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Quality Assurance Training Programme

HIS MAJESTY'S GOVERNMENT


Ministry of Health | Department of Health Services
National Public Health Laboratory
Phone 4252421 Fax 4252375 Address Teku, Pachali, Kathmandu

Quality Assurance
Training Programme

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Preface
Since the beginning of the nineties the National Public Health Laboratory has been
involved in quality assurance for medical laboratories in Nepal. Out of this a Quality
Assurance Unit has developed with the support of the Laboratory Assistance
Programme (LAP) of the International Nepal Fellowship. This now runs regular training
courses. For these courses training materials have been produced and compiled by
the Medical Technologists of LAP to form this present manual.
The main aim of this manual is to standardise commonly used laboratory techniques
while putting a special emphasis on internal quality control procedures (IQC).
We wish this manual to be a bench aid as well as a reference for the laboratory
workers in their day to day management of their work.

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Acknowledgements
We wish to thank all those who have contributed to this work.
Special thanks are due to:
• Dr Sarala Malla, Director, National Public Health Laboratory, Department of Health
Services, Teku Kathmandu
• Gabriele Mallapaty who inspired us to produce this material by providing the
format and producing the first handout on internal quality control procedures.
• The Technical Assistance Programme of the International Nepal Fellowship for the
section on maintenance and their regular input to the training of laboratory staff in
simple maintenance of equipment.
• Dot and Lines Graphics Art for their design work and drawings.
• W.H.O. Manual of Basic Techniques for a Health Laboratory (1980) from which
most of the pictures have been taken.
• All our colleagues in the National Public Health Laboratory who contributed with
valuable suggestions and comments.

For the Laboratory Assistance Programme


Marianne Brocqueville, Heike Priebe
July 2003

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Contents

Chapter 1 Quality Assurance Programme 1


Diagram of a Quality Assurance Programme 4
Standard Operating Procedures (SOPs) 7
Diluting Solutions Correctly 12
Universal Precautions 13
General Code of Conduct 13
Pipetting Techniques 15
Maintenance 19

Chapter 2 Quality Assurance in Haematology 23


Sample Collection 25
Prepration of EDTA anit-coagulant bottles 26
Sample storage and transportation 26
Waste Disposal and Cleaning 27
IQC Procedures 27

Chapter 3 Quality Assurance in Biochemistry 31


Sample Collection 33
Sample Storage and Transportation 34
IQC Procedures 34
Quality Assurance in the analytical stage 35
Establishing Performance Standard 38

Chapter 4 Quality Assurance in Bacteriology 47


Sample collection 49
Sample storage and Transportation 52
Waste disposal 52
Sterilisation 53
IQC Procedures 54
Gram Stain 55
Specimen Sites 64

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Chapter 5 Quality Assurance in Urine 69
Sample collection 71
Sample storage and transporation 71
IQC Procedures 72
Urine deposit 73

Chapter 6 Quality Assurance in Parasitology 85


Stool Parasites 87
Blood Parasites 89
Samples Collection 93
Collection of pinworm eggs 95
Stool examination - methods of concentration 97
Direct Slide Preparation 102
Eggs and larvae of intestinal parasites 104
Parasitic worms whose eggs are found in stools 109
Examination of segmented flat worms - Taeniae 115
Common Parasitic Protoza 126
Differences between Amoebic and Bacterial 131
Malaria : life cycle 135
Malaria : keys to laboratory diagnosis 137
Identification of Malaria Parasites 139
Malaria : Laboratory diagnosis - Giemsa Stain 142
Identification of Malaria parasties 144

Chapter 7 Quality Assurance in CSF 153


Sample Collection 155
Sample storage and transporation 155
IQC Procedures 155

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Chapter 1
QUALITY
ASSURANCE
PROGRAMME

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The ultimate goal of any quality assurance programme is to obtain test
results that are reliable, relevant and reproducible.

A quality assurance programme consists of three parts:


• Internal Quality Control (IQC) Procedures
• External Quality Assessment (EQA)
• Quality Management

Internal Quality Control (IQC) Procedures are done during daily routine work. They
are applied to all work procedures and to every test done in the laboratory. They
provide an immediate control so that errors can be corrected immediately.
External Quality Assessment (EQA) evaluates past performance by testing
unknown samples and comparing the performance with others. It provides a forum for
improvement and correction of errors
Quality Management involves training of the laboratory staff, the use of
Standard Operating Procedures (SOPs), a standard supply of equipment and
materials, supervision in the laboratory and the organisation of the laboratory

Why is Internal Quality Control needed?


It is needed to ensure that test results are reliable and reproducible. it is carried out
as part of the daily work routine.

Why is External Quality Assessment needed?


It is needed to detect hidden problems and to compare the performance of one
laboratory with another to improve the quality of the results produced. Help and
support is received from NPHL.

Why is Quality Management needed?


It is needed to allow the laboratories to produce quality results that are affordable
and relevant and to ensure that test results are interpreted correctly.

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Diagram of a Quality Assurance Programme

Quality Management

National Public Health Laboratory


External Quality Assessment Programme

Results to NPHL
Training SOP's
LaboratoriestosamplesControl

NPHLfromadviceandFeedbac
Supply

Supervision
k

Equipment

Your laboratory
ICQ Procedures
DAILY

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Example of the form you will receive with EQA samples.


National Public Health Laboratory, Quality Assurance Programme,
Teku, Kathmandu
Test Code : QA-12 Date Sent: 15/06/2003 Code: ....................... Lab: ..................
District: ......................... Your EQA No.: ......................

Test Your Result Comment Name

Haemoglobin (Units: g/dl) QA-12.1

Total WBC count (Units: /mm3) QA-12.1


Platelet count (Units: /mm3) QA-12.1
Differential WBC Count (Units: % for Poly
items 1 to 9) QA-12.1 Lymphcytes
Monocytes
Eosinophils
Basophils
Blast cells
Promyelocytes
Myelocytes
Metamyelocytes
NRBC
MP

Morphology

Blood Glucose (Units: mg/dl) QA-12.3


Blood Urea (Units: mg/dl) QA-12.3
Serum Albumin (Units: g/dl) QA-12.3
Serum Creatinine (Units: mg/dl) QA-12.3
Serum Uric Acid (Units: mg/dl) QA-12.3
total Protein (Units: g/dl) QA-12.3
Serum Sodium (Units: mmol/l) QA-12.3
Serum Potassium (Units: mmol/l) QA-12.3
Gram Stain QC 4.4

Sputum AFB QA-12.5

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Example of the feeback form you will receive after sending your EQA results
National Public Health Laboratory,

Quality Assurance Training Programme


Quality Assurance Programme, Teku, Kathmandu
Test Code: ..............................
Name of the Laboratory: ....................... Your EQA number: .................... Date received: 09/10/2002
Test Target Acceptable range Your results Marks Comments
value
Haemoglobin (units:g/dl) QC-10.1 11.50 9.20 to 13.80 12.50 0.90 Well done, excellent result
Total WBC count (units: /mm3) QC-10.1 4730.00 2838.00 to 6622.00 3000.00 1.80 _
RBC Total Count (unit: Million/mm3) QC-10.1 3.80 2.28 to 5.32 0.00 0.00 _
Platelet count (unit: /mm3) QC 10.1 134.00 80.40 to 187.60 0.00 0.00 _
Blood Glucose (units: mg/dl QC-10.3 27.20 23.50 to 30.89 28.00 0.00 Well done, excellent result
Blood Urea (nits: mg/dl) QC-10.3 14.20 11.36 to 17.04 9.00 3.70 do pipette carefully, check
that the reagent is clear and
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not too old. Check that the


standard is not contaminated
Serum Uric Acid (units: mg/dl QC-10.3 1.30 1.04 to 1.56 0.00 0.00 _
Serum Creatinine (units: mg/dl) QC-10.3 0.73 0.58 to 0.87 0.00\ 0.00 _
Serum Albumin (units: g/dl) QC-10.3 1.48 1.25 to 1.70 0.00 0.00 _
Total Protein (units:g/dl) QC-10.3 3.75 3.18 to 4.31 0.00 0.00 _
Serum Sodium (units: mmol/l) QC-10.3 100.30 95.68 to 104.91 0.00 0.00 _
Serum Potassium (units: mmol/l) QC-10.3 4.60 0.00 0.00 _
Gram stain QC-10.4 Gram Positive Cocci Gram positive cocci & 3.00 Please keep the slide to look
Gram negative cocci at it with a supervisor
Sputum AFB QC-10.5 No AFB seen AFB not seen 0.00 Well done, excellent result
Overall Performance

Average marks : 1.57 Signature


Quality Assurance Training Programme
Three are three stages of Quality Assurance

1. The pre-analytical stage


The pre-analytical stage includes the management and organisation of the
laboratory, the usefulness of the requested test, the patient preparation , and the
sample collection, storage and transportation.
2. The analytical stage
The analytical stage includes the organisation of the routine work, the reagents
and test method used, the state of the equipment, the use of Internal quality
control procedures and the following of the standard operating procedures.
3. The post-analytical stage
The post-analytical stage includes the organisation of result recording and reporting,
the interpretation of results and the speed at which the results are obtained.

Standard Operating Procedures (SOPs).


SOPs are written instructions that include all aspects of laboratory work practices.
They help to prevent mistakes rather than detecting them. They are an important part
of the quality assurance programme

SOPs have the following general features:


• SOPs are written in accordance with a standard format.
• SOPs are written in simple language, readily understood by employees.
• SOPs contain sufficient procedural details to enable trained staff to perform the
task without supervision.
• SOPs are written by qualified and experienced laboratory officers.
• SOPs must be followed exactly by all staff.
• SOPs are given a title, identification number and date.
• SOPs are reviewed and updated on a regular basis.

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Erythrocyte Sedimentation Rate (ESR)


Westergren Method
A-001a
Authorised signature: Dr. Sarala Malla Issuing Date May 2003
NPHL Director Next Revision Date May 2006

Staff able to perform test: Laboratory Assistant and higher

Principle of the Test Method


The ESR expresses in mm the rate at which red blood cells settle when anticoagulated
blood is allowed to stand in a narrow tube (Westergren). It is shown by the height of the
column of clear plasma at the top of the tube after exactly one hour.

Clinical Significance of the Test


ESR is used as a screening method for all diseases that are associated with a
modification in the ratio of the plasma proteins like globulin, albumin and
fibrinogen. ESR is not a very reliable screening method as it can be raised when
there is no disease and can be normal when disease is present. It does not
indicate the type of disease but has prognostic value.

Specimen
2 ml fresh EDTA Blood or2 ml fresh Citrate Blood (not older than 24 hr. if kept at 4°C)

Equipment Requirements
• Westergren Rack
• Westergren Tubes, internal diameter 2.5 mm
• Dilution bottles to hold 2 ml (4 volumes of blood/1 volume of anticoagulant
diluent solution
• Timer (1hour)

Reagents & Stain Requirements


3.8% Tri-sodium Citrate Solution

Preparation of 3.8% Tri-sodium Citrate Solution

Tri-sodium Citrate, anhydrous……………………………………………………..3.8 g


Distilled Water………………………………………………………………..up to100 ml

IMPORTANT: Keep the solution in the refrigerator. If the solution is cloudy or


contains particles, discard it and prepare a fresh solution!

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Erythrocyte Sedimentation Rate (ESR)


Westergren Method
A-001a
Test Procedure Instructions

1. Measure exactly 0.4 ml of the 3.8% Tri-sodium


Citrate Solution with the help of a pipette or a
syringe into a clean and dry small bottle.

2. Draw 2 ml of venous blood and immediately place


1.6 ml into the tri-sodium citrate solution. Note:
You can also use EDTA blood. If kept at 4°C, it can
be used up to 24 hours. In this case mix the EDTA
blood well and place 1.6 ml into the tri-sodium
citrate solution.

3. Mix the blood and tri-sodium citrate solution well.

4. Fill a clean, dry Westergren ESR tube with the


sample, exactly to the 0 mark. Do not mouth
pipette. Use a pipetting device.

5. Wipe the outside of the Westergren tube with a


tissue and make sure no air bubbles enter the tube.
Recheck that the tube is filled exactly to the 0 mark.

6. Close the top of the tube firmly and place the


tube into the tube holder. ( the mixture will
escape from the tube if not firmly closed.)

7. Immediately set your timer for 1 hour or write


down the time on a sheet of paper.

8. After exactly 1 hour read the level of the


plasma. Give the results in mm per hour.

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Erythrocyte Sedimentation Rate (ESR)


Westergren Method
A-001a
Reporting and Interpretation of Results
Normal Value: Male: 1 – 10 mm in first hour
Female: 3 – 14 mm in first hour

Increased Values of ESR


Are found with all diseases associated with a modification in the ratio of the plasma
proteins like globulin, albumin and fibrinogen. ESR shows especially high values in
• Tuberculosis
• Leishmaniasis
• Malignant conditions
• Hepatic Amoebiasis
• Acute and Chronic Inflammations

Special Note:
While reading the result you should also pay attention to the following: The colour
of the plasma:
• Dark yellow, indicates hepatitis
• Clear as water, indicates lack of iron
• White and turbid, indicates nephrosis, diabetes, lipaemia.
• Increased layer of white blood cells just above the red blood cells indicates
leukocytosis

Internal Quality Control Procedures and Sources of Error


• Correct dilution of blood and tri-sodium citrate solution
• Mix the sample well before use
• Store tri-sodium citrate solution in the refrigerator
• The tri-sodium citrate solution should not be turbid
• Avoid air-bubbles in the Westergren tube
• Place the Westergren tube in a vertical position
• Temperatures above 23°C increase the ESR. Therefore keep the ESR rack at
the coolest place of the lab and out of direct sunlight.
• Do not use haemolysed or partially clotted blood
• Do not use wet ESR tube

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Erythrocyte Sedimentation Rate (ESR)
A-001a
Westergren Method

References
1. A handbook of Medical Laboratory TechnologyHaematology for Students and
Practitioners. Dr. Ramnik Sood M.D..Jaypee Brothers medical publishers.

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V1 = ........................

Diluting Solutions Correctly


In the laboratory it is frequently necessary to dilute solutions. To dilute a solution
means to reduce its concentration.
A weaker solution can be made from a stronger solution using the following formula:
C1 x V1 = C2 x V2
C1 = Concentration of the stronger solution
V1 = Volume (ml) of the stronger solution needed to make the
weaker solution (Unknown)
C2 = Concentration of the weaker solution required
V2 = Volume of the weaker solution required

V1 = C2 x V2
C1
Example:
To make 1ml of 100mg% Glucose solution from a 500mg% stock solution:
C1 = 500mg%
V1 = ? 100 mg% x 1 ml
C2 = 100mg% V1 = = 0.2 ml
500mg%
V2 = 1 ml

Therefore measure 0.2 ml of the 500 mg% solution and make up to 1 ml with
distilled Water.

Exercise:
A. To make 1 litre of Hydrochloric acid (HCl), 0.01 mol/l from a 1 mol/l solution:
C1 =
V1 = ?
C2 = =
V2 =

Always add acid to water and never water to acid


Therefore add ..........ml HCl 1mol/l to 500ml of the water and then make up to 1 litre with
distilled Water.

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B. To make 25 ml of 350 mg% Glucose solution from a 500mg% stock solution:
C1 =
V1 = ?
C2 =
V1 = ........................ =
V2 =
Therefore measure ...............ml of the 500 mg% solution and make up to 25 ml with

distilled Water.

Universal Precautions:
• Treat all blood specimens as potentially infectious!
• Consider all equipment that has been in contact with blood specimens as potentially
infectious.
• Keep the laboratory clean. After work wipe the benches with disinfectant!
• Do not mouth pipette and wear protective coats at all times.
• Do not eat, drink or smoke in the laboratory. Cover open wounds.

General Code of Conduct


• Keep your laboratory clean and in order! For example: clean the workbench every
day after work with disinfectant, label your chemicals, and cover your microscope
with a protective cover.
• Keep your glassware and instruments clean and handle them with care!
• You are dealing with infectious material, carelessness can harm you and
your patients!
• Examine each specimen with the same accuracy and care, as you would
examine your own!
• Never give false reports! If you are not sure about a result, repeat it or tell the
doctor that you are not sure and that you would like to receive a fresh sample!

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Pipetting techniques

1. The use of glass


pipettes Introduction
Whenever volumes between 1 ml and 25 ml need measuring in the lab, the lab
assistant will use a glass pipette. Pipetting with those glass pipettes needs to be
accurate and safe. We recommend the use of a medium size rubber bulb with a ‚blue‘
plastic tip to avoid mouth pipetting.
Mouth-pipetting is the cause of many infections and injuries due to hazardous
chemicals. A cotton wool plug in the top of a pipette is not an effective microbial filter.

Important hints for pipetting


• Always hold the pipette straight
• Do not blow into the solution
• Do not blow out the last drop when emptying the pipette
• Always use the lower edge of the meniscus to measure the volume – unless
the solution has a very dark colour which makes the meniscus hard to see.
• Do not mouth pipette.

