stirring.
1.5.2.8. Observe color development for 1 minute.
1.5.2.9. Interpret the results as follows:
Faint green- T
Green – (+)
Greenish blue – (++)
Blue – (+++)
Deep blue – (++++)
1.4. Microscopic Examination Of The Urine Sample
1.4.1. Sediment Preparation
1.4.1.1. Be sure to bring urine to room temperature before preparing the
sediments.
1.4.1.2. Transfers 10-15 ml(12 ml is recommended) well-mixed sample in a
conical centrifuge tube.
1.4.1.3. Centrifuge the specimen for 5 minutes using an RCF of 400.
1.4.1.4. Decant the supernatant.
1.4.1.5. Gently suspend the pellet by mild agitation.
1.4.1.6. Place 2o ul if the sediments at the center of a glass slide and cover with a
22 x 22 mm glass cover slip.
1.4.2. Sediment Examination
1.4.2.1. Check for the general composition of the sediment using LPO while
keeping an eye for elements that are of clinical significance. Upon
encountering such elements, change the setting to HPO to identify them.
1.4.2.2. Make sure that the bright-field microscope is operating under a reduced
light intensity.
1.4.2.3. Examine a minimum of 10 fields under both low and high power field.
1.4.3. Reporting Of Results
AND PROCEDURES

1.4.3.1. The following are the common ways of reporting microscopic elements:
1.4.3.1.1. Range- range between the lowest and the highest number of a
particular element counted in 10 fields.
1.4.3.1.2. Average number- average of all numbers obtained per field.
1.4.3.1.3. Estimated count: use semi-quantitative terms
Rare or +1 – zero to five per field
Few or +2 – six to ten per field
Moderate or +3 – eleven to twenty per field
Many or +4 – more than twenty per field
Obscuring TNTC - too numerous to count, other elements
are observed.
1.4.3.2. The following structures are reported using LPO :
1.4.3.2.1 Estimated amount/lpf
- Epithelial cells
- Crystals (identify type)
- Mucus threads
- Cylindroids
- Amorphous sediments
1.4.3.2.2. Range/ lpf: casts (identify type)

1.4.3.3. The following structures are reported using HPO:
1.4.3.3.1. Estimated amount/hpf: Yeast Cells, Bacteria
1.4.3.3.2. Range/hpf: RBC, WBC
1.5. Elements Found In Urine:
1.5.1. Erythrocytes
1.5.1.1. Erythrocytes in urine may be:

1.5.1.1.1. Intact: small yellowish discs, darker at the edges (8mm)
1.5.1.1.2. Crenated: spiky edges, reduced diameter (5–6mm)
1.5.1.1.3. Swollen: thin circles, increased diameter (9–10mm)
1.5.1.2. The shape of the cells often changes during storage of urine and does not
have any diagnostic importance. There are normally very few erythrocytes
in urine.
AND PROCEDURES

Note: Erythrocytes may be found in the urine of women if the specimen has been taken during the
menstrual period.
1.5.2. Leukocytes
1.5.2.1. Leukocytes found in urine may be:

1.5.2.1.1. Intact: clear granular discs, 10–15mm (the nuclei may be
visible);
1.5.2.1.2. Degenerated: distorted shape, shrunken, less granular;
1.5.2.1.3. Pus: clumps of numerous degenerated cells.
1.5.2.2. The presence of many leukocytes, especially in clumps, indicates a urinary
tract infection.
1.5.3. Ureteral and renal pelvic cells
1.5.3.1. Medium-sized oval cells with a distinct nucleus.

1.5.3.2. If many cells are present together with leukocytes and filaments, they may
be from the ureter. If a few are present, with no leukocytes, they may be cells
from the renal pelvis.
1.5.4. Renal cells
Renal cells are smaller than renal pelvic cells (the size of 1–2 leukocytes) and are very
granular. The nucleus is shiny and clearly visible. Renal cells are almost always
present with protein in the urine.
1.5.5. Casts
1.5.5.1. Casts are cylindrical in shape and long, crossing almost the whole field
when examined under the x40 objective.
1.5.5.2. Hyaline casts are transparent and slightly shiny; the ends are rounded or
tapered. They may be found in healthy persons after strenuous muscular
effort and have no diagnostic significance.
1.5.5.3. Granular casts are rather short casts filled with large granules, pale yellow
in colour, with rounded ends. The granules come from degenerated
AND PROCEDURES

epithelial cells from the tubules of the kidney and have no diagnostic
significance.
1.5.5.4. Fine granular casts have smaller granules that do not fill the cast. Do not
confuse with hyaline casts, partly covered by amorphous phosphate
crystals.
1.5.5.5. Blood casts are filled with more or less degenerated erythrocytes,
brownish in colour. They are found in acute kidney disease.
1.5.5.6. Pus casts are completely filled with leukocytes. Do not confuse with hyaline
casts, which may contain a few leukocytes. Pus casts are found in patients
suffering from kidney infection.
1.5.5.7. Epithelial casts are filled with pale yellow epithelial cells . (To make the
cells more distinct, add a drop of 10% acetic acid (reagent no. 2) to the
deposit.) Epithelial casts have no diagnostic significance.
1.5.5.8. Fatty casts are very shiny yellowish casts; the edges are indented and
distinct and the ends are rounded They are soluble in ether but not in
acetic acid. Fatty casts are found in patients with severe kidney disease.
1.5.5.9. False casts
1.5.5.9.1. clumps of phosphate crystals, short and clear-cut;
1.5.5.9.2. aggregations of translucent mucus, the ends tapering into
threads.
1.5.6. Miscellaneous foreign substances
1.5.6.1. If dirty receptacles or slides are used or if the urine specimen is left
exposed to the air, the following may be found:
1.5.6.1.1. Oil droplets (shiny)

1.5.6.1.2. Starch granules (which will be stained blue–black with lugol
iodine, 0.5%
1.5.6.1.3. Solution;
1.5.6.1.4. Grains of pollen from flowers;
1.5.6.1.5. Hairs ;
1.5.6.1.6. Cotton fibres
1.5.6.1.7. Air bubbles
1.5.7. Crystals
AND PROCEDURES

Crystals have regular geometric shapes (a), unlike amorphous debris, which is
made up of clumps of small granules with no definite shape (b). Except in very
rare diseases, crystals in urine have no diagnostic significance.
1.5.7.1. Normal crystalline deposits

1.5.7.1.1. Calcium oxalate (acid urine)
1.5.7.1.1.1. Size: 10–20mm or about 50mm
1.5.7.1.1.2. Shape: envelope-shaped or peanut-shaped
1.5.7.1.1.3. Color: colorless, very shiny.
1.5.7.1.2. Uric acid (acid urine)
1.5.7.1.2.1. Size: 30–150mm.
1.5.7.1.2.2. Shape: varies (square, diamond-shaped, cubical or
rose-shaped).
1.5.7.1.2.3. Color: yellow or brownish-red.
1.5.7.1.3. Triple phosphates (neutral or alkaline urine)
1.5.7.1.3.1. Size: 30–150mm.
1.5.7.1.3.2. Shape: rectangular or like a fern leaf or star
1.5.7.1.3.3. Color: colorless, shiny.
1.5.7.1.4. Urates (alkaline urine)
1.5.7.1.4.1. Size: about 20mm.
1.5.7.1.4.2. Shape: like a cactus or a bundle of needles
1.5.7.1.4.3. Color: yellow, shiny.
1.5.7.1.4.4. Urates are often found together with phosphates.
1.5.7.1.5. Calcium phosphate (neutral or alkaline urine)
1.5.7.1.5.1. Size: 30–40mm.
1.5.7.1.5.2. Shape: like a star.
1.5.7.1.5.3. Color: colorless.
1.5.7.1.6. Calcium carbonate (neutral or alkaline urine)
1.5.7.1.6.1. Size: very small.
1.5.7.1.6.2. Shape: similar to millet or corn grains, grouped in
pairs.
1.5.7.1.6.3. Color: colorless.
1.5.7.1.6.4. If acetic acid, 10% solution (reagent no. 2) is added,
the crystals dissolve, giving offbubbles of gas.
1.5.7.1.7. Calcium sulfate (acid urine)
1.5.7.1.7.1. Size: 50–100mm.
AND PROCEDURES

1.5.7.1.7.2. Shape: long prisms or flat blades, separate or in

bundles. Calcium sulfate crystals can be
distinguished from calcium phosphate crystals by
measuring the ph of the urine.
1.5.7.1.8. Amorphous debris
1.5.7.1.8.1. Amorphous phosphates (alkaline urine)
Amorphous phosphates appear as small, whitish

granules, often scattered.They are soluble in acetic
acid, 10% solution (reagent no. 2) (one drop per
drop ofdeposit).
1.5.7.1.8.2. Amorphous urates (acid urine)
Amorphous urates appear as very small, yellowish

granules, which are grouped incompact clusters.
They are not soluble in acetic acid, 10% solution
(reagent no. 2), but dissolve if theurine is gently
heated.(Urine kept in the refrigerator often shows a
heavy precipitate of urates.)
1.5.7.2. Other crystalline deposits
The following are rarely found in the urine. When present, however, they are
foundin large quantities in patients with certain diseases.
1.5.7.2.1. Cystine (acid urine)
1.7.5.2.1.1. Size: 30–60mm.
1.7.5.2.1.2. Shape: hexagonal plates.
1.7.5.2.1.3. Colour: colourless, very shiny.
1.7.5.2.1.4. Cystine crystals are found only in fresh urine as
they are soluble in ammonia.
1.7.5.2.1.5. They are found in patients with cystinuria, a very
rare hereditary disease.
1.5.7.2.2. Cholesterol (acid urine)
1.5.7.2.2.1. Size: 50–100 mm.
1.5.7.2.2.2. Shape: squarish plates, with notches on one side.
1.5.7.2.2.3. Colour: colourless, shiny.
1.5.7.2.2.4. Cholesterol crystals are found in the urine of
patients with nephrotic syndrome
AND PROCEDURES

1.5.7.2.3. Bilirubin (very rare)

1.5.7.2.3.1. Size: about 5mm.
1.5.7.2.3.2. Shape: square or like beads or needles.
1.5.7.2.3.3. Colour: brown.
1.5.7.2.3.4. (The chemical test for bile pigments is positive.)
1.5.7.2.4. Acetyl sulfonamides (neutral or acid urine)
1.5.7.2.4.1. Shape: varied, but often similar to sheaves of
needles.
1.5.7.2.4.2. Acetyl sulfonamide crystals are found in the urine
following treatment with
Sulfonamide drugs. The presence of these crystals
should be reported as they can
Cause kidney damage.
1.5.8. Fungi
1.5.8.1. Size: 5–12mm.

1.5.8.2. Shape: round or oval bodies of various sizes found together. Do not
confuse withErythrocytes. Budding may be seen. Fungi are not soluble in
acetic acid.
1.5.8.3. Fungi are occasionally present in urine containing glucose. Check that the
urine
Specimen is fresh.
1.5.9. Parasite eggs and larvae
1.5.9.1. The following may be found:

1.5.9.1.1. Eggs of Schistosoma haematobium: found together with
erythrocytes.
1.5.9.1.2. Microfilariae of Wuchereria bancrofti ;the urine appears
whiteand cloudy.
2.0. GRAM STAIN
The Gram Stain is used to numerate cells and numerate and identify the microbiological
flora present. Although the exact mechanism of the Gram Stain is unknown, it is thought
AND PROCEDURES

that the bacterial cell wall composition (specifically the amount of lipid present) is
responsible for the characteristic Gram Stain reactions. Crystal violet is the primary stain
that all the bacteria take up and iodine acts as a mordant that fixes the crystal violet to the
bacteria. When the decolorizing agent (acetone) is applied, the cells with a high lipid
content in their cell wall lose this lipid and become more permeable, allowing the purple
crystal violet-iodine complex to escape. The bacteria without the high lipid content is
dehydrated by the decolorizing agent, become less permeable, and do not lose the dye
complex. These dehydrated cells cannot pick up the Safranin counter-stain and will remain
purple. These bacteria are referred to as “Gram positive”. The bacteria that become
permeable as a result of the decolorizing agent will now stain with Safranin and the
resulting color is red. These are “Gram negative” bacteria.
2.1. Specimen Preparation
2.1.1. Write the specimen accession number on the frosted end of the slide with a lead
pencil. Do not use a pen or felt tip pen because these inks will wash off during the
decolorization step.
2.1.2. Specimens submitted on swabs require no sample preparation before making the
Gram Stain slide. Make the Gram Stain slide before inoculating bacteriological media.
Gently roll the swab over the inscribed circle on the slide taking care to spread
thinly. Rolling prevents the disruption of the cellular components in the specimen
and the creation of aerosols when working with pathologic material.
2.1.3. Mix urine specimens by swirling for 15 seconds. Change the direction of swirling 3
times before placing an aliquot of the urine within the inscribed circle on the slide.
2.1.4. Use a 4 mm loop to obtain the necessary aliquot. Smear one loopful of urine over the
entire area of the inscribed circle.
Note: Do not centrifuge urine specimens before Gram Stain preparation or inoculation
of media.
2.1.5. Centrifuge body fluids (cerebral spinal fluids (CSF), synovial fluid, peritoneal, etc.) at
1000 g for 15 minutes.
2.1.6. Observe the following guidelines:
2.1.6.1. Centrifuge in a sealed sterile tube.
AND PROCEDURES

2.1.6.2. Do not centrifuge purulent (grossly turbid) material. Prepare Gram Stains
directly from the uncentrifuged material.
2.1.6.3. Centrifuge cloudy or slightly turbid fluids and examine the sediment by
Gram Stain.
2.1.6.4. Volume guidelines: 15 ml or less of fluid--the entire specimen is
centrifuged to obtain the working sediment.Greater than 15 ml of fluid--
the entire specimen is mixed (20 rapid inversions) and 10-15 ml are
withdrawn and centrifuged.
2.1.6.5. Decant the supernatant into a sterile tube. Mix the sediment with the small
amount of fluid that drains back from the sides of the tube. Forcefully
aspirate the sediment up and down in a sterile pipette several times to
adequately disperse the organisms and cells that remain adhered to the
bottom of the tube after centrifugation.
Note: Mixing of the sediment is critical. Use a microbiological loop or sterile Pasteur pipette
to prepare the slide and inoculate media.
2.2. Materials
2.2.1. Clean slides with a frosted end inscribed with a circle by a diamond point pen and
sterilized.
2.2.2. Staining rack or area
2.2.3. Forceps or tweezers
2.2.4. Microscope
2.3. Reagents
2.3.1. Crystal Violet

2.3.2. Gram’s Iodine
2.3.3. Acetone
2.3.4. Safranin
2.3.5. Methanol
2.4. Procedure:
2.4.1. Allow the slide to air dry at room temperature.
AND PROCEDURES

Note: Do not apply heat to speed up the drying process because this causes distortion
of cells and may cause aberrant Gram Staining.
2.4.2. Fix the slide after drying using either the heat-fix or methanol-fix method:
2.4.2.1. Heat-Fix Method:
2.4.2.1.1. Slowly pass the slide through the flame of a Bunsen burner (2
seconds) three times. (The slide should be hot enough to cause
discomfort when placed on the back of the hand but it should not
burn the hand.)
1.4.2.1.2. Allow the slide to cool.
1.4.2.1.3. Another technique may be used. Place the slide for one minute
on top of a Bacti-Incinerator that has been preheated for 15
minutes. Allow the slide to cool.
Note: The one minute time period is an approximation-a time
much shorter than this may result in improper fixing and loss of
the specimen during staining. A time much longer than this may
result in disintegration of cellular morphology and aberrant Gram
Stain results.
2.4.2.2. Methanol-Fix Method:
2.4.2.1.1.
Place the slide on a staining rack and flood the slide with
methanol. Let the slide fix for 1 minute.
2.4.2.1.2. Drain the alcohol from the slide and allow the methanol to
evaporate prior to Gram staining.
2.4.2.1.3. Another technique may be used. Place the slide in a Copeland
jar filled with methanol for 1 minute. Allow the methanol to
evaporate off the slide before Gram staining.
2.4.3. Flood the smear with crystal violet solution and let stand for one minute.
Note: The crystal violet stage of staining is critical. If the stain is allowed to partially
dry and precipitate, artifacts resembling Gram positive cocci may occur.
2.4.4. Pick the slide up with a forceps or tweezers. Hold the slide parallel to a stream of
gently flowing tap water or Type I water and rinse for 5-10 seconds. Drain off any
excess water. Place the slide back on the staining rack after rinsing.
2.4.5. Flood the slide with iodine and let stand for one minute. Wash with tap water or
Type I water.
AND PROCEDURES

Note: This stage of the Gram stain is least critical in terms of timing. However, the
Gram’s iodine cannot be allowed to completely evaporate from the slide because of
artifact formation. If this occurs, discard the slide.
2.4.6. Hold the slide with a tweezers or forceps. Allow acetone to run over and off the
entire slide until no further color flows from the slide. This usually takes 5-10
seconds, depending on the thickness of the smear. Rinse the slide with tap water or
Type I water.
2.4.7. Place the slide back on the rack after rinsing.
Note: The decolorization process is the most critical step in the Gram Stain
procedure.Over or under decolorization may result in misinterpretation, improper
patient therapy, and personal embarrassment.
2.4.8. Flood the slide with Safranin and let it stand for 10 seconds in order to counterstain
the smear. Rinse the slide with tap water or Type I water.
Note: Safranin counterstaining is critical because the precipitate may form red rod-
like crystals that resemble Gram negative rods.
2.4.9. Allow the slide to air dry before examination. If examination is needed before air
drying is complete, use the following procedure to blot the slide dry:
2.4.9.1. Lay a paper towel on the countertop.
2.4.9.2. Lay the slide on the paper towel with the stained side up.
2.4.9.3. Gently fold the towel over the slide and lightly press down on the slide to
blot any excess moisture from the slide. Do not rub the towel on the front of
the slide as this may result in loss of stained specimen.
2.4.9.4. Blotting slides is less desirable than air drying because of the risk of
specimen loss and artifact introduction.
2.4.10. Examine the stained dry smear under oil immersion.
2.4.10.1. Gram positive organisms are purple.

