THE AGA KHAN HOSPITAL KISUMU.

PATHOLOGY DEPARTMENT

AKHK SOP NO.:

CRITICAL VALUE REPORTING- AKHK Kenya
EMT 903
Version: 00

Prepared by: Patience Kache Verified by: Milton Isanya

Date: Date:
Reviewed by: Jacob Seroni Approval authority: Dr. B. V. S. Prasad
Date: Date:
Next Review Date:
Effective Date:

DOCUMENT CHANGE RECORD

Date Rev. Section Description/ Reason for Change

1.0. PURPOSE AND APPLICABILITY

1.1.0. To outline the procedure of performing differential count on a PBF
2.0. SUMMARY
Examination of the well-prepared and well-stained blood smear is one of the most
efficient and important diagnostic screening tests used in the evaluation and detection of
abnormal blood counts. Equally important is the blood morphology, which can often
reveal important diagnostic or therapy-related information. Therefore, the manual
differential white blood cell count is performed to determine the relative number of each
type of white blood cell present in the blood. At the same time, a study of red blood cell,
white blood cell, and platelet morphology and maturity is performed. This includes an
approximation of the platelets count.

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3.0. DEFINATIONS AND ABREVIATIONS
4.0. EQUIPMENT/TOOLS/SPECIMEN
4.1.0. A proper wedge peripheral blood smears stained in accordance with the
Wright Stain (a Romanowsky type blood stain) manual or automated
methods.
4.2.0. Microscope
Low power 10X objective and 100X oil immersion objectives
4.3.0. Wright stained peripheral smear with identification label (attached to the
smear side only)
4.4.0. Cell differential counter
4.5.0. labeled automated CBC instrument report with histograms
4.6.0. Immersion Oil

5.0. RESPONSIBILITIES
6.0. PROCEDURE

6.1.0. Quality Control:

6.1.1. The following steps must be taken during differential testing to ensure
uniform reporting within the group:
Agree upon the definition of all terms used.
Circulate unfamiliar slides among the group members and compare
results.
Establish a required level of accuracy for new laboratory personnel and
assess competency before performing patient differential count testing.
Monitor uniformity standards through scheduled competency assessment.
Establish criteria for peripheral smear review by the department supervisor
and pathologist

6.2.0. Reporting:
6.2.1. Verify that the automated CBC report and blood smear identification
match.
6.2.2. Place the stained slide (specimen side up) on the microscope stage.
The 10X low power objective. Examine the feathered edge (figure 1) of
the smear to determine blood film adequacy.
The edges of even the best-prepared blood smear have accumulations of
leukocytes. However, if there are an increased number of leukocytes in
this area, the differential count may be inaccurate. Incorrectly prepared
films may have too many large cells at the film edges, leaving relatively
smaller cells, such as lymphocytes, in the center and results in inaccurate
manual differential counts. Evaluate the number of white cells per 100X
at the feathered edge, if it exceeds 2-3 times the number per field in the
body of the film, another blood smear must be prepared and evaluated.

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 If platelet clumps are identified in this area, the automated platelet count
will be falsely decreased. Do not perform a platelet estimate.
Follow the procedure for platelet clumping as identified later.

6.2.3. Review the area where the red cells are evenly distributed and begin to
overlap. Scan for abnormalities such as platelet clumping, rouleaux,
agglutination or abnormally large leukocytes. If platelet clumping,
rouleaux or agglutination is identified, refer to platelate clumping in this
SOP for the appropriate corrective action. Review all abnormally large
leukocytes under 100X for appropriate classification and follow up testing.

6.2.4. Select the best area for detailed morphological evaluation and differential
count. The erythrocytes will be evenly distributed and not distorted. The
field will also be devoid of broken areas caused by improper smear
preparation.

6.3.0. MANUAL DIFFERENTIAL:

6.3.1. Perform the 100 cell differential count.
Select the optimal counting area. This is the area where the erythrocytes
are adjacent but do not overlap. The 40x objected may be used to perform
the differential count but the 100x objective must be utilized to classify
any immature cells to include myelocytic cells more immature than the
segmented neutrophil.
6.3.2. Use a differential cell counter, count and classify 100 white blood cells
using the cross-sectional technique where the white cells are counted in
consecutive fields as the blood film is moved from side to side as pictured

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 6.3.3. Classify the cells as follows: segmented neutrophil, banded neutrophil,
metamyelocyte, myelocyte, eosinophil, basophil, lymphocyte, or
monocyte.