Steps in Pipetting
1. Get a testube rack ready with as many
test tubes as needed
2. Get the solution ready
3. Take a clean glass pipette
4. Take a rubber bulb with ‚blue‘ tip
5. Blow all the air out of the bulb by pressing
very hard
6. Set the bulb – with the tip pointing into the
pipette – on top of the top end of the pipette
7. Hold the pipette upright

8. Put the bottom end of the pipette into


the solution
9. Slowly let go of the pressure in the bulb
and suck the solution into the pipette

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10. Suck up the solution up to a little bit above


the „0" mark

11. Remove the bulb and place the index finger


of your other hand on the top of the pipette,
closing the hole
12. Lift the pipette out of the solution but not out
of the bottle
13. Let the solution drop until the lower edge of
the meniscus meets with the „0" mark or the
volume that you want to measure

14. Lift the pipette out of the bottle


15. Wipe the outside of the pipette and place it
into the first tube with the tip of the pipette
touching the inner wall of the test tube
16. Let go of the solution until the lower edge of
the meniscus meets with the mark of the
volume that you want to measure
17. Lift the pipette out of the first tube into
the second tube
18. Repeat step 15 into the next tube

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2. The use of automatic pipettes
1. Preparation 2. Aspiration 3. Distribution 4. Purge 5. Home

Hold the pipette in a Immerse the pipette in the Place the pipette tip at an Wait one second, then Allow the plunger to
vertical position. Depress liquid. Allow the plunger angle against the inside depress the plunger to move up to the rest
the plunger smoothly to to move up smoothly to wall of the receiving tube. the second stop position. position.
the first stop position the rest position. Wait Depress the plunger This removes any
one second so that all the smoothly to the first stop remaining sample from
liquid has time to move position. the tip. Remove pipette
up into the tip. Keeping tip end from sidewall by
the pipette upright sliding it up the wall.
remove the pipette from
the liquid and wipe the
outside of the tip without
touching the open end.
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1

Rest Position

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Stop
Second Stop
or purge
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Maintenance
Good functioning laboratory equipment is an important part of quality assurance. All
our equipment has to be kept in good order, which is achieved by maintenance.

Maintenance includes
• Appropriate setting up of new equipment
• Proper daily handling
• Regular cleaning
• Routine maintenance (e.g. oiling of mechanical parts, change of bulb after x hours)
• Appropriate repairs
Remember maintenance is NOT ONLY repair.

Colorimeter

The essential part of a colorimeter are:

Light Source Filter Cuvette Detector Multiplier Recorder


Elements of a Photometer

Each of these elements can be prone to defect.

1. Light source
The light bulb gets slowly weaker usually indicated by difficulty in zeroing or instability of
the absorption signal. To prolong the life of the lamp, switch of the colorimeter after use.

When changing the bulb:


• check that the specifications of the bulb are according to the user manual
• follow the manual’s instruction to change the bulb
• do NOT touch the bulb with your fingers
• make sure that the bulb is well fixed and give the maximum transmission
• Always keep a spare bulb in your laboratory.

2. Filter
• Check regularly that your filters are clean and not scratched or broken.
• Broken and scratched filter must be replaced.

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• Dirty filters can be cleaned with care to avoid scratches
• Always check that you are using a filter of the correct wavelength for the test method.

3. Cuvette
The quality of glass cuvettes is superior to plastic cuvettes. They can be cleaned
better and reused.
To clean off protein precipitates you can soak them in concentrated sulphuric acid/
potassium dichromate solution overnight and then rinse them with abundant
distilled water before drying.
If you are using plastic cuvettes clean them immediately after use with distillated
water or detergent solution followed by abundant distilled water. Keep them up side
down to dry. The sulphuric acid/potassium dichromate solution cannot be used, as it
would damage the plastic.
• Always check before use that cuvettes are clean and not scratched.
• Use the right kind of cuvette for your colorimeter
• Put the cuvette in the right position

4. Detector, multiplier and recorder


These elements are electronic. If they are damaged the repair needs to be done by
a qualified technician.
You can avoid most damage by:
• using according to the manual’s instructions
• transporting with care
• using an appropriate power supply
The colorimeter should be kept in a safe place and protected against the dust.
Calibrate the colorimeter for each test method.

Microscope

• The microscope is the most important instrument in a peripheral laboratory.


• It allows us to diagnose numerous infectious diseases and count and identify cells
in many specimens.

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• Staff should know how to use the microscope properly and handle it with care
• Each day before use the microscope should be cleaned and checked by
an experienced person
• The microscope should be stored safely at the end of each day
• Never place the microscope into direct sunlight.

1. Cleaning of optics
Condenser, objectives and eye pieces should be clean with a soft camel-hair brush or
a blower. Always wipe off immersion oil from the 100x objective with lens paper, soft
paper or cotton-wool. The following cleaning solution can be used for oil dried onto the
objective: Methanol 30%, ether 70%.

2. Cleaning of the body


Clean with a soft, dry cloth.
Heavy contamination can be removed with mild soapy solutions or with
water/ 96%ethanol (50/50) solution.

3. Dry climate precautions


To protect the microscope against the dust:
• Cover properly with dust cover
• Clean the dust off every day with a brush or a blower

4. Humid climate precautions


To prevent fungus growth:
• Clean the dust off every day with a brush or a blower
• When not in use, keep the microscope under an airtight plastic cover with some
blue silica. Check the color of the silica and if it becomes red it should be
regenerated in the hot air oven.
• The microscope can also be put in a cupboard with a light bulb left on.
In humid climate if nothing is available it is better to leave the microscope on the
table, covered with a cloth which allows the air through. Never put it in a closed box.

5. Changing the bulb


• Check that the specifications of the bulb are according to the user manual
• Do not touch the bulb with you fingers
Before switching on the microscope turn the lamp brilliance control to its
minimum and then increase progressively. This will prolong the life of the bulb.
• Always keep a spare bulb and a spare fuse in your laboratory.

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Centrifuge

Appropriate use of the centrifuge will maintain it


in good condition for a long time.
• Place it on a firm level bench, away from the edge.
• Make sure the load is balanced at all time.
• Only use thick-walled centrifuge tubes for centrifugation.

• Turn the speed control up and down slowly.


• Stop the centrifuge immediately if it makes
an abnormal noise.
• Never use a higher speed than necessary.
• Always close the lid during centrifugation to avoid
dangerous aerosol
• Never attempt to stop the moving centrifuge
by hand. (Injury and aerosol).
• When using a swing-out rotor centrifuge, make
sure the size of the tubes is correct, otherwise
the tubes can break when the bucket swings out
to its horizontal position.
• Clean any sample spillage and disinfect
immediately.
• Clean regularly with disinfectant (once a week)
• Check carbon brush every 3 months and replace
if necessary. Always keep a spare carbon brush in
your laboratory.
• Check for corrosion and cracks every 3 months
and clean and paint with anticorrosive paint when
necessary.

The Auto-pipette

• Auto-pipettes will only function accurately if they


are well maintained and kept clean.
• Auto-pipettes are expensive, handle them with care.
• Use only one kind of tips.
• Auto-pipettes should be dismantled and cleaned
from time to time. Follow manufactures
instructions.
• Plastic pipette tips can be reused but must be
disinfected and well cleaned before reuse.

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Chapter 2
Quality Assurance
in Haematology

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Sample collection

Haematology tests are carried out on fresh capillary blood or anti-coagulated


venous blood.

Capillary blood
Capillary blood is obtained by pricking the finger, the ear lobe or in infants the heel of the
foot. Capillary blood is used immediately and therefore does not need anticoagulants to be
added. It can be used for haemoglobin estimations, total WBC-counts, differential WBC-
counts, platelet counts, reticulocyte counts and for Malaria or Filaria films

Capillary blood collection


Step one: Arrange all items required:
• Sterile lancets
• Dry cotton wool
• Cotton wool soaked in 70% alcohol
• Clean glass slides or 20 µl pipettes
• Reagents as needed
Step two: Collect the blood
• Clean the skin thoroughly with an alcohol swab and allow to dry.
• Prick the finger deeply enough that the blood flows freely.
• Wipe the first drop of blood away with a dry cotton swab.
• Use the next drop for your sample.
NEVER use absolute alcohol for disinfecting the skin. Use only 70% alcohol !
NEVER use blood that has been squeezed out by force. If the blood doesn’t flow easily,
rub the hand and prick a different finger.

Venous blood
Venous blood is obtained by vene-puncture. An anti-coagulant must be added to the
blood to prevent it clotting. Commonly used anti-coagulants for haematology are
EDTA (Ethylene diamine tetra-acetate), heparin and trisodium-citrate.

Blood anti-coagulated with EDTA


The correct dilution of EDTA and blood is very important. Blood films for differential
WBC and for malaria parasites should be made within one hour of collecting the
blood into EDTA.

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Preparation of EDTA anti-coagulant bottles

Step one: Prepare 10% EDTA solution


EDTA powder ......................................................................... 10 g
Distilled water ....................................................................... 100ml

Weigh out the powder and dissolve it in 100 ml of distilled water. Pour the solution into
a labelled reagent bottle.

Step two: Prepare the sample collection (Bijou) bottles


Place the clean, dry sample bottles (Bijou) on the bench. Pipette exactly 0.04ml of
10% EDTA solution into each bottle. Store the sample bottles open in a dust-free
place over night to dry completely. When the bottles are dry cap them and label them
“EDTA”. The bottles can be stored at room temperature for up to one year.

Step three: Collect the patients blood into the bottles Add
about 2.5 ml of blood and mix gently but thoroughly.
NEVER add a pinch of EDTA powder directly to the sample bottles. - High
concentrations of EDTA lead to the RBCs shrinking and destroy the structure of the
WBCs and platelets. NEVER add the blood before the EDTA solution is completely dry
as it will dilute the blood and destroy the RBCs.

Blood anti-coagulated with trisodium-citrate


Trisodium citrate is used to dilute blood for ESR (erythrocyte sedimentation rate)and
for coagulation testing. ESRs can be done with EDTA blood but the blood must still be
diluted with trisodium citrate.

Sample storage and tansportation

Blood films
• Blood films should be prepared within one hour of collection when EDTA blood is
used.
• Store unstained blood films in a dry place and protected from direct sunlight, dust
and flies.
• Stain blood films as soon as possible.
• Malaria and filaria positive slides should be kept in a box for future inspection by
a supervisor.
• Doubtful slides should be kept in a separate box for inspection by the supervisor.
• For transportation each slide should be wrapped in a piece of paper and kept in a
box to avoid breakage.

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Quality Assurance Training Programme
EDTA anti-Coagulated Venous blood
When EDTA anti-coagulated blood cannot be tested within 1 to 2 hours it must be
refrigerated at 4-8oC to prevent cellular changes affecting test results. Manual or
automated blood cell counts, reticulocyte counts, and PCV change little in EDTA blood
at 4-8oC when stored for up to 24 hours. Haemoglobin concentration is stable for 2 to
3 days at 4-8oC providing there is no haemolysis.

Waste Disposal and Cleaning

Blood
• Where running water is available, pour the blood into a sink that is connected to a
soak pit. Where running water is not available pour blood into a bucket that contains
a 10% lysol solution, soak over night and dispose off with solid waste (bury).
• 1% Virex solution can also be used. Soak for half an hour and dispose off with
solid waste.

Glass slides
• Soak used glass slides in 5% lysol solution at least over night. Clean and rinse next
day.

Needles and Lancets


• Store used needles and lancets in an empty metal container. When full burn and
bury it.

IQC Procedures

Westergren ESR
• Ensure that the correct dilution of blood and tri-sodium citrate solution is used
• Store the trisodium citrate solution in the refrigerator
• The trisodium citrate solution should not be turbid
• Avoid air-bubbles in the Westergren tube
• Place the Westergren tube in a vertical position
• Temperatures above 23°C increase the speed of the ESR. Therefore keep the ESR
rack in the coolest place of the lab and out of direct sun light.

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Quality Assurance Training Programme

Total White Blood Cell Count


• Mix the blood and white cell diluting fluid well before filling the counting chamber and
allow the white cells to settle for 2 minutes before counting.
• Place the cover-slip firmly onto the chamber. You should see rain-bow colours at
both sides of the cover slip.
• Only use the cover-slips of the counting chamber. (Do not use ordinary
glass cover-slips).
• Do not let air-bubbles enter while filling the counting chamber
• Count the WBC in all four corners of the chamber
• Calculate the results properly

Differential White Blood Cell Counts


• The slide must be clean and free from dust, oil or grease, and finger marks.
• The slide spreader must clean and the edge of the spreader must be smooth.
• The film should not have ridges, waves, or holes. It should be smooth and even.
• Avoid poor staining, which makes the film too blue, by altering the staining times
and gain good colour differentiation.
• Neutral or slightly acid water (pH 6.8) is essential for good cell colour differentiation.
Acid water produces a picture that is too red; alkaline water, a picture that is too
blue. The pH of water should therefore be checked regularly and Wright stain and
buffer solution should be kept away from acid or alkaline solutions.
• Avoid stain precipitate on the film by filtering the stain regularly.
• Examine the slide at the right place, as big cells are more in the thin parts of
the smear and lymphocytes more in the thick parts of the smear.
• Count at list 100 cells
• Comment on morphology is an important part of blood slide examination

Haemoglobin Sahli Method


• Remove the first drop of blood after pricking the finger.
• Avoid blood clots while collecting the blood.
• Do not allow air-bubbles to enter the pipette.
• Wipe the tip of the pipette before entering the hydrochloric acid solution.
• Add the hydrochloric acid drop by drop and compare the colour after each drop.

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Quality Assurance Training Programme
• If the haemoglobin concentration is very low inform the doctor or health-care
worker immediately.
Haemoglobin Colorimetric method
A new calibration graph must be prepared whenever the colorimeter, cuvette type or
the test method is changed.
• Always allow the colorimeter to warm up before measuring the test samples.
• Ensure that the correct filter (540nm wavelength—yellow/green filter) is in place.
• To test for accuracy use a control sample of known value.
• Control samples are either commercially available control samples or samples
of known (true) value measured by a very reliable laboratory.
• Repeat the test on a single specimen to control for reproducibility (precision).
• A new stock of Drabkin’s solution must be checked against the old solution with
samples of known value before it is used for patients
• Drabkin’s solution should be clear and pale yellow in colour. If it is turbid or loses its
colour it must be discarded.
• Handle Drabkin’s solution with care. It is very poisonous. Do not mouth pipette.
Leave the blood mixed with Drabkin’s solution for 10 minutes before reading the
optical density so that the haemoglobin can convert to cyanmethaemoglobin.
• Be careful with cuvettes with frosted sides. The clear side must face the light path.
• Be careful to avoid air bubbles in the cuvette.

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Quality Assurance Training Programme

Chapter 3
Quality Assurance
in Biochemistry

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Sample Collection

Venous blood sample collection for biochemistry:


Serum is used for most biochemistry tests carried out at a district hospital.
• Do not leave the tourniquet on the arm for more than 2 minutes, as this will affect
the concentration of cells and substances in the blood.
• Do not collect the blood from an arm in which an intravenous infusion is being given.
• Remove the needle from the syringe before transferring the blood into the
collection tube. Transferring the blood through the needle may cause haemolysis,
breaking of the red cells.
• The collection tube must be clean and dry.
• Allow the blood to clot at room temperature away from direct sunlight.
• Centrifuge the blood and separate the serum as soon as possible, and not more
than 1 hour after collection.
• After centrifugation remove the serum carefully without sucking up red cells.

Blood for sugar estimation is normally requested on a fasting specimen. You should be
familiar with the following terms:

Fasting specimen
• This means no food or drink except water has been taken since the night before
and the blood sample is collected in the morning before any drink or food is taken.

Post-prandial specimen (PP)


• This means the specimen has been taken about two hours after the last meal.

Random
• This means the specimen has been collected at any time of the day, irrespective
of food intake.
The normal range of blood sugar differs in serum & whole blood, whole blood levels
are about 15% higher.

Below are normal values for adults:


Fasting Random
Serum Less than 120 mg% Less than 180 mg%
Whole blood Less than 140 mg % Less than 200 mg %

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Quality Assurance Training Programme
Sample Storage and Transportation:

• Carry out the biochemistry test as soon as possible after separation of the serum
from the blood clot.
• Not all analytes are stable, which means their concentration will reduce over time.
• Generally, stability is prolonged if the serum is kept in the refrigerator.
• Some analytes such as bilirubin are also affected by light.