2.4.10.2. Gram negative organisms are red.
2.4.10.3. Cellular components (epithelial cells and white blood cells) stain red.
AND PROCEDURES

2.4.10.4. White blood cells will be best differentiated in thin portions of the smear.
Do not try to “guess” the type of white blood cells in thick area
2.5. Results Derivation
2.5.1. Some infectious agents can be readily identified on a Gram Stain. These agents are
presumptively identified and reported to the physician. The bacterial agents
commonly seen in infections and the presumptive reports are listed in Table 1.
2.5.2. The technologist must be aware of specimens, which contain normal bacterial flora
(sputum, vagina) in contrast to those specimens that are normally sterile. This
knowledge is used to determine if the bacteria present on a Gram Stain are
pathogens or normal endogenous flora. If a specimen contains normal
microbiological flora and no obvious predominant bacterial pathogen is seen on the
Gram Stain, a report, “mixed bacterial flora” is issued. Table 2 contains a list of
specimen sources and pathogenic organisms most likely to be seen on a Gram Stain.
2.5.3. Differentiation of white blood cells found on the Gram Stain provides useful
information for the clinician. The presence of polymorphonuclear cells may indicate
a bacterial infection while the presence of mononuclear cells may indicate a viral,
tuberculous, or fungal agent. The CSF glucose is usually depressed in cases of acute
bacterial or tuberculous meningitis and normal in the other meningitides. CSF
protein is usually elevated in meningitis. Correlate the results of the cell count and
chemistries with the Gram Stain results before reporting the Gram Stain results.
2.5.4. Use the following guidelines to numerate polymorphonuclear cells, mononuclear
cells, and microbiological flora found on a Gram Stain:
Numeration of Cellular Elements and Microbiological Flora
Number / Oil Field Report as:
AND PROCEDURES

0 No
1 Rare
2–4 Few
5 – 10 Moderate
≥ 11 Many
Table 1: Bacterial Agents Commonly Seen in Infections
Organism: Microscopic Morphology: Report As:
Staphylococcus Gram positive cocci in clumps. Gram positive cocci

(suggestive of
Staphylococcus).
Streptococcus Gram positive cocci in chains. Gram positive cocci

(suggestive of
Streptococcus).
Streptococcus pneumonia Gram positive lancet-shaped Gram positive cocci

cocci in pairs and short chains, (suggestive of Streptococcus
pneumoniae).
Haemophilus Tiny Gram negative rods Tiny Gram negative rods

(suggestive of
Haemophilus).
Coliforms Large Gram negative rods Gram negative rods
AND PROCEDURES

(suggestive of coliforms).
Neisseria sp.
Neisseria gonorrhoeae Gram negative intracellular For males:

(from genital specimens) diplococci. (See note 3) “Gram negative intracellular
diplococci (suggestive of
Neisseria gonorrhoeae)”
For females:
“Gram negative intracellular
Neisseria gonorrhoeae)
Culture confirmation is
necessary.”
Gram negative extracellular For males:

diplococci “Gram negative extracellular
Neisseria gonorrhoeae)
Culture confirmation is
necessary.”
For females:
(If rare, and associated with
other bacteria, usually Gram
positive rods, report only
as):
“mixed bacterial flora.”
Neisseria meningitidis Gram negative diplococci Gram negative diplococci

(suggestive of Neisseria sp.).
AND PROCEDURES

Candida albicans Yeast with pseudomycelium Yeast (suggestive of Candida

sp.)
Cryptococcus Encapsulated yeast Positive for encapsulated

yeast (suggestive of
Cryptococcus sp.).
Clostridium perfringens Gram positive rods Gram positive rods

(suggestive of Clostridium
perfringens).
Gardnerella vaginalis Presence of clue cells; vaginal Small gram negative rods
epithelial cells covered by small (suggestive of Gardnerella
gram negative rods. vaginalis).
Note(1): In clinical material, spores are rarely seen with C. perfringens. If spores are found, report out
Clostridium sp. Clostridium is only reported in specimens from areas that are normally sterile,
report as mixed bacterial flora and quantitate.
Note(2): Encapsulated yeast is seen with an India Ink preparation. Spinal fluids are the most common
specimen in which encapsulated yeast are seen.
Note(3): A statement regarding the absence or possible presence of Neisseria gonorrhoeae is
reported on all genital specimen Gram Stains except placentas. Because of the difficulty of
interpretation (eg. overdecolorized Staphylococcus in males and non-Neisseria Gram negative cocci
in females) and the social ramifications, a pathologist must be consulted about any Gram Stain
report suggestive of Neisseria gonorrhoeae before reporting. If extracellular but no intracellular
Gram negative diplococci are present in a specimen from a male patient, the diagnosis should be
confirmed by culture. Refer to Table 1item F for examples of reporting. For smears of male urethral
exudates, positive correlation with culture of N. gonorrhoeae is greater than 95% for smears
containing intracellular Gram negative diplococci. In females, however, smears of endocervical
secretions detect only 50-70% of culture positive specimens. Thus, both Gram Stained smears and
cultures of endocervical secretions should be done.
Table 2: Specimen Sources and Pathogens Most Often Seen as Determined by Gram Stain
AND PROCEDURES

Specimens: Pathogens:
Lower RespiratorySee note 1 Streptococcus pneumoniae

and note 2 Haemophilus
Coliforms
Yeast
Staphylococcus
Urinary System Coliforms

Streptococcus
Genital System See note 1 Candida albicans

Neisseria gonorrhoeae
Gardnerella vaginalis
Gastrointestinal See note 1 Yeast

Staphylococcus
Abscesses (pelvic, Anaerobes

culdocentesis, brain, etc…) Streptococcus
Staphylococcus
Wounds
Deep Anaerobes
Superficial Staphylococcus
Streptococcus
Cerebral Spinal Fluid See Haemophilus

note 3 and note 4 Streptococcus pneumoniae
Streptococcus
AND PROCEDURES

Cryptococcus neoformans
Neisseria meningitidis
Listeria monocytogenes
Note(1): Normal flora, which consists of many different mixed bacterial types, is seen in the majority
of the specimens.
Note(2): Pathogens most commonly found in spinal fluids are related to the age of the patient.
Note(3): A pathologist must be consulted before any CSF Gram Stain or Methylene Blue Stain
containing organisms is reported. If there are >5 polys/mm 3 and no organisms are seen, report the
results without calling a pathologist and have a pathologist review the slide as soon as possible
during regularly scheduled hours.
2.6. Quality Control
2.6.1. Stain a control slide each time a new lot of reagent is put into use and weekly
thereafter.
2.6.2. Prepare control slides in the following manner:
2.6.2.1. From subcultures:
2.6.2.1.1. Use subcultures (no older than 48 hours) of S. aureus and E. coli
or P. aeruginosa to prepare quality control smears.
2.6.2.1.2. Place two small drops of water onto each of two inscribed circles
on a slide labeled + and -.
2.6.2.1.3. Use a wooden applicator stick to place a portion of the S. aureus
subculture into the + circle and E. coli or P. aeruginosa in the -
circle. Spread within the respective circles.
2.6.2.1.4. Allow the slide to dry and stain according to the Gram Stain
procedure.
2.6.2.2. From suspension in water:
2.6.2.2.1. Prepare a heavy suspension of Staphylococcus aureus and E. coli

or Pseudomonas aeruginosa in water.
AND PROCEDURES

2.6.2.2.2. Place a drop of the suspensions onto each of two inscribed circles
on a slide labeled + and - in the respective circles.
2.6.2.2.3. Allow the slide to dry and stain according to the Gram Stain
procedure.
2.6.3. Acceptable results:
2.6.3.1. S. aureus will stain as purple cocci, which are referred to as Gram positive.
2.6.3.2. E. coli or P. aeruginosa will stain as red rods and are referred to as Gram
negative.
2.6.3.3. Record the results on the Quality Control card.
2.6.4. Corrective action:
2.6.4.1. If the slide does not stain as expected, the following course of action is
followed:
2.6.4.1.1. Do not report outpatient results.

2.6.4.1.2. Prepare and stain a repeat control slide paying particular
attention to the decolorization process.
2.6.4.1.3. Carefully check the reagents to make sure they are acceptable.
2.6.4.1.4. Follow the “Incidence Recording” procedure.
2.6.5. Save Gram Stained smears for a minimum of 30 days. Place the smears, by accession
number in a slide box. Date the outside of the box once it is full and place in the
appropriate drawer. Discard after 30 days have elapsed.
3.0. Routine Fecal Analysis
Parasitic diseases are often present with non-specific symptoms and signs. Most parasitic
diseases cannot be diagnosed by physical examination alone, and laboratory investigation
is necessary to decide whether or not the patient is infected with a parasite and what
species of parasite is present.
3.1. Collection of Stool Specimen
AND PROCEDURES

3.1.1. The reliability of the results obtained will depend largely on the care taken in
collecting the specimen. The following precautions should be taken in collecting
specimens for parasitological examination.
3.1.1.1. Permit detection of parasites, if present, in low concentration.
3.1.1.2. Prevent rapid drying of stools. The specimen should contain at least
4cucm.
3.1.2. Provision of a container for the patient's use. The patient should be given a waxed
cardboard/light plastic box (container) for collection of the specimen.
3.1.3. Examination of stools while fresh:a) Stools must be examined within one hour of
collection.b) If a number of specimens are received at the same time, pick out: -
Liquid stools and those containing mucous or blood. Examine them first, because
they may contain motile amoebae (Trophozoite) that die quickly.
3.1.4. Things not to do:
3.1.4.1. Never leave stool specimens exposed to the air in containers without lids.
3.1.4.2. Never set aside stool specimens for examination after 2 to 3 hours.
3.1.4.3. Never accept stools mixed with urine or water.
3.1.4.4. Never place the container of the stool specimen on the examination request
form.
3.1.5. Quality of Specimens:
3.1.5.1. Specimen should be obtained before any type therapy initiated, since
antibiotics, antihelmintics, antidiarrhoeal drugs, antacids, laxatives, soap,
and hypertonic salt enemas suppress parasites. At least 1 week should be
allowed to elapse after treatment before reexamination of the stool for
parasites.
3.1.6. Preservatives used to preserve stool specimens:
3.1.6.1. Formalin
3.1.6.2. Polyvinyl alcohol (PVA) Fixative.
3.2. Macroscopic (Gross) Examination

3.2.1. Note the following characteristics of the specimen:
3.2.1.1. Consistency of stool:
3.2.1.1.1. Formed : may contain cysts, eggs, and larvae.
3.2.6.1.1. Soft : may contain trophozoite, cysts, eggs and larvae.
3.2.6.2. Character of stool: may be bloody, mucoid, watery or pussy.
3.2.6.3. Presence or absence of whole worms or parts of worms in strained
specimen, e.g., proglottides, scolices, or adult organisms such as pinworms,
roundworms, tapeworms, hookworms.
AND PROCEDURES

3.2.2. Microscopic Examination

3.2.2.1. Direct Method:
3.2.2.1.1. Saline wet mounts : Trophozoite (motility), cysts, eggs and larvae
3.2.2.1.2. Eosin in saline: Trophozoite (motility), cysts, eggs and larvae – an
optional technique
3.2.2.1.3. Iodine wet mounts .: Cysts, eggs and larvae
3.2.2.2. Procedure:
3.2.2.2.1. Mark the number of the specimen on the slide.
3.2.2.2.2. Put two drops of normal saline in the middle of the slide.
3.2.2.2.3. Using an applicator, take a small portion of the stool from inside and
from the surface of the specimen.
3.2.2.2.4. Mix the sample with the drops of saline solution on the slide.
3.2.2.2.5. Place a coverslip over the mixture.
3.2.2.2.6. Examine the preparation under the microscope use the 10x and 40x
objectives. Reduce the amount of light by lowering the condenser to
increase the contrast.
3.2.2.2.7. Repeat the steps (a) to (f) by using a drop of working iodine instead of
normal saline if you suspect the presence of ova, cyst or trophozoite.
3.2.2.3. Reagents:
3.2.2.3.1. Normal Saline: Dissolve 8.5g sodium chloride (NaCl) in one litre of
distilled water.
3.2.2.3.2. Lugol Iodine (stock):
Iodine Potassium 1 g
Iodide (KI) 2 g
Distilled water (DW) 100 ml
Dissolve the KI in about 30ml D.W., add the iodine and mix until
dissolved, complete to 100ml with D.W. and store in a brown bottle.
3.2.2.3.3. Working Iodine Solution:
Dilute 5 times the stock iodine with D.W.
3.2.2.4. Concentration Method:
This procedure should be used whenever possible especially if the number of
organisms in the stool is low and direct wet mount may not detect parasites.The
direct wet mount examination is necessary because the protozoan trophozoite
will not be seen in the concentration method.
3.2.2.5. Can be performed by two preparations:
3.2.2.5.1. Saline wet mounts : Cysts, eggs and larvae.
3.2.2.5.2. Iodine wet mounts : Cysts, eggs and larvae.
AND PROCEDURES

3.2.2.6. Procedure:
3.2.2.6.1. Add 5ml of 10% formalin to about 1g of faeces.
3.2.2.6.2. Mix until you get a suspension.
3.2.2.6.3. Filter the suspension through a gauze filter into a clean centrifuge tube.
Discard the gauze filter with residue.
3.2.2.6.4. Add 2ml of ether or ethyl acetate and mix well for at least one minute.
3.2.2.6.5. Centrifuge for one minute.
3.2.2.6.6. Loosen the fatty plug at the top with a stick applicator, pour away the
supernatant by inverting the tube.
3.2.2.6.7. Mix the sediment at the bottom of the tube.
3.2.2.6.8. Transfer one drop to a slide for examination.
3.2.2.6.9. Use the X10 and X40 objectives to examine the whole area under the
cover slip for ova, cysts or larvae.
4.0. OCCULT BLOOD

Blood in the stool may be grossly visible or it may be in small amounts and revealed only
by specific test - occult blood. The presence of blood in the faeces is always abnormal and
denotes haemorrhage into the alimentary tract.
4.1. Principle:
Heme compounds catalyze the oxidation of organic substances such as benzidine,
Orthotolidine or 4-aminophenazone by hydrogen peroxide.
4.2. Reagents:
4.2.1. Glacial Acetic Acid 30% Glacial Acetic Acid Distilled water
4.2.2. 4-Aminophenazone 5% 4-Aminophenazone Ethyl Alcohol (95%)
4.2.3. Hydrogen peroxide 3% Hydrogen peroxide concentrated Distilled water
4.3. Procedure:
4.3.1. Place a small portion of faeces on a piece of filter paper and spread it on a small area.
4.3.2. Add one drop of Glacial acetic acid on the stool area.
4.3.3. Add on drop of 4-Aminophenazone on the same area.
4.3.4. Add one drop of hydrogen peroxide reagent on the same area.
4.3.5. A positive reaction is shown by the appearance of a blue ring at the junction of the
two fluids.
NOTE: The following procedures stated on the next pages may be used as referral procedures
for examination of stool samples. The guidelines were taken from the recent WHO
Bench Aids for Parasitology.
AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

AND PROCEDURES

Various structures that may be seen in stool preparations:
I-8. Various yeast cells. 9. Macrophage with nucleus. 10-11. Deteriorated macrophage without
nucleus. 12-13. Polymorphonuclear leucocytes.14-15. Pollen grains.16. Charcot-Leyden crystals.
2 ACID-FAST BACILLI STAIN
Acid-fastness is a physical property of certain bacteria (and, less commonly, protozoa),

specifically their resistance to decolorization by acids during staining procedures. Acid-fast
organisms are difficult to characterize using standard microbiological techniques (e.g.
AND PROCEDURES

Gram stain - if an acid-fast bacillus (AFB) was gram stained, the result would be an
abnormal gram positive organism, which would indicate that further testing was
necessary), though they can be stained using concentrated dyes, particularly when the
staining process is combined with heat. Once stained, these organisms resist the dilute acid
and/or ethanol-based de-colorization procedures common in many staining protocols,
hence the name acid-fast
The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is
responsible for the staining pattern of poor absorption followed by high retention. The
most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen
stain, in which the acid fast bacilli are stained bright red and stand out clearly against a blue
background.
5.1. Clinical Significance
Most Mycobacteria grow at a relatively slow rate; therefore, the acid-fast smear plays an
important role in the early diagnosis of Mycobacterial infections. The acid-fast smear
should not be used in place of culture. It is an adjunct, since studies have shown that 5,000
- 10,000 bacilli per ml of sputum are required for recognition by direct microscopy; culture
detects as few as 10 - 100 viable Mycobacteria.
5.2. Specimen collection
5.2.1. Label innermost tube with patient name and date of birth before obtaining
specimens and before giving container to patient for home sampling.
5.2.2. Instruct patient to:

