MLAB 1415: Hematology WBC and PLT Estimates and Morphology

Laboratory #7

Laboratory: WBC AND PLATELET ESTIMATES AND MORPHOLOGY

Skills= 20pts

Objectives:

1. Identify and grade, within one qualitative unit of the instructor, the white
cell and platelet morphology of each blood smear.
2. Count and calculate the WBC and platelet estimate using a given formula
within 25 minutes per specimen.
3. Perform WBC and PLT estimates within +30% of the automated WBC and
PLT counts
4. Describe the procedure for resolving a specimen exhibiting platelet
satellitism due to EDTA.
5. Calculate the corrected automated platelet count when performed on a
sodium citrate specimen.
6. Describe and appropriately report abnormal WBC morphology and inclusions
7. Describe and appropriately report abnormal platelet morphology.
8. Appropriately report smears with microscopic platelet clumps.

Principle:
Peripheral blood smears are evaluated to determine cell morphology, verify
automated cell counts, and determine the percentage of each type of WBC.
Today’s lab focuses on verifying the automated cell counts for white blood
cells and platelets and documenting abnormal WBC and platelet morphology.

White Blood Cells (WBCs): Adult Reference Range = 4.5-11.0 X 10 3/µL

The automated WBC count is verified by counting the number of WBCs in a
specified number of fields within the examination area of the blood smear
and comparing it to the instrument’s WBC result. A well made blood smear
with proper distribution of WBCs throughout the examination area and
feathered edge is essential for WBC estimation (see Blood Smear
Preparation Lab). A discrepancy between the automated WBC count and the
WBC estimation greater than 30% should be investigated. Occasionally, CLL
(Chronic Lymphocytic Leukemia) patients will produce abnormally small
lymphs that are not counted by the instrument resulting in a falsely low WBC
count. Nucleated red cells can be counted as WBCS yielding a falsely
elevated WBC count. The automated WBC must be corrected when >5 nRBCs
are seen per 100 WBC differential. This calculation will be discussed in the
upcoming “Manual Differential” Lab.

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Any abnormal WBC morphology or inclusions must be documented

appropriately to alert the physician to potential underlying conditions.

*Note*
In conditions such as severe infections, burns, cancer, and toxic drug
administration, DÖhle bodies are seen in conjunction with cytoplasmic
vacuolization and toxic granulation of neutrophils.

Platelets (PLTs): Reference Range = 150-450 X 103/ µL

If the instrument gives an abnormal platelet flag, the platelet count is low,
and/or the specimen is in a pediatric tube, the EDTA sample must be
checked for clots. If visible clots are observed, the specimen is
UNACCEPTABLE and CBC results cannot be resulted. Best practices state
the specimen should be rejected and recollected.

If there are no macroscopic clots, the platelet count is verified by counting

the number of platelets on the blood smear in a specified number of fields
and comparing the manually calculated estimate with the automated result.
Platelet counts are falsely decreased when microscopic platelet clumps are
present.

Platelet satellitism and Na citrate correction

Platelet satellitism refers to the EDTA-anticoagulant induced formation of a
platelet rosette which is characterized by four or more platelets around a
neutrophil or band neutrophil. Platelet satellitism falsely decreases platelet
counts. If the technologist determines that the clumping is due to EDTA, a
Sodium citrate (blue top) specimen can be used for the platelet count only.
In this circumstance, an automated platelet count is run on the Na citrate
tube, and then the platelet result must be multiplied by 1.1. This factor of
1.1 accounts for the dilutional effect of citrate in the Na citrate tube.

Example: Initial EDTA platelet count= 70 X 103/µL. Na citrate

platelet result =180X 103/µL. The reportable platelet count is
calculated by multiplying the sodium citrate result of 180 by 1.1 (180
x 1.1=198); therefore the Reported platelet result = 198 X 103/µL

Specimen:
Peripheral blood smear made from EDTA-anticoagulated blood. Smears
should be made within 4 hours of blood collection from EDTA specimens
stored at room temperature to avoid distortion of cell morphology.
Unstained smears can be stored for indefinite periods in a dry environment,

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but stained smears gradually fade unless coverslipped.

Reagents, supplies, and equipment:

1. Prepared slides
2. Manual cell counter designed for differential counts
3. Microscope
4. Immersion oil
5. Lens paper

WBC estimate Procedure: (Reference: Rodak, et al. Hematology: Clinical Principles and Applications, 4thd ed.)

1. Take the first of two of the instructor-supplied prepared slides.

2. Set a timer for 15 minutes.
3. Focus the microscope on the 10X objective (low power). Scan the smear
to check for cell distribution, clumping, and abnormal cells. In scanning
the smear it is important to note anything unusual or irregular, such as
rouleaux or RBC clumping.
2. Examine the peripheral edge of the smear the smear. The number or
WBCs should NOT exceed 3X the number of cells in the proper
examination area. Large cells such as neutrophils and monocytes can be
pushed to the edges. If this occurs, the distribution of the cells is poor
and the smear is unacceptable.
4. If the smear is acceptable as determined by observation on 10x, change
to the 50x oil objective. Find an area of the smear where ½ the RBCs are
overlapping and ½ are not overlapping.
5. Begin the count in the thin area of the slide and utilize the battlement
track to read the slide as shown below.