Below is a list indicating the stability of analytes in serum:


Test Stability at Room Affected by Remarks
Temperature Haemolysis
Glucose Few hours only Yes Red blood cells cause
glycolysis
Urea 1 day No
Creatinine Few hours only Yes Examine as soon as
possible
Bilirubin 1-2 hours onlyAway from light Yes Light reduces the
concentration of bilirubin
Albumin Few hours only Yes
Amylase 1-2 days No

IQC Procedures:

General
• Do not examine the specimen when the blood is haemolysed.
• Do not examine the specimen when the time of the sample collection is not clear.
• Label the specimen with patient name and lab number to avoid confusion.
• Take great care when pipetting samples and reagents.
• Care for your colorimeter. Cover it with a protective cover when not in use.
• Handle filters with great care. Do not touch the filter with fingers, always hold from
the side.
• Prepare standard curves whenever you use new reagents or equipment.
• If possible use commercially available control sera to control accuracy.
• Do repeated tests or redo tests from the day before to check your precision.
• Be careful when repeating and redoing tests as some analytes are not stable.
• The serum for sugar has to be separated from the blood clot within one hour after
the collection and the test should be done within two hours after collection as the
concentration of glucose decreases over time due to glycolysis.

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Quality Assurance Training Programme
Quality assurance in the analytical stage

Reliable test results depend on the detection and correction of errors at an early
stage. Good laboratory practice and quality control helps keep errors of imprecision
and inaccuracy low.

Accuracy is defined as the closeness of agreement between a test result and


the accepted true value.
Precision shows how well a value is reproduced if done repeatedly.
The diagram below illustrates the difference between accuracy and precision.

Mr. Poudyal Mr. Shrestha Mr. Yadav


1kg

1kg

Mr Poudyal regularly checks his equipment and closely supervises his shop assistant
so the bags always contained the same amount of salt and as close as possible to the
exact weight. They were accurate and precise.
Mr Shrestha and his assistant were careless and imprecise when filling the bags, which
did not contain the correct weight but varied from day to day. They were neither
accurate nor precise.
Mr Yadav’s assistant was careful in filling the bags, but Mr Yadav never checked his scales,
which were inaccurate and weighing too little. They were inaccurate but precise.

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Quality Assurance Training Programme
When to check for accuracy?
Always check when changing reagent, instrument and methodology, as a routine once
a week or as appropriate and when there is doubt about the result.
Errors of accuracy are also known as errors of bias, they are consistent and
systematic. They can be caused by incorrect calibration, due to inaccurate standard
and pipette, incubation at the wrong temperature, reading the test at the wrong
wavelength and the use of an incorrect calibration factor. The use of poor quality
reagents and standards also leads to errors of accuracy.
Errors of precision are also known as errors of scatter, they are irregular and random.
They can be caused by inadequate mixing of the sample and reagent, inconsistent
pipetting, incubation at variable temperature and dirt or air bubbles in the colorimeter.
Incorrect storage and handling of samples and dirty glassware will also lead to errors
of precision.
Accuracy is best checked with commercially available quality control sera and
through the External Quality Assurance Programme control sera. At least two levels
of control sera should be used, one normal and one pathological.
Errors of precision can be avoided by internal quality control procedures and
quality management.

How to economise on commercially available control sera.


• Dissolve the control serum as directed
• Place into small containers about double the quantity required for the test
• Label each container and deep-freeze immediately
• When required remove from freezer
• Allow the sera to reach room temperature and mix well before use

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Quality Assurance Training Programme
Expected result of Blood Glucose 120 mg%

Example One
• Repeated Results of the Blood Glucose show the following values:
101, 103, 99, 101, 102 mg %

140 mg %

120 mg %

100 mg %

EXCELLENT PRECISION, POOR ACCURACY STOP

Example Two
• Repeated Results of the Blood Glucose show the following values:
122, 119, 121, 120, 123 mg %

140 mg %

120 mg %

100 mg %

EXCELLENT PRECISION, EXCELLENT ACCURACY

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Quality Assurance Training Programme
Establishing Performance Standard

Mean
Describes the midpoint of a population or in other words gives the calculated average
of a set of values.

I.e.: If 10 tests have been performed, the 10 results are added and this sum is divided
by 10.
No.
1 .............................................. 107 mg%
2 .............................................. 118 mg%
3 .............................................. 102 mg%
4 .............................................. 114 mg%
5 .............................................. 110 mg%
6 .............................................. 108 mg%
7 .............................................. 117 mg%
8 .............................................. 109 mg%
9 .............................................. 112 mg%
10............................................ 113 mg%
Total 1110 mg%

1110 mg% ÷10 = 111 mg% (Mean)

Before any test method is used for patients we must first make sure that the method
is reliable and that it can be performed within acceptable limits of variation.
Once a test has been introduced it must be controlled routinely.
To assess reliability first determine the
• Standard Deviation (SD) then the
• Optimal Conditions of Variance.

1. Standard Deviation
• In statistical terms the distribution or scatter of values around the mean can
be expressed as standard deviation.
• A range of 2 standard deviations (± 2 SD) is generally considered as the limit for
a control value to be acceptable.

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Quality Assurance Training Programme
Usually
• 68% of results will fall within ±1SD
• 95% of results will fall within ±2SD
• 99.7% of results will fall within ±3SD
• To establish the SD perform 20 measurements on the same control serum.
• List the values for each of the 20 estimations as shown in the example below.
• Calculate the mean of these 20 values
• For each result work out the difference in value from the mean and record it
in column 2
• Multiply each difference value by itself (to give the squared difference value).
Enter these values in column 3
• Add up the values in the third column to obtain the sum of the squared differences.

Test no. 1 2 3
Test results Diffrence from the mean Squared differences
1 72 74-72= 2 22 4
2 74 74-74= 0 02 0
3 75 74-75= -1 -12 1
4 74 74-74= 0 0
02
5 72 74-72= 2 4
22
6 78 74-78= -4 -42 16
7 74 74-74= 0 02 0
8 72 74-72= 2 22 4
9 73 74-73= 1 12 1
10 74 74-74= 0 02 0
11 75 74-75= -1 -12 1
12 76 74-76= -2 -22 4
13 73 74-73= 1 12 1
14 74 74-74= 0 02 0
15 72 74-72= 2 22 4
16 74 74-74= 0 02 0
17 72 74-72= 2 4
22
18 77 74-77= -3 -32 9
19 74 74-74= 0 02 0
20 75 74-75= -1 -12 1
Mean 74 Sum = 54

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Quality Assurance Training Programme
• Calculate the Standard Deviation from the formula :
SD = sum of the squared differences
n-1 n = number of results.
E.g.: SD = 54 = 1.69

19 n = 20

2. Optical Conditions Variance (OCV)


If you stop after calculating the SD, the value obtained can be misleading as the
same SD can be obtained in the case of a high mean or a low mean.
As an example let’s take the SD we have obtained above 1.69 for a glucose mean of 74
mg%. The same SD of 1.69 could be obtained for a calcium mean of 8.3 mg%. In the
first case this is acceptable, in the second case it is much too high. The SD value
doesn’t give a good idea of the reliability of the method.
The OCV gives the SD as a % of the mean.
SD
OCV = mean x 100
E.g.:
• Glucose test OCV = 1.69 / 74 x 100 = 2.28 This is acceptable. The method can
be used.
• Calcium test OCV = 1.69 / 8.3 x 100 = 20.36 This is much too high and
totally unacceptable.

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Quality Assurance Training Programme

Test Suggested OCV maximun %


S-ALT (GPT) 7.5%
S-Albumin 3%
S- Alkaline Phosphatase 7.5%
S-Amylase 10%
S-AST (GOT) 7.5%
S-Bicarbonate 10%
S-Bilirubin total 10%
S-Calcium 2%
S-Chloride 1.5%
S-Cholesterol HDL 6%
S-Cholesterol LDL 7.5%
S-Cholesterol total 3.5%
S-CK total 10%
S-CK MB 12.5%
S-Créatinine 7.5%
S-Iron 4%
S-Glucose 3.5%
S-γ-GT 7.5%
S-IgA 10%
S-IgG 10%
S-IgM 10%
S-LDH 7.5%
S-Lithium 3%
S-Magnesium 2%
S-Phosphate 3%
S- Potassium 1.5%
S-Protein total 1.5%
S-Sodium 1%
S-Thyroxine (T4) 7.5%
S-Transferrin 10%
S-Triglyceride 7.5%
S-Triiodothyronine (T3) 7.5%
S-Uric acid 5%
S-Urea 3.5%

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Quality Assurance Training Programme
The above OCV values are suggested OCV maximum % that you may use to access
the analytical methods you are using. Different books and Quality Control web sites
may give slightly different values. These are considered suitable for a district laboratory.
OCV (Optimal Conditions Variance) can be obtained only in ideal conditions, the same
technician carefully performing 20 measurement of the same control serum, at a given
time.
The RCV (Routine Conditions Variance) is obtained in routine conditions when different
technicians are performing the same test at different times. The acceptable value may
be up to twice the OCV.

3. To Establish a Daily Quality Control Chart Run


a control serum (CS) with every batch of tests.
Most faults can be identified and corrected at an early stage by plotting these
control serum results on a graph.
To Set Up A Daily Control Chart
• Take a sheet of graph paper.
• Mark the vertical axis with the mean, +2 SD and - 2 SD, which are supplied by the
Quality Assurance Programme or the commercial supplier.
• Draw horizontal lines from these corresponding points.
+ 2 SD= mean + 2SD. Above this value is a TAKE ACTION zone.
- 2 SD= mean - 2 SD. Below this value is a TAKE ACTION zone.
• The horizontal axis is numbered with the date the test is performed.
• Using the same CS , for each batch of tests , plot the values on the control chart.
• Each day after charting the control value, check that it is within the acceptable
limits of ±2 SD and that there is no marked change in the distribution of results
above or below the mean or a drift towards the TAKE ACTION zones.

42
Month: October
Analyte: Urea Low
45
44.5
44
43.5
43
42.5
42
41.5
41
40.5
40
39.5
39
38.5
38
37.5
37
36.5
36
35.5
35
34.5
34
33.5
33
32.5
32
31.5
31
Mean
43

30.5
30
29.5
-2 sd

TrainingAssuranceQuali
29
28.5 +2sd
28
27.5
27
26.5
26
25.5
25
24.5
24
23.5
23
22.5
22
21.5
21
20.5
20
19.5
19

ty
18.5
18
17.5
17
16.5

Progra
16

mme
15.5
15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Day
Quality Assurance Training Programme
Interpretation of Results
• Control Serum value within + 2 SD limits is a good sign and it can be assumed that
the patient results are reliable and can be reported with confidence.
• Control Serum value outside + 2 SD is unacceptable and the patient results must not be
reported. Take a fresh control serum and repeat with a few of the patient samples.
- If the repeat CS is within +2SD and the repeat tests agree with those of the
first testing all the patients’ results can be reported.
- If the repeat CS is within +2SD but the repeat test results do not agree with those
of the first testing all the patients’ tests should be repeated.
- If the CS is still not acceptable do not report patient results. Check for errors -e.g.
reagent deterioration, incorrect preparation of reagents, faulty equipment, or
wrong filter in the colorimeter. Correct the error and repeat the batch with CS.
• Serum Control Values moving towards the TAKE ACTION ZONE report patient
results, but a drift upwards or downwards is a warning that the test is
becoming unreliable and the cause must be investigated.

analyte: month:
I II III IV V

2SD

mean

2SD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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Quality Assurance Training Programme

Control serum

I. Results are close to and equally I. gtLhfx? cf}zt cyjf cf}ztsf]j/Lk/L


distributed around the mean value glhs}5l/Psf 5g\h;n]k|of]uzfnfsf]
indicating good laboratory
performance. sfd /fd|f]5 eGg]hgfpF5 .
II. A result outside the 2 SD limits, II. Pp6f gtLhf b'O Standard deviation =
indicating that the analytical system is 2SD eGbf aflx/ 5 h;n] Nofjsf] sfd
out of order. Immediate action should v/fj ePsf]hgfpF5 . s]sf/0fn]sfd lju|]sf]
be taken to identify the cause.
xf]t'?Gt sfjf{O cyjf vf]hL ug'{k5{.
III. Sequence of results within the limits
of acceptance but uniformly III. gtLhfx? dfGo kl/wLdf 5g\t/ Psgf;n]
distributed above (or below) the Mean -cf}zt_ eGbf dfly -cyjf tn_
mean, indicating a bias which needs ePsfn] 7Ls gtLhf eGbf s]xL km/s
to be identified and corrected. b]lvG5g\. s]sf/0fn]of]km/s
b]lvPsf]xf]Tof] kQf nufO{;Rofpg'k5{.
IV. A sequence of seven results with a IV. ;ftj6f gtLhfx? cf}ztjf6 kf/ u/]tfkgL
tendency towards decreasing (or 36\bf]e'msfjsf dfg b]vfO/x]sf 5g\h;n]
increasing) values, even when
ljZn]if0f k|s[ofdf nuftf/ 36\bf]e'msfj 5
passing through the mean, indicating
eGg]hgfpF5 . -v/fjL pks/0fdf, l/Ph]G6df,
a steady tendency to the analytical Standard df cyjf df

system (= instrument performance,


reagents, calibrators, control material) x'g;S5 . sf/0f kQf nufP/ ;Rofpg'k5{.
to depart from control. Causes need
to be identified and corrected.
V. Broad scattering of the results around V. dfgx? dfGo kl/wL leq 7"nf]If]qdf 5l/Psf
the mean indicates low precision of 5g\ h;n] Measurement df Low
measurement, the causes of which precision ePsf] hgfpF5 . o;sf]
need to be identified. sf/0f kQf nufpg'k5{.
(Taken from Basics of Quality Assurance for intermediate and peripheral laboratories, WHO, 1992)

45
Quality Assurance Training Programme

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Quality Assurance Training Programme

Chapter 4
Quality Assurance
in Bacteriology

47
Quality Assurance Training Programme

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Quality Assurance Training Programme
Sample Collection
If the pathogens are to be isolated successfully, the type of specimen, method of
collection, the time and method of dispatch to laboratory must be appropriate.

Things to be remembered
A. Use of aseptic collection technique
B. Correct type of specimen: Eg, MSU, Endocervical swabs, Sputum
etc. C. Time of Collection
D. Containers/swabs for specimen collection
E. Proper labeling of specimen

Sputum for TB
As a rule three sputum samples are collected:
1. When the patient comes to the health centre.
2. The following morning at home
3. At the clinic during the second visit at the health centre.
• It is best to collect the first sputum in the morning
• Before collecting the sputum, the patient may drink a glass of hot water.
• The patient should stand if possible and take a few very deep breaths filling the lungs.
• The lungs should be empty in one breath coughing as hard and deeply as possible
• The patient should spit what he/she brings up into the clean collection container.
• Write the name and patient number on the container to avoid confusion.

Instructions for sputum smear preparation


• Only use new slides.
• Label the slide with a diamond pencil.
• Prepare the smear looking especially for the mucus particles in the sputum.
• Leave slides to air-dry. Protect the slides from flies, insects and dust while drying.
• Heat-fix the smear only after the slide is completely dry to avoid aerosols.
(Health hazard)
• Stain the smear with Ziehl Neelsen stain.

Skin scraping for leprosy


• Label a new glass slide with a diamond pencil.
• Take the smears from the right ear lobe, the left ear lobe, any anaesthetic patches
or around the right elbow and the left knee

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Quality Assurance Training Programme
• Clearly mark from which site each smear is taken
• Take a fold of skin between your index finger and thumb and squeeze tightly
to prevent the blood flow and make a cut without releasing the pressure
• Collect the tissue fluid
• If there is lots of blood do not collect the sample but make a new cut again
squeezing the skin tightly

Skin scraping for fungus


• Place a drop of 40% potassium hydroxide on a clean slide and with a scalpel blade
• Mix the skin scales with the potassium hydroxide and cover with a cover slip
• Heat gently over a spirit lamp without allowing it to boil
• Place the slide in a dampened petri-dish for 30 minutes

Pus
• Collection by experienced medical staff
• Possible pathogens:
Staphylococcus aureus Pseudomonas aeruginosa
Streptococcus pygoens Proteus spp.
Other streptococci Klebsiella
Clostridium tetani E. coli
Clostridium perfringens Bacteroids spp. etc.

• Pus from an abscess is best collected at the time it is incised and drained, or after
it has ruptured itself.
• When collecting pus from abscesses, wounds or other sites, special care should be
taken to avoid contaminating the specimen with commensal organisms from the skin.
• A specimen from a wound should be collected before antiseptic dressing is
applied/ before antimicrobial therapy is started.

Collection and transport:


• Drain the abscess with a sterile needle/syringe and transfer to a sterile leak
proof, screw capped container.
• If pus is not being discharged, use a sterile swab to collect the sample from the
in-fected site. (It is recommended to take 2 swabs if possible)
• Process for culture/gram stain etc if possible.
• Ortherwise, insert the swab into Stuart's/Amies transport medium, breaking off
the swab stick to allow the bottle top to be replaced tightly.

Urine
• Patients should be instructed about proper urine collection technique (MSU).
• Procedure for Mid Stream Urine Collection

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Quality Assurance Training Programme
• Possible pathogens from urine:

E. Coli Staphylococcus saprophyticus


Pseudomonas aeruginosa Klebsiella spp.
Salmonella typhi and paratyphi Neisseria gonorrhoeae
Proteus spp.
• Bacteriuria/pyuria
• Urine itself is a good culture media for most organisms
• Examination/processing should be done immediately
• Refrigeration of sample in case of unavoidable delay: boric acid.