Note:
If blasts or promyelocytes are identified on the peripheral smear, do not
certify the manual differential until it can be verified by the pathologist.
If the automated WBC count is less than 2.0 K/uL, a 50 cell count can be
performed and converted to 100% by multiplying all values by 2. Add the
following comment on the report 50 cells counted during the manual
differential and converted to percentage.
If the automated WBC count is greater than 30.0 K/uL, a 200 cell count
must be performed and converted to 100% by dividing all values by 2.
Round accordingly and report only whole numbers. Add the following
comment on the report 200 cells counted during the manual differential
and converted to percentage.
If nucleated red blood cells (NRBC) or Megakaryocytes are seen during
the differential count, enumerate them separately from the white blood cell
count.

6.3.4. If the manual differential count does not correlate to within 10% (10 cell
difference) of the automated differential count for any one cell class,
review the slide for accurate wedge smear preparation. If the slide quality
is questionable, prepare, stain and recount the differential on the new slide.
If the slide is acceptable, repeat the count. If the recount is out , proceed as
follows:
Enter the manual differential count on the Patient report.
Calculate absolute cell counts using the manual differential.
Add each cell class to obtain a total number for that class, i.e.
Lymphocytes + Atypical Lymphocytes = Total number of Lymphocytes,
Neutrophils + Bands + Meta’s, etc = Total number of Neutrophils.
Convert the total percentage to decimal, i.e. 45% lymphs = 0.45.
Multipy the decimal by the total white count, i.e., 10.0 K/uL WBC x 0.45
Lymphs = 4.5 K/uL Lymphocytes.

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 Add the totals from each cell class calculation and they will equal the total
WBC count. If not, the calculations were performed incorrectly and must
be repeated.

6.3.5. Enter the manually calculated absolute counts from step above. in place of
the automated absolute count fields on patient result with the following
comments: Absolute counts calculated based on manual differential.

6.3.5 If the CBC was certified, do not amend the results. Result the manual
differential

6.4.0. RBC Morphology and WBC Review

The RBC Morphology and WBC Review will be performed with every
manual differential. The test will be performed using the high power
(100X) oil immersion objective. Begin the examination in an area where
the red cells are touching but not overlapping. There are approximately
300 RBC’s present in this area in patients with a normal hematocrit.
Locate a mature (normal and not atypical) lymphocyte for comparative
purposes and review the smear for the following:

6.4.1. Red Blood Cell Size – Normal erythrocytes are 6 to 8 micron in diameter
or approximately the same size as the nucleus of a normal lymphocyte.
The cells are classified and reported as follows:
Normocytic - normal size
Microcytic - smaller than normal
Macrocytic - larger than normal

6.4.2. Review a minimum of 10 fields, using the 100X oil immersion objective,
and compare the RBC size to that of a normal lymphocyte. Tally the
number of each classification per field and report as follows:

1+ 2+ 3+
Occasional 5-25% Moderate 25-50% Many >50%
10 to 50 cells 51 to 100 cells >100 cells
Normocytic cells are not graded.
Report Normocytic if microcytosis and/or macrocytosis are not present
or they are present at less than 10 cells per field.

6.4.3. Anisocytosis - Variation in cell size is measured using the Red Cell
Distribution Width (RDW) and is graded during the RBC Morphology
Examination. Grade the variation using the guide listed below:

1+ 2+ 3+
Occasional Moderate Many
RDW 15.0 to 17.9 18.0 to 20.9 >21

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 6.4.4. Red Blood Cell Color - Normal RBC’s are red to buff pink with more
color intensity at the marginal areas than in the central portion when
stained with Wrights Stain. Cells are classified as follows:
Normochromic - normal color
Hypochromic - pale color and large central pallor
Polychromasia – bluish color and usually macrocytic

Review the RBC color in a minimum of 10 fields using the 100X oil
immersion objective. Tally the number of each classification per field and
report as follows:

Hypochromia
1+ 2+ 3+
Occasional 5-25% Moderate 25-50% Many >50%
10 to 50 cells 51 to 100 cells >100 cells

Polychromasia
1+ 2+ 3+
Occasional 1% Moderate 1 – 2.5% Many >2.5%
<1 to 2 cells 2 to 5 cells >5 cells

Normochromic cells are not graded.

Report Normochromic if Hypochromia is not present or it is present at less
than 10 cells per field. Polychromasia can be reported with both
normochromic and Hypochromic reports.