BATTLEMENT pattern start

*Note*:
Start counting in the thinnest area available with approximately ½ of the RBCs are
touching and ½ are not and then proceed to the thicker area; however,

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morphology cannot be assessed in areas where all RBCs are overlapping.

5. Estimate the white cell count by counting the number of WBCs in ten (10)
50X fields, and then apply the calculation below.

WBC estimate= Total number of leukocytes counted X 3000 = ? /µL

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Example: You counted a total of 30 WBCs in 10 fields on 50X and

calculated the WBC estimate as follows:

30
10 X 3000= 9 x 103/µL

*Note*:
The estimate should be within ±30% of the actual automated white cell
count and performed within 15 minutes. If the estimate is NOT within
this range, the estimation should be repeated.

6. Record the WBC estimate, with appropriate units, on the report form
provided.

WBC Morphology and Inclusion Evaluation Procedure

1. Change the objective to 100X oil and focus.

2. Select an area of the slide where ½ the RBCs are overlapping and ½
are not overlapping.

3. Evaluate the general appearance of all types of WBCs present.

(Review the Blood Smear Preparation and Staining Lab for
descriptions of appropriately stained WBCs and atlas for normal
granulation seen in granulocytes). When looking at all white cells
evaluate the cytoplasm and chromatin for the following components:

Cytoplasm Nuclear chromatin

Vacuoles Lobation

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Inclusions Nucleoli

Granulation

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4. Evaluate the WBCs in a minimum of ten (10) 100x (hpf) fields.

5. Grade the WBC morphology and inclusions according to the table
found at the end of the procedure portion of this lab. If no inclusions
or WBC morphology is seen, report “normal”.
6. Document the graded WBC morphology/inclusions on the report form
provided.

PLT Estimate Procedure:

1. Set a timer for 10 minutes.

2. Using the 100x oil objective (hpf), place a small drop of oil on the slide and
examine the smear for platelets. Find an area of the smear where ½ the
RBCs are overlapping and ½ are not overlapping. Count the number of
platelets on 5 successive fields, then apply the formula below:

PLT estimate = Total number of platelets counted X 15 = ? X 10 /µL

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Example: You counted a total of 85 platelets in five (5) 100X fields and
calculated the PLT estimate as follows:

85

5 X 15 = 255 X 103/µL

3. Record the calculated platelet estimate, with appropriate units, on the

report form provided.

*Notes*:
 The estimate should be within ±30% of the actual automated platelet count and
should be completed within 10 minutes. If it is not within this range, the platelet
estimation should be repeated.

 An appropriate field on a smear from an individual with a platelet count within

the reference range (150-450 X 103/µL) will have approximately 9-26 platelets
per 100X field (hpf).

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Platelet Morphology Procedure (cont’d):

1. Continuing on 100x oil objective, select an area of the slide where ½

the RBCs are overlapping and ½ are not overlapping.

2. Evaluate the size and appearance of the platelets in a minimum of ten (10)
100X fields.

*SIZE Notes*
 The reference range for the Mean Platelet Volume (MPV) is
6.8-10.2 fL.
 The diameter of a NORMAL platelet is 1.5-3 microns. The diameter
of a RBC is approximately 6-9 microns.
 Platelets that are slightly smaller than a RBC (but larger than normal)
or the same size as a RBC are considered LARGE platelets
 Platelets that are larger than RBCs are considered GIANT platelets

*GRANULARITY Notes*
 Normal platelets do not have a nucleus but have numerous granules.
They look textured and stain light blue to dark blue or purple. Review
your atlas for images of normal platelets.
 Lighter staining, smooth-looking platelets are HYPOGRANULAR
platelets and must be noted.

*Clumping Notes*
 If you observe more than 1 clump of >4 platelets stuck together or 1
large clump, a comment regarding platelet clumping must be noted as
detailed in the chart below. The terms adequate, decreased, and
increased are compared to the reference range of 150-450 X
103/µL.

3. Evaluate a minimum of ten (10) 100X fields and determine if there are
platelet clumps.

4. If the size and granularity of the platelets appear abnormal and/or platelet
clumps are observed, use the Platelet Chart below to determine the
appropriate comment.

5. Document appropriate platelet comments on the report form provided. If

platelet morphology is normal, report “normal” on the report form.

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6. Repeat all steps of all procedures on second slide.