Note: It is advisable to screen all urine samples sent for culture with a screening
method for urinary tract infection (UTI). The screening method avoids unnecessary
culture of uninfected samples and saves time and money. (see SOP-C002)

Instructions for collecting mid-stream urine


The patient should wash their hands with soap and water.

Female
Spread the legs apart and with your left hand spread the labia apart. Keep holding
the labia apart during the entire collection process.
Male
Pull back the foreskin hold it and back during the entire collection period.
Male and female
Pass a small amount of urine but do not collect it. Move the collection container under
the stream of urine and collect some into the container. Do not collect the last few
drops of urine. Close the lid of the container and take the urine sample immediately to
the laboratory for examination.

Blood
Blood for culture must be collected under comletely sterile conditions to
avoid contamination.
• Always collect blood for culture before antibiotic treatment has been started
• If possible collect the blood at the time when the patient’s temperature is rising
• Remove the blood culture bottles from the fridge and allow them to reach
room temperature.
• If the culture broth appears turbid do not use for blood culture
• Possible pathogens:
Salmonella typhi, paratyphi E. coli
Streptococcus viridans Klebsiella
Streptococcus pneumoniae Proteus
Staphylococcus aureus Haemophilus influenzae etc.
• Bacteraemia/septicaemia

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Quality Assurance Training Programme
Method of Collection:
Must be collected asceptically
• Make the patient sit comfortably.
• Locate the suitable vein in the arm.
• Cleanse thoroughly the skin over the vein with 70% ethanol
• Do not touch the skin after disinfection.
• Using a sterile syringe, draw 5 ml of blood and immediately dispense into blood
culture bottle containing 50 ml culture broth, (for children, 3 ml into 30 ml of broth).
• Recap the bottle, label the specimen and dispatch to microbiology laboratory.

Sample Storage and Transportation


For bacteriological processings : Proper handling of sample after its collection
and transportation is very important.
• It is better to process the samples soon after its receival if facilities are available.
• Proper storage : depending upon the type of specimen and expected pathogens
• Use of transport media.
• Proper labeling of specimen for dispatch and sending any additional information
available.

Sputum for TB
• Smears are prepared and heat-fixed when the sputum is received at the laboratory.
• Usually slides are prepared and heat-fixed before sending them to a
reference laboratory. Sputum is only sent when culture is required.
• For transportation wrap each individual slide in a piece of paper or use a slide box.
• Store slides in a box away from flies, dust and direct sunlight.

Waste Disposal
• Used sputum containers, slides and wooden applicators must be collected in a
waste container that is covered with a lid.
• Dig a deep pit of 1 metre and throw the waste into the pit. Cover the pit with soil
when it is about half full.
• All the waste material must be burnt.
• Do not leave the waste in the open uncovered.
• Do not throw the waste into a river.
• Do not reuse slides of TB and leprosy smears.

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Quality Assurance Training Programme
Sterilisation
Sterilisation is the killing of all living micro-organisms including bacterial spores.

Methods of sterilisation
1. Steam under pressure (autoclave, steam steriliser or pressure cooker).
• Sterilisation is achieved through pressure and temperature.
• Recommended conditions are 121°C for 15 minutes at a pressure of 15 psi.
• It is used for sterilising culture media and instruments.
2. Incineration
• Incineration is burning of material. It is the most effective method of
destroying infected disposable material.
• Waste material for awaiting incineration should be kept covered and well protected
to prevent access by people, animals and insects.
3. Flaming
• Fire kills all living organisms.
• This is use to disinfect reusable metal or glass objects, like wire-loops, glass slides
and the necks of blood culture bottles.

Disinfection
Disinfection is the killing or removal of pathogenic (causing disease) micro-
organisms. Methods of Disinfection
1. Chemicals
The chemicals most commonly used for disinfection are Phenols,
Aldehydes, Alcohols and Halogens.
2. Boiling in water
Boiling in water for 20 – 30 minutes can be used. At altitudes above 2000 feet 30
minutes is recommended.

Where to use chemical disinfectants


• Work benches, floors, instruments and equipment
• Spills of potential infectious and infectious material
• Skin, hands, body fluids
• In basins used to collect used glass slide, cover slips, pipettes and other
reusable items.
Note: Do not use weak phenols such as Dettol and Savlon for disinfection in
the laboratory.
Most commonly used chemicals for disinfection

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Dilution Use
Phenols • 5% solution of Lysol to Glassware / Floor /Benches
disinfect glassware and
other reusable items.
• 10% solution of Lysol to Body fluids.
disinfect body fluids.
Aldehydes • 10% Formalin Preservation of stool
• Not commonly use for parasites.
disinfection of instruments
as it is highly irritating to
skin, eyes and lungs.
Alcohols • 70% alcohol Skin disinfection
• Methylated spirit (no need Smear fixation
to dilute)
Halogens • 0.5% Hypochlorite solution Hand disinfection
(Bleach)
• 0.5% Iodine solution Glassware Skin disinfection
• 1% Virex Glassware, bench top

IQC Procedures
Gram stain
• Follow proper sample collection procedures.
• Do not overheat the slide when heat-fixing the smear.
• Filter the crystal violet before use.
• Discard Lugol’s iodine when the colour has faded.
• Decolourise carefully. Just few seconds are requesred then wash off with water.
• Stain known samples of Gram-negative and Gram-positive bacteria once a week
and whenever new stains have been prepared.

Ziehl Neelsen Stain


• Follow the proper sample collection procedures.
• Label slides with diamond pen, (grease pencil will wash off).
• Do not recycle slides for Ziehl Neelsen stain. Use only new slides.
• Do not overheat the slide when heat-fixing the smear.
• Filter the carbol fuchsin stain onto the slide.
• Stain known sample of AFB positive sputum once a week or whenever using new
carbol fuchsin stain.
• Store all positive slides and 10% of the negative slides in a box for Quality Control.

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Gram Stain

Clinical Significance
1. Gram stain results can have a dramatic effect on patient care.
2. Hospitalisation may be required when the Gram stain results indicate bacteria
are present in a normally sterile body fluid.
3. The initial choice of antibiotic therapy is guided by Gram stain results.

Gram Stain Procedure


1. The Gram stain is a technique for staining and detecting bacteria and yeast.
2. Four reagents are used to perform a Gram stain
• crystal violet
• Gram’s iodine
• acetone-alcohol
• safranin

Direct Smear Preparation


If the specimen is a fluid, place a drop of the fluid
on a clean glass slide and allow to air dry.

If the specimen is received on a swab, gently roll


the swab on a clean glass slide to avoid rupturing
host cells. Allow to air dry.

In both cases, the specimen is fixed to the glass


slide by passing it a few times over a flame.

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Staining Procedure

1. Flood the heat-fixed slide with crystal violet for 1


minute. Almost all bacteria are stained by crystal
violet.

2. Rinse with water

3. Flood the slide with Gram’s iodine for 1 minute.


Gram’s iodine does not cause a colour change.

4. Rinse with water.

5. Decolorize the slide by gently rinsing with an


acetone-alcohol solution for 10 seconds. Then
rinse with water.Gram-positive bacteria retain
the crystal violet stain. Gram-negative bacteria
do not.

6. Flood the slide with safranin for 1 minute.


Safranin counterstains Gram-negative
bacteria.Rinse with water and air dry.

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Theory
Gram-positive and Gram-negative bacteria stain differently because of the structure
of their cell walls.

Microscopy
Examine the slide under oil immersion and record the presence of any host
cells, bacteria, or yeast.

Reporting Results
Use systematic, descriptive terminology to report Gram stain results.

For example, this Gram stain is properly


described as “Gram-positive cocci (clusters) and
white blood cells present.”

It is NOT possible to determine the species of bacteria from Gram stain results
alone! Definitive identification requires culture and biochemical testing

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Morphology

Cells

White Blood Cells


To correctly identify white blood cells, it is necessary for the white blood cells to
be intact.
White blood cells will remain intact if the specimen is handled gently during
smear preparation.
White blood cells will rupture if the specimen is handled roughly during smear
preparation.

1. Polymorphonuclear Neutrophils
A polymorphonuclear neutrophil (PMN) is
distinguished from other white blood cells by its
segmented nucleus.PMN’s are attracted to the site
of infection in response to bacterial and host
inflammatory products.PMN’s are short-lived,
phagocytic, and usually appear in the acute stages
of bacterial infection.

2. Lymphocytes
Lymphocytes are smaller than PMN’s. The nucleus
is round and has a thin border of
cytoplasm.Lymphocytes are uncommon on direct
Gram stains except from cerebrospinal fluid.A
smaller number of lymphocytes are present in CSF
from healthy individuals. A large number of
lymphocytes usually indicates viral meningitis.

3. Macrophages
Macrophages are slightly larger than PMN’s. The
nucleus is large, non-segmented and slightly
indented.Macrophages are polymorphic; they may
resemble large, atypical lymphocytes.Macrophages
are widely distributed in tissues throughout the
body.Macrophages are long-lived, phagocytic, and
are more prevalent in chronic infections.

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Epithelial Cells

1. Squamous Epithelial Cells


Squamous epithelial cells are commonly found on
direct Gram stains from a variety of sites.They
are distinguished by their comparatively large
size, small nucleus, and cytoplasm.Their
diagnostic relevance is dependent on the site.

2. Ciliated Columnar Epithelial Cells


Ciliated columnar epithelial cells are occasionally
seen on Gram stain smears from the respiratory
tract.They are distinguished by the presence of cilia
at on end of the cell and their elongated
shape.Their presence suggests that the specimens
is from lower respiratory tract

Sperm Cells

Sperm Cells may be present on Gram stains


from genital or urine specimens.

Bacteria

Gram-positive Cocci
The Gram-positive coccus is a spherical bacterium.
Gram-positive cocci may appear in four groupings.

When cocci appear in pairs they are called


diplococci. Gram-positive diplococci may by
elongated in shape.

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Cocci may also form chains of variable length.

Groups of exactly four cocci are called tetrads.

Groups of cocci are referred to as clusters. The


number of cocci in a cluster may vary considerably.

Gram-positive Rods
The Gram-positive rod or bacillus is a rectangular shaped bacterium.
Rods are variable in length, width, and staining characteristics.
There are four clinically significant shapes of Gram-positive rods....

Long, wide rods usually have blunt ends.

Long, narrow rods also have blunt ends. They


may form chains of variable length.

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Coccobacilli share characteristics of rods an cocci.


They are very short, coccoid rods.

Branched rods are long and narrow, and may have


a beaded staining pattern. Note: do not confuse with
chains of bacteria.

Gram-negative Cocci
Gram-negative cocci may appear in two clinically significant groupings...

The Gram-negative coccus is spherical.

When cocci appear in pairs they are called


diplococci. Gram-negative diplococci may be
kidney-shaped.

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Gram-negative Rods
The Gram-negative rod or bacillus is a rectangular shaped bacterium.
Rods are variable in length, width, and staining characteristics.
There are five clinically significant shapes of Gram-negative rods....

Long, narrow rods typically show uniform staining.


They may appear in short chain.

Coccobacilli share characteristics of rods and cocci.


They are very short, narrow rods. Note: do not
confuse with Gram-negative cocci.

Curved rods are very short, narrow, and curved.


They usually show minimal staining, and may form
short chains.

Fusiform rods are long and narrow with


pointed ends.

Spiral rods are long, narrow, with multiple curves.

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Yeasts

Budding Yeasts
Yeasts commonly appear as “budding yeasts.”
Note: do not confuse single yeasts with sperm cells

Pseudohyphae
Yeasts may also appear as pseudohyphae which
are elongated projections from yeasts.
Pseudohyphae have tapered ends.

Artifacts

Crystal Violet Precipitate


Crystalline precipitate forms in crystal violet
reagent over time. This precipitate may be seen on
a Gram stain.Crystal violet precipitate is blue to
purple in colour, varies in shape and size, and may
be refractile.Crystals may be distinguished from
bacteria by focusing up and down.

Cellular Debris
Direct Gram stains often show a variety of
background debris which must be distinguished
from host cells, fungi, or poorly staining
bacteria.The debris can include the following:
mucus strands, ruptured or disintegrating cells, and
protein precipitate.

Decolorization
Overdecolorization
Gram-positive organisms may appear partially or completely Gram-negative if they
have been overdecolorized.Overdecolorization may be due to poor staining technique.
The timing of the acetone-alcohol step is critical.Overdecolorization may occur if the
Organisms are old or dead. This is due to damage to the organism’s cell wall.

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Underdecolorization
Epithelial cells, WBC’s, mucus, and cellular debris should stain Gram-negative.Thick
areas of a Gram stained smear are more likely to be undercolorized.
Antibiotic Effects
The presence of antibiotics may alter the size, shape, and arrangement of some
species of bacteria.
For example, the bacteria may appear swollen or elongated.

Specimen Sites

Cerebrospinal Fluid
Normal
Cerebrospinal fluid is normally sterile.
Rarely lymphocytes may be present.
PMN’s are not normally present.
Infection
Any organism is considered significant in CSF.
Usually only a single morphotype of bacterium or yeast is present.
The presence of PMN’s and/or mononuclear cells usually indicates infection. PMN’s
predominate in bacterial infection.

Note: Gram negative bacteria can be very difficult to see on a CSF gram stain, it is
therefore compulsory to do a simple Methylene blue stain first to detect the presence
of any bacteria and then to do a Gram stain.

Blood
Normal
Blood is usually not directly Gram stained. Rather, it is cultured in a blood culture bottle,
and the fluid from a positive culture is Gram stained.
Blood is normally sterile. Depending on the blood culture media used, white blood cells
can be seen in a Gram stain from a normal blood culture.
Septicemia
Any organism is considered significant in blood. Interpretation of positive blood cultures
is complicated by the possibility of contamination during specimen collection.

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Contamination
Contamination during collection may complicate interpretation of blood culture results.
Skin flora (eg. Staphylococci, micrococci, corynebacteria) can be pathogenic in certain
circumstances and therefore cannot be automatically disregarded as contaminants.

Sputum
Contaminated Sputum
Gram-stained sputum specimens should be examined under low power to determine
if the specimen has been contaminated with saliva.
A contaminated sputum specimen is characterized by abundant squamous
epithelial cells and very few PMN’s.
Identification of bacteria from contaminated specimens will provide misleading
results. Acceptable Sputum
An acceptable sputum specimen should contain very few squamous epithelial cells.
Acceptable sputum specimens are more likely to provide accurate culture results.
Only acceptable sputum specimens should be cultured.
Pneumonia
The lower respiratory tract is normally sterile.
The presence of ciliated columnar epithelial cells helps to confirm that the specimen
is from the lower respiratory tract.
Large number of PMN’s and bacteria are usually present in sputum specimens
from patients with bacterial pneumonia.

Urine
Normal
Urine is normally sterile.
Rare epithelial cells may be present.
PMN’s are normally present in urine specimens.
Infection
PMN’s are common in the urine of patients with urinary tract infections.
The presence of a single bacterium per oil immersion (100x objective) field
indicates infection with approximately 100 000 organisms per millilitre of urine.
The absence of PMN’s and bacteria does not rule out a urinary tract infection.
Contamination
Urine is easily contaminated with bacteria from the periurethral area.
Contaminated urine contains squamous epithelial cells and a mixture of Gram-
positive organisms.
A few Gram-negative rods may be present in a contaminated urine specimen.
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Genital
Normal vagina
Squamous epithelial cells are commonly seen on normal vaginal smears.
Lactobacilli are the predominant normal flora present in the vagina.
Occasional PMN”s are considered normal.
Vaginosis
“Clue cells” are squamous epithelial cells with large numbers of adherent coccobacilli.
“Clue cells” are associated with bacterial vaginosis. Gram-variable
coccobacilli are a predominant organism in bacterial vaginosis. Urethritis
(male)
Intracellular Gram-negative diplococci from a male urethral specimen suggest
a diagnosis of gonorrhea.
PMN”s are commonly present on a urethral discharge smear.
Trichomonas vaginalis
Typical Morphology:
oval-shaped, can resemble a white blood cell or a small epithelial cell.

Wound
Not Infected
If the wound specimen is from a non-sterile site, normal flora an be present.
If the wound specimen is from a normally sterile site, no bacteria should present.
Epithelial cells may be present.
Infected
Large numbers of PMN’s are usually present in wound infections.
A wide variety of bacterial species can cause wound infections.
Wound infections caused by a mixture of bacteria are common.

Eye
Normal
The conjunctiva of the eye is normally colonized with non-pathogenic corynebacteia and
staphylococci from the skin.
PMN’s are rarely seen under normal conditions.
Infection
The presence of PMN’s indicate an infection.
Usually only a single morphotype of bacteria is present.