6.4.5. Red Blood Cell Shape – Normal RBC’s are round with an area of central
pallor that occupies one-third of the cell. Poikilocytosis is the term used to
describe variation in cell shape. Grade poikilocytosis using the following
cell shape classifications identified below.
Acanthocytes
Bite Cells
Blister Cells
Codocytes – Target Cells
Dacryocytes – Teardrops
Drepanocytes – Sickle Cells
Echinocytes - Crenated or Burr Cells
Elliptocytes or Ovalocytes
Envelope forms or folded cells
Schistocytes
Spherocytes
Stomatocytes
Review the RBC shape in a minimum of 10 fields using the 100X oil
immersion objective. Tally the number of each classification per field and
report as follows:

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 Codocytes (Target Cells), Echinocytes (Burr or Crenated),
Elliptocytes (Ovalocytes) & Stomatocytes
1+ 2+ 3+
Occasional 5-25% Moderate 25-50% Many >50%
10 to 50 cells 51 to 100 cells >100 cells

Acanthocytes, Bite Cells, Blister Cells, Dacryocytes (Teardrops),

Drepaocytes (Sickle Cells), Envelope forms or Folded Cells,
Schistocytes, & Spherocytes
1+ 2+ 3+
Occasional 1% Moderate 1 – 2.5% Many >2.5%
<1 to 2 cells 2 to 5 cells >5 cells

If none of the shapes identified above are present or they are present at
less than the minimum criteria, grade and report poikilocytosis as 1+
occasional, 2+ Moderate, 3+ Many.

6.4.6. Red Blood Cell Inclusions – Various RBC inclusions can occur for a
variety of reasons. Scan a minimum of 10 fields using the 100X oil
immersion objective. Tally the number of each inclusion classification per
field and report as follows:
Basophilic Stippling
Howell Jolly Bodies
Pappenheimer Bodies
Cabot Rings
Hemoglobin C Crystals
Bacteria
Parasites
Mauer’s Dots
Schüffner’s Dots
Nucleus – Refer to paragraph
for procedure.
Artifacts
Review the RBC shape in a minimum of 10 fields using the 100X oil
immersion objective. Refer to appendix 3 for a brief summary of the
various inclusion appearances, using Wright Giemsa Stain, and the
associated clinical conditions. Tally the number of each classification per
field and report as follows:

RBC Inclusions
1+ 2+ 3+
Occasional 1% Moderate 1 – 2.5% Many >2.5%
<1 to 2 cells 2 to 5 cells >5 cells

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 Bacteria, parasite, Mauer’s Dots, and Schuffner’s Dots are critical
findings. If any of these inclusions are present, THEY REQUIRE
IMMEDIATE PATHOLOGIST CONFIRMATION before they can be
reported. Notify the pathologist immediately to arrange verification and
provider notification.

6.4.7. White Blood Cell Color

CELL NUCLEUS COLOR, CYTOPLASMIC

TYPE TEXTURE, & SHAPE COLOR
& SIZE
Two to five nuclear lobes Pinkish tan to light lilac;
Segmented connected by filaments. specific non-visible
Neutrophil Purple chromatin coarsely neutrophil granules; may
10-15 m clumped with randomly contain few red-purple
distributed open areas. primary granules.
Similar to mature neutrophil
Banded except nuclear shape is a non- Same as above but may be
Neutrophil segmented continuous band of more basophilic with more
10-18 m chromatin that may contain primary granules.
more open areas.
Eosinophil Similar to mature neutrophil Numerous bright orange
10-15 m except the nucleus is bi-lobed. spherical granules
Large purple-black
Basophil Similar to mature neutrophil. granules that may obscure
10-15 m the nucleus

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 CELL NUCLEUS COLOR,
TYPE TEXTURE, & SHAPE
& SIZE
Mononuclear with a high N/C
ratio. Nucleus is oval, round,
or slightly indented.
Lymphocyte Chromatin is diffusely dense
or coarse with clumpy masses.
7-15 m
Nucleoli are non-visible. A
small pale chromocenter may
be present.
Irregularly shaped or unusual
Atypical chromatin pattern. Nucleus
Lymphocyte may be stretched like a band
10-25 m across the cell. The nucleus
may have visible nucleoli.
Round to ovoid, usually
Plasma Cell eccentric; may be binucleated;
10-20 m rarely multinucleated.
Chromatin is coarse and
clumped with dense masses
arranged in a wheel-like
pattern. Low N/C ratio.
Mononuclear; usually
Monocyte convoluted or horseshoe-
12-20 m shaped; evenly distributed
pale and delicate chromatin.
CYTOPLASMIC
COLOR
Pale or medium blue,
translucent, and may
contain several azurophilic
granules
Abundant cytoplasm with
vacuoles; radial and
peripheral basophilia;
increased number of
azurophilic granules.
Medium to deep blue and
occasionally flame color
with a prominent peri-
nuclear clear area or hof;
agranular; may have
vacuoles of varying size.
Usually abundant, dull
gray- blue; vacuolated;
sparse lilac granules