5.2.2.1. Rinse mouth well with water to avoid contamination with food particles
and mouth flora. NOTE: Rinsing may result in contamination with m.
gordonae if the water is contaminated. Not rinsing at all may result if
excessive overgrowth of normal flora.
5.2.2.2. If patient will provide specimens from home, instruct to obtain specimen
before eating and to store specimen in refrigerator until transported to
clinic.
5.2.2.3. Inhale deeply 2-3 times, breathe out hard each time.
5.2.2.4. Cough deeply from the chest.
AND PROCEDURES

5.2.2.5. Place the open container close to the mouth to collect the specimen.
5.2.2.6. Avoid contaminating the inside of the container and lid.
5.2.2.7. If the patient is unable to raise early morning sputum, suggest that he/she
stand or sit in a steamy environment for 15 minutes first by running hot
water in the shower. If that still fails, obtain a physician’s order for
sputum induction.
Note:
Offer to have the patient empty bladder just prior to procedure. Place waterproof shield on
chair if patient will be seated during procedure.
The nurse will supervise collecting the first specimen to ensure that the patient
demonstrates correct technique.
The ideal specimen size is 5 to 10 mL. The absolute minimum specimen size is 2 mL.
A deeply coughed specimen is the preferred specimen (not saliva or nasal secretions).
Alternately, transtracheal aspirate, gastric aspiration or bronchial brushing are methods
that may be used in other settings.
For initial sampling, it is recommended that three (3) specimens are collected for AFB smear
and culture at least eight hours apart within a 48 hour period. The first specimen is collected
on the spot when the patient is first encountered, regardless of time of day in order to
expedite management among ill suspects and unreliable patients. At least one specimen
should be a first morning specimen. The nurse may obtain additional specimens if there is a
question about the integrity of a given specimen, but not more than 5 to 6 specimens.
For follow-up sampling for patients with active TB disease, collect a specimen for AFB smear,
culture and AST) once the patient has completed two months of appropriate therapy.
When the patient will be collecting specimens at home, give him/her a labeled container and
instruct patient to collect an early morning specimen before eating. Instruct patient to store
specimen in the refrigerator and return specimen to the clinic as soon as possible after
collection, preferably within 1 to 2 hours after collection.
5.3. Materials
5.3.1 Microscope slide with frosted end, 75 x 25 mm
AND PROCEDURES

5.3.2 Secure line red pen
5.4. Reagents
Store reagents at 15 - 30°C in the flammable storage cabinet in the supply room.Store
working reagent in the cabinet under the staining sink. Store all reagents in the dark after
removing from the box.
5.4.1. Carbolfuchsin
Warning: Flammable liquid and vapor. May cause eye, skin, or respiratory irritation.
Possible cancer hazard.
5.4.2. Decolorizer
Harmful if swallowed.
5.4.3. Methylene blue
5.4.4. Methanol
5.5. Procedure
5.5.1. Write the specimen accession number on the frosted end of the slide with a lead
pencil or Secureline red pen. Do not use a pen or a felt tip pen because these inks
will wash off during the decolorization step.
AND PROCEDURES

5.5.2. Draw 2 circles with the Secureline red pen, one for a concentrated specimen (direct
and concentrated) and one for an unconcentrated specimen.
5.5.3. Vigorously vortex the sediment from concentrated specimens. If possible, thoroughly
mix (vortex or stir) specimens that have not been concentrated.
5.5.4. Transfer a representative portion of the specimen to the slide by using a pipette or
swab. Use the sediment obtained from the concentration process for concentrated
specimens. Use bloody or necrotic material, if available, for unconcentrated
specimens.
5.5.5. Smear the specimen on the slide over an area about the size of a dime.
5.5.6. Allow the slides to air dry at room temperature in the biological safety cabinet.
Note: Do not apply heat to speed up the drying process because this causes distortion of cells
and may cause aberrant staining.
5.5.7. Heat-fix the slides after drying. Slowly pass the slides through the flame of a Bunsen
burner (2 seconds) three times. Avoid excessive heating. The slides should be hot
enough to cause discomfort when placed on the back of the hand but should not
burn the hand. Let the slides cool until no longer warm to the touch.
5.5.8. Place the slides in a jar of absolute methanol for 1 minute since heat-fixing may not
kill all Mycobacteria, or flood the slides on the staining rack with absolute methanol
for 1 minute.
5.5.9. Place the slides on a staining rack.
5.5.10. Flood the slides with Carbolfuchsin for ten minutes.
5.5.11. Rinse gently in running Type I water.
5.5.12. Decolorize withDecolorizer for 3 to 5 seconds or until no more pink color appears
in the washing.
5.5.13. Rinse gently in running Type I water.
AND PROCEDURES

5.5.14. Counter-stain with Methylene blue and let stand for 1 minute.
5.5.15. Rinse gently with Type I water.
5.5.16. Air dry over gentle heat. Do not blot.
5.6. Results Derivation
5.6.1. Examine the smear with oil immersion lens, (1000X) taking care to wipe the lens
well after each examination, especially if the smear is positive. Make a series of three
parallel longitudinal sweeps over the entire smear. A minimum of 300 fields is
examined before a smear is reported as negative.
5.6.2. The acid-fast bacilli stain red. Acid-fast bacilli are approximately 1 - 10 µm long and
typically appear as slender, rod-shaped bacilli, but they may appear curved or bent.
Individual bacteria may display heavily stained areas referred to as beads and areas
of alternating stain producing a banded appearance. Some mycobacterium other
than M. tuberculosis may appear pleomorphic, ranging in appearance from long rods
to coccoid forms, with uniform distribution of staining properties. Nonacid-fast
organisms stain blue. The background material stains blue.
5.6.3. Reports state the staining method used and the quantitation (number) of acid-fast
bacilli seen on smear. A typical report is as follows: Kinyoun Acid-Fast Smear: No
acid-fast bacilli seen.
5.6.4. United Clinical Laboratories guidelines for reporting smears are as follows:
Note: The smears are scanned with a 100X objective.
AND PROCEDURES

Number of bacilli / 1000X Report
0 Negative for acid-fast bacillus
1 - 2 / 300 fields (entire smear) Perform repeat smear from the same specimen.
If 1 - 2 are present, report the number found
and request another specimen.
1 – 9 / 100 fields Rare
1 – 9 / 10 fields Few
1 – 9 / field Moderate
> 9 / field Many

5.7.1. Slide Preparation:
5.7.1.1. Prepare separate suspensions approximately equal to a No. 1

McFarland turbidity standard of E. coli or P aeruginosa (negative
control) and M. gordonae (positive control) in 1 ml of sterile saline
or Type I water in a biological safety cabinet.
Note: Do not use Mycobacteria considered to be rapid growers, since they

demonstrate an inconsistent ability to retain acid-fast stains.
5.7.1.2. Label the control slides and use a pipette to place one drop of the
negative control suspension on one end of the slide and the
positive control in the area between the frosted end of the slide and
the negative control.
5.7.1.3. Allow the slides to air dry in the biological safety cabinet.
AND PROCEDURES

5.7.1.4. Fix using heat and absolute methanol as described

previously.
5.7.1.5. Store slides in a labeled slide box.
5.7.2. Smear Evaluation:
5.7.2.1. Known acid-fast positive and negative control organisms

are stained with each batch of patient acid-fast slides.
5.7.2.2. A notation is recorded in the accession log including the date, the
statement “Control Smear: Satisfactory (OK)” and initials when
the acid-fast organisms stain red and the nonacid-fast organisms
stain blue.
6.0. KOH
The KOH Prep is used to detect fungal elements in clinical specimens by microscopic examination.
Cellular material may mask fungal elements. KOH will dissolve the keratin in these specimens, making
any fungi more visible.
6.1. Clinical Significance
Microscopic detection of a fungus in clinical specimens can alert the physician to the etiology of the
disease, help establish the significance of the fungus, and provide early information that may be crucial
for determining appropriate therapy.
6.2. Collection of Specimen
Collect specimen using a sterile scraper (tongue depressor, scalpel or tweezers). Sponge
the infected area with 70% alcohol and allow to dry. Scrape the active (leading) edge of the
lesion or top of vesicle with the sterile scraper into a sterile container. Label appropriately
AND PROCEDURES

(including source, date and time of collection). Transport specimen to the laboratory
immediately after collection.
6.3. Materials
6.3.1. Glass microscope slide
6.3.2. Wooden applicator stick
6.3.3. 22 x 22 mm coverslip
6.3.4. Heating element
6.3.5. Microscope
6.4. Reagent
6.4.1. 10% KOH
6.5. Procedure
6.5.1. Place a drop of 10% KOH in the center of a glass microscope slide.
6.5.2. Place a fragment of tissue, purulent material or scrapings in the KOH and tease the
material with a wooden stick to give a thin preparation.
6.5.3. Mount with a cover slip and gently heat the prep by passing it through a Bunsen
burner flame two or three times or setting the slide on a Bacti-incinerator for one
minute. Do not boil. This process will dissolve keratinized cells.
6.5.4. When the specimen has cleared, (about 20 minutes) examine under low power with
reduced light. Confirm observations with high power. It is rarely necessary to use
oil immersion to recognize a mycotic agent in a KOH preparation. Look for hypae,
AND PROCEDURES

arthroconidia and yeast. On hair specimens, determine if the fungus is growing

outside the hair shaft (ectothrix invasion) or inside the hair shaft (endothrix
invasion).
6.6. Reporting of Results
6.6.1. If fungal elements are not observed report: Fungal elements: None seen.
6.6.2. If fungal element are observed report the type of fungal elements seen:
6.6.2.1. Fungal elements: Mycelium present
6.6.2.2. Fungal elements: Yeast present.
7.0. PREGNANCY TEST
Human Chorionic Gonadotropin (hCG) is a hormone that is elevated during pregnancy, it

could be detected in urine or blood. Its detection aid in early prediction of pregnancy in
general. The test is a rapid chromatographic immunoassay for the qualitative detection of
(hCG). The test utilizes a combination of antibodies, including a monoclonal (hCG)
antibody, to selectively detect elevated levels of (hCG).
7.1. Specimen Collection
7.1.1. Method of collection The urine specimen must be collected in a clean, dry container.
7.1.2. Volume of specimen required- 5 pipette drops (included) or approximately 2ml.
7.1.3. Special patient preparation requirements
Specimens collected at any time of day may be used; however, the first morning
urine generally contains the highest concentration of hormone. Fresh urine does not
require anyspecial pretreatment.
7.1.4. Handling Conditions of specimen
AND PROCEDURES

Urine specimens may be refrigerated (2-8°C) and stored up to 72 hours prior to

testing. If samples are refrigerated, they must be equilibrated to room temperature
before testing.
7.1.5. Criteria
Urine samples exhibiting visible precipitates should be filtered, centrifuged or

allowed to settle, and clear aliquots are obtained for testing.
7.2. Materials
7.2.1. 50 individually wrapped test devices. Each device contains an anti-alpha hCG capture
antibody coated membrane and colloidal gold particles coated with mouse anti-beta
hCG monoclonal antibody.
7.2.2. 50 disposable dropper pipettes
7.2.3. One instruction insert
7.2.4. Specimen collection container
7.2.5. Timer
7.3. Procedure:
Timing is critical. For procedural steps, please refer to the manufacturer’s instruction as
per the kit insert.
7.4. Interpretation
7.4.1. POSITIVE: Two distinct pink-colored bands appear, one in the patient test region
(T) and one in the control region (C).
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7.4.2. NEGATIVE: Only one pink-colored band appears in the control region (C). No
apparent pink band appears in the patient test region (T).
7.4.3. INVALID: A total absence of pink-colored bands in both regions is an indication of

procedural error or that test reagent deterioration has occurred.
Notes on the interpretation of results
Negative test results in patients suspected to be pregnant should be retested with a sample
obtained 48 to 72 hours later, or by performing a quantitative assay. When testing with a
urine specimen, the first morning specimen would contain the highest concentration of hCG.
The shade of pink in the test band region (T) will vary depending on the concentration of hCG
present. However, neither the quantitative value nor the rate of increase can be determined
by a qualitative test.
7.5. Limitations
7.5.2. A number of conditions other than pregnancy including trophoblastic disease and
certain nontrophoblastic neoplasms cause elevated levels of hCG. These diagnoses
should be considered if appropriate to the clinical evidence.
7.5.3. If a urine specimen is too dilute (i.e. low specific gravity) it may not contain
representativelevels of hCG. If pregnancy is still suspected, a first morning urine
should be obtained from the patient 48-72 hours later and tested.
7.5.4. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the
results of a single test, but should only be made by a physician after all clinical and
laboratory findings have been evaluated.
7.5.5. Immunologically interfering substances such as those used in antibody therapy

treatments may invalidate the test result.
7.5.6. Some samples containing very high levels of hCG (≈ 200,000 mlU/ml) may yield a
test line with a color intensity lighter than what is expected. When high dose “hook
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effect” is suspected a 1:10 dilution of the specimen with DI H20 is recommended.

The test should then be repeated with the diluted specimen.
A procedural control is included in the test. A colored band appearing on the control region
(C) is considered an internal positive procedural control, indicating proper performance
and reactive reagents. A clear background in the results window is considered an internal
negative procedural control. If the test has been performed correctly and reagents are
working properly, the background will clear to give a discernible result.
It is recommended that two levels of control specimens be used with each new kit,
however, each laboratory should follow their state and local requirements. For this
purpose, we recommend the use of SD Bioline hCG (Positive and Negative) controls.
8.0. SPERM ANALYSIS
Semen analysis comprises a set of descriptive measurements of spermatozoa and seminal

fluid parameters that help to estimate semen quality.
Conventional semen analysis includes measurement of particular aspects of spermatozoa

such as concentration, motility and morphology and of seminal plasma. Quantification and
identification of non-spermatozoidal cells and detection of antisperm antibodies are also
part of basic semen analysis.
Normal values of semen parameters issued by the World Health Organisation (WHO) in
1992 are generally used as reference values (Table I).
Ideally, each laboratory should set its own normal values, reflecting the specific population
analyzed.
Table I. Normal values of semen variables (WHO 1992)
Standard tests
Volume 2.0 ml or more
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pH 7.2-8.0
sperm concentration 20x106 spermatozoa/ml or more
total sperm count 40x106spermatozoa per ejaculate or more
Motility 50% or more with forward progression(categories a and
b)or 25% or more with rapid progression(category a)within
60 minutes of ejaculation
Morphology 30% or more with normal forms
Vitality 75% or more live,i.e.,excluding dye
white blood cells fewer than 1x106/ml
immunobead test fewer than 20% spermatozoa with adherent particles
MAR test fewer than 10% spermatozoa with adherent particles
Optional tests
a -Glucosidase(neutral) 20 mU or more per ejaculate
zinc(total) 2.4 m -mol or more per ejaculate
citric acid(total) 52 m -mol or more per ejaculate
acid phosphatase(total) 200 U or more per ejaculate
fructose(total) 13 m -mol or more per ejaculate
Table II. Nomenclature for semen variables (WHO 1992)
normozoospermia normal ejaculate as defined in table I

oligozoospermia sperm concentration fewer than 20x106/ml
asthenozoospermia fewer than 50% spermatozoa with forward
progression(categories a and b)or fewer than 25%
spermatozoa with category a movement
teratozoospermia fewer than 30% spermatozoa with normal morphology
oligoasthenoteratozoospermia signifies disturbance of all three variables(combination
of only two prefixes can be used)
azoospermia no spermatozoa in the ejaculate
aspermia no ejaculate
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Normal semen is an admixture of spermatozoa suspended in secretions from the testis and
epididymus which are mixed at the time of ejaculation with secretions from the prostate,
seminal vesicles, and bulbourethral glands. The final composition is a viscous fluid that
comprises the ejaculate
8.1. Sample collection and delivery
The following instructions for sample collection and delivery are based on WHO
recommendations. The subject should be provided with clearly written or oral instructions
concerning the collection and, if required, transport of the semen sample.
8.1.1. The sample should be collected after a minimum of 48 hours and no longer than 7
days of sexual abstinence. The name of the man, period of abstinence, date and time
of collection should be recorded. The time interval between the last ejaculation and
sample collection should be well defined and preferentially as constant as possible
in order to allow a reliable interpretation of the results of, in particular, sperm
concentration and motility. When the duration of abstinence is more than 7 days,
sperm motility, i.e. the proportion of spermatozoa with rapid progressive motility,
may decline. If the duration of abstinence is <48h, sperm concentration may be
reduced, but motility will probably not be affected.
8.1.2. Two semen samples should be collected for initial evaluation. The interval of time
between the collections will depend on local circumstances but should not be less
than 7 days or more than 3 months apart. If the results of these assessments are
remarkably different, additional semen samples should be tested because marked
variations in sperm output may occur within the same individual. Analysis of
multiple semen specimens provides a reliable screen in the evaluation of male factor
infertility. Information and support are important since semen analysis cause a
moderate amount of stress.
8.1.3. Ideally the sample should be collected in the privacy of a room near the laboratory. If
not, it should be delivered to the laboratory within 1h after collection.
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8.1.4. The sample should be obtained by masturbation and ejaculated into a clean, wide-
mouthed glass or plastic container. If plastic is used, it should be checked for lack of
toxic effects on spermatozoa. The container should be warm to minimize the risk of
cold shock.
8.1.5. Ordinary condoms must not be used for semen collection because they may
interfere with the viability of spermatozoa. In cases in which masturbation is not
possible or against an individual’s values, the specimen can be collected in a non-
spermicidal condom following intercourse. It has been shown that semen samples
collected during intercourse using a special plastic condom or a silastic collection
device tend to have better parameters. Other authors, referring to their experience,
hold the view that the quality of the specimen when collected in this way is generally
compromised. This way of collection should be considered for a second sample if the
first one shows a relatively low volume. Coitus interruptus is not acceptable as a
means of collection because it is possible that the first portion of the ejaculate,
which contains the highest concentration of spermatozoa, will be lost. Moreover,
there will be cellular and bacteriological contamination of the sample and the acid
pH of the vaginal fluid will adversely affect sperm motility.
8.1.6. Incomplete samples should be not analyzed, particularly if the first portion of the
ejaculate is lost. The sample should be protected from extremes of temperature (not
less than 20°c and not more than 40°c) during transport to the laboratory. The
sample should be examined immediately after liquefaction and certainly within 1h
of ejaculation.
Note:Laboratory technicians should be aware that semen samples may contain