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Joint Fluid
Normal
Synovial fluid is normally sterile.
PMN’s are not normally present.
Cellular debris may be present.
Infection
Any organism is considered significant in synovial fluid.
Usually only a single morphotype of bacteria is present.
The presence of PMN’s usually indicates infection.

Stool
Normal
In some circumstances, Gram stains of stool specimens may be useful; however,
they are not commonly performed.
Normal stool specimens contain abundant Gram-positive and Gram-negative organisms.
PMN’s are not usually present in normal stool specimens.
Infection
The presence of PMN’s in stool usually indicates that an invasive pathogen is present.
Reduction of the normal faecal flora is associated with diarrhoea.
It is difficult to distinguish pathogens from normal faecal flora on Gram
stains. Bacterial stool pathogens are usually Gram-negative rods

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Chapter 5
Quality Assurance
in Urine

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Sample Collection

Biochemical tests
• For chemical tests such as sugar, protein or bile pigment the method of collection is
less important. Any random urine sample can be used. It is always best to request
for a mid-stream urine sample.
• The concentration of analytes will be highest in the first morning urine.
• Tell the patient to collect about 20 ml of urine into a clean, dry container.
• Label the container immediately with the patient’s name and the laboratory number.
• Examine as soon as possible

Urinary deposit
• For urinary deposit you must instruct the patient to collect a clean urine sample or
a so-called mid-stream urine sample (MSU) See separate sheet!

Urine culture
• For urine culture you need a mid-stream urine sample collected into a
sterile container.

Sample Storage and Transportation


Biochemical tests
• After receipt at the laboratory keep the urine samples in a cool place away from
direct sunlight.
• Urine sugar must be tested as soon as possible because of glycolysis, caused
by bacteria. Bacteria multiply rapidly in urine if kept at room temperature.
• Bile pigment is affected by light so examine urine as soon as possible.
Urinary deposit
• Urine for urinary deposit must be examined within one hour of collection.
• Prolonged storage at room temperature causes bacteria to multiply which leads
to chemical changes in the urine. This causes cells to degenerate and crystals to
increase, making it difficult to examine the deposit under the microscope.
Urine culture
• Urine for urine culture must be examined within 30 minutes after collection.

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IQC Procedures
• Always note the colour of the urine and report unusual colours. For example, urine
can show a red colour when containing blood, or brownish green colour in patients
with hepatitis, or pale almost white colour in patients with diabetes, or milky white
colour when urine contains lymph gland fluid (chyluria – search for microfilaria).
• The urinary deposit must be examined within one hour of collection as bacteria
will multiply, cells become unclear & crystals increase.
• Examine urine for culture within 30 minutes after collection.
• When preparing new reagents, test the reagent with a known positive urine sample
or prepare your own positive control.
• Always refer to a picture atlas if you find structures that you do not know very well.

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Urine Deposits

Examination of urine is most important in patient care in that it aids diagnosis, assists
in monitoring disease and treatment and helps provide valuable information on the
patient’s well being.
The kidney has a prime role in maintaining normal healthy life, therefore many
early changes owing to disease may be reflected in the urine well before they
become clinically obvious.
Urine examination is also essential for the diagnosis of Urinary Tract Infection.

Principle
Routine urine analysis consists of physical, chemical and microscopic analysis.
Normal urine is almost clear and contains very few microscopic elements. In
some diseases the appearance of urine, chemical content and content of
microscopic elements changes. These changes are examined and reported.

Specimen Collection – mid stream sample


1. Give the patient a clean sterile collection container.
2. Tell the patient to discard the first portion of urine released, and then to catch the
next stream of urine into a container. They should also discard the last portion of
urine passed.
A badly collected specimen will give results, which are difficult to interpret.
• Keep the urine in the refrigerator if analysis can not be done immediately. Bacteria grow
quickly and are the main cause of deterioration of the specimen. Cells, casts and
chemical constituents are lost if the urine is allowed to stand at room temperature.

Urine contains elements in suspension. The following can be found:


• red blood cells
• leucocytes
• yeast
• trichomonas
• spermatozoa
• epithelial cells
• casts
• parasitic eggs and larvae
• crystals

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CELLS

Normal urine may contain a variety of cellular elements in small numbers, but
an increased number of any types can have diagnostic significance. The cells
must therefore be identified and quantified.

White Blood Cells


Presence of WBC in urine may be indication of
infection or inflammation. They are reported as
number per high power (400x) field.
1+ (6-20 per field)
2+ (21-50 per field)
3+ (greater than 50 per field)
4+ (packed field)

The WBC may be:


a. Intact: clear granular discs, 10-15 µm (the
nuclei may be visible).
b. Degenerated: distorted shape, shrunken,
less granular.
c. Pus: clumps of numerous degenerated cells.

Red Blood Cells


RBC may be present in urine due to injury or
disease of the urinary tract. In females, they may
also be present due to menstrual contamination.
They are reported as number per high power (400x)
field. More than 2 to 3 is considered abnormal.
1+ (3 to 8 per field)
2+ (9 to 30 per field)
3+ (more than 30 but not packed)
4+ (packed)

RBC may appear as:


a. Intact: small yellowish disks, darker at the
edges (8 µm)
b. Crenated: cell has losed water and shrinked,
the surface is covered with spiny projections.
c. Ghost: swollen, thin circles, increased in
diameter (9-10 µm).

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Epithelial Cells
Epithelial cells may be seen in small numbers in
normal urine. The presence of increased numbers
of transitional or renal cells may indicate disease. To
differentiate the cells we look at the size, shape,
appearance and nuclear to cytoplasmic ratio. They
are usually reported as number per high power (40
x objective) field.
Squamous epithelial cells
Large, flat, irregularly shaped cells with small nuclei.
Large number of squamous epithelial cells may
indicate improper specimen collection. In female
patient it is usually of vaginal contamination.
Transitional cells:
Round, oval or pears shaped cells usually have a
centrally located nucleus. They come from the
renal pelvis to the terminal urethra, they have
ability to absorb large amounts of water and
therefore may appear swollen.
Renal epithelial cells:
The shape can vary depending on the location of
origin within the kidney. Renal cells may be very
difficult to distinguish from small transitional cells.
They are slightly larger than leukocytes and
contain a large, usually eccentric, round nucleus.
Increased numbers of renal cells may indicate
tubular necrosis.

Oval Fat Bodies


Oval Fat bodies are renal tubular epithelial cells that
contain lipids. Round highly refractile inclusions in the
cell. Oval fat bodies may be seen together with free
fat droplets in the urine and a strongly positive
biochemical test for protein. A stain must be used to
confirm the identification of an oval fat body. They are
frequently associated with nephrotic syndrome.

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CASTS

• Casts are structures formed in the distal tubules and collecting ducts. The cells of
these parts of the renal system form a simple protein. In certain conditions this
protein forms fibrils which become attached to the tubule walls. If urinary flow is
decreased, more protein accumulates around the fibrils forming casts.
• If cells are present they may be incorporated into the cast matrix.
• Like cells, casts need to be quantified. They are quantified by number per low
power field (10 x objective).

Hyaline Casts
Hyaline casts are composed primarily of simple
proteins.
This kind of cast may be very difficult to see.
Lowering the light level, focusing up and down will
make it easier to see hyaline casts clearly.
Hyaline casts can be seen following fever, stress,
exercise or postural changes in normal individuals.
They are not indicative of any particular renal
disorder but increased numbers may be seen in all
diseases of the kidney.

Granular casts
Granular casts may be the result of degeneration of
a cellular cast or the results of direct aggregation of
serum protein granules into the matrix of protein.
They may be present in normal persons after
strenuous exercise. They may be found in a wide
variety of renal diseases, often associated with
the presence of cellular casts.

Waxy casts
Waxy casts represent the last stage in the
degeneration of hyaline, granular or cellular casts.
They are highly refractile and more easily seen than
hyaline casts. They have usually smooth blunt ends
and sharply defined edges.
Waxy casts are always accompanied by
a positive biochemical test for protein.
They usually indicate end stage renal disease.

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Fatty Casts
Fatty casts may contain lipid droplets or oval fat
bodies. They may be seen together with free fat
droplets in the urine and a strongly positive
biochemical test for protein. They are
frequently associated with nephrotic syndrome.

Renal Casts
They are hyaline casts with renal cells incorporated in
the matrix. They may occasionally be seen in normal
person due to normal fall off of dead tubular cells. The
presence of more than an occasional renal cast per
low power field is indicative of tubular injury.

RBC Casts
RBC casts contain red blood cells in the cast matrix
and are usually red or reddish brown in colour.
They are the most diagnostic of all elements in
urinary sediment and their presence is always
pathological. They indicate acute disorder of the
glomerulus. RBC casts indicate that other free
RBCs in the sediment came from the kidney.
You must see a sharp red cell outline in at least
part of the cast to identify it as a red blood cell
cast. Usually accompanied by positive blood and
protein tests.

WBC Casts
WBC casts contain white blood cells in the cast
matrix. You must see a sharp cell outline in at
least part of the cast to identify it as WBC cast. They
are associated with inflammation or infection within
the nephron.

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CRYSTALS

• Most crystals, which appear in the urine, are normal and are of little
clinical significance.
• There are a few abnormal crystals, which the microscopist must be able
to differentiate and identify.
• Normal crystals may be found at either acid or alkaline pH. All abnormal crystals
and most drug crystals are found in urine with an acid pH.

Normal crystals

Uric acid
pH of urine : acid
Soluble in : alkali and heat
Insoluble in : hydrochloric acid and acetic acid
• Urine acid may be found in multiple forms
• Uric acid is highly birefringent

Hippuric acid
pH of urine: acid but may also be found in neutral
or alkaline urine.
Soluble in : alkali
Insoluble in : acetic acid
• Hippuric acid crystals are colourless six-sided
prisms, needles or plates.
• They are uncommon and sometimes
confused with triple phosphate crystals.

Calcium Oxalate Crystals


pH of urine: acid but may be seen in neutral or
slightly alkaline pH
Soluble in : hydrochloric acid and 90% ethyl
alcohol
Insoluble in : acetic acid
• Calcium Oxalate crystals are octahedral
(envelope shape), dumbbells, ovoid or round in
shape. They are sometimes confused with RBC.
• This type of crystal is a common cause of
kidney stones.

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Triple Phosphate
pH of urine: alkaline
Soluble in : acetic acid
Insoluble in: sodium hydroxide and ammonium
hydroxide.
• The typical appearance of triple phosphate
crystals is a coffin-lid form (1) but they may have
a fern leaf (2) appearance if freshly formed.

Calcium Carbonate
pH of urine: alkaline
Soluble in : effervesces with hydrochloric or acetic
acid
Insoluble in: alkali
• Calcium carbonate crystals are seen as
granules or dumbbells.

Calcium Phosphate
pH of urine: alkaline
Soluble in : dilute acetic acid
Insoluble in: alkali
• Calcium phosphate crystals are large flat-shaped
or wedge-shaped prisms.

Ammonium biurate
pH of urine: alkaline
Soluble in : acetic acid
Insoluble in: ammonium hydroxide
• Ammonium biurate crystals are yellowish-brown
in colour with a characteristic round with thorny
projection shape.

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Abnormal Crystals

Leucine
pH of urine : acid
Soluble in : sodium hydroxide, hot water
Insoluble in : hydrochloric acid
• Leucine crystals are round with concentric
striations, yellow-brown, and oily looking.
• They may be seen in a wide variety of
liver disorders.
• Leucine crystals are often seen with
tyrosine crystals.

Tyrosine
pH of urine : acid
Soluble in : hydrochloric acid and sodium
hydroxide
Insoluble in : alcohol, acetic acid
• Tyrosine crystals will appear as colourless to
yellow-brown single needles or clumped or
rosettes.
• They may be seen in a wide variety of liver
disorders or in the genetic condition tyrosinemia.
• Tyrosine crystals often appear with leucine
crystals.

Cystine Crystals
pH of urine : acid
Soluble in : hydrochloric acid, sodium hydroxide
and ammonium hydroxide
Insoluble in : acetic acid
• Cystine crystals are thin, colourless, hexagonal
plates and may appear wrinkled when dissolving.
• Cystine crystals are found in an
inherited condition known as cystinuria.

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Bilirubin Crystals
pH of urine: acid
Soluble in : acetic acid, hydrochloridric acid,
sodium hydroxide and acetone
Insoluble in : alcohol
• Bilirubin crystals are typically yellow-brown
needles or sometimes granules.
• They should be accompanied by a positive
biochemical test for bilirubin.
• Bilirubin crystals are found in a wide variety
of hepatic disorders.
• Bilirubin may stain the entire sediment yellow.

Cholesterol crystals
pH of urine : acid
Soluble in : chloroform or ether
Insoluble in : dilute acids / alkalis
• Cholesterol crystals appear as regular or
irregular flat plates.
• Increased urinary protein, increased
serum cholesterol, and decreased serum
albumin usually accompany them.
• Cholesterol crystals are associated with nephrotic
syndrome.

Sulfonamide Crystals
pH of urine : acid
Soluble in : acetone and alkali
Insoluble in : acetic acid
• Sulfonamide crystals may be seen in a wide
variety of shapes.
• They should be confirmed with biochemical test
before reporting.
• Sulfonamide crystals are seen in the urine as
a result of drug therapy.

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Radiopaque Dye Crystals


• Radiopaque dye crystals are associated with very
high specific gravity measurements (>1.035) by
refractometer.
• They are variable in form, but usually appear as
flat needles accompanied by round globules.
• Radiopaque dye crystals are highly birefringent.
• They may be mistaken for tyrosine or
sulfonamide crystals.

Organisms and Artifacts

Yeast
• Yeast is a common cause of urinary tract infection
in immunocompromised or diabetic patients.
• The usual organism is Candida albicans.
• Candida albicans may be seen as budding or
non-budding or may show pseudohyphae.
• Non-budding forms may be confused with red
blood cells. These may be differentiated by using
dilute acetic acid which will lyse the red blood
cells but leave the yeast intact.

Parasite
• Trichomonas vaginalis is common parasite seen
in urine. It is most frequently seen in females
but may also be seen in males.
• Trichomanas may be confused with white blood
cells.
• Flagella motility is necessary for positive
identification.

Sperm
• May be seen in urine from males or females.
• Sperm is not usually considred a clinically
significant finding.

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Bacteria
• The presence of bacteria in a fresh, clean catch
urine may indicate a urinary tract infection,
especially if white blood cells are also present.
• The most common bacteria causes of urinary
tract infection also react to cause a positive
nitrate biochemical test.
• Large amounts of bacteria without white
blood cells present may indicate poor
specimen handling.

Fibers
• Fibers may be present as contaminants
from clothing or faecal material.
• Fibers may be confused with hyaline or waxy
casts. Look carefully at the end of the fiber.

Starch
• Starch crystals may frequently be seen in
the urinary sediment as a contaminant from
powdered gloves.
• Starch crystals frequently have a characteristic
greenish appearance and a t-shaped notch in
the centre.
• Small starch crystals may be confused with
fat droplets.

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Chapter 6
Quality Assurance
in Parasitology

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STOOL PARASITES

Stool Sample Collection


• Provide the patient with a suitable wide-mouthed, container with a lid.
• Ask the patient to collect a walnut size piece or about 10ml of a watery specimen. It
is not necessary to fill the whole container.
• Ask the patient to keep the outside of the container clean. (Health hazard)
• Do not accept stool samples collected in matchboxes, newspapers or
other containers that leak. (Health hazard)
• Ask the patient not to mix urine with the stool, as this destroys trophozoites present.
• When a fresh specimen is required, tell the patient to bring the sample to the
lab within 1 hour of collection.
• Label the container clearly with the patient name and laboratory number.

Sample collection for Giardia


• If the first sample is negative request at least two more samples on alternate days.
• Advice the patients it is better to bring the sample during a diarrhoeal episodes.

Sample collection for Enterobius vermicularis(Pinworm)


• Collect an anal swab, as ova are concentrated in the anal skin area.

Sample collection for Amoeba


• A fresh sample is required and examination must be done immediately.

Sample Storage and Transportation


How to preserve stool samples in 10% Formalin
Step one: Prepare 10% Formalin solution
Formalin (neutral formaldehyde, at least 37%) ............................ 10 ml
Distilled water .............................................................................. 90 ml

Step two: Label sample bottles


Label a screw-capped brown glass bottle as 10% Formalin and write the patient
name and lab number.
Step three: Add patient sample
• Put about 2 ml of 10% formalin into the bottle.
• Add a tip of little finger size piece of stool and close the screw cap tightly.
Keep refrigerated or in a cool place.