6.4.8. Neutrophilic Inclusions – Various neutrophil inclusions can occur for a

variety of reasons. Scan a minimum of 10 fields using the 100X oil
immersion objective. Tally the number of each inclusion classification per
field and report as follows:
Döhle Bodies
Toxic Granulation
Alder’s Anomaly
Chédiak-Higashi Anomaly
Auer bodies
Vacuoles
Phagocytized material
Review the Neutrophils in a minimum of 10 fields using the 100X oil
immersion objective. Refer to appendix 4 for a brief summary of the
various inclusion appearances and morphology, using Wright Giemsa
Stain, and the hematopoietic cells involved. Tally the number of each
classification per field and report as follows:

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 Neutrophilic Inclusions
1+ 2+ 3+
Occasional 1% Moderate 1 – 2.5% Many >2.5%
<1 to 2 cells 2 to 5 cells >5 cells

6.4.9. Dysplastic Features– The following list of abnormalities can occur in the
following cell types:
Granulocytes:
Agranular
Abnormally large granules
Hyposegmentation (bi-lobed, pince-nez or mononuclear)
Hypersegmentation (≥6 lobes)
Monocytoid change
Monocytes
Granulocytic change
Hyperlobulation
If any of the dsyplastic features are present, tally the number of each
classification per field and report.
Provide the number and type of cells as a percentage of 100 cells. For
example, if 4 hypersegmented neutrophils are identified during the 100
cell differential count, report “4% Hypersegmented neutrophils present”
and include the 4 cells in the neutrophil count for the 100 cell differential
cell count. Repeat for each dysplastic feature noted above.

6.5.0. Spurious result identification – Engage the 40X oil immersion objective
and review the slide for the any of the abnormalities identified below.
6.5.1. Agglutination – This occurs as cells aggregate into random clusters or
masses when exposed to various red cell antibodies. It can be observed in
certain hemolytic anemias, atypical pneumonia, staphylococcal infections
and trypanosomiasis..
6.5.2. Rouleaux Formation – This is observed in the normal examination area.
The erythrocytes appear in short or long stacks resembling coins or flat
plates. Rouleaux occurs as a result of abnormal RBC arrangement due to
biconcave surface opposition. It is commonly seen in multiple myeloma
and hyperproteinemia. Review the hemogram indicies for rule of three
acceptability. If the indicies are within range, rouleaux formation can be
reported as present. If they are not appropriate corrective action must.
6.5.3. Hemolysis – Mild hemolysis is frequently undetected in most CBC tests.
Severe hemolysis usually manifests with increased platelet counts that
cannot be confirmed by the peripheral smear examination. Reject
hemolyzed samples and refer to the Spurious Results SOP for further
guidance.
6.5.4. If there is considerate difference between the RBC indicies and blood
smear presentation, prepare and evaluate a new blood smear.

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 6.6.0. Platelet Estimation
6.6.1. Perform a platelet estimate on any differential or RBC Morphology review
smears. This is done to correlate the automated platelet count with the
estimate and check the smear for giant platelets, clumps or platelet
satellitism.
To do this, locate an area of approximately 150 red blood cells. The cells
should be just touching or barely overlapping.
Count all the platelets in that area.
Follow the above step until 10 fields have been counted. Keep the total
number of all 10 areas counted. Divide the total number by 10 to obtain
the average platelet per field, then multiply by (x) 20,000 to obtain the
platelet estimate. Refer to Calculation Section of this SOP for example.
If the platelet count from the instrument does not correlate with the
platelet estimate on the smear; the instrument count, the smear, and the
platelet estimate process will be repeated.
Occasionally platelets will be satellite around the neutrophils, giving a
halo or a platelet clumping appearance. This is due to thrombocytopenia
or micro fibrin clotting. If this occurs follow the steps below:
Values greater than 50 x 10/L: Document that "Platelet count inaccurate
due to platelet clumping or platelet satellitism present".
Notify the Ward/OPD, Clinic or Physician that the platelet count is
inaccurate. Request new EDTA and Citrate tubes for evaluation
Document that "Platelet count inaccurate due to platelet clumping or
platelet satellitism present. Another specimens requested for evaluation".