harmful viruses (e.g., HIV and viruses causing hepatitis and herpes ) and should
therefore be handled with due care.
8.2 Macroscopic Analysis
8.2.1. Appearance
The semen sample is first evaluated by simple inspection. A normal sample has a
grey-opalescent appearance, is homogenous and liquefies within 60min at room
temperature under the influence of enzymes of prostatic origin. In some cases,
liquefaction does not occur within the normal time period and this fact should be
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recorded, as it may suggest functional disturbance of the prostate. Normal semen

samples may contain jelly-like grains which do not liquefy.
The sample may appear clear if the sperm concentration is too low. It may also
appear brown when red blood cells are present in the ejaculate (haematospermia).
The presence of mucous streaks may interfere with the counting procedure and
suggests inflammation or abnormal liquefaction.
Samples which do not liquefy need additional treatment such as exposure to

bromelin, to make the sample amenable to analysis.
The sample should be well mixed in the original container. Incomplete mixing is
probably a major contributor to errors in determining sperm concentration.
8.2.2. Consistency
The consistency, also called viscosity, of the liquefied sample can be estimated by
gentle aspiration into a 5-ml pipette and then allowing the semen to drop by gravity
and observing the length of the thread formed. A normal sample leaves the needle as
small discrete drops, while in cases of abnormal consistency the drop will form a
thread of >2 cm. Another method to estimate consistency does not use needles and
is performed by introducing a glass rod into the sample and observing the thread
that forms on withdrawal of the rod. Again the thread should not exceed 2 cm.
Increased consistency has the same clinical meaning as abnormal liquefaction, and
may be related to prostate dysfunction resulting from chronic inflammation.
Very viscous specimens can impair the availability of fertile sperm at the site of
fertilization .
8.2.3. Volume
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The major component of the ejaculate volume is made up of secretions from the
accessory glands. The bulk of the volume is secreted by the seminal vesicles and
between 0.5 and 1 ml originates from the prostate. The volume of the ejaculate
should be measured either with a graduated cylinder or by aspirating the whole
sample into a wide-mouthed pipette by means of a mechanical device.The sample
volume can also be determined directly in the collection tube by weighing, assuming
1ml equals 1g.. Thereby, loss of volume associated with transfer from the collection
tube to either another tube or a pipette can be avoided
A low ejaculate volume can reflect abnormalities in accessory sex gland fluid
synthesis or secretion. It can also be indicative of a physical obstruction somewhere
in the reproductive tract, or may occur in cases of incomplete or (partially)
retrograde ejaculation.
Large volumes are sometimes found in association with varicocele or after relatively
long periods of sexual abstinence.
8.2.4. pH
The pH is determined by acidic secretions of the prostate and alkaline secretions of

the seminal vesicles. It should normally be in the range of 7.2-8.
Recently, one author has shown that the mean pH values are consistently well above
8.0 regardless of the method of analysis and the time of examination and has
suggested that the range of normal values needs to be revised further.
To test pH, pH paper range 6.1 to 10.0 is used. Whatever type of pH paper is used for
this analysis, its accuracy should be checked against known standards before the use
in routine semen analysis.
If the pH exceeds 8.0, infection should be suspected with decreased secretion of

acidic products by the prostate, such as citric acid. Abnormal pH may also be
recorded in cases of incomplete ejaculation. Extremely acidic pH (<6.5) is found in
cases of agenesis (or occlusion) of the seminal vesicles.
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8.3. Microscopic Analysis
During the initial microscopic investigation of the sample, estimation of motility and
concentration of spermatozoa is performed. The presence of cells other than spermatozoa
and of agglutination of spermatozoa are determined.
8.3.1. Motility
In recent years, a number of techniques for objective assessment of movement

characteristics of human spermatozoa have been introduced by using computer-
assisted semen analysis (CASA) systems. For the purpose of conventional analysis, a
simple classification system which provides the best possible assessment of sperm
motility without resorting to complex equipment is recommended.
A fixed volume of semen (not more than 10 m l ) is delivered onto a clean glass slide
and covered with a 22x22 mm coverslip. It is important that the volume of semen
and the dimension of the coverslip are standardized so that the analyses are always
carried out in a preparation with fixed depth (i.e., 20m l). This depth allows full
expression of the rotating movement of normal spermatozoa. The preparation is
then examined at a magnification of x400-600. An ordinary light microscope can be
used for unstained preparations, particularly if the condenser is lowered to disperse
the light. However, a phase-contrast microscope is preferable.
The weight of the coverslip spreads the sample for optimal viewing. The freshly
made, wet preparation is left to stabilize for approximately one minute. Motility
estimation can conveniently be carried out at a room temperature between 18 and
24°c. At temperatures outside this range, some alteration in sperm motility will
occur and this must be standardized in the laboratory.
The microscopic field is scanned systematically and the motility of each

spermatozoon encountered is graded a, b ,c or d according to whether it shows:
 (a) rapid progressive motility.

 (b) slow or sluggish progressive motility
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 (c) non-progressive motility.

 (d) immotility.
Spermatozoa graded (a) are supposed to display rapid progressive motility along a
linear track, covering a distance of at least 20 m m (half the length of a
spermatozoon) per second.
At least 100 spermatozoa are classified in this way. Visual field close to the border of
the coverslip should be avoided.
It is advisable to repeat the procedure on a second drop of semen processed in the

same way.
8.3.2. Estimation of sperm concentration
The concentration can be estimated roughly during the initial examination in order
to determine the dilution procedure to be used and to indicate whether
centrifugation may be required to prepare an adequate smear for morphologic
analysis.
8.3.3. Cells other than spermatozoa
The ejaculate usually contains cells other than spermatozoa. These include
polygonal cells from the urethral tract. If many of these are present, and they are
covered with bacteria then it is probably that the sample was obtained by coitus
interruptus and the cells originate from the vagina. Spermatogenic cells and white
blood cells (WBC), which are often referred to as "round cells", are present in almost
every semen sample. By conventional light microscopy or sperm staining techniques
it is not possible to reliably differentiate WBC from immature germ cells in semen. In
contrast, the cytochemical peroxidase method reliably identifies granulocytes, the
most prevalent WBC type in semen. The method is cheap, fast and easy to perform.
The gold standard for the detection of all WBC populations in semen is immuno-
cytology using monoclonal antibodies. However, it is expensive and time-consuming,
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thus remaining a research tool at present. For clinical purposes, the peroxidase
method is ideally suited to detect granulocytes.
The method aims at the counting of peroxidase-positive round cells in a

haemocytometer. The working solution is prepared by combining 1ml of saturated
NH4Cl solution, 1ml of 5% of Na2 EDTA solution, 9ml of orthotoluidine solution and
1 drop of H2O2. This solution is mixed before use and can be conserved for 24h after
preparation. The procedure consists of mixing 0.1ml of semen with 0.9ml of the
working solution to achieve a total volume of 1ml. This mixture is shaken for 2 min.
It is then left for 20-30 min at room temperature and mixed again by shaking. The
mixture is now transferred onto a haemocytometer chamber for leukocytes and the
number of peroxidase-positive cells which stain brown is counted. Peroxidase-
negative cells remain unstained and are counted in the haemocytometer chamber.
The differentiation of round cells into either peroxidase-positive polymorphonuclear
granulocytes or peroxidase-negative spermatogenic cells or lymphocytes is of
clinical relevance. The presence of an excessive number of peroxidase-negative
mostly spermatogenic cells suggests pathology at the level of the seminiferous
epithelium with inadequate spermatogenesis and premature release of spermatids
spermatocytes or, rarely, spermatogonia. The pathological meaning of the presence
of an elevated number of WBC is still a matter of dispute. Some reports have
demonstrated that leukocytospermia appears to be of no diagnostic value to identify
men with actual microbial infections. Also, measurement of seminal leukocytes in
routine semen analysis appears to be of little prognostic value with regard to male
fertilizing potential.
Others hold the view that the presence of an elevated number of WBC may be
associated with infection or inflammation of the accessory glands and that the
unfavorable effect on spermatozoa of hydrogen peroxide secreted by peroxidase-
positive WBC has clearly been proven.
A comprehensive approach that considers other clinical and laboratory findings

seems to be more reliable in detecting male accessory gland infection.
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8.3.4. Agglutination
Agglutination of spermatozoa means that motile spermatozoa stick to each other,

head to head, midpiece to midpiece, tail to tail, or mixed, e.g. midpiece to tail. The
adherence of either immotile or motile spermatozoa to mucus threads, to cells other
than spermatozoa, or to debris is not considered agglutination and should not be
recorded as such.
The presence of agglutination is suggestive of, but not sufficient evidence to prove
the existence of an immunological factor of fertility . The extent of agglutination may
be important but even the presence of only a few groups of small numbers of
agglutinated spermatozoa should be recorded. In case of agglutination, sperm
culture must be performed in order to exclude infection with e.g. Escheria coli.
Sperm agglutination could be used also as indication for antisperm antibody testing
of infertile men.
8.3.5. Sperm viability
Vital staining of the spermatozoa allows quantification of the fraction of living cells
independently of their motility. Live and dead sperm are distinguished by adding
one drop of eosin y stain to one drop of semen at room temperature (one to two
minutes) and smearing the mixture on a microscopic slide. 100 spermatozoa are
classified as either colored orange-red, if the stain has passed through the
membrane and therefore the cell is considered dead, or non-stained, the cell than
being considered alive.
This staining technique makes it possible to differentiate spermatozoa that are

immotile but alive from those that are dead. Reduced percentage of motility with a
high percentage of viable sperm may reflect structural or metabolic abnormalities of
sperm that are derived from abnormalities in testicular function or antimotility
factors in the seminal plasma.
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This technique also provides a check on the accuracy of the motility evaluation, since
the percentage of dead cells should not exceed the percentage of immotile
spermatozoa.
8.3.6. Hypo-osmotic swelling (HOS) test
The hypo-osmotic swelling (HOS) test measures sperm membrane integrity by

examining its ability to swell when exposed to hypo-osmotic media, and has been
claimed to be relevant to fertilizing ability. The rationale of the test is based on the
assumption that an undamaged sperm tail membrane permits passage of fluid into
the cytoplasmic space causing swelling and the pressure generated leads to curling
of tail fibers, while the damaged or chemically inactive membrane allows fluid to
pass across the membrane without any accumulation and accordingly no
cytoplasmic swelling and curling of the tail occur.
The HOS test should not be used as a sperm function test but may be used as an
optional, additional vitality test. It is simple to perform and easy to score and gives
additional information on the integrity and the compliance of the cell membrane of
the sperm tail.
8.3.7. Counting the spermatozoa
The concentration of spermatozoa should be determined using the haemocytometer

method.
In this procedure a 1:20 dilution from each well-mixed sample is prepared by

diluting 50 m l of liquefied semen with 950 m l diluent. The latter is prepared by
adding 50 g of sodium carbonate (NaHCO3), 10ml of 35% (v/v) formalin and,
optionally, 0.25 g of trypan blue or 5ml of saturated aqueous gentian violet to
distilled water and making up the solution to a final volume of 1000ml. The stain
needs not to be included if a phase-contrast microscopy is used. If the preliminary
examination of the semen indicates that the concentration of spermatozoa present is
either excessively high or low, then the extent to which the sample is diluted should
AND PROCEDURES

be adjusted accordingly. For samples containing less than 20x10 6 spermatozoa/ml,

a 1:10 dilution should be used; for samples containing more than 100x10 6
spermatozoa/ml, a 1:50 dilution may be appropriate. Both chambers of the
haematocytometer are scored and the average count is calculated, provided that the
difference between the two counts does not exceed 1/20 of their sum (i.e., less than
10% difference). If the two counts are not within 10%, they are discarded, the
sample dilution re-mixed and another haemocytometer prepared and counted.
An optional procedure for determining sperm concentration employs specialized

counting chambers, e.g. Makler chamber.
The total number of spermatozoa per ejaculate reflects spermatogenesis and is

related to the duration of sexual abstinence.
Perhaps the most widely utilized semen parameter is sperm count. Men with
<20x10 6 spermatozoa per ml are typically deemed sub-fertile, and men with counts
<5x10 6 spermatozoa/ml are often considered infertile.
Other authors have confirmed that in patients with sperm counts <20x10 6/ml the
fertility potential is significantly impaired. However, it must be emphasized that
patients with sperm counts <20x10 6 are not infertile. It simply takes them a
substantially longer period of time to achieve pregnancies
8.3.8. Analysis of the morphological characteristics of spermatozoa
Sperm cells represent a unique population in which up to 50% (up to 70% according
to WHO criteria 1992 and up to 86% according to strict criteria) of the cells can have
morphological defects in normal fertile individuals (4). Although the morphological
variability of the human spermatozoon makes differential sperm morphology
evaluation very difficult, observations on the selection of spermatozoa recovered
from the female reproductive tract (especially in post coital cervical mucus) helped
to define the appearance of a normal spermatozoon. The normal head should be oval
in shape. Allowing for the slight shrinkage that fixation and staining induce, the
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length of the head should be 4.0-5.5 m m, and the width 2.5-3.5 m m. The length-to-
width ratio should be 1.50 to 1.75. There should be a well-defined acrosomal region
comprising 40-70% of the head area. There must be no neck, midpiece or tail defects
and no cytoplasmic droplet more than one-third the size of a normal sperm head.
This classification scheme requires that all borderline forms be considered
abnormal.
8.3.8.1. The following categories of defects should be scored.
8.3.8.1.1. Head shape/size defects, including large, small, tapering,

pyriform, amorphous, vacuolated (>20% of the head area
occupied by unstained vacuolar areas), or double heads, or any
combination of these.
8.3.8.1.2. Neck and midpiece defects, including absent tail, non inserted or
bent tail (the tail forms an angle of about 90° to the long axis of
the head),distended/irregular/bent midpiece, abnormally thin
midpiece or any combination of these.
8.3.8.1.3. Tail defects, including short, multiple, hairpin, broken, irregular
width, or coiled tails, tails with terminal droplets, or any
combination of these.
8.3.8.1.4. Cytoplasmic droplets greater than one-third of the area of a
normal sperm head.
The traditional feathering technique (whereby the edge of a second slide is used to
drag a drop of semen along the surface of the cleaned slide) may be used to make
thin smears of spermatozoa. The Papanicolaou smear for staining of spermatozoa is
the method most widely used in andrology laboratories. In our practice we have
tried simpler methods: Meyer’s haematoxiline, Harris haematoxiline and Giemsa. We
have reported that these methods are not as elegant as the Papanicolaou method but
allows the classification of the spermatozoa in the main groups with the same
accuracy.
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Sperm morphology gives information for the function of the reproductive tract and
is a predictor of man’s fertility potential.
Physical sperm aberrations may occur during the production of sperm or during
storage in the epididymus. In cases of teratozoospermia, one should start first by
excluding the presence of monomorphic genetic syndromes such as
globozoospermia, microcephaly and short tail spermatozoa. The increased number
of immature spermatozoa may be due to epididymal dysfunction or is a consequence
of frequent ejaculations. The increased number of spermatozoa with tapering heads
are found in association with varicocele. In a recent study we have reported that the
percentage of tapered spermatozoa, spermatozoa containing cytoplasmic droplets
and spermatozoa with bent tail are significantly increased in varicocele patients
compared to controls .
The usefulness of sperm morphology assessment as a predictor of a man’s fertilizing

potential has often been challenged due to different classification systems, various
slide preparation techniques and problems with reproducibility because of observer
variations.
According to the literature the importance of sperm morphology as a single and

independent predictor of in-vivo and in-vitro fertilization seems to be proven.
AND PROCEDURES