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Stool examination flow-chart

Macroscopic examination
Note the colour and consistency
Look for adult worms and segments of tapeworms

‹ ‹
Formed or soft stools
Liquid, watery or bloodstained stools
Examine within 24 hours! Examine within 1 hour of collection
Keep in cool place!
‹ ‹

‹ ‹
Microscopic examination

Direct Smear Preparation


Normal Saline/Iodine • Normal Saline/iodine
• Sodium dichromate 2.5% solution
• Modified Ziehl Neelsen

Search for helminths eggs or larvae and Search for helminths eggs or larvae,
protozoa cysts protozoa, Amoebic trophozoites and
Cyclospora

Report

Write the name of the intestinal parasite found and the grade of infection (few, some,
many).Report the presence of red blood cells and faecal leucocytes.

Waste Disposal and Precautions


• Consider all stool samples as highly infectious.
• Avoid contact with bare fingers by wearing gloves if possible or handle with extreme care.
• Do not reuse stool containers. Burn stool sample containers and wooden applicators.
• Soak glass slides in 5 % Lysol solution at least over night.
• Cover slips break easily and may cause injuries, therefore soak in a
separate container in 5 % Lysol solution at least over night.

Chemical waste disposal


• Pour old and used chemicals into the sink and flush with water if the sink
is connected to a soak pit.
• Otherwise pour chemicals directly into the soak pit. Ensure that the soak pit is
not near a natural water source.
• Formalin is irritating to the skin and the vapour should not be inhaled.
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IQC Procedures

Intestinal Parasites
• Follow proper collection procedures to ensure accurate diagnosis, e.g.
amoebic trophozoites begin to degenerate within 1/2 hours after collection.
• Cysts, flagellates and eggs also undergo changes especially if the stool is left at
high temperatures.
• Label the specimen properly with patient name and lab number to avoid confusion.
• Only accept fresh specimens and refuse specimens contaminated with dirt or urine.
• If you cannot examine specimens immediately, leave them in a cool place
not exposed to sunlight.
• Always examine watery and blood-stained specimens first.
• Store Lugol’s iodine in brown bottles. Prepare Lugol’s iodine fresh every two weeks.
• Select portions of the stool that are coated with blood or mucus when preparing
the smears.
• Keep prepared slides in a wet chamber to prevent them from drying.
• Do not touch the stool or the smear with your bare fingers. Stool may
contain infectious material. (Health hazard)
• If you are in doubt about structures that resemble eggs or cysts refer to the
pictures and charts.
• Preserve the stool in 10% formalin for examination by a visiting expert or for referral
of the specimen if in doubt as to what it contains.
• Always examine the slide systematically.

BLOOD PARASITES

Sample Collection
Malaria parasites & Microfilariae
Capillary blood is usually used and the following preparations are examined:

Thick blood film


• Screening for Microfilariae
• Screening for Malaria parasites
Thin blood film
• Identification of Microfilariae
• Identification of Malaria parasites

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Direct wet preparation
• Screening for Microfilariae
Note: You can prepare a thick and a thin film on one slide together.

Mark with pencil

Ram 23
Thick Film n n
Thin Film

Sample Collection

Leishmania
It is difficult to find Leishmania amastigotes in capillary blood.
It is best to examine the layer of white cells (buffy coat) after centrifugation of EDTA
anti-coagulated blood.

Buffy coat
• EDTA blood is used for buffy coat examination.

Aldehyde test –screening for an increase in


IgG • Venous blood is used for the Aldehyde test.

Collection time
The number of certain parasites in the blood depends on the time of collection.

Malaria
• Highest number of parasites is found during fever attacks and before the start
of treatment

Microfilariae
• Take the specimens at night between 10 p.m. – 2 a.m. (W.bancrofti and
Brugia malayi)

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IQC Procedures

Smear for Malaria Parasites


• Follow proper collection procedures.
• Glass slides must be clean and free from grease.
• Thick films and thin films must be prepared properly.
• While drying protect blood films from dust, flies and insects.
• Do not dry films in direct sun light
• When fixing the thin film, be careful not to let methanol touch the thick film.

Smear for Microfilaria


• Filaria are seldom found in the early and late stages of the disease.
• The proper collection time is important (10 p.m. to 2 a.m. )
• Unsheated non-pathogenic filaria can be found any time of the
day. (Refer to picture atlas)
• Patients with filaria in the blood also show an eosinophilia.

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Medical Parasitology studies human illnesses caused directly or indirectly by parasites.

What is a parasite ?
A parasite is an organism that lives in or on a living
organism of a different species and obtains its
food from it.
Host ?
The host is the organism from which the parasite
takes is food. The definitive host carries the adult
stage of the parasite. The intermediate host
carries the larval stage of the parasite.
Vector ?
The vector carries the parasite from one host to
another, it can sometimes also be an
intermediate host.
Classification of parasites
By habitat
• Ectoparasite
• Endoparasite
By phylum
• Protozoa (which can be categorized by the
way they move.)
• Helminthes with the 3 following groups:
- Nematodes (round worms)
- Cestodes (tape worms)
- Trematodes (flukes)

Samples for parasitological examination

What samples?
• Stool
• Urine
• Sputum
• Blood
• Cerebrospinal fluid (CSF)
• Other secretions or tissues

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How do you collect samples ?
Generally speaking, samples for parasitology do not need to be collected in a sterile
way. However if the same sample has to be examined for bacteria, it must be
collected under sterile conditions. The sample should always be sent first for
bacteriological examination before parasitological processing.

Stool
Use the right container
• Waxed cardboard box
• Tin with a lid·Plastic box
• Glass container

Collect a sufficient quantity


• To have enough for concentration
• To prevent rapid drying of the stool
• Take at least 4 ml

Examine the stool when it is fresh


• Stools must be examined within 1 hour.
• Liquid stools and those containing blood or
mucus should be examined first to check
for motile protozoa.

Things not to do
• Never leave stool specimens exposed to the air
in containers without lids.
• Never set aside stool specimens for examination
at the end of the morning (i.e. 2-3 hours later)·
Never accept stools mixed with urine.
• Never place the stool specimen container on
the examination request form.

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Collection of pinworm eggs


Enterobius vermicularis (pinworm) eggs are usually not found in stool samples.
They are collected from the folds of skin around the anus.

1. Place a strip of adhesive tape, sticky side down,


on a slide.

2. Place a spoon handle flat against the underside of


the slide.

3. Gently pull the tape away from the slide and loop it
over the end of the spoon handle.

4. Hold the completed tape swab in one hand,


pressing the slide firmly against the spoon.

5. Separate the patient’s buttocks with the other


hand. Press the end of tape swab against the
skin around the anus in several places.

6. Take the slide and fold the tape back on to it,


sticky side down.

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7. Make sure that the tape is firmly stuck flat to the


slide by pressing it with a piece of cotton wool.

8. Examine under the microscope, using 10x


objective.

9. If no adhesive tape is available use a cotton


swab to wipe around the anus and dip it into
a test tube with saline.

10. Place a drop of saline on a slide, cover with


a coverslip and examine under the microscope
using the 10x objective.

Urine
In parasitology urine samples are collected to
check for filariasis or schistosomiasis.
If there are a lot of RBCs without WBCs or other
signs of infection in any urine deposit, you
should look for schistosomiasis.
• The patient should be asked to do some physical
exercises before collecting the urine.
• Collect the final urine passed.
Sputum
In rare cases sputum may reveal
amoebic abscesses of the lung.
• Collect sputum as for AFB.

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Blood
Blood is collected for research of malaria, filaria
or leishmania.
Make a thick and a thin film. (EDTA anticoagulated
blood can be used, however it is better to use fresh
blood collected from the finger prick.)
For details on the method, see the course
on Malaria.

Other
CSF and other secretions and tissues are generally collected by the doctor.

Stool Examination - Methods of Concentration

Concentration of stool specimens is necessary for detection of worm eggs, larvae,


and cysts of protozoa. The two most commonly used concentration methods are:
• Sodium chloride method
• Formaldehyde-ether method
Sodium chloride concentration method
• Recommended for eggs of:
- hookworm
- roundworm
- H.nana
- Taenia
- whipworm
• NOT suitable for:
- eggs of flukes and schistosomes
- larvae of threadworm
- protozoa

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Principle:
The stool are mixed with saturated solution of sodium chloride (increasing the
specific gravity). The eggs are lighter in weight and float to the surface where they
can be collected.

Materials:
• 10ml penicillin vials
• wooden applicator
• coverslip
• ethanol
• petri dish
• saturated saline solution

Reagent preparation:
• Saturated sodium chloride solution
Sodium chloride....................................................................... 125 g
Distilled water ........................................................................ 500 ml

Dissolve the sodium chloride by heating the mixture to boiling point. Leave standing
to cool. Check that some of the salt remains undissolved. If it has all dissolved add
an extra 50 g. Filter and store.

Method:
1. Prepare grease-free coverslips by cleaning them
with 95% ethanol. Air dry completely.

2. Place a portion of stool (1 cm2) in the


penicillin vial. Fill a quarter of the vial with the
saturated saline solution.

3. Using an applicator, crush the stool and mix it


well with the solution. Then fill the bottle to the
top with the solution. The suspension should be
completely uniform.

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4. Place the coverslip over the mouth of the bottle.

5. Make sure there are no air bubbles present.


Leave for 10 minutes.

6. Carefully remove the coverslip; a drop of liquid


should remain on it. Place the coverslip on a slide
and examine under a microscope immediately.

FORMALDEHYDE - ETHER CONCENTRATION METHOD

• Recommended for:
- all eggs
- larvae
- cysts of protozoa
• NOT suitable for:
- motile forms of amoebae
- flagellates

Materials:
• Electric centrifuge
• 15 ml conical centrifuge tubes with caps
• Funnel
• Gauze
• Graduated cylinder
• Wooden sticks
• Formaldehyde solution (10%)
• Pure ether
• Lugol iodine solution

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Reagent preparation:

• Formaldehyde solution (10%)


Formaldehyde (>37%) ........................................................... 100 ml
Distilled water ........................................................................ 300 ml
WARNING: Formaldehyde is corrosive and poisonous!
• Lugol iodine solution
Iodine 1g
Potassium iodide (KI) 2g
Distilled water 100 ml

Measure 100 ml of water in a cylinder. In 30 ml of this water first dissolve the


potassium iodide. Add the iodine and mix until dissolved. Add the remainder of the
water and mix well. Store in brown bottle.

Method:
1. Take 1 cm2 of stool. Crush and mix it in 10ml
of 10% formaldehyde solution.
2. Stir the mixture well and let it stand for 5 minutes.

3. Filter through two layers of gauze into a


centrifuge tube

4. Add 3 ml ether.
WARNING: Ether is highly flammable. Make
sure there is no open flame in the laboratory.

5. Cap the tube and shake vigorously for


30 seconds.

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6. Remove the cap carefully. Centrifuge for 1 minute


at low speed. There will be 4 layers in the tube:-
• 1st layer : ether
• 2nd layer : debris
• 3rd layer : formaldehyde solution
• 4th layer : deposit containing parasites

7. Free the layer of debris by rotating a wooden


stick between it and the sides of the tube. Pour
off the supernatant. Remove any debris stuck to
the side of the tube.

8. Mix the remaining fluid well with the deposit by


tapping the tube gently.

9. Place 2 drops of the deposit on a slide. Add a


drop of the iodine solution to one of the drops.

10. Place coverslips over both drops. Examine


under the microscope.- Preparation 1
(unstained): use 10x and 40x objectives.
Preparation 2 (stained): use 40x objective

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DIRECT SLIDE PREPARATION

Materials:
• Microscope slides
• Coverslips, 20 mm x 20 mm
• Wooden applicators or wire loops
• Sodium chloride solution
• Lugol iodine solution, diluted 1:5

Reagent Preparation
• Sodium chloride solution (8.5 g/L)
Sodium chloride........................................................................ 8.5 g
Distilled water ...................................................................... 1000 ml
Mix sodium chloride until fully dissolved.
• Lugol iodine solution
Iodine ........................................................................................... 1g
Potassium iodide (KI) .................................................................. 2g
Distilled water ........................................................................ 100 ml

Measure 100 ml of water in a cylinder. In 30 ml of this water first dissolve the


potassium iodide. Add the iodine and mix until dissolved. Add the remainder of the
water and mix well. Store in a brown bottle.

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Method
1. Take a slide and put :
• 1 drop of sodium chloride on the left half
• 1 drop of the iodine solution on the right half

2. Using an applicator, take a small portion (2-


3mm is diameter) of stool.
• If the stool is formed, take a portion from
inside the sample and one from the surface.
• If the stool contains mucus or is liquid, take
a portion from the mucus on the surface or
from the surface of the liquid.

3. Mix a portion of stool with the drop of


sodium chloride solution on the slide.

4. Using the same applicator, take a second portion


of stool from the specimen and mix it with the
drop of iodine solution.

5. Place a coverslip over each drop (apply


coverslips as shown to avoid the formation of
air bubbles). Label the slide.

6. Examine the preparations under the microscope.


To see eggs and cysts better, lower the
condenser to increase the contrast.- For the
saline preparation use 10x and 40x objectives.-
For the iodine preparation use 40x objective

7. Examine the saline preparation as indicated in


the picture.

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EGGS AND LARVAE OF INTESTINAL PARASITES

A. Characteristics Of Eggs
Eggs laid by parasitic worms and found in stools
are identified by:
• size
• shape
• shell
• content
and occasionally by
• colour
• external features
For example: the egg of Schistosoma mansoni
Size : 150 mm
Shape : oval
Shell : external shell, thin and turgid;
internal shell, thin, membranous
and less distinct
Content : 1 ciliated embryo
Colour : pale yellow
External feature: 1 lateral spine

B. Size of Eggs
The size can be estimated by comparison with
that of a red blood cell, which measures 7.5-8 mm
1 micrometre (1 mm) = 1/1000 of a mm
The size in mm given in this manual is that of
the long side of the egg.

The size can also be assessed in relation to


the microscopic field:
• using a x 10 objective, this egg takes up 1/10
of the field
• using a x 40 objective, this egg takes up 1/3 of
the field

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The egg can be measured by inserting a micrometer


scale slide in the eyepiece of the microscope.

Another method is to compare the egg with one of


another species common in the locality whose size
under the microscope is known (hookworm, round
worm, etc.).

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Ova of Helminths Inhabiting Human Intestine

UNFERTILIZED TRICHURIS UNDEVELOPED EMBRYUNATED


FERTILIZED
FERTILIZED
DECORTICATED TRICHIVRA ENTEROBUUS VERMICULARIS

WITH FOUR EMBRYONATED TRICHO TAENIS HYMENOLEPIS HYMENOLEPIS UNDEVELOPMENT IN


METAPHORES STRONGYLUS SAGINATA NANA DIMINUTA DEVELOPMENT
ANCYLOSIOMA DUODENALE\ SP TAENIA DIPHYLLOBOTHRIUM LATUM
SOLIUM
AND NECATORAMERICANUS HETEROPHYES
METAGONIMUS
HETEROPHYES YOKAGAWAL

FASCIOLOPSIS ECHINOSTOMA SP ENLARGEMENTS


BUSKI CLONORCHIS
SINENSIS

OVA OF HELMINTHS NOT INHABITING HUMAN INTESTINE BUT PASSED IN


FECES (LIVER, BLOOD AND LUNG FLUKES)

FASCIOLA PARAGONIMUS SCHISTOSOMA SCHISTOSOMA SCHISTOSOMA


HEPATICA WESTERMANI MAANSONI HAEMATOBIUM JAPONICUM
OVA OF PSEUDOPARASITE ANCHYLOSTOMA RHABDITIFORM LARVAE

DUODENALE
MOUTH INTESTINE RUDIMENDOD GENITAL ORGAN

ESOPHAGUS

STRONGLYLOIDES
EMBRY STERCORALIS
WITH POLAR GLOBULES
ONATED
HETERODERA MARIONI

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PARASITIC WORMS WHOSE EGGS ARE FOUND IN STOOLS

Hook worm
1. Ancylostoma duodenale
Size : 50-60 µm
Shape : oval
Shell : very thin, appears as a black line
Colour : the inside cells are pale grey
Contents : varies as the egg matures
Type A (fresh stool): 4,8 or 16 granular cells
Type B (stool a few hours old): uniform mass
of many small grey granular cells
Type C (stool 12-48 hours old): the egg is filled with
a small larva folded around itself

2. Ancylostoma duodenale larvae


Size :200-300 µm, 15 µm thick
Tail :tapered
Mouth : long, 15 µm
Genital part : small, 7 µm

Thread worm
1. Strongyloides stercoralis
The larvae are highly motile in the stools.
Size : 200-300 µm, 15 µm thick
Tail : tapered
Mouth : short, 4 µm
Genital part : long, ~22 µm

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Round worm
1. Ascaris lumbricoides
There are four types of Ascaris eggs:
Type A : Fertilised eggs with double shell
Size : about 60 -70 µm
Shape : oval or sometimes round
Shell : There are two distinct shells
• External shell - rough, brown, and covered
with little lumps
• Internal shell - smooth, thick, colourless

Type B : Fertilised egg with single shell


Size : about 60-70 µm
Shape : oval
Shell : single, smooth, thick, and colourless
Contents : a single, round, colourless, granular,
central mass

Type C : Unfertilised eggs with double shell


Size : about 80-90 µm
Shape : longer than Type A
Shell : Two shells are NOT distinct
• External shell - is brown with rough bumps
• Internal shell - is very thin and may not be visible

Type D : Unfertilised eggs with single shell


Shell : single, smooth, thin, colourless shell
(looks like a double line)
Content : large round, colourless, shiny
granules
CAUTION : Do not confuse Type D with the Type B
hook worm egg (shell appears as a single line;
inside cells are not shiny) or giant fluke egg (much
bigger in size).