6.6.2. If a significant numbers of giant platelets and/or platelet clumps are

detected, a peripheral estimate of WBCs should be done to prevent
reporting spuriously high white blood cell count.
Do a manual platelet estimate count if a significant numbers of Microcytic
red blood cells and/or small cell fragments are detected during the RBC
morphology.

Platelets Between 2-3 m in Cytoplasmic fragments

diameter with red-purple granules
and no nucleus
Megakaryocytes Between 3-6 m in Usually round, containing
diameter multiple red-purple
granules

6.7.0. Calculations:
6.7.1. WBC Correction for NRBC or Megakaryocyte presence - If 10 or more
NRBCs or Megakaryocytes are present use the below formula:
WBC count X 100
= Corrected WBC Count
# NRBCs or Megakaryocytes + 100

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 example:
20 X 100
WBC count = 20 X 10/L = 18.2 X 10/L Corrected WBC
NRBCs = 10 10 + 100

Document that the WBC count was corrected due to NRBCs or

Megakaryocytes present.

6.7.1. To perform platelet estimation, follow the below example:

Platelet estimate = Average of platelets in 10 field counted X 20,000

For example: Total of 10 field counted = 100 platelets

Average platelet count = 100 / 10 = 10

Platelet estimate = 10 X 20,000 = 200 X 10/L

Peripheral estimation of the WBCs should be done to check the accuracy

of the actual count. To do this, follow the below steps:
This estimate is done on 100X oil immersion where the red blood cells are
just beginning to touch each other.
Count the number of WBCs in ten (10) fields of a stained smear. Take the
average of this count and use the following table:

6.8.0. Interpretation & Reporting Results:

After the 100 cells count, RBC morphology and platelet estimation is
completed, reviews the results and report.

Note:
Pyknotic cells: The nuclear chromatin condenses into a solid,
structureless mass. THEY ARE NOT COUNTED IN THE
DIFFERENTIAL. They are usually found in old blood i.e., from outlying
clinics. Outlying clinics are required to submit smears they have prepared
from fresh specimens when requesting CBCs through this laboratory.

Smudge or Basket cells: Cells that are obviously white blood cells in
origin, resulting in a loss of characteristic shapes and forms thereby
precluding identification of the cells. An increased number of these cells
are seen in lymphocytic leukemia. If necessary prepare a new blood film
using a 1:5 ratio of 22% albumin to whole blood. This will markedly
reduce smudge formation and aid in diagnosis. Smudge or basket cells are
reported as Present.
Refer any unusual smear to the supervisor and/or the pathologist.
Peripheral smears will be kept for seven (7) days to assure further .

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 verifications of the smear if requested by the physician. Abnormals kept
longer

6.9.0. Sources of error:

Certain cell types, particularly eosinophils and Basophils, may be
distributed in a nonrandom manner on a blood film. High percentages of
these cell types, especially if found in a limited area of the film, should be
rechecked before the numbers are reported.

6.10.0. Limitation of the procedure:

Although morphologic evaluation of a stained blood film is one of the
most important hematologic procedures, the manual differentiation of
leukocytes is an inaccurate and un-reproducible method.
It is subject to errors that cannot be eliminated such as sampling errors,
uncontrollable errors in cell distribution on the blood film, and human
inconsistency in cell interpretation.

7.0. REFERENCES:
7.1.0. Reference Leukocyte Differential Count, NCCLS Document H20-A, Vol.
12 No. 1, March 1992.
7.2.0. Brown, Barbara A., Hematology: Principles and Procedures, Lea and
Febiger Book Publisher, Sixth Edition, 1993, Pages 102 to 105.
7.3.0. Lee, Richard G., Windrobe’s Clinical Hematology; Lea and Febiger Book
Publisher, Ninth Edition, 1993, Pages 223 to 238.
7.4.0. Hoffman, Ronald, Hematology: Basic Principles and Practice, Second
Edition, Churchill Livingston Inc., 1995, Pages 308 to 312.

8.0. DISTRIBUTION LIST

Section/Area No. issued Date Issued Sign.

Quality Management
Front office/Dispatch
Specimen Management
Hematology Section
Clinical Chemistry
Blood Bank
Microbiology
Histology & Cytology

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9.0. TRAINING LOG

Document read by Signature Date

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