STANDARD OPERATING
PROCEDURES
AND PROCEDURES

IMMUNOLOGY AND SEROLOGY
IMMUNOLOGY AND SEROLOGY
Many of the diagnostic techniques applied in immunology are based on the fact that antigens and
antibodies interact. Isolating and identifying the infectious microorganism in a specimen from the
patient diagnose most infectious diseases. In some cases microorganisms are difficult to culture
and isolate or may require special and often expensive techniques that are not available for
routine diagnosis. In other immunological disorders there is no microorganism per se to identify
or isolate. There are also some “naturally” occurring immune diseases, often classified as
autoimmune diseases, which are not caused by a microorganism but can be detected by some of
the diagnostic techniques applied in immunology. Several of these techniques detect specific
metabolic products or specific antibodies and antigens. In those disease states where a
microorganism is involved, these immunology tests do not detect the microorganism directly, but
provide evidence of its presence.
Serology tests are those tests based on the antigen-antibody reactions. They include
Agglutination, Complement Fixation, Immunodiffusion, Precipitation, Neutralization inhibition,
Enzyme-Linked Immunosorbent Assay(ELISA), Immunofluorescence and
Radioimmunoassay(RIA) tests.
AND PROCEDURES

1.0. Determination of rheumatoid factors by the latex-agglutination technique

1.1. Materials and reagents
1.1.1. Test plates (preferably with a dark background)
1.1.2. Stirring rods, wooden orange sticks or rotator
1.1.3. Test-tubes,5ml
1.1.4. Test-tube rack
1.1.5. Micropipettes
1.1.6. Latex rheumatoid factor (RF) reagent (aqueous suspension of latex particles
coated with human IgG)
1.1.7. Negative and positive control sera
1.1.8. Sodium chloride, 0.85% solution (reagent no. 53).
NOTE: The above-mentioned materials and reagents are usually supplied as part of a commercial
test kit.
1.2. Method
1.2.1. Bring the test and control sera and latex RF reagent to room temperature.
1.2.2. Dilute the test and control sera 1:5 with sodium chloride solution.
1.2.3. Apply one drop of each dilution to the test plates.
1.2.4. Shake the vial of latex RF reagent and add one drop to each of the samples on the
test plates.
1.2.5. Mix well with stirring rods or orange sticks (one for each sample) and rotate the
plates gently (about 10 times), or place on a rotator.
1.2.6. After 2 minutes, examine the plates and compare the reactions of the test sera
with those of the control sera.
1.2.7. If any sera are positive, repeat steps 3–6, using a twofold dilution.
1.2.8. The highest dilution of serum that causes agglutination is the titer.
2.0. Tests for the determination of anti-streptolysin O antibodies

2.1. Anti-streptolysin O test (ASOT)
Streptolysin O is a toxin produced by haemolytic streptococci.The anti-streptolysin O test
(ASOT) is the most commonly used laboratory test for following a streptococcal infection
and its sequelae (rheumatic fever and acute post-streptococcal glomerulonephritis). Other
approaches are now available, but the “standard” ASOT is based on the fact that
streptolysin O will lyse human or sheep erythrocytes unless neutralized by anti-
streptolysin O antibodies present in the patient’s serum.
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One streptolysin O unit is the minimum amount of streptolysin O that will lyse 0.5ml of a
freshly prepared 5% suspension of sheep erythrocytes when incubated for 1 hour at 37
°C. One Todd unit is defined as the amount of anti-streptolysin O antibody that will
neutralize 0.5 streptolysin O units.
2.2. Principle
The test is performed by incubating a constant amount of standardized streptolysin O
with serial dilutions of heat-inactivated patient’s serum (containing anti- streptolysin O
antibodies) at 37 °C for 15 minutes. A freshly prepared suspension of 5% sheep
erythrocytes is then added to all the tubes and incubation is continued for a further 45
minutes. After centrifugation at 500g, the highest dilution of patient’s serum that still has
a clear supernatant (no haemolysis) is the end-point and its Todd unit value (reciprocal of
the dilution) is reported. This technique is rather time-consuming and simpler and more
rapid methods using latex agglutination are now available.
2.3.1. Volumetric flask, 1000 ml
2.3.2. Test-tubes, 75 mm ¥ 12 mm
2.3.3. Test-tube racks
2.3.4. Serological pipettes
2.3.5. Water-bath
2.3.6. Centrifuge
2.3.7. Phosphate-buffered water, pH 6.8 (reagent no. 43)
2.3.8. Sodium chloride, 0.85% solution (reagent no. 53)
2.3.9. 5% Suspension of washed sheep erythrocytes in sodium chloride, 0.85% solution
2.3.10. Reduced streptolysin O (instructions for the preparation of reduced streptolysin O
from the unreduced form are usually provided by the manufacturer).
2.4. Procedure:
2.4.1. Make three dilutions of the patient’s serum (inactivated by heating at 56°C for 30
minutes) as follows: 0.5ml of serum + 4.5ml of phosphate buffer = 1:100.5ml of
1:10 serum + 4.5ml of phosphate buffer = 1:100 1ml of 1:100 serum + 4ml of
phosphate buffer = 1:500
2.4.2. From these master dilutions, make an extended series of dilutions as shown in
Table 11.1. For screening purposes, use only the first seven tubes and the control
tubes (13 and 14).
2.4.3. Add 0.5ml (equivalent to 1 International Unit (IU)) of reduced streptolysin O to all
the tubes except tube 13. Mix and incubate in a water-bath at 37 °C for 15
minutes.
AND PROCEDURES

2.4.4. Add 0.5ml of the 5% suspension of sheep erythrocytes to each tube. Mix and
incubate in a water-bath at 37 °C for 45 minutes, mixing again after the first 15
minutes of incubation.
2.4.5. Centrifuge the tubes gently at 500g for 3 minutes and observe for haemolysis. The
end-point is the last tube (i.e. the highest dilution) showing no haemolysis.
Control tube 13 should show no haemolysis. If there is haemolysis in this tube the
test should be repeated. Control tube 14 should show complete haemolysis
2.4.6. Preparation of dilution series for the anti-streptolysin O test
2.5. Late
x agglutination
2.6.1. Test plates
2.6.2. Stirring rods, wooden sticks or rotator
2.6.3. Test-tubes,5ml
2.6.4. Test-tub
2.6.5. Micropipettes, 50 ml
2.6.6. Anti-streptolysin O latex reagent: suspension of latex particles coated with strep-
tolysin O
2.6.7. Negative control serum
2.6.8. Positive control sera (strongly and weakly positive)
2.6.9. Sodium chloride, 0.85% solution.
AND PROCEDURES

2.7. Procedure:
2.7.1. Bring the reagents and test and control sera to room temperature.
2.7.2. Apply one drop of each of the test and control sera to the test plates.
2.7.3. Shake the anti-streptolysin O latex reagent to mix; add one drop to each of the test
and control sera.
2.7.4. Mix well with stirring rods or wooden sticks (one per sample) and rotate the
plates gently about 10 times, or place on a rotator.
2.7.5. After 2 minutes, examine the plates and compare the reactions of the test sera
with those of the controls. A positive reaction is indicated by the presence of
agglutination. A negative reaction is indicated by the absence of agglutination.
2.7.6. If any sera are positive, repeat steps 2–5, using a twofold dilution. The highest
dilution that causes agglutination is the titre. Most anti-streptolysin O reagents
have a detection limit (e.g. 200IU/ml) that is usually multiplied by the dilution
factor to give the serum concentration of anti-streptolysin O in IU/ml.
3.0. Tests for hepatitis virus infection
Routine tests for hepatitis include the use of markers for hepatitis A, B and C viruses.
Hepatitis A is most common in children, especially in nurseries; however, it is not
routinely tested for, except in cases of epidemics. Hepatitis B and C viruses are transmitted
through blood products, body fluids, contaminated needles and other contaminated
materials. Hepatitis B virus has several markers which include:
Surface Antigen (Hbsag)
Antibody To Surface Antigen (Anti-Hbs)
Envelope Antigen (Hbeag)
Antibody To Envelope Antigen (Anti-Hbe)
Antibody To Core Antigen (Anti-Hbc).
The concentrations of these markers vary during the course of an infection. The antigen
markers appear first or earlier on after exposure to the virus. Seroconversion (antibody
production) often occurs several weeks or months after exposure.
Hepatitis testing is routinely done by solid-phase ELISA and radioimmunoassay methods.

Commercial kits for detection of hepatitis markers are available and specific criteria and
instructions are provided with each kit. The general principles of the ELISA technique for
one of the markers for hepatitis B virus are outlined below.
AND PROCEDURES

3.1. ELISA for hepatitis B surface antigen

3.2.1. Micropipettes
3.2.2. Incubator or water-bath
3.2.3. Washer or vacuum pump
3.2.4. Spectrophotometer (reader)
3.2.5. Commercially available test kit containing solid-phase support system,
reagents and controls
3.2.6. Distilled or deionized water.
3.3. Procedure:
3.3.1. Add the test (serum) samples and controls to the anti-HBs precoated solid- phase
support system and incubate according to the manufacturer’s instructions.
3.3.2. Using a vacuum pump or automated washer, carefully aspirate the liquid from the
solid phase and wash the system.
3.3.3. Add the specified amount of conjugate (enzyme-linked anti-HBs) and incubate
according to the manufacturer’s instructions.
3.3.4. Aspirate the liquid and wash to remove any unbound conjugate.
3.3.5. Add the specified amount of substrate (usually o-phenylenediamine) and incu-
bate in the dark. (This is the colour development stage and should be protected
from light.)
3.3.6. Add the stopping solution as specified. The stopping solution (usually an acid)
inhibits any further reaction between the enzyme and the substrate.
3.3.7. Read the results in a spectrophotometer at the specified wavelength.
3.3.8. Calculate the cut-off value for the test run as instructed by the manufacturer.
3.3.9. The test run is invalid if the positive control values are less than the cut-off value.
In such cases the assay must be repeated.
3.4. Precautions; The ELISA method is fairly easy to perform, but pay attention to the following:
3.4.1. Make sure that the reagents and samples are brought to room temperature.
3.4.2. Make appropriate dilutions of reagents or specimens if required.
3.4.3. Make sure that the pre-coated antigen or antibody (solid phase) is not disturbed
during the addition of the sample or of beads.
3.4.4. Prepare only enough chromogen solution for a single test run. Store the solution
in a closed container, in the dark. If colour develops prior to application, a new
solution should be prepared.
3.4.5. Avoid cross-contamination of samples.
3.4.6. Adhere strictly to the incubation times, temperatures and other conditions
specified in the manufacturer’s instructions.
AND PROCEDURES

3.4.7. In addition to the controls in the kit, it is generally recommended to include an in-
house control of known optical density for quality control purposes.
3.5. Dipstick test for hepatitis B surface antigen Principle

The dipstick test for the detection of hepatitis B surface antigen (HBsAg) takes advantage
of the formation of a visible spot by precipitating immunocomplexes. Conjugates of
monoclonal antibodies against HBsAg coupled to colloidal Zone B gold particles are
adsorbed to one area of a nitrocellulose strip (zone A in the Figure below).
Zone B
Zone A
Polyclonal antibodies against HBsAg are chemically fixed to

another area of the strip A drop of human serum is applied to zone A. The HBsAg antigen
in the serum binds to the antibody conjugate and the gold–HBsAg immunocomplex
migrates along the strip until it reaches the fixed polyclonal antibodies in zone B. The
polyclonal antibodies precipitate the gold–HBsAg immunocomplex, and form a visible
blue/red band in zone B. No colored band is formed if the serum does not contain HBsAg.
Commercially available test kit containing dipsticks, reagents and controls.
3.7. Procedure:
3.7.1. Label the test strip with the patient’s name and/or number.
3.7.2. Add a drop of serum to zone A as recommended by the manufacturer.
3.7.3. Allow the serum fluid to migrate to zone B on the test strip.
3.7.4. Inspect zone B after 10–20 minutes for the appearance of a spot indicating a
positive reaction.
4.0. Tests for syphilis infection
Syphilis is caused by Treponema pallidum. There are four stages of syphilis infection:
primary, secondary, latent and tertiary, and a special condition of maternal–fetal
AND PROCEDURES

transmission termed congenital syphilis. Immune responses to syphilis can be grouped

into two categories: non-specific (or reaginic) and specific.
The non-specific reagin is of the IgM class and reacts with an alcoholic extract of beef heart
known as cardiolipin (a phospholipid). Since the reaginic antibody lacks specificity, it
shows up in many other conditions and disease states unrelated to treponemal infection. In
these cases false-positive reactions can occur. Specific antibodies to treponemes (both to T.
pallidum and to nonpathogenic treponemes) of the normal bacterial flora of the oral or
genital tract can also develop. These antibodies are of the IgG class and remain detectable
throughout the life of the patient despite treatment. Routine tests for syphilis include the
rapid plasma reagin (RPR) test, the fluorescent treponemal antibody-absorbed (FTA-Abs)
test and the T. pallidum haemagglutination (TPHA) test.
4.1. Principle
4.1.1. RPR test
The RPR test has replaced the Venereal Disease Research Laboratory (VDRL) test, as
a rapid screening test for the following reasons:
4.1.1.1. There is no need for daily preparation of reagents.
4.1.1.2. No microscope is required.
4.1.1.3. Heat inactivation of serum is not required.
4.1.1.4. The RPR test uses the VDRL antigen modified with choline chloride to
inactivate complement, and charcoal particles to allow the results of the
reaction to be read without a microscope. The RPR test can also be applied
as a semi-quantitative test.
4.1.2. FTA-Abs test
The FTA-Abs test is used in the confirmation of syphilis. In the first step of the test,
serum is diluted in a concentrated culture filtrate of Reiter treponemes to absorb
any antibodies to nonpathogenic treponemes. The serum is then layered over a glass
slide on which killed T. pallidum organisms (Nichols strain) have been
affixed.Theslide is incubated, washed and overlaid with a fluorescent-labelled anti-
human immunoglobulin antibody. If the test result is positive the treponemes will
fluoresce.
This indirect immunofluorescence technique is highly sensitive in all stages of

syphilis, especially in the very early and very late stages. Once positive this test
remains positive for the life of the patient. It is not used as a screening test for
syphilis because it does not detect reinfection and it is time-consuming and costly (a
fluorescence microscope with a dark-field condenser is required). The results of a
AND PROCEDURES

test for syphilis must be interpreted according to the type(s) of test employed and
the stage of the disease the patient has reached. Remember that a positive result
from a screening test for syphilis may be due to other heterophile antibodies, faulty
technique or to the presence of other treponemal antibodies. A negative result may
mean one of the following:
 The infection is too recent to have produced detectable levels of antibodies.
 The test is temporarily non-reactive because of treatment the patient is
receiving.
 The test has been rendered temporarily non-reactive because the patient has
consumed alcohol prior to testing.
 The disease is latent or inactive.
 The patient has not produced protective antibodies because of
immunological tolerance.
 The technique is faulty.
Weakly positive results may be due to:

 Very early infection;
 Lessening of the activity of the disease after treatment;
 Nonspecific immunological reactions;
 Incorrect technique.
The greatest value of the non-treponemal tests is in screening following therapy and
in the detection of reinfection.
4.1.3. TPHA test

The TPHA test is also used in the confirmation of syphilis. In the first step of the test,
diluted serum is mixed with absorbing diluent containing non-pathogenic Reiter
treponemes. The serum is then transferred to a microtitre plate and erythrocytes
sensitized with killed T. pallidum organisms (Nichols strain) are added. If the test
result is positive the erythrocytes will form a smooth mat of agglutinated cells.
4.3. Procedures:
4.3.1. RPR test
4.3.1.1. Materials and reagents
4.3.1.1.1. Test plates
4.3.1.1.2. Disposable Pasteur pipettes
4.3.1.1.3. Serological pipette
AND PROCEDURES

4.3.1.1.4. Test-tubes, 75 mm ¥ 12 mm
4.3.1.1.5. Test-tube rack
4.3.1.1.6. Rotator
4.3.1.1.7. RPR antigen
4.3.1.1.8. Negative, weak-positive and strongly positive controls
4.3.1.1.9. Sodium chloride, 0.85% solution
NOTE: The above reagents are usually supplied as part of a test kit.
4.3.1.2. Method
4.3.1.2.1. Bring the test and control sera and RPR antigen to room
temperature. Dispense one drop of each of the test and control
sera on to the test plates and spread carefully in the individual
wells.
4.3.1.2.2. Add one drop of RPR antigen to each well of the test plates.
4.3.1.2.3. Place the test plates on the rotator and rotate for 8 minutes at
100rpm. (The recommended speed is between 95 and
105rpm and this should be checked daily as part of quality
control.) If a mechanical rotator is not available, tilt the plates
back and forth and rotate the plates carefully for 8 minutes at
80–85rpm.
4.3.1.2.4. Examine the test plates for flocculation and compare the
reactions of the test sera with those of the controls.
4.3.1.2.5. Prepare a twofold dilution of any positive sera and examine
the dilutions as described in steps 2–5. The highest dilution of
serum to give flocculation is the titer.
4.3.2. TPHA test
4.3.2.1. Materials and reagents
4.3.2.1.1. Test-tubes
4.3.2.1.2. Test-tube rack
4.3.2.1.3. Commercially available TPHA test kit containing microtitre
plates, micropipettes (with disposable tips), absorbing
diluent, erythrocytes sensitized with T. pallidum, unsensitized
erythrocytes, positive and negative control sera
4.3.2.1.4. Distilled water.
NOTE: The reagents and controls should be reconstituted before use

according to the manufacturer’s instructions.
4.3.2.2. Method
AND PROCEDURES

4.2.2.2.1. Dilute the test and control sera 1:20 with absorbing diluent.
4.2.2.2.2. Using a micropipette, dispense 25ml of the negative control
serum into wells 1 and 2 of the first horizontal row of the
microtitre plate.
4.2.2.2.3. Dispense 25ml of the positive control serum into wells 1 and 2
of the second horizontal row of the microtitre plate.
4.2.2.2.4. Dispense 25ml of the first test serum into wells 1 and 2 of the
third horizontal row of the microtitre plate.
4.2.2.2.5. Repeat the procedure with the remaining test sera. If
necessary, use the adjacent wells.
4.2.2.2.6. Add 75ml of the control erythrocytes to the wells in the first
vertical row (1) and every other row (3, 5, 7, 9 and 11), as
appropriate.
4.2.2.2.7. Add 75ml of the sensitized erythrocytes to the wells in the
second vertical row (2) and every other row (4, 6, 8, 10 and
12), as appropriate.
4.2.2.2.8. Rotate the plates carefully, cover and leave to stand at room
temperature for the time recommended by the manufacturer.
The plates should be protected from vibration, radiant heat
and direct sunlight.
4.2.2.2.9. Place the plates carefully on a white background or a sintered
glass plate illuminated from below or a viewing device that
allows the sedimentation pattern to be seen from below
through a mirror.
4.2.2.2.10. If the result is positive a smooth mat of agglutinated cells will
be seen. The cells may be surrounded by a red circle, or may
even cover the entire base of the well. If the result is negative a
compact red button of non-agglutinated cells will be seen,
with or without a very small hole in its center. If the result is
doubtful (borderline) a button of non-agglutinated cells with a
small hole in its center will be seen.
Note: The results should be interpreted according to the criteria

provided by the manufacturer.
AND PROCEDURES

STANDARD OPERATING
PROCEDURES
CHEMISTRY
AND PROCEDURES

CHEMISTRY
Clinical chemistry examinations are one of the mostly requested in the laboratory. It is also one of
the most crucial examinations performed. This operations manual shall consist of the basic
principles utilized in the analysis of analytes. The laboratory shall be using fully automated
chemistry analyzers (Cobas C1-11) as its main analyzer (Refer to Manufacturer’s Manual).
1.0 GLUCOSE
Glucose is the major carbohydrate present in the peripheral blood. Oxidation of glucose is
the major source of cellular energy in the body. Glucose derived from dietary sources is
converted to glycogen for storage in the liver or to fatty acids for storage in adipose tissue.
The concentration of glucose in the blood is controlled within narrow limits by many
hormones, the most important of which are produced by the pancreas.
1.2. Test Principle
Enzymatic Reference Method With Hexokinase
Hexokinase catalyzes the phophorylation of glucose to glucose-6-phophate by ATP.