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Pin worm
1. Enterobius vermicularis/Oxyuris
Size : 50-60 µm
Shape : asymmetrical oval which is flattened
on one side and rounded on the
other
Shell : smooth and thin, but a double line is
visible
Contents :
Type A : small, granular, oval mass
Type B : small folded larva
Colour : transparent, colourless

Whip worm
1. Trichuris trichiura
Size : 50 µm
Shape : large lemon-shaped
Shell : very thick, smooth with two layers
Colour : brown shell, yellow contents
Other features : transparent plug at each pole
Contents : uniform granular mass
IMPORTANT : Specify whether there are many or
few whip worm eggs present.

Schistosomes
1. Schistosoma haematobium
Eggs are found in urine, and occasionally in stools.
Size : 120-150 µm
Shape : oval; poles are different
Spine : terminal
Shell : smooth, very thin
Colour : grey or pale yellow
Contents : well-formed, ciliated embryo,
surrounded by an internal
membrane

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2. Schistosoma mansoni
Size : 150 µm
Shape : oval; one pole more round than the
other
Spine : lateral spine
Shell : smooth, very thin
Colour : pale yellow
Contents : ciliated embryo, surrounded by a
membrane

3. Schistosoma japonicum

Size : 70-80 µm
Shape : oval, almost round
Colour : transparent or pale yellow
Spine : difficult to see, lateral and very small
Contents : ciliated embryo

Tape worms
1. Taenia saginata (beef tape worm)/ Taenia
solium (pork tape worm)
The eggs of these two species are almost identical.
Size : 30-40 µm
Shape : round
Shell : very thick, lined, and smooth
Colour : yellowish brown shell, light yellowish
grey contents
Contents : granular mass surrounded by a
membrane containing 3 pairs
of hooklets
External sac: sometimes the egg is enclosed in
a transparent sac

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2. Hymenolepis nana (dwarf tape worm)


Size : 45-50 µm
Shape : oval, almost round
Shell : double shell
• External : thin membrane
• Internal : thick membrane, thicker at the
poles with filaments coming away
from both poles
Colour : very pale grey
Contents : rounded mass containing six shiny
hooklets. Granules also seen in
centre.
IMPORTANT: Report whether there are many or
few eggs present.

Flukes
1. Fasciola hepatica (Giant liver fluke)
Size : 130-150 µm
Shape : oval with rounded poles
Shell : smooth and fine, with double line
Colour : yellow to dark brown
Other features : operculum may be seen at one
pole; thickening at the other pole
Contents : a large mass of cells that are
not clearly defined. Cells
appear granulated.

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Not to be mistaken for eggs and larvae


1. Starch granules from plants
Size : 50-100 µm
Shape : round, long oval; but the outline is
always irregular
Shell : thick in places, very irregular, with
cracks
Colour : whitish or greyish yellow; iodine
solution turns it violet
Contents : irregular-shaped masses packed
closely

2. Digested meat fibres

Size : 100-200 µm
Shape : oval or rectangular with rounded
corners
Colour : yellow
Contents : transparent with no granulations;
lines may or may not be present

3. Soaps

Size : 20-100 µm
Shape : round, oval, or irregular
Colour : brownish yellow or colourless
Contents : lines around the edge, pointing
inwards; nothing in the centre

4. Air bubbles
Size : variable, can be any size
Shape : perfectly round
False shell : a circular ring, very shiny (several
rings in the case of oil bubbles)
Contents : none

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5. Plant hairs
Size : very variable, 50-300 µm
Shape : rigid, often curved; clear cut at
one end and tapered at the other
Colour : pale yellow
Contents : a narrow, empty central tube
between two transparent
shiny layers
WARNING : Do not mistake plant hairs for
Strongyloides larvae

6. Pollen grains and fungus spores


Size : very variable
Shape : variable, see pictures

Examination of Segmented Flat Worms - Taeniae


Identification
Colour : white or pale blue
Length : total worm, 3-10 m; single mature
segments, 1-3 cm long; sometimes
fragments of chains are present
IMPORTANT: If there is a delay in examination,
separate segments may dry up and roll around
themselves, looking like roundworms. Wet them
slightly to restore their shape.

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Examination
1. Examine a chain of segments to identify the
arrangement of the pores along the side of
the segments.
2. Examine a single segment gently
flattened between two slides.
3. Hold the slide against the light to count the
number of uterine branches with the naked eye.

4. To examine the head (scolex):


- place the whole worm in a petri dish (or
plate) filled with water
- starting with the thicker end, transfer the
worm little by little into another dish

5. The head is found at the end of a very narrow


section (neck) and looks like a swelling the size of
a small pinhead. Examine this under the 10x
objective.
Note: The head is rarely found.

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Nematode (Round) Worms


Ascaris lumbricoides ( The round worm )

Head of adult to show arrangement


of the three lips Hands carry infective ova
form soil contaminated
with human extreta
Pneumonitis vegetables, dust etc.

s
h
t

n
o
m
2
n
a
m
n
larvae penetrate mucosa, enter
i

e l
c
y lymphatics and venules, to right
c
n heart and lungs break out into
o
i
t
Alveoli, mount twice, ascend
a
r
u
respiratory tree, descend
oesophagus to mature in the
t

a
M
intestine.
Adult life span1 0
-
1
2
m

n
h
s t

a
rs
e

Eosinophilia
y

-
s
n
th
o

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le
b

ia
V
.
s
k
e
e

w
2
-
1
in
re
a
tu

OVA
ADULTS
150 - 200 X 2 - 4 mm.

Narmal form Decorticated Embryonated Unfertilized

60 x45 um 200 - 350 X 4 - 6 mm.

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The Hookworms
Ancylostoma duodenale Necator americanus

Pneumonitis
Old world
New world

Buccal capsule Buccal capsule


2 pairs of teeth Cutting plates

Occult
blood s
y
a
d
5 Eosinophilia
3
Bursa n Anaemia Bursa
a
Dorsal ray, shallow cleft, tips m Dorsal ray, deep cleft, bifid tips
tridigitate i n

n spicules fused and barbed


o
i
t
a
r
u
t
Enter circulation and
60 x 40 µm a
Filarform
M via heart, lungs 70 - 38 µm
Maturation respiratory tree and
in soil besophagus reach
R
7 - 8 days intestine
h
a
b
d
i o
Allergy
t

Ovum f
m
r

Ground etch Ovum


Filariform

Life size
8 - 11 x 0.45 mm. 10 - 13 x 0.6 mm.

250 µm -700 µm

Rhabditiform larva Filariform larva

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Strongyloides stercoralis

Rhabditiform larva
250 x 20 µm

Filariform larva
600 x 20 µm

Larvae mature in
duodenum (or
bronchus ) Fertilised enters mucosa,
lays eggs which hatch to
rhabditiform larvae, these
then make their ways to bowel
lumen
s
y
a
d
Eosinophilia
7
1
n
a
m
i
n
e
g
a
t

s
n Rhabditiform larvae
o
Enter circulation i
t

a
and via heart, lungs, r

u passed in stool Rhabditiform larvae metamorphose


respiratory tree and t

a in bowel to filariform larvae


oesophagus reach M
intestine

Fil

Survive weeks
Fil in soil
Under unfavourable New host Same host Same host
Rhab
F skin Bowel wall
environment
i

l
conditions meta-
marphose to infective h 12 - 24 h h
a

R
R

filariform larvae a b
Free living b
12 - 24 h
mou

lts
Direct cycle Direct cycle Autoinfection Hyperinfection
Indirect cycle

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Trichuris trichiura ( The Whip Worm )

Maturation
in man
3 months

Contaminated
soil, food, etc.
Mainly caecum

Ovum

50 - 22 µm

Life size

35 - 50 mm.

30 - 45 mm.

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Taenia saginata ( The beef tape worm)

Man infected by eating uncooked beef

Life cycle

Cattle Motile seqments rupture

and release eggs


Scolex Ovum
1 - 2 mm. 30 - 40 µm Scolex evaginates in
small intestine, attaches
itself to mucosa of
jejunum.
4 Suckers No hooklets Gravid segment

5 - 10 metres

1000 - 2000 segments

Strobila

Uterus with 15 - 30 lateral branches Maturation time 8 - 10 weeks


16 - 20 x 5 - 7 mm.
Life span up to 25 years

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Cestode ( Tape) Worms


Teania solium ( The pork tape warm)
Usual
Definitive host
Intermediate host ( and reservoir)
Occasional liberated embryo,via bloodstream
Intermediate host to tissue especially muscle.

Life cycle

Ovum

31 - 43 µm
Human cystricercosis Infection with adult

AUTO - INFECTION
Cysticercus is liberated,
scolex evaginates, attaches
itself to mucosa of small
intestine. Develops to adult.
Maturation time 3 months.
Life span up to 25 years.

Development of cysticercus
(Cysticercus cellulosae - 5 x 8 - 10mm.)

Scolex Strobila Progiottid

2 - 8 metres.

1 mm. 800 - 1000


segments

4 suckers - 2 rows of
large and small hooks
25 - 30 7 - 12 uterine
branches on
Section of human brain showing each side.
viable larva of T. solium

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Dwarf Tapeworms
Hymenolepis nana (Vampirolepts nana)

Adult
4 Suckers 20 - 30 hooks
Autoinfection Ova ingested in
in children contaminated food
via hands etc. 40 X 0.5 - 0.9 mm.

Life cycle
200 segments
No intermediate host required
(RODENTS) Segment

Broader than long


Ova passed in faeces Ovum
30 days after infection Natural Poiar filaments
mammalian
host

45 X 35 µm
Liberated embryo penetrates villus
and becomes cysticercoid in 4 days.
Cysticercoid re-enters lumen, attaches
itself to mucosa nd develops into
adult worm in 10 - 12 days

Hymenolepis diminuta (rat tapeworm)


Adult
INTERMEDIATE HOST
HOST No hooks 4 suckers
Accidental ingestion RAT Ingested by

by man of infected 300-600 X 4 mm.


FLEA rodents
insect
Life cycle 800-1000 segments
Cysticercoid liberated,
attaches itself to mucosa,
develops to adults.
Ovum
Segment

Embryo hatched in gut, Resembles H. nana


penetrates intestinal wall, OVUM
Bishnu Dots & lines
develops into cysticercoid
in body cavity

70 X 50 µ
NO polar
filament
(hexacanth
embryo)

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Common Parasitic Protozoa


Intestinal parasites include both helminthes (worms) and protozoa. Protozoa are
single-celled microorganisms, which are generally classified by the way they move:
• Amoebae - cells move slowly with pseudopodia, no flagella or cilia
• Flagellates - with flagella (long extensions)
• Ciliates - with cilia (short hairs)
Parasitic worms are detected in stools as eggs, larvae, or segments of adult forms. In
contrast, protozoa may be found in stools as trophozoites (motile form) or as cysts
(non-motile form). Of the following species, only Trichomonas vaginalis is not found in
stools but in urine and genital discharge.

Common Features Used To Identify Trophozoites Of Intestinal Protozoa

Cytoplasm
(ecto and endoplasm)
Nucleus
Pseudopodium (used for movement)

Ectoplasm

Endoplasm

Vaculoes

Contents:
a) red blood cells
b) bacteria, yeast cells, debris

Nuclear membrane

Nuclear Karyosome (black dot)

Flagellum

Undulating membrane

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Common features used to identify cysts of intestinal protozoa

Cytoplasm

Nuclei

Nuclear membrane
(chromatin)
Regular, thin Irregular,
chromatin rough chromatin

Karyosome
Small, central Large, off-center
Karyosome karyosome

Chromatoid body
(shiny)
Rounded Sharp
chromatoid body chromatoid body

Vacuole (reddish brown)


Fibrils (from flagella)

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Amoebae
1. Entamoeba histolytica (dysentery amoeba)
This amoeba can cause dysentery or intestinal abscesses. It is the only
intestinal amoeba that causes disease in man. E. histolytica can be found in
liquid or diarrhoeal stool.

TROPHOZOITE
• Size :12-35 µm (about as long as 3 to 4 red blood
cells)
• Shape :when moving, irregular and changing;
when not moving, round
• Motility :moves in one direction
• Cytoplasm :
- Ectoplasm = transparent
- Endoplasm = greyish and granular, may have
yellowish-green areas that contain vacuoles
• Nucleus : not seen unless stained with iodine;
nucleus has a single circular
membrane with a black dot in the
centre (karyosome)
• Vacuoles : contains red blood cells

CYST
• Size : 12-15 µm
• Shape : round
• Nuclei : 1-4 nuclei

- membrane : thin, dotted, circular


- karyosome : central black dot
• Cytoplasm : (iodine) yellowish-grey and granular
• Chromatid bodies : sausage-shaped (not always
present)
• Vacuole : sometimes a large vacuole (reddish-
brown in iodine) is present with 1-2
nuclei

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2. Entamoeba coli
TROPHOZOITE
• Size : 20-40 µm
• Shape : oval or long oval, irregularly-shaped
• Motility : often not moving, or moving very
slowly; pseudopodia go in all
directions
• Cytoplasm : ectoplasm and endoplasm look the
same; both are granular
• Inclusion bodies : numerous and varied (bacteria,
yeast cell, debris), but there are
never any red blood cells
• Nucleus : visible without staining;
membrane is irregular and looks like
a beaded necklace, the karyosome
is large and off-centred

CYST

• Size : 12-20 µm
• Shape : round or slightly oval, sometimes
irregular
• Nuclei : 1-8 nuclei
- membrane : irregular, thick in parts like a
beaded necklace
- karyosome : large, not clearly seen, off-centred
• Cytoplasm : (iodine) yellow, brighter than in E.
histolytica
• Chromatid bodies : sharp, knife-shaped or
needle-shaped; not found in all cysts
• Vacuole: sometimes a large vacuole is seen
in between two nuclei

WARNING : Identification of E. histolytica must NOT be confused with E. coli,


a common intestinal amoeba that does NOT cause disease in man.

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Comparison of Entamoeba histolytica and Entamoeba coli
TROPHOZOITES E. histolytica E. coli
Motion in one direction in all directions
Motility motile not usually motile
Ectoplasm transparent; different from little or no difference
endoplasm between the endoplasm
and ectoplasm
Inclusion bodies red blood cells bacteria; yeast cells;
debris; NO red blood cells
Nucleus (fresh state) invisible visible
Nucleus (iodine) regular membrane; small, irregular membrane;
dense, central karyosome large, off-centred
karyosome

CYSTS E. histolytica E. coli


Size 12-15 µm 12-20 µm
Shape round round, slightly oval
Nuclei 1-4; membrane is thin; 1-8; membrane is thick in
may appear dotted parts like a beaded
necklace
Karyosome small, dense, in the centre large, not clearly seen,
off-centred
Cytoplasm (iodine) pale yellow brighter yellow
Chromatid bodies sausage-shaped sharp, knife- or needle-
shaped

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Differences Between Amoebic (Entamoeba histolytica)
and Bacterial (Shigella dysenteriae) Dysentery by Stool
Examination
Amoebic dysentery Bacterial dysentery
Naked eye
pH reaction Acidic alkaline
smell extremely strong usually does not smell
stool always present maybe absent; blood and
mucus only
mucus small amount large amount
Microscopic
Bacteria (1) Numerous may be few
Pus cells (2) few, cells intact very numerous, cells
damaged
Red blood cells (3) often in rolled formation scattered
(rouleaux)
Large macrophages (4) None may be numerous; may
have ingested red cells
(do NOT mistake for
amoebae)
Charcot-Leyden crystals(5) may be present absent
E. histolytica (6) Present absent
Amoebic Dysentery Bacterial Dysentery

6 1 2 1
2 3 4

5 6 3

4
2

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Flagellates
1. Giardia lamblia
TROPHOZOITE
• Size : 10-18 µm
• Shape :
- Front view : tear drop-shaped; numerous
pairs of flagella
- Side view : spoon-shaped
• Motility : moves in one direction, sometimes
turning in a loop; movement is only seen in
fresh liquid stools
• Contents : two large, oval nuclei; slightly visible

CYST
• Size : 8-12 µm
• Shape : oval
• Shell : thick shell with a double wall
• Nuclei : 2-4 oval nuclei
- membrane : very fine
- karyosome : small, central, slightly coloured
• Cytoplasm : clear, shiny when unstained; pale
yellowish-green (iodine)
• Fibril : shiny, looks like hair; can be folded in half
or S-shaped

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2. Trichomonas hominis
T. hominis is the most resistant flagellate, it can
be found as a trophozoite even in old stools.