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 MLAB 1415: Hematology WBC and PLT Estimates and Morphology

ABNORMAL PLATELET CHART

Name Description Comment/Action
Lightly stained, smooth, no
Hypogranular platelets Hypogranular platelets present
granularity
Platelets 4-8 microns in diameter
Large Platelets or slightly smaller to the same Large platelets present
size as RBCs. MPV of >10-100 fL
Platelets >9 microns in diameter or
Giant Platelets Giant platelets present
larger than RBCs. MPV >100 fL

>4 platelets surrounding a cell of Do not report platelet count on the EDTA tube. Redraw in Na Citrate for
Platelet Satellitism
neutrophilic origin platelet count only. **Remember to multiply citrate result by 1.1**

Within the
Report: “Platelet count may not be accurate due to clumping. Platelets
reference
appear adequate.” (between 150-450 X 109/L)
range 150-
450 X 109/L
Less than
Report: “Platelet count may not be accurate due to clumping. Platelets
150 X 109/L
Microscopic clumps of platelets appear decreased” (<150 X 109/L)
Platelet
along fibrin strands or sticking
Clumping Report: Platelet count may not be accurate due to clumping. Platelets
Greater than together.
appear increased.” (>450 X 109/L)
450 X 109/L
Report: “Platelet count may not be accurate due to clumping. Platelets
ALL Platelets appear
clumped too clumped for accurate estimate.”

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 MLAB 1415: Hematology WBC and PLT Estimates and Morphology

WBC Morphology and Inclusions

Name Description Grade/Comment/Action

Light gray-blue oval inclusions in the cytoplasm of neutrophils and eosinophils

DÖhle bodies DÖhle bodies present
(composed of aggregates of Rough endoplasmic reticulum)

Large, deep, blue-black primary granules in the cytoplasm of neutrophils, bands

Toxic granulation Toxic granulation present
and metamyelocytes

Neutrophil cytoplasmic
Clear unstained areas often as a result of phagocytosis Cytoplasmic vacuolization present
vacuolization

Degenerating nuclei of granulocytes that appear as condensed, smooth and dark-

Pyknotic nuclei Not reported unless >10% of granulocytes
staining spheres (can be confused with nRBCs)

Smudge cells In the clinical setting an albumin smear should be

Cell whose cytoplasmic membrane has ruptured leaving a bare nucleus made if the WBC smudging is >12-15% of the cells
(Basket cells) present and comment “Smudge cells present”

Neutrophilic
Neutrophil nucleus has >5 lobes Hypersegmented neutrophils present
hypersegmentation

Pelger-Huet (pince nez) Neutrophil with less than 3 lobes “Pelger-Huet or PseudoPelger-Huet cells present”
Count cells as either segs/bands
Alder-Reilly Large, dark cytoplasmic granules in ALL types of leukocytes Pathology review for Alder-Reilly anomaly

Reddish-blue staining needle-like inclusion within the cytoplasm of leukemic Auer Rods present (cells with Auer Rods count as
Auer Rod
myeloblasts as a result of abnormal granule formation “Others” because they are blasts)

Chediak-Higashi Giant fused granules in neutrophils and lymphs Pathology review for Chediak Higashi syndrome

May-Hegglin Blue DÖhle-like cytoplasmic inclusions in ALL granulocytes Pathology review for May-Hegglin anomaly

Small dark blue-purple round or rod-like inclusions within the cytoplasm of

Intracellular bacteria Intracellular bacteria present
phagocytes (monocytes and neutrophils)

Intracellular morulae Dark blue, large inclusions that are microcolonies of Ehrlichia found in leukocytes Pathology review for morulae

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MLAB 1415: Hematology WBC and PLT Estimates and Morphology

Name:_______________
Date:________________

Laboratory: WBC/PLT Estimates and Morphology Grading Report Form

Skills= 20 Pts.

WBC Estimate PLT Estimate

Slide number/Patient Platelet morphology WBC morphology
Calculation and result Calculation and result
Name and ID comments comments
(show your work and units) (show your work and units)

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MLAB 1415: Hematology WBC and PLT Estimates
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Name:_______________________

Date:________________________

Laboratory: WBC AND PLATELET ESTIMATES AND MORPHOLOGY

Study Questions
20pts
Each question is worth one point, unless otherwise stated.

1. What is the adult reference range for a WBC count?

2. What is the reference range for a Platelet count?

3. What objective is used when counting platelets for the platelet estimate?

4. Platelets surrounding many neutrophils were observed on a patient smear made from a purple top
specimen. What tube should be collected?

5. A technician is performing a WBC estimate on 50X oil. She counts 115 WBCs in 10 fields. What is the
WBC estimate for the patient? (2pts)

6. A platelet count of 225 X 103/µL is obtained from a Sodium (Na) Citrate tube. What result would be
reported for the platelet count?

7. What is the calculation used in determining the platelet estimate?

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MLAB 1415: Hematology WBC and PLT Estimates
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8. Name the inclusion that is comprised of remnants of rough endoplasmic reticulum.

9. Name three (3) conditions associated with the triad of Dohle bodies, toxic granulation, and
vacuolization. (3pts)

10. Hyposegmented neutrophils are called ______________________ cells.

11. We evaluate the cytoplasm of WBCs based on what 3 components? (3pts)

12. How do you report platelets that are larger than a RBC with an MCV of 100 fL?

13. How many fields are counted for a WBC estimate performed on 50X?

14. How many fields are evaluated for the detection of platelet abnormalities?

15. You perform a smear review and note that there are large clumps of platelets in each field. You can
tell that there are 30-36 platelets per 100x field. What comment would you report to the physician?

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