Glucose -6-phosphate dehydrogenase oxidizes glucose-6-phosphate in the presence of
NADP to gluconate-6-phosphate. No other carboyhdrate is oxidized. The rate of NADPH
formation during the reaction is directly proportional to the glucose concentration and
is measured photometrically.
2.0 CHOLESTEROL
Cholesterol is a steroid with a secondary hydroxyl group in the C3 position. It is

synthesized in many types of tissue, but particularly in the liver and intestinal wall.
Approximately three quarters of cholesterol is newly synthesized and a quarter
originates from dietary intake. Cholesterol assays are used for screening for
atherosclerotic risk and in the diagnosis and treatment of disorders involving elevated
cholesterol levels as well as lipid and lipoprotein metabolic disorders.
AND PROCEDURES

2.1. Test Principle
Cholesterol esters are cleaved by the action of cholesterl esterase to yield three
cholesterol and fatty acids. Cholesterol oxidase then catalyzes the oxidation of
cholesterol to cholest-4-en-3-one and hydrogen peroxide. In the presence of peroxidase,
the hydrogen peroxide formed effects the oxidative dupling of phenol and 4-
aminoantipyrine to form a red quinone-imine dye.
The color intensity of the dye formed is directly proportional to the cholesterol
concentration. It is determined by measuring the increased in absorbance.
3.0 LOW DENSITY LIPOPROTEIN ( LDL)
Low density lipoproteins (LDL) play a key role in causing and influencing the progression
of atherosclerosis and , in particular, coronary sclerosis. The LDLs are derived from VLDLs
(Very Low Density Lipoprotein ) rich in triglycerides by the action of various lipolytic
enzymes and are synthesized in the liver. The elimination of LDL from the plasma takes
place mainly by the liver parenchymal cells via specific LDL receptors. Elevated LDL
concentrations in blood and an increased in their residence time coupled with an increase
in the biological modification rate results in the destruction of the endothelial function and
a higher LDL-cholesterol uptake in the monocyte/ macrophage system as well as smooth
muscle cells in vessel walls. The majority of cholesterol stored in atherosclerotic plaques
originates from LDL. The LDL-cholesterol value is the most powerful clinical predictor
among all of the single parameters with respect to coronary atherosclerosis. Therfore,
therapies focusing on lipid reduction primarily target the reduction of LDL- cholesterol
which is then expressed an improvement of the endothelial function, prevention of
atherosclerosis and reducing its progression as well as preventing plaque rupture.
3.1. Test Principle
In the presence of peroxidase, the hydrogen peroxide generated reacts with 4-

aminoantipyrine and HSDA to form a purple blue dye. The color intensity of this dye is
directly proportional to the cholesterol concentration and is measured photometrically.
AND PROCEDURES

4.0 HIGH DENSITY LIPOPROTEIN ( HDL)
High Density Lipoprotein (HDL) are responsible for the reverse transport of cholesterol
from the peripheral cells to the liver. Here, cholesterol is transformed to bile acids which
are excreted into the intestine via the biliary tract. Monitoring of HDL- cholesterol in serum
is of clinical importance since an inverse correlation exist between serum HDl- cholesterol
concentrations and the risk of atherosclerotic disease. Elevated HDL- cholesterol
concentrations are protective against coronary heart disease, while reduced HDL- cholesterol
concentrations, particularly in conjunction with elevated triglycerides, increase the
cardiovascular risk. Strategies have emerged to increase the level of HDL- cholesterol to treat
cardiovascular disease.
4.1. Test Principle
Homogeneous enzymatic colorimetric test.

In the presence of magnesium ions, dextran sulfate selectively forms water-soluble
complexes with LDL, VLDL and chylomicrons which are to PEG-modified enzymes.
The cholesterol esters are broken down quantitatively intop free cholesterol and fatty
acids by cholesterol esterase.
In the presence of peroxidase (POD), teh hydrogen peroxide generated reacts with 4-amino-
antipyrine and HSDA to form a purple-blue dye. The color intensity of this dye is directly
proportional to the HDL-cholesterol concentration and is measured photometrically.
5.0. TRIGLYCERIDES
Triglycerides protein is essential for growth, the production of new tissues, and the repair of
injured tissue. An increase in triglyceride levels may ne the result of nephrosis, cholestasis,
pancreatitis, cirrhosis, diabetes mellitus, and hepatitis. A decrease is seen with
malnutrition.
5.1. Principle:
AND PROCEDURES

The analysis is based upon the enzymatic hydrolysis of Triglycerides with lipase in a
quinoneimine formed from hydrogen peroxide used as a color indicator. He intensity of
color is proportional to the amount of Triglycerides in the sample.
6.0. CREATININE
Chronic kidney disease is a worldwide problem that carries a substanial risk for
cardiovascular morbidity and death. Current guidelines define chronic kidney disease as
kidney damage or glomerular filtration rate (GFR) less than 60mL/min per 1.73m 2 for three
months or more, regardless of cause. The assay of creatinine in serum or plasma is the most
commonly used test to asses renal function. Creatinine is a break-down product or creatine
phosphate in muscle, and is usually produced at a filtered by the flomeruli and under
normal conditions, is not re-absorbed by the tubules to any appreciable extent. A small but
significant amount is also actively secreted.
Since a rise in blood creatinine is observed only with marked damage of the nephrons, it is
not suited to detect early stage of kidney disease. A considerably more sensitive test and
better estimation of glomerular filtration rate (GFR) is given by the creatinine clearance test
based on creatinine’s concentration in urine and serum or plasma, and urine flow rate. For
this test a precisely timed urine collection (usually 24 hours) and a blood sample are
needed. However, since this test is prone to error due to the inconvenient collection of
timed urine, mathematical attempts to estimate GFR based only on the creatinine
concentration in serum or plasma have been made. Among the various approaches
suggested, two have found wide recognition: that of Cockroft and Gault and that based on
the results of the MDRD trial. While the first eqaution was derived from data obtained with
the conventional Jaffee method, a newer version of the second is usablefor IDMS- traceable
creatinine methods. Both are applicable for adults. In children, the bedside schwarts
formula should be used.
In addition to the diagnosis and treatment of renal disease, the monitoring of renal dialysis,
creatinine measurements are used for the calculations of the fractional excretion of the
urine analytes (e.g albumin, α-amylase). Numerous methods were described determining
creatinine. Automated assays established in routine laboratory include the Jaffe alkaline
picrate method in various modifications, as well as enzymatic tests.
AND PROCEDURES

6.1. Test principle:
This enzymatic method is based on the conversion of creatinine with the aid of creatininase,
creatinase, and sarcosine oxidase to glycine, formaldehyde and hydrogen peroxide.
Catalyzed by peroxidase the liberated hydrogen peroxide reacts with 4-aminophenazone
and HTIBa to form a quinone imine chromogen. The color intensity of the quinone imine
chromogen formed is directly proportional to the creatinine concentration in the reaction
mixture.
7.0 ASPARTATE AMINOTRANSFERASE
The enzyme aspartate aminotransferase (AST) is widely distributed in tissue , principally

hepatic, cardiac, muscle, and kidney.
Elevated serum levels are found in disease involving these tissues: Hepatobillary Diseases:
Cirrhosis, Metastatic carcinoma, and Viral Hepatitis, Myocardial infarction (peaks two days
after onset).
Decreased levels may be observed in patients undergoing dialysis, those who has Vit. B
deficiencies
7.1. Principle:
AST in the sample catalyzes the transfer of an amino group between L-aspartate and 2-
oxoglutarate to form oxaloacetate and L-glutarate. The oxaloacetate then reacts witn NADP,
in the presence of malate dehydrogenase (MDH), to form NAD+. Pyridoxal phosphate serves
a coenzyme in the amino transfer reaction. T ensures full enzyme activation. The rate of
NADH oxidation is directly proportional to the catalytic AST activity. T is determined by
measuring the decrease in absorbance.
8.0 ALANINE AMINOTRANSFERASE
AND PROCEDURES

Alanine aminotransferase (ALT) is an enzyme found mainly in the liver. Elevated levels are
seen in patients with hepatitis and mononucleosis.
8.1. Principle:
The analysis is based on the transfer of amino group L-alanine to a-oxoglutarate in the
presence of GPT to produceglutamate and pyruvate. The pyruvate formed in the
deamination of L-alanine is converted by lactate dehydrogenase (LDH) in the presence of
NADH, which is oxidated to NAD+. The rate of change in reflection density measured.
9.0 UREA
The determination of serum urea is presently the most widely used screening test for the
evaluation of kidney function. The test is frequently requested along with the serum
creatinine test since simultaneous determination of these two compounds appears to aid
in the differential diagnosis of prerenal, renal and postrenal hyperuremia. Hyperuremia
may also indicate liver disease or dehydration. Elevated levels are most commonly
associated with renal disease, but may also be from dehydration, a high-protein diet,
excess destruction of body proteins, and gastrointestinal diseases, especially with
intestinal obstruction.
9.1. Principle:
The analysis is based on hydrolysis of urea to ammonia and cardon dioxide. The ammonia
reacts with an indicator to produce a highly colored dye. Urea is hydrolysed in the
presence of water and urease to produce ammonia and carbon dioxide. In a modified
Berthelot reaction the ammonium ions react with hypochlorite and salicylate to give a
green dye. The increase of absorbance at 578 nm is proportional to the urea concentration
in the sample.
10.0 URIC ACID
Determination of uric acid is of great value in the diagnosis of gout and in the assessment
of renal function.
10.1. Principle:
AND PROCEDURES

Uric acid is oxidized in the presence of uricase which is used to improve specificity. The
hydrogen peroxide produced reacts with 3.5-dichloro-2-hydroxybenzene sulfonic acid
(DCHBS) and 4-aminophenazone in the presence of catalase to produce a red-violet
quinoneimine complex. The increase in absorbance produced by quinoneimine production
is proportional to the amount of uric acid in the sample.
AND PROCEDURES

STANDARD OPERATING
PROCEDURES
MICROBIOLOGY
MICROBIOLOGY
AND PROCEDURES

Direct microscopic examination of smears is generally not sufficient to identify a bacterial

species; precise identification can only be obtained by culture. The collection and dispatch
of specimens to referral laboratories is, therefore, of utmost importance. Nevertheless,
direct microscopic examination of stained smears is an efficient way of studying the
presence of bacteria in biological fluids that are normally sterile and in specimens from
other sources. It may provide information of great value for the diagnosis, immediate
treatment and control of the disease. For example:
 Specimens from male patients with urethritis at an early stage can be used to diagnose
gonococcal infection with reasonable certainty (in females it is much more difficult).
 Microscopic examination of sputum smears is a practical and effective technique for the
detection of infectious cases of tuberculosis.
 Microscopic examination of CSF is used in identifying the bacteria or fungi that cause
meningitis. The diagnosis of some diseases is also possible through serology; an
example is syphilis. Serological techniques are also important for epidemiological
surveillance and early detection of diseases caused by bacteria that are difficult to
culture (e.g. Mycobacterium tuberculosis).
1.0. PREPARATION AND FIXATION OF SMEARS

1.1. Principle
The sample to be examined (pus, sputum, urine centrifugate, CSF, etc.) is prepared as
follows:
1.1.1. The specimen is spread in a thin layer on a glass slide.
1.1.2. It is allowed to dry completely.
1.1.3. It is fixed with 70% methanol or by heating before being stained.
1.2.1. Inoculating loop: this is a metal wire (usually made of nickel–chromium alloy)
fixed on to a handle and bent into a loop at the other end. Make the loop with
forceps, taking care that it is center. The actual diameter of the loop should be
2mm.
1.2.2. Microscope
1.2.3. Microscope slides
1.2.4. Coverslips
1.2.5. Bunsen burner or spirit lamp
1.2.6. 70% Methanol.
1.3. Preparation of smears
1.3.1. Flame the loop until it is red-hot: hold the loop just above the blue part of the
flame, as close to the vertical as possible. Allow it to cool (count to 20).
AND PROCEDURES

1.3.2. Take a portion of the specimen to be examined by placing the loop flat on the
surface of the liquid.
1.3.3. Number a slide, then press the loop flat on to the centre of the slide .
1.3.4. Still holding it flat against the slide, move the loop in an oval spiral, outwards
from the centre .Leave a space between the specimen and each of the four
sides of the slide. Let the slide dry completely in the air.
1.3.5. Repeat step 1.
NOTE: Unmarked smears are sometimes received in the laboratory from outside
sources.
To find out on which side of an unmarked slide the smear has been made,
turn the slide so that it reflects the light from the window:
● The side without the smear will shine.
● The side with the smear will not reflect the light.
1.4. Fixation of smears
When the smear has dried completely, fix it by covering the slide with a few drops of
70% methanol for 2 minutes or by quickly passing the back of the slide through the
flame three times.It is sometimes useful to draw a circle around the smear with a
grease pencil, so that it can be seen more easily.
2.0 STAINING TECHNIQUES
2.1. Gram staining

Gram stain will enable the smear to be examined by microscopy for the presence of
bacteria, pus cells,Vincent’s bacilli and Candida albicans. Commensal bacteria, which
are always present, are not important. They do not need to be considered for fur-
ther examination or reported.
2.1.1. Principle
5.2.1.1. Crystal violet stains all bacteria deep violet .Iodine solution
fixes the violet colour more or less strongly in the bacteria
5.2.1.2. 95% Ethanol:
5.2.1.2.1. Decolorizes certain bacteria when the crystal violet is
not strongly fixed by iodine solution
5.2.1.2.2. Does not decolorize other bacteria when the crystal
violet is strongly fixed by iodine solution Carbol
AND PROCEDURES

fuchsin, neutral red or safranine solution (pink): re-

stains (pink) the bacteria discoloured by ethanol has
no effect on the other bacteria, which remain dark
violet
2.1.2. Materials and reagent
2.1.2.1. Microscope
2.1.2.2. Slide rack
2.1.2.3. Crystal violet, modified Hucker
2.1.2.4. Lugol iodine, 0.1% solution
2.1.2.5. Acetone–ethanol decolorizer Carbol fuchsin solution for Ziehl
Neelsen stain (diluted 10- fold with 95% ethanol), neutral red,
0.1% solution or safranine solution .
2.1.3. Method
2.1.3.1. Fix the smear
2.1.3.2. Cover the smear with crystal violet stain for 60 seconds.
2.1.3.3. Wash off the stain with clean water. Drain the slide and cover
the smear with iodine for 60 seconds.
2.1.3.4. Wash off the iodine with clean water. Decolorize rapidly with
acetone–ethanol. Only 2–3 seconds are needed.
2.1.3.5. Cover the smear with carbol fuchsin for 2 minutes.
2.1.3.6. Wash off the stain with clean water and place the slide upright
in a slide rack to drain and air-dry.
2.1.4. Microscopic examination
First examine the slide using the x 40 objective to see how the smear is
distributed and then use the x 100 oil-immersion objective.
2.1.4.1. Gram-positive organisms
Gram-positive organisms appear dark purple (e.g. staphylococci,
streptococci, micrococci, pneumococci, enterococci.
2.1.4.2 Gram-negative organisms
Gram-negative organisms appear red (e.g. gonococci,
meningococci, coliform bacilli, shigellae, salmonellae, cholera
vibrios).
2.1.4.3. Identification of specific organisms
2.1.4.3.1. Candida albicans appears as large (2–4mm in diameter)
oval or round Gram- positive spores with mycelium-like
filaments of varying length with rounded ends.
AND PROCEDURES

2.1.4.3.2. “Actinomycetes” are seen as large granules, sometimes

visible to the naked eye (white to yellow colour). The
center is Gram-negative, the periphery Gram- positive
.They are found in pus from skin, sputum, etc.
2.1.4.3.3. Vincent’s bacilli are seen as Gram-negative spirochaetes
and fusiform rods.No other bacteria should be reported
as there are many commensal bacteria which may be
confused with pathogens.
2.2. Sources of error
2.2.1. A false Gram-positive reaction may occur because:
2.2.1.1. The smear was fixed before it was dry.
2.2.1.2. The smear was too thick.
2.2.1.3. There was sediment in the bottle of crystal violet (filter before
using).
2.2.1.4. The iodine solution was not thoroughly washed off the slide.
2.2.1.5. The acetone–ethanol was not left on the slide long enough.
2.2.1.6. The carbol fuchsin (or safranine or neutral red) solution was too
strong or left on the slide too long.
2.2.2. A false Gram-negative reaction may occur because:
2.2.2.1. The iodine solution was not left on the slide long enough.
2.2.2.2. The acetone–ethanol was left on too long or not washed off properly.
3.0 STAINING WITH ALBERT STAIN (FOR THE DETECTION OF CORYNEBACTERIUM

DIPHTHERIAE)
If diphtheria is suspected a sputum smear should be stained with Albert stain. This stain is
used to show the dark-staining volutin granules that appear in Corynebacterium
diphtheriae bacilli.