TROPHOZOITE
• Size : 10-15 µm
• Shape : oval with two pointed poles; usually four
flagella
• Motility : turns in all directions with vibrations
• Undulating membrane : on one side only;
moves in a fast wave-like manner
• Nucleus : one nucleus, difficult to see

3. Trichomonas vaginalis
IMPORTANT: T. vaginalis is found only in urine or
genital discharge specimens.
• Size : 15 µm
• Shape : round; 4 flagella on one end
• Motility : motile in fresh urine, turning motion
• Undulating membrane : on one side only

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Cilates
1. Balantidium coli
B. coli is a very common parasite in pigs. It rarely
causes disease in humans. When in humans, it
is associated with pig farming.

TROPHOZOITE
• Size : very large; 50 µm (similar size to
Ascaris egg)
• Shape : oval; transparent
• Cilia : covered with very small cilia which move
very quickly
• Motility : moves very rapidly in stools; moves in
definite direction; sometimes turning in circles
• Nucleus : large kidney-shaped nucleus next to
a small round nucleus
• Mouth : has a “dent” on one side that is used
for feeding

CYST
• Size : 50-70 µm
• Shape : round
• Shell : thin, double wall
• Nuclei : one large kidney-shaped nucleus, one
small nucleus like a thick dot
• Cytoplasm : granular, greenish, filled with
inclusion bodies

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Malaria : Life Cycle


Four species of protozoan parasites cause malaria:
• Plasmodium falciparum
• Plasmodium vivax
• Plasmodium malariae
• Plasmodium ovale
The malaria parasite has a complex life cycle with different stages in the
Anopheles mosquito and man:

Life cycle of the malaria parasite

(1) During a bite by an infected


mosquito, sporozoites are
injected into the human
bloodstream through the
saliva. (2) Sporozoites enter
liver cells where they mature
over a period of two weeks into
schizonts (3) Some parasites
remain dormant within the liver
as hypnozoites, which are the
cause of relapses of malaria.
In the red blood cells,
merozoites mature into the ring
form (4) finally rupturing to
produce 10,000-40,000
merozoites (5) These enter red
blood cells and begin the
asexual blood stage. (7)
trophozoite (8) and schizont

(9)which complete the cycle by


releasing merozoites for reinfection
of more red blood cells. Some
merozoites
mature into female and male Fig. 1 Life cycle of the malaria parasite
gametocytes within the red
blood cells (11) which can be
taken up by the Anopheles mosquito on feeding. The gametocytes enter the gut
of the mosquito where the male gametocyte exflagellates (12) to form male
microgametes, which fertilise the female gamete to form the zygote (13)The
zygote invades the gut (14) where it develops into an oocyst (15) Inside the
oocyst thousands of sporozoites (16) develop, which are released into the gut
(17) and migrate to the salivary glands ready to re-infect another person.

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Malaria : Clinical diagnosis


• The patient will not show any symptoms for approximately 7 - 12 days after
the original mosquito bite.
• Clinical symptoms include anaemia, enlargement of the spleen, and the classic
cycle of coldness, fever peaks and sweats.
• Malaria can mimic many other diseases, such as gastroenteritis,
pneumonia, meningitis, encephalitis, typhoid or hepatitis.
• Other possible symptoms include lethargy, loss of appetite, nausea,
vomiting, diarrhea, and headaches.
• A patient with malaria usually has a decreased Total Leucocyte Count. Although,
occasionally an increased TLC with a left shift (immature granulocytes seen in
the blood film) may be seen.
• The Differential Count of a patient with malaria will often show an increase in
the number of eosinophils or a decrease in the number of thrombocytes.
There are two species of malaria parasites commonly found in Nepal - Plasmodium
vivax and Plasmodium falciparum. Plasmodium vivax is the most common malaria
parasite in Nepal. It causes an easily treatable form of malaria that is rarely fatal. On
the other hand, Plasmodium falciparum causes a form of malaria that can be rapidly
fatal. Listed below are some of the clinical differences between P. vivax and P.
falciparum forms of malaria:

Plasmodium vivax
• The first symptoms usually occurs from 7-10 days after a mosquito bite.
• Sometimes, symptoms such as headache, sensitivity to light, muscle aches, loss
of appetite, nausea, and vomiting may occur before parasites can be detected in
the bloodstream.
• Sometimes parasites can be found before symptoms occur.
• During the first few days, the patient may not exhibit regular fever cycles but may
have a low grade fever. Once established, fevers recur in a cycle of 48-hours.
• Relapses may occur after weeks, months, or up to 5 or more years.
• P. vivax affects only the reticulocytes (immatue RBCs), therefore the presence
of parasites is usually limited to a maximum of 2% of the available RBC’s.
• Enlargement of the spleen occurs during the first few weeks of the infection, and
the spleen will progress from being soft and palpable to hard.
• A decreased Total Leucocyte Count is usually present, but sometimes an increased
TLC is present during episodes of fever.
• Serum potassium may be increased as a result of RBC lysis.

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Plasmodium falciparum
• Invades all ages of RBC’s and the proportion of infected cells may be between
30 and 40%.
• Onset of an attack occurs from 8-12 days after infection and is preceded by 3-4 days
of vague symptoms such as pains, headache, fatigue, loss of appetite, or nausea.
• The onset is characterised by fever, a more severe headache, and nausea
and vomiting.
• If a fever cycle is established it is less than 48hrs.
• Severe or fatal complications of P.falciparum malaria can occur at any time during
the infection and are related to the plugging of vessels in the internal organs by the
multiplying merozoites (see Fig. 1)
• Disseminated Intravascular Coagulation – vascular endothelial damage from
endotoxins and bound blood cells containing parasites may lead to clot formation
in small vessels.
• Cerebral malaria is most often seen in P. flaciparum malaria. If the onset is
gradual, the patient may become disorientated or violent or may develop severe
headaches and pass into coma.
• Extreme fevers, 107oF (41.7oC) or higher may develop. Without vigorous therapy,
the patient usually dies.
• Remittent fever involves the liver with symptoms including abdominal pain,
nausea, and severe and persistent vomiting containing evidence of bile and fresh
blood. There may be severe diarrhea or dysentery with resulting dehydration. The
liver is large and tender, the skin becomes yellowish, and the urine contains bile.
• Diarrhea or dysentery without liver involvement or yellowing of the skin may be seen.
• Blackwater fever, which is often fatal, can occur in patients with a history of
previous malarial attacks. Sudden, intravascular haemolysis results in a dramatic
colour change in the urine.
• Acute renal failure may also occur.

MALARIA: Keys to laboratory diagnosis


Malaria is life threatening, therefore laboratory requests for blood smear examination
should be treated as URGENT requests! A patient with the diagnosis of P.falciparum
malaria should be considered a medical emergency because the disease can be
rapidly fatal.
For each patient:
• Prepare 1 thin film - for identification of the parasite species, which is important for
the treatment of the disease.
• Prepare 1 thick film - for quick detection and diagnosis of malaria.
• When there is a high number of patients for malaria diagnosis, it is possible to
prepare the thin and thick films on the same slide (see method for thick and thin
films on the same slide).

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• Always prepare blood smears before antimalarial drugs have been given.
• Stain thin and thick films with Giemsa stain. However, Wright’s or Wright-Giemsa
stain can also be used.
• For thin films, 200 – 300 oil immersion fields should be examined before the
blood film is considered negative. NOTE: The presence of a small number of
malaria parasites in the blood is easily missed.
• Additional blood specimens should be examined over a 36 hr time frame, since
one set of negative films will not rule out malaria
• Note the following information which will be very useful in the diagnosis of malaria:
- Has malaria been diagnosed in the patient before? If so, what species
was identified.
- What medication (prophylaxis or other) has the patient received, how often
and when was the last dose?
- When was the blood specimen drawn and was the patient symptomatic at
the time? (E.g. with fever).

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Malaria : Laboratory diagnosis - Preparation of thin films

Making a glass spreader


1. Take a slide with a perfectly smooth edge.
2. Make a diagonal mark across the 2 corners at
one end with a diamond pencil.
3. Snap off the two marked corners.
NOTE: The long edge of a clean glass slide may
also be used as a spreader.

Preparing the slide


• Use only clean grease-free slides.
• If necessary, clean slides with ethanol/ether using
a soft cloth.

Collecting the blood specimen


• Take blood from the side of the 3rd or 4th finger.
• In babies < 6 months, prick the heel or the big toe.
• Blood anticoagulated with dried EDTA
dipotassium salt solution can also be used.
However it is better to use fresh blood for
thin films.

Making the film


1. Collect a drop of blood (size = 3-4 mm) by
touching the finger lightly to one end of the slide.

2. Hold the slide in one hand and angle (45°)


the spreader just in front of the drop of blood.

3. Pull the spreader back until it touches the drop


of blood.

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4. Allow the blood to run along the edge of the


spreader. Note: Never let the blood reach
either edge of the slide (indicated by arrows).

5. Gently push the spreader to the opposite end of


the slide with a smooth and rapid movement. (All
the blood should be used up before you reach
the end of the slide.)

6. A satisfactory film should:


• be completely dry
• be smooth at the end (A) and not damaged (B)
• not have lines running across or down
through the film
• not be too thick or too long
• not contain holes due to grease on the slide

Malaria : Laboratory diagnosis - Preparation of thick films


Making the film
1. Collect a drop of blood (size = diameter of a
pen) by touching the finger lightly to one end of
the slide.
2. Make a thick smear in the centre of the slide
using the corner of a clean slide to spread the
blood drop to an even thickness. Allow the smear
to air dry completely.
NOTE : The correct thickness enables you to see
printed material through the smear, without being
able to read

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Preparation of thin and thick films on a single slide
During the malaria season, there are a large number of patients needing to be
screened for malaria. During this period it is possible to make the thin and thick films
on a single slide as shown below:

1. Collect capillary blood as described above.


Place a small drop of blood (3-4 mm) in the
centre of the slide and a larger drop (diameter
of a pen) 1.5 cm to the right.

2. First, using the small drop of blood, spread the


thin film to the left. Then, using the larger drop of
blood, make the thick smear. Allow the film to air
dry completely, keeping it free from dust or dirt.
The slide should look as shown:

3. Using a swab, place a small drop of absolute


methanol on the thin film. DO NOT let the
methanol touch the thick film. Fix the thin film for
1-2 minutes. The slide is now ready to be
stained with Giemsa.

Malaria : Laboratory diagnosis - Giemsa Stain


Reagents needed
• Giemsa stock solution
Powdered Giemsa stain ................................................................... 0.75 g
Methanol ........................................................................................... 65 ml
Glycerol ............................................................................................ 35 ml

Place the ingredients in a bottle containing glass beads and shake. Shake the
bottle 3 times a day for 4 consecutive days. Filter.
• Measuring cylinders (10, 100 ml)
• Methanol in drop bottle
• Buffered water

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Procedure
1. Mix 1.5 ml Giemsa stock solution in 50 ml of
buffered water (3% solution).

2. Cover thick films and fixed thin film with diluted


Giemsa stain for 30 minutes.

3. Wash off the stain with buffered water. Do NOT


tip off the stain then wash, as this will leave a
deposit of stain over the smear.

4. Drain off the water and place slides in drying rack


with stained sides facing downwards.

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Identification of malaria parasites
Plasmodium falciparum
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infection
• Multiple ring forms
• Double chromatin dot
• ring form lying on red cell membrane surface
• Cytoplasm : small thin pale blue ring
• Chromatin : 1-2 small red dots

MATURE TROPHOZOITE
• Not usually seen

SCHIZONT
• Not usually seen

GAMETOCYTE
• Fairly frequently found in severe infections
• Shape : like a banana
• Colour : reddish-blue (male) or dense blue
(female)
• Nucleus : reddish-pink
• Pigment : a few blue-black granules in the
centre of the cytoplasm or scattered

RED CELLS
• Normal in size

PARASITE DENSITY
• Often high percentage of cells infected
with parasites (30-40%)

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Plasmodium vivax
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infections
• Ring forms are larger than in P. falciparum
• May resemble a perfectly shaped ring
• Cytoplasm: irregular blue ring, quite thick
• Chromatin: 1 rather large dot

MATURE TROPHOZOITE
•· Sometimes seen
• Cytoplasm : large, blue, irregular
(sometimes divided into 2,3, or 4)
• Chromatin : 1 red dot

SCHIZONT
• Fairly frequently found in severe
infection· Merozoites 12-24

GAMETOCYTE
• Fairly frequently found in severe infections
• Female : oval or rounded, dense blue; dense red
triangular nucleus often at one end; many
orange pigment particles in cytoplasm
• Male : rounded, pale blue; round central pale
red nucleus; some orange pigment particles in
cytoplasm

RED CELLS
• Enlarged in size
• Schuffner’s dots

PARASITE DENSITY
• Up to 2% of cells can be infected

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Plasmodium malariae
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infections
• Bird’s eye ring form may be present
• Cytoplasm : thick, dense, blue ring with
some granules of black pigment
• Chromatin : 1 large red dot

MATURE TROPHOZOITE
• Cytoplasm
1. round, compact, dark blue, with many
black pigment particles;
2. in band form
• Chromatin : a round dot or red band

SCHIZONT
• Fairly frequently found in severe infections
• Merozoites 6-12

GAMETOCYTE
• Fairly frequently found in severe infections
• Shape : large, oval or rounded
• Colour : dense blue (female) or pale blue (male)
• Nucleus : 1 round red spot (chromatin) near
one edge
• Pigment : large black granules in cytoplasm

RED CELLS
• Normal size

PARASITE DENSITY
• Rarely more than 1% can be infected.
Therefore, it can be easily missed!

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Plasmodium falciparum

TROPHOZOITES

SCHIZONTS

GAMETOCYTES
Thin film Thick film

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Plasmodium vivax

TROPHOZOITES

SCHIZONTS

GAMETOCYTES
Thin film Thick film

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Plasmodium malariae

TROPHOZOITES

SCHIZONTS

GAMETOCYTES
Thin film Thick film

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Plasmodium ovale

TROPHOZOITES

SCHIZONTS

GAMETOCYTES
Thin film Thick film

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P. falciparum
Diagnostic points
1. Red Cells are not enlarged.
2. Rings appear fine and delicate and there may be several in one cell.
3. Some rings may have two chromatin dots.
4. Presence of marginal or applique forms.
5. It is unusual to see developing forms in peripheral blood films.
6. Gametocytes have a characteristic crescent shape appearance.
However, they do not usually appear in the blood for the first four weeks of infection.
7. Maurer’s dots may be present.

P. vivax
Diagnostic points
1. Red cells containing parasites are usually enlarged.
2. Schuffner’s dots are frequently present in the red cells as shown above.
3. The mature ring forms tend to be large and coarse.
4. Developing forms are frequently present.

P. malariae
Diagnostic points
1. Ring forms may have a squarish appearance.
2. Band forms are a characteristic of this species.
3. Mature schizonts may have a typical daisy head appearance with up to
ten merozoites.
4. Red cells are not enlarged.
5. Chromatin dot may be on the inner surface of the ring.

P. ovale
Diagnostic points
1. Only found in Africa.
2. Red cells enlarged.
3. Comet forms common (top right)
4. Rings large and coarse.
5. Schuffner’s dots, when present, may be prominent.
6. Mature schizonts similar to those of P. malariae but larger and coarser.
(Division of Laboratory Medicine at Royal Perth Hospital, Malaria an On-line Resource)
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Chapter 7
Quality
Assurance in CSF

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Sample collection

Cerebrospinal fluid must be collected by an experienced medical officer. It must be


collected aseptically. The first 1ml should be collected into a sterile tube and
marked No. 1. A further 2-3 ml should be collected into a sterile tube labeled No.2.
Purulent or cloudy C.S.F indicates the presence of pus cells suggestive of
acute bacterial meningitis.
Blood in the C.S.F. This may be due to a traumatic lumbar puncture or less commonly to
haemorrhage into the central nervous system. When it is due to a traumatic lumbar
puncture the second specimen will be less blood stained than no. 1. Following a
subarachnoid haemorrhage, the fluid may appear xanthochromic ie. yellow-red.
Clots in the C.S.F. indicate a high protein concentration with an increase in fibrinogen.
This can occur in bacterial meningitis or when there is spinal constriction.
Cobweb formation may be noticed in Tubercular meningitis

Sample Storage and transportation

All CSF specimens should be considered as urgent. No storage of specimen should


be allowed. Glucose level can drop rapidly due to glycolysis and therefore this test
should be done as soon as possible.
Specimens to be send for culture should be placed in Stuart transport media, as N.
meningitidis, the most commonly found bacteria in CSF is highly fragile and may die
out during transportation.

IQC PROCEDURES

• Always consider CSF as urgent and report the results as soon as possible
• Handle the sample carefully as CSF collection is difficult and quite often cannot be
repeated
• Physical inspection (colour, turbidity) should be performed immediately after
puncture at the patient’s side.

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