MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and MorphologyLaboratory #8: WBC AND PLATELET ESTIMATES AND

MORPHOLOGYSkills= 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

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and Morphologythe upcoming “Manual Differential” Lab.

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 10 3 / 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 correctionPlatelet 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 10 3 /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

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and Morphologydry environment, but stained smears gradually fade unless coverslipped.

Reagents, supplies, and equipment:

1. Prepared slides2. Manual cell counter designed for differential counts3. Microscope4. 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

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and MorphologyRBCs are touching and ½ are not and then proceed to the thicker area; however, 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

10

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 10 3 /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: Vacuoles Inclusions

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and Morphology Granulation

Nuclear chromatin: Lobation Nucleoli

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.6. Document the graded WBC morphology/inclusions on the report

form provided.

P LT 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 3 /µL 5

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 10 3 /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.

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and Morphology 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).

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.

MLAB 1415: Hematology Laboratory #8

Laboratory #7

WBC and PLT Estimates and Morphology5. Document appropriate platelet comments on the report form

provided.

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

MLAB 1415: Hematology Laboratory #8

WBC and PLT Estimates and Morphology

ABNORMAL PLATELET CHARTName Description Comment/Action

Hypogranular plateletsLightly stained, smooth, no

granularityHypogranular platelets present

Large Platelets Platelets 4-8 microns in diameter or slightly smaller to the same

size as RBCs. MPV of >10-100 fLLarge platelets present

Giant PlateletsPlatelets >9 microns in diameter or larger than RBCs. MPV >100 fL

Giant platelets present

Platelet Satellitism>4 platelets surrounding a cell of

neutrophilic originDo not report platelet count on the EDTA tube. Redraw in Na Citrate for

platelet count only. **Remember to multiply citrate result by 1.1**

Platelet Clumping

Within the reference

range 150-450 X 109/L

Microscopic clumps of platelets along fibrin strands or sticking

together.

Report: “Platelet count may not be accurate due to clumping. Platelets appear adequate.” (between 150-450 X 109/L)

Less than 150 X 109/L

Report: “Platelet count may not be accurate due to clumping. Platelets appear decreased” (<150 X 109/L)

Greater than 450 X 109/L

Report: Platelet count may not be accurate due to clumping. Platelets appear increased.” (>450 X 109/L)

ALL Platelets clumped

Report: “Platelet count may not be accurate due to clumping. Platelets appear

too clumped for accurate estimate.”

MLAB 1415: Hematology Laboratory #8

WBC and PLT Estimates and Morphology

WBC Morphology and Inclusions

Name Description Grade/Comment/Action

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

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

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

and metamyelocytesToxic granulation present

Neutrophil cytoplasmic vacuolization

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

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

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

Smudge cells

(Basket cells)Cell whose cytoplasmic membrane has ruptured leaving a bare nucleus

In the clinical setting an albumin smear should be made if the WBC smudging is >12-15% of the cells

present and comment “Smudge cells present”

Neutrophilic hypersegmentation

Neutrophil nucleus has >5 lobes Hypersegmented neutrophils present

Pelger-Huet (pince nez) Neutrophil with less than 3 lobes “Pelger-Huet or PseudoPelger-Huet cells present”

Alder-Reilly Large, dark cytoplasmic granules in ALL types of leukocytes Pathology review for Alder-Reilly anomaly

Auer RodReddish-blue staining needle-like inclusion within the cytoplasm of leukemic

myeloblasts as a result of abnormal granule formationAuer Rods present (cells with Auer Rods count as

“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

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

phagocytes (monocytes and neutrophils)Intracellular bacteria present

MLAB 1415: Hematology Laboratory #8

WBC and PLT Estimates and MorphologyIntracellular morulae Dark blue, large inclusions that are microcolonies of Ehrlichia found in leukocytes Pathology review for morulae

MLAB 1415: Hematology Laboratory #8

WBC and PLT Estimates and MorphologyName:_______________Date:________________

Laboratory #8: WBC/PLT Estimates and Morphology Grading Report Form

Skills= 20 Pts.

Slide number/Patient Name and ID

WBC Estimate

Calculation and result

(show your work and units)

PLT Estimate

Calculation and result

(show your work and units)

Platelet morphology comments

WBC morphology comments

MLAB 1415: Hematology

Laboratory #8

WBC and PLT Estimates and Morphology

Name:_______________________

Date:________________________

Laboratory #8: WBC AND PLATELET ESTIMATES AND MORPHOLOGY

Study Questions20pts

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?

8. Name the inclusion that is comprised of remnants of rough endoplasmic reticulum.

MLAB 1415: Hematology

Laboratory #8

WBC and PLT Estimates and Morphology

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?