3.1.2. Microscope
3.1.3. Slide rack
3.1.4. Albert stain
3.2. Method
3.2.1. Fix the smear
3.2.2. Cover the smear with Albert stain for 3–5 minutes.
AND PROCEDURES

3.2.3. Wash off the stain with clean water and place the slide upright in a
slide rack to drain and air-dry.
3.3. Microscopic examination
First examine the slide using the x 40 objective to see how the smear is distributed
and then use the x 100 oil-immersion objective. Corynebacterium diphtheriae appears
as green rods containing green–black volutin granules. The rods may be arranged in
rows or in V-formation , or joined at angles, giving the appearance of Chinese
characters . The presence of slender rods containing volutin granules is sufficient
evidence for starting treatment for diphtheria. If diphtheria is suspected, a specimen
should be sent to the bacteriology laboratory for culture.
4.0 STAINING WITH ZIEHL–NEELSEN STAIN (FOR THE DETECTION OF ACID-FAST

BACILLI)
Ziehl–Neelsen stain is used to identify mycobacteria and oocysts of CryptosporidiumSpp.
4.1. Principle
When mycobacteria and oocysts of Cryptosporidium spp. are stained with a hot strong
solution of carbol fuchsin, they resist decolorization with a solution of acid or acid–ethanol
and stain red. Tissues and other organisms are decolorized by the acid–ethanol solution
and are demonstrated by a counterstain such as methylene blue, which stains them blue.
Mycobacterium lepraeand oocysts of Cryptosporidium spp. only resist decolorization with
weak solutions of acid or acid–ethanol. They are demonstrated using the modified Ziehl–
Neelsen technique .Mycobacterium spp. and oocysts of Cryptosporidium spp. are referred to
as “acidfast”due to their resistance to decolorization with acid solution. They do not stain
well with Gram stain or simple stains such as methylene blue.
4.2.1. Microscope
4.2.2. Spirit lamp or Bunsen burner
4.2.3. Slide rack
4.2.4. Forceps
4.2.5. Carbol fuchsin solution for Ziehl–Neelsen stain(filtered before use)
Organisms stained by Ziehl–Neelsen stainSample Organism
AND PROCEDURES

SAMPLE ORGANISM
Sputum M. tuberculosis
M. bovis
Skin M. leprae
M. ulcerans
Urine M. tuberculosis
M. bovis
Stool Cryptosporidium spp.
Gastric lavage M. tuberculosis
M. bovis
Reporting the number of acid-fast bacilli present

Number of acid-fast bacilli present Result
per
microscope field
< 0.1 (< 10 per 100 fields) Specify number present per 100 fields
0.1–1 (10–100 per 100 fields) +
1–10 ++
> 10 +++
4.2.6. Acid–ethanol for Ziehl–Neelsen stain

4.2.7. Malachite green, 1% solution), diluted 1:1 with distilled
water, or methylene blue solution
4.3. Method
4.3.1. Fix the smear
4.3.2. Cover the smear with filtered carbol fuchsin stain. Using forceps, gently heat the
slide over a spirit lamp or Bunsen burner until the stain starts to evaporate (at about
60°C — do not overheat.
4.3.3. Leave the stain on the slide for 5 minutes.Wash off the stain with clean water and
cover the smear with acid–ethanol for 5 minutes or until the smear is pale pink.
4.3.4. Wash the slide well in clean water and cover the smear with malachite green or
methylene blue for 1–2 minutes.
AND PROCEDURES

4.3.5. Wash off the stain with clean water and place the slide upright in a slide rack todrain
and air-dry. Do not blot the smear.
Examine the slide under the microscope; first use the X 40 objective to see how thesmear is
distributed. Then systematically examine the slide with the X 100 oil immersionobjective
to look for acid-fast bacilli (red bacilli). Examine the slide from end to end in steps until the
whole smear is covered. Count the number of acid-fast bacilli present per microscope field
(or per 100 microscope fields, if very few acid-fast bacilli are present).
Before moving to another slide, wipe the objective clean with lens tissue to prevent
transfer of acid-fast bacilli to another slide. If red bacilli can be seen, report as “acid-fast
bacilli present”. Report the numbers of acid-fast bacilli present. If no acid-fast bacilli are
seen, report as “no acid-fast bacilli found”.
5.0. STAINING WITH LOEFFLER METHYLENE BLUE (FOR THE DETECTION OF BACILLUS
ANTHRACIS)
Loeffler methylene blue is used to stain Bacillus anthracis, which causes anthrax
Note: Anthrax is a highly contagious disease. Gloves and protective clothing should
therefore be worn when specimens suspected of being infected with anthrax are
handled. The staining procedure should be carried out in a safety cabinet.

5.1.1. Microscope
5.1.2. Slide rack
5.1.3. 4% solution Potassium permanganate
5.1.4. Loeffler methylene blue
5.2. Method
5.2.1. Cover the slide with potassium permanganate for 10 minutes.
5.2.2. Wash the slide in clean water and cover the smear with Loeffler methylene
blue for 1 minute.
5.2.3. Wash off the stain with clean water and place the slide upright in a slide rack
to drain and air-dry.
First examine the slide using the X 40 objective and then use the X 100 oil-
immersion objective. Bacillus anthracisappears as large blue rods surrounded by a
mauve capsule; the bacilli appear in chains.
AND PROCEDURES

6.0. EXAMINATION OF SPUTUM SPECIMENS AND THROAT SWABS

The presence of pathogenic organisms is revealed by microscopic examination ofsputum
specimens and throat swabs. The organisms include:
 Bacteria: Gram-positive and Gram-negative acid-fast bacilli.
 Fungi or yeasts: filaments of mycelium with or without pores. They may be
pathogenic or saprophytes that have multiplied in the sample after collection
(correct identification by a specialized laboratory necessary).
 Actinomycetes: granules
 Parasites: eggs of pulmonary flukes and, very rarely, eggs of schistosomes and adult
worms of Mammomonogamus laryngeus.
Culture is often necessary for the identification of the infective agents.

6.1.1. Microscope
6.1.3. Wide-necked, leakproof containers for sputum specimens, such as jars or
stiff paper boxes
6.1.4. Sterile cotton wool swabs
6.1.5. Tongue depressor or spatula
6.1.6. Test-tubes
6.1.7. Sodium chloride crystals
6.1.8. N-cetylpyridinium chloride
6.1.9. Distilled water.
6.1.10. If possible, sterile cotton wool swabs should be prepared at a central-level
laboratory; otherwise, the following technique may be used.
6.1.10.1. Prepare some thin sticks of wood (or aluminium wire), 18 cm
long and 2mm indiameter. Prepare strips of cotton wool, 6
cm long by 3cm wide and as thin as possible.
6.1.10.2. Roll the cotton wool round one end of the stick (or wire).
6.1.10.3. Mould the swab into a conical shape.
6.1.10.4. Place in a glass test-tube. Plug with non-absorbent cotton wool.
6.2. Method
6.2.1. Collection of specimens
6.2.1.1. Sputum Specimens
AND PROCEDURES

6.2.1.1.1. Sputum specimens should be collected early in the

morning.
6.2.1.1.2. Ask the patient to take a deep breath and then cough
deeply, spitting what he or she brings up into the container.
Secure the top and label the container with the name and
number of the patient. Check that a sufficient amount of
sputum has been produced.If the specimen is to be
dispatched to a laboratory for culture of
Mycobacteriumtuberculosis , ask the patient to expectorate
directly into a wide-mouthed, screw-topped jar containing
25 ml of the following solution:
 N-cetylpyridinium chloride 5 g
 Sodium chloride 10g
 Distilled water to 1000ml.
 Screw on the top and label the jar with the patient’s
name and the date of collection of the specimen.
Important: Liquid frothy saliva and secretions from the nose and pharynx are
not suitable for bacteriological examination. Ask the patient to produce another
specimen.
6.2.1.2. Throat Specimens

6.2.1.2.1. Using a tongue depressor or a spatula to press the tongue
down, examine the back of the throat.
6.2.1.2.2. Look carefully for signs of inflammation and any exudate,
pus, membraneous deposits or ulcers.
6.2.1.2.3. Use a sterile cotton wool swab to swab the infected area.
Take care not to contaminate the swab with saliva. Return
the swab to the sterile test-tube.
Examine the sputum with the naked eye and then by microscopy.The sputum of a
person suffering from a bacterial infection usually contains:
6.3.1 thick mucus with air bubbles
6.3.2. Threads of fibrin
6.3.3. Patches of pus
6.3.4. Occasional brownish streaks of blood.
After visual inspection report the appearance of the sputum as:
AND PROCEDURES

6.3.5. Purulent: greenish, containing pus;

6.3.6. Mucopurulent: greenish, containing both pus and mucus;
6.3.7. Mucoid: containing mostly mucus;
6.3.8. Mucosalivary: containing mucus with a small amount of saliva.
NOTE:If there is blood present, this must also be reported.A sputum sample composed mostly of
saliva will not be useful either for culture orfor direct examination. Examine the smear stained with
Albert stain . If greenrods containing green-black volutin granules are seen, report as
“Corynebacterium diphtheriae present”.
Examine the smear stained with Ziehl–Neelsen stain. If red bacilli can be seen, report as “acid-fast
bacilli present”. Report the numbers of acid-fast bacilli present as described in If no acid-fast bacilli
are seen, report as “no acid-fast bacilli found”
7.0. DISPATCH OF SPECIMENS FOR CULTURE

7.1. Sputum Specimens
Sputum specimens are sent to a bacteriology laboratory for culture of
Mycobacteriumtuberculosis, antimicrobial susceptibility testing and inoculation into
guinea-pigs.The specimen should be collected in a transport medium and dispatched
immediately to the laboratory. Maximum preservation time: 10 days.
7.2. Throat Specimens
7.2.1. For routine investigation
As soon as the specimen has been collected, replace the swab in the sterile
test-tube and send it immediately to the bacteriology laboratory.
7.2.2. For confirmation of Corynebacterium diphtheriae infection
If diphtheria is suspected, the specimen should be sent in a sterile tube
containing coagulated serum (which must be stored in a refrigerator). Rub the
swab over the slanted surface of the serum, starting from the bottom and not
applying pressure. Send the same day. Maximum preservation time: 24 hours.
7.2.3. For detection of meningococci

This is seldom necessary, except for epidemiological surveys aimed at identifying
carriers of meningococci. If possible, use a transport medium such as Stuart
transportMedium. Rub the swab over the surface of the medium from one side of
the bottle to the other, starting from the bottom. Send the same day. Maximum
preservation time: 3 days.
8.0 EXAMINATION OF UROGENITAL SPECIMENS FOR GONORRHOEA
AND PROCEDURES


8.1.1. Microscope
8.1.3. Bottle, 100ml
8.1.4. Pasteur pipette
8.1.5. Cotton wool
8.1.6. Amie’s transport medium
8.2. Collection of specimens
8.2.1. From male patients
8.2.1.1. If possible collect the specimen first thing in the morning before the
patient passes urine.
8.2.1.2. Clean around the urethral opening with sterile saline.
8.2.1.3. Apply gentle pressure on the penis so that a drop of pus appears on the
meatus; if no pus appears, gently massage the urethra from above
downwards.
8.2.1.4. Collect a sample of the pus using a sterile cotton wool swab on a stick.
Insert the swab into a small bottle containing Amies transport medium.
Cut the stick to allow the top to be tightened.
8.2.1.5. Use another swab to collect a drop of the pus for Gram staining.
8.2.2. From female patients
The specimen should be taken from the cervical canal by a physician or specialist
nurse. In cases of chronic gonorrhoea, the specimen should be taken just before
or just after the menstrual period.
Microscopic examination is of great value in the diagnosis of gonorrhoea in males: it is
much less so in females. Culture is therefore necessary to isolate and identify the
gonococci in specimens from females. Examine the slides using the X 100 oil-
immersion objective. Pay particular attention to the edges of the smears, where the
elements are spread more thinly and are easier to see and the stain is less
concentrated.
8.3.1. Pus cells

Pus cells have a pink nucleus and a colourless cytoplasm. The nucleus may
appeardegenerated.
8.3.2. Gonococci
AND PROCEDURES

Gonococci appear as Gram-negative diplococci (in pairs). Cocci appear oval and
kidney-shaped. Extracellular Gram-negative diplococci should also be reported. A
presumptive diagnosis of gonorrhoea can be made if Gram-negative intracellular
diplococci are seen in smears from male patients. Extracellular Gram-negative
diplococci may be seen if the pus cells are damaged.
Report as:
— Gram-negative intracellular diplococci present;
— Gram-negative extracellular diplococci present;
— no Gram-negative diplococci found.
8.3.3. Other bacteria that cause infections in male patients
Numbers of the following may occasionally be seen in smears of urethral pus:
— Gram-positive cocci (e.g. staphylococci);
— Gram-positive bacilli (e.g. diphtheria bacilli);
— Gram-negative bacilli (e.g. coliform bacilli).
8.3.4. Other bacteria that cause infections in female patients
All kinds of organisms are found in the smears, particularly:
— Gram-positive bacilli;
— Gram-negative cocci (saprophytes).
9.0 DISPATCH OF SPECIMENS FOR CULTURE

9.1. Using Stuart transport medium
Sending the specimen in Stuart transport medium, is the best method, if the medium
can be obtained from a specialized laboratory. It is usually supplied in 30-ml flat
bottles that contain 8 ml of solid medium (along one side of the bottle) and are filled
with a mixture of air (90%) and carbon dioxide (10%). Round bottles may also be
used. The bottle should remain open for as short a time as possible to prevent the
escape of gas.
9.1.1. Method
9.1.1.1. Place the bottle of medium upright. Collect the pus specimen on a
swab asdescribed. Unscrew the bottle cap.
9.1.1.2. Holding the bottle as upright as possible (to prevent the gas
escaping), rub theswab of pus over the whole surface of the solid
medium, from one side of the bottle to the other, starting from the
bottom.
9.1.1.3. Replace the cap on the bottle at once. Dispatch the bottle (at ambient
temperature)immediately. Maximum preservation time: 3 days, but
AND PROCEDURES

the shorter the delay the better.This transport medium is also

suitable for meningococci.
9.2. Using a Pasteur pipette
9.2.1. Method
9.2.1.1. Collect the pus specimen on a sterile cotton wool swab
9.2.1.2. Draw the pus specimen into a sterile Pasteur pipette plugged with
cotton wool.
9.2.1.3. Place the pipette in a sterile test-tube, padded and plugged with
cotton wool. Maximum preservation time: 6 hours (at ambient
temperature).
10.0 EXAMINATION OF GENITAL SPECIMENS FOR SYPHILIS

Syphilis is a sexually transmitted disease caused by Treponema pallidum and occursin
three clinical stages. The primary stage is characterized by a painless genital ulcer
(syphilitic chancre), sometimes with enlargement of the lymph nodes in certain regions of
the body. The chancre heals spontaneously, even when untreated. In some patients the
disease progresses to the secondary stage.
The secondary stage results in:
— skin rash
— mouth ulcers
— genital warts
— generalized enlargement of lymph nodes.
The tertiary stage is very rare and is characterized by central nervous system involvement
and cardiac disease. Secondary or tertiary syphilis may be transmitted to a fetus in utero
(congenital syphilis).
11.0 EXAMINATION OF ASPIRATES, EXUDATES AND EFFUSIONS

Aspirates, exudates and effusions are collected by inserting a sterile needle into the
appropriate cavity. This can only be done by an experienced physician as there is a risk of
introducing infection. The cavities from which effusions can be collected include the
following:
— pleural (chest)
— peritoneal (abdominal)
— pericardial
— synovial joint
— bursa.
AND PROCEDURES

Bubo aspirates are examined for Yersinia pestis, which causes bubonic plague. The
organism is carried from the sites of inoculation to the lymph glands in the axillae, groin
and neck, where it causes localized swellings or buboes.
11.1.1. Microscope
11.1.3. Centrifuge
11.1.4. Centrifuge tubes
11.1.5. Specimen containers
11.1.6. Inoculating loop
11.1.7. 70% Methanol
11.1.8. Giemsa stain
11.1.9. Gram stain
11.1.10. Wayson stain
11.1.11. Ziehl–Neelsen stain
11.2. Method
Aspirated cavity fluid is collected into clean, dry, sterile containers. Report
the appearance of the fluid. Cavity fluid is normally straw-colored (yellow),
but can appear turbid or bloodstained.
11.2.2. Preparation Of Slides
11.2.2.1. Aspirated Cavity Fluid
11.2.2.1.1. Using an aseptic (sterile) technique, transfer 10 ml of
the fluid to a centrifuge tube and centrifuge at
moderate speed (2000g) for several minutes.
11.2.2.1.2. Remove the supernatant, re-suspend the deposit and
use an inoculating loop (sterilized by flaming) to

prepare three smears. Spread the fluid thinly over each
slide .
11.2.2.1.3. Allow the smears to air-dry and fix with methanol.
1.2.2.1.4. Stain the slides with:
— Gram stain
— Ziehl–Neelsen stain
— Giemsa stain
11.2.2.2. Bubo Aspirates
11.2.2.2.1. Prepare a smear from the aspirated fluid
AND PROCEDURES

11.2.2.2.2. Fix the smear in methanol for 2 minutes and stain with
Wayson stain.
11.3.1. Aspirated Cavity Fluid
Examine each slide using the x 40 objective and the x 100 oil-immersion
objective.
Look for any bacteria present on the slide stained with Gram stain. Look for
acid-fast bacilli (mycobacteria) on the slide stained with Ziehl–Neelsen stain.
When examining the slide stained with Giemsa stain, identify the
predominant type of blood cell present — leukocytes, lymphocytes or
mesothelial cells (from the lining of the cavity) and any atypical cells, which
may suggest cancer cells.
If there are more than few cells present or if the fluid is bloodstained, send the
slide to a bacteriology laboratory for culture.
11.3.2. Bubo Aspirates
First examine the slide using the x 40 objective to check the distribution of the
material and then use the x 100 oil-immersion objective to look for Yersinia
pestis. Yersinia pestis is seen as bipolar organisms which stain blue with pink
ends.
12.0 EXAMINATION OF PUS FOR BACILLUS ANTHRACIS

Bacillus anthracisis a pathogen of several types of animal. It is responsible for cutaneous
anthrax where it shows in its early form as a blister on the skin often called a malignant
pustule.
12.1.1. Protective clothing
12.1.2. Gloves
12.1.3. Microscope
12.1.5. Inoculating loop or sterile cotton wool swabs
12.1.6. Loeffler methylene blue (reagent no. 35)
12.1.7. Potassium permanganate, 4% solution
12.2. Method
Warning: Anthrax is highly contagious. Gloves and protective clothing must
therefore be worn when specimens are collected. Using an inoculating loop or a
AND PROCEDURES

cotton wool swab, collect a few drops of pus or fluid from malignant pustules.
Leave the smear to air-dry in a safety cabinet.
12.2.1.1. Preparation of slides
12.2.1.1.1. Prepare a smear from the pus or fluid
12.2.1.1.2. Fix the smear with potassium permanganate for 10
minutes, and then stain withLoeffler methylene blue
First examine the smear using the x 40 objectives to check the distribution of the
material and then use the x 100 oil-immersion objective to look for Bacillusanthracis.
Bacillus anthracis is seen as large blue rods surrounded by a mauve capsule; the
bacilli are arranged in chains.
AND PROCEDURES

STANDARD OPERATING
PROCEDURES
MYCOLOGY
1.0 Examination Of Skin And Hair For Fungi

Ringworm or tinea is a fungal infection of the skin. It can be found on the surface of the
body, the scalp and the nails and between the toes. Cross-infection between humans
frequently occurs and infection can also be acquired from infected animals or soil. The
circular lesions on the skin consist of a mass of branching hyphae; infected hair and nails
may also contain spores of fungi.
AND PROCEDURES


1.1.1. Microscope
1.1.2. Microscope slides (or dark paper)
1.1.3. Coverslips
1.1.4. Scalpel
1.1.5. Tweezers
1.1.6. Petri dish
1.1.7. Bunsen burner or spirit lamp
1.1.8. Cotton wool swab
1.1.9. Cotton wool
1.1.10. 70% Ethanol
1.1.11. Lactophenol cotton blue mounting solution
1.1.12. Potassium hydroxide, 20% solution
1.2.Method
1.2.1.1. Clean the infected area with a cotton wool swab soaked in ethanol.
1.2.1.2. Use a sterile scalpel to gently scrape the edge of a lesion and collect
some skin scales on to a glass slide or on to a piece of dark paper on
which the scales can be more easily seen. Also collect a few broken or
damaged hairs from the infected areas of the scalp using broad
tweezers and place them on the slide.
1.2.1.3. Place a drop of lactophenol cotton blue mounting solution and a drop of
20% potassium hydroxide on to the scales and hair. Cover with a
coverslip. The strong alkali will dissolve the keratin in the tissue,
enabling hyphae and spores to be seen.
Note: Potassium hydroxide is a corrosive fluid and should not be allowed to
touch the skin.
1.2.1.4. Place the slide in a covered Petri dish with some damp cotton wool to
prevent the specimen drying out. Leave the specimen to clear for 5–30
minutes, depending on the thickness. Alternatively, clear the specimen
by holding the slide above a Bunsen burner or spirit lamp flame for 1
minute.
Examine the cleared specimen using the x 10 and x 40 objectives. Adjust the iris
diaphragm of the condenser to give good contrast. Branching hyphae and chains of
angular rounded arthrospores may be seen. Fungal hyphae can be differentiated from
other tissue structures by their branching and cross walls or septa. They stain blue
AND PROCEDURES

with lactophenol cotton blue. Spores (large round granules with transparent
membranes) may be seen around the outside of the hairs . These spores are known
as ectothrix.
Spores found inside the hairs are called endothrix . Report as “fungal hyphae or
spores present” or “not found”.
2.0 Examination Of Pus For Mycetoma

Mycetoma is a chronic granulomatous disease of the subcutaneous and deep tissue. The
most commonly infected site is the foot, where it is called “Madura foot”. Other possible
sites of infection include the hands, the head and the chest wall. Mycetoma produce small
granules which are discharged through sinuses to the surface. These granules are used in
the diagnosis of the disease.
2.1.1. Microscope
2.1.3. Coverslips
2.1.4. Sterile needles
2.1.5.Distilled water
2.1.6. 70% Methanol
2.1.8. sodium chloride, 0.85% solution
2.1.9. Potassium hydroxide, 20% solution
2.1.10. Reagents for Gram staining
2.2. Method
2.2.1.1. Use a sterile needle to lift the surface crust over a sinus.
2.2.1.2. Carefully remove some of the discharging pus on to a slide.
2.2.1.3. Add a drop of saline or water, spread the pus gently and look for
granules. Granules vary in colour, size, shape and degree of hardness.
2.2.1.4. Crush a few granules in some distilled water and place on two slides.
2.2.1.5. Allow one slide to air-dry, fix with methanol for 2–3 minutes and
stain with Gram stain.
2.2.1.6. Place a few drops of 20% potassium hydroxide on to the second
slide and cover with a coverslip. Leave the specimen to clear for 10
minutes.
Examine the cleared specimen using the x 10 and x 40 objectives. Adjust the iris
diaphragm of the condenser to give good contrast. Look for branching and twisted
AND PROCEDURES

hyphae or fragmented threads. Gram-stained granules may show thin or fragmented

Gram-positive threads.
Report as:
_ “pus from sinus containing granules [specify colour and size] present”;
_ “Gram staining shows Gram-positive thin hyphae” or “Gram staining does not
show Gram-positive thin hyphae”.
3.0 Examination Of Skin For Pityriasis Versicolor

Pityriasis versicolor is a common skin disease in hot climates; it is caused by the fungus
Pityrosporum furfur. The face and body are covered with patches, which appear:
— pale and discoloured in dark-skinned patients;
— yellowish-brown in white-skinned patients.

3.1.1. Microscope
3.1.3. Adhesive cellophane tape
3.1.4. Tongue depressor or glass rod
3.1.5. Forceps
3.1.6. Gauze pads
3.1.7. Eosin, 1% solution , if possible (otherwise, examine without staining).
3.2 Method
3.2.1.1. Choose a rapidly developing patch of infected skin. Moisten it with
a gauze pad dipped in the eosin solution . Leave to dry for 1 minute.
(Do not take the specimen if talcum powder has been used on the
skin. Wash it off first.)
3.2.1.2. Cut a strip of adhesive tape about 5cm long. Place it over the patch
so that it overlaps one edge.
3.2.1.3. Stick the tape on the skin and press firmly from one end to the
other, passing a tongue depressor or glass rod over it several times.
Pull the adhesive tape away with forceps. Place it immediately on a
microscope slide, sticky side down.
AND PROCEDURES

AND PROCEDURES

LABORATORY
Result Forms
Figure 1.0 URINALYSIS RESULT FORMAT: 10 parameter macroscopic examination
AND PROCEDURES

Figure 2.0 URINALYSIS RESUTL FORMAT: 4 parameter Macroscopic Examination
AND PROCEDURES

Figure 3.0 PREGNANY TEST RESULT FORMAT:
AND PROCEDURES

Figure 4.0 FECALYSIS RESULT FORMAT:
Figure 5.0 FOBT RESULT FORMAT:
AND PROCEDURES

Figure 6.0 URINE KOH RESULT FORMAT:
Figure 7.0 SPERM ANALYSIS RESULT FORMAT:
AND PROCEDURES

Figure 8.0 COMPLETE BLOOD COUNT RESULT FORMAT:
AND PROCEDURES

Figure 9.0 COAGULATION TESTING RESULT FORMAT:
AND PROCEDURES

Figure 10.0 ORAG GLUCOES TOLERANCE TEST RESULT FORMAT:
AND PROCEDURES

Figure 11.0 CHEMISTRY TEST RESULT FORMAT:
AND PROCEDURES

Figure 12.0 CHEMISTRY TEST RESULT FORMAT 2:
AND PROCEDURES

Figure 13.0 HbA1c TEST RESULT FORMAT:
AND PROCEDURES

Figure 14.0 THYROID FUNCTION TEST RESULT FORMAT:
AND PROCEDURES

Figure 15.0 PSA RESULT FORMAT:
AND PROCEDURES

LABORATORY
FLOW CHARTS
Flowchart: Laboratory Procedures

FLOWCHART OF LABORATORY PROCEDURES
Receive Request
I. Receipt and Validation of Laboaratory Requests
Check/Validate Request
AND PROCEDURES

Call Department (Nurse-on-Duty) and ask

In-Patient for needed Information OR Ask Patient
during extraction
Does it contain N0
complete
Ask Patient of needed Information
information? Out-Patient
YES
Inform Nurse-on-Duty of availability of

procedure and suggest referral to another
In-Patient
laboratory/Inform necessary Patient
preparation
Is the procedure NO/YES

available? Does it
need Inform availability of procedure/Inform
Preparations? necessary Patient preparation; Schedule
Out-Patient
specimen collection if possible

Continuation
II. Charging and Logging of Requests
In-Patient Charge slips will be submitted to the Billing Dept. Every after an
Charge the Procedures
8-Hour shift
Out-Patient Patient must pay the procedure prior collection
Follow-up and checking of charging accomplishments per shift

In-Patient
Checking of Official
Receipt/Charging
AND PROCEDURES

Out-Patient Check Official Receipt
Record Request in
designated LOGBOOK
Blood extraction will be performed by Phlebotomists/Med.techs. in the different
departments; *** Specimen which were sent to the Laboratory shall have a
different Procedural protocol
Specimen Collection
All specimens for OP will be collected within the laboratory

Continuation
III. Acceptance and Running of Specimen
Inform Nurse-on-Duty; Repeat

Is the specimen In-Patient Collection
quantity and N0
quality fit for Inform or all Patient; Repeat
Out-Patient
Collection
Testing?
YES
Run Testing
AND PROCEDURES

Is the result N0
ready for
release? ***TROUBLESHOOTING
PROCEDURES (refer to Lab.
Manual)
(Repeat Testing unsuccessful)
YES

Continuation
IV. Releasing of Results

Record results in designated Result
LOGBOOK
Encode results in computer; Save a soft

copy of result
Record time of release and results to be

released in Releasing LOGBOOK;
Release results to Nurse-on-Duty;

Require name, signature, and time of
In-Patient receipt
Release results
Out-Patient Release results to Patient; Require OR
(note release of result);Require name,
signature, and time of receipt
QUALITY CONTROL
Income Proof Sheet per Shift
AND PROCEDURES

Census and Workload Report per Shift
END
Flowchart: Referral Examinations
Inform Patient/Department of Scheduled

release of result and the send-out protocol
of the department
Prepare specimen for transport
Inquire for the availability of the

examination for referral
Inform referral laboratory of tests to be sent

out
Accomplish Petty Cash Voucher (Billing

Departmen)
Transport specimen to referral laboratory
AND PROCEDURES

Wait for release of result
Release result to Patient/Department once

available
Flowchart: Patient Instructions
SUBMIT REQUEST FORM TO

YOU WILL BE INFORMED OF THE

AVAILABILITY, PREPARATIONS (if any)
AND PRICE OF THE TESTS REQUESTED
YOU WILL BE GIVEN A CHARGE SLIP
PLEASE PAY THE CHARGED AMOUNT

ING THE CASHIER
SHOW THE OFFICIAL RECEIPT TO THE

SPECIMEN COLLECTION WILL BE MADE
PLEASE WAIT FOR THE RESULT
AND PROCEDURES

Flowchart: Purchase Request and Receiving of supplies
Accomplish Purchase Request Form
Attach Monthly Inventory Report
Submit request to Purchasing Department
Follow-up approval of Purchase request
Receive delivery of supplies
Accomplish and record supplies received in

designated logbook
Update Inventory list
Store supplies properly

END
AND PROCEDURES

Flowcharts: Needs Assessment Program

This is the program framework for the Department’s Annual Training Program. Needs Assessment
shall be the basis of personnel needs, which will be subjected to remedial and corrective
actions.
AND PROCEDURES

Flowcharts: Application for Laboratory Annual Training Program

This is the step-by-step procedure in accomplishing approval for the Annual Training
Program.
AND PROCEDURES

Machine Breakdown
Initial Troubleshooting by RMT
Yes FIXED No
Proceed to Analysis Inform Depts of status of Call Service Engineer

machine
Flowcharts: Machine Malfunction

This is the step-by-step procedure in handling Machine Breakdown in the
Laboratory.
AND PROCEDURES

RECEIPT OF REQUEST FORM
STAT/ER IN-PATIENT OUT-PATIENT
ISSUE CHARGE SLIP ISSUE CHARGE SLIP ISSUE CHARGE SLIP
RECORDING OF LABORATORY RECORDING OF LABORATORY RECORDING OF LABORATORY

REQUEST REQUEST REQUEST
RECEIVING OF SPECIMEN SPECIMEN COLLECTION SPECIMEN COLLECTION

I
I
REJECTIONNI
REPEAT COLLECTION FROM SPECIMEN PREPARATION

PHLEBOTOMIST
PROCESSING OF SPECIMEN
CHECKING OF RESULTS _ _ _ _ _ _ _ _ _ _ _ _ _ _ QUESTIONABLE

I
DOUBLECHECK/DO MANUAL
PROCEDURES
RECORD AND PRODUCE HARD COPY SIGN OUT
RESULTS
RELEASING OF RESULTS
Flowcharts: Laboratory Standard Protocol

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Document Name: LABORATORY POLICIES

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

GENERAL POLICIES AND

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

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Timed Specimens/ Multiple Specimens 52

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

GENTRI MEDICAL CENTER AND HOSPITAL

Doc. Property: LABORATORY DEPARTMENT

Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

The pathology laboratory provides and interprets analytical and morphological

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5.0 DEFINITION OF TERMS:

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Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

5.4. Department: term used to denote the Laboratory Department.

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7.0 JOB RESPONSIBILITIES:

Doc. Property: LABORATORY DEPARTMENT

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Effective Date: JUNE 2013

7.2 CHIEF MEDICAL TECHNOLOGIST

7.3 MEDICAL TECHNOLOGIST:

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Santusan st., Manggahan, Gen Trias Cavite

Effective Date: JUNE 2013

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7.4.8. Files examination results, receiving and releasing logbooks

8.0. SCHEDULES AND SHIFTING

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Santusan st., Manggahan, Gen Trias Cavite

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Doc. Property: LABORATORY DEPARTMENT

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Effective Date: JUNE 2013