Hematology: Normal Differentials/ RBC Morphology

CLS 312/322

Specific Laboratory Objectives:

At the completion of this laboratory exercise, the student will be able to:

1. Perform 10 normal differentials according to the procedure provided.

2. Correctly recognize criteria for performing a 200 cell differential.

3. Make the necessary corrections for the presence of nucleated RBC's.

4. Calculate MCV, MCH, and MCHC and evaluate results.

5. Recognize abnormal WBC morphology.

6. Accurately determine WBC estimates and Platelet estimates.

Specific Lecture Objectives

Upon completion of the lecture on smear examination the student will be able to:

1. Estimate the total WBC count based upon the number of WBC's seen per low power field.

2. Evaluate the distribution of cells seen on the smear to determine if a representative differential may
be performed.

3. Select the proper area of the slide for performing a differential.

4. Identify the following cell types and state their function and normal peripheral blood values:

a. Neutrophil
b. Basophil
c. Eosinophil
d. Polychromatophilic erythrocyte
e. Platelet
f. Monocyte
g. Lymphocyte

5. Define and explain the significance of each of the following terms:

a. Normocytic Red Cell n. Basophilic Stippling

b. Microcytic Red Cell o. Howell Jolly Body
c. Macrocytic Red Cell p. Siderotic Granules
d. Anisocytosis q. Acanthocyte
e. Normochromic Red Cell r. Reticulocyte
f. Hypochromia s. NRBC
g. Poikilocyte t. Agglutination
h. Ovalocyte u. Rouleaux
i. Sickle Cell v. Crenated Red Cell
j. Schistocyte w. Target Cell
k. Helmet Cell
l. Spherocyte
m. Stomatocyte

6. List and explain how to prevent 3 conditions which would lead to artifactual changes in red cells
resulting in crenation.

7. List and explain how to prevent 3 conditions which would lead to artifactual changes in red cells
resulting in schistocytes.
NORMAL DIFFERENTIALS

Procedure

1. Estimate WBC Count

- Use the low power objective of the microscope. (20x)

- Look at 5-6 fields and determine the average number of WBC's per field
- divide the average # by 4 and multiply by 1000 to get your estimated WBC count
NOTE: For student microscopes with 10x objectives, divide the above average by 8 instead of 4
EXAMPLE: WBC's Seen: 5 4 6 4 5
Average: 4.8
Calculation: (4.8/4) X 1000 = 1,200 WBC

2. Categorize 100 white cells

- In the feathered edge, where the RBC's lay side by side (just touching one another), count 100
white blood cells.

3. Estimate the Platelet Count

- Use the oil immersion lens estimate the number of platelets per field
- Look at 5-6 fields and take an average
- Multiply the average by 20,000
- Note any macroplatelets

4. Evaluate WBC Morphology

Note if any abnormal white cell morphology is present

- Hypersegmented poly's (5 or more lobes)

- Vacuolation of neutrophils
- Toxic granulation of neutrophils
- Dohle bodies
- Atypical Lymphocytes
- Smudge cells

5. Make sure cells add up to 100

6. If 10 or more nucleated RBC's (NRBC) are seen, correct the

white count using this formula:

WBC x 100 = Corrected WBC count

NRBC + 100

7. Count 200 WBC's if:

A. WBC count is > 15,000/mm3

B. 3 or more Basophils are seen
Tips on Diff's

a. Do not count cells that are disintegrating

- smudge cells
- eosinophil with no cytoplasmic membrane and with scattered granules
- Pyknotic cell (nucleus extremely condensed and degenerated, lobes condensed
into small, round clumps with no filaments interconnecting).
- Basket cells

b. If a cell does not fit into a category or you are unsure of what the cells is, ask someone to look at it.
(may be abnormal or immature)

c. Make sure to count in an area where cells are distributed evenly and the central pallor of RBC's is
present. RBC's should touch or only slightly overlap. The pattern of scan in the diagram below is
not to scale. It is intended to show direction of movement only. (ref: NCCLS Standards, Fig.1 p. 14
Sec.5.6.2 Vol. 12 No. 1)

d. Always check the edge of a smear for platelets if the platelet estimate is decreased.

e. If a cell is borderline between 2 cells, call it the more mature.

f. Compare the WBC estimate with the automated count.

g. Always check WBC morphology. Don't assume the diff is finished after classifying 100 cells.
Depending on your findings, you might have to count 200 cells. See section on Abnormal
Differentials.

8. Evaluate RBC Morphology

a. Variation in Size - MCV (mean corpuscular volume)

a. MCV = Hct x 10
RBC

b. Measured in femtoliters (fl) or cubic microns (µ3)

c. Normals:

Females: 88 +/- 9 fl
Males: 87 +/- 7 fl

d. Eight theoretical morphological types:

Microcytic,
hypochromic
normochromic
hyperchromic
 Normocytic,
hypochromic
normochromic
hyperchromic

Macrocytic,
hypochromic
normochromic
hyperchromic

e. N,N = 33 - 36% saturated with Hgb. This is the maximum level; if higher would precipitate
out, therefore RBC's cannot actually be hyperchromic.

f. Anisocytosis - presence of varying sizes of RBC's in a single smear.

b. Variation in color - Hgb content MCH (Mean Corpuscular Hemoglobin)

MCHC (Mean Corpuscular Hemoglobin Concentration)

1. MCH = mean corpuscular Hgb - absolute amount of Hgb/RBC - measured as

weight

a. MCH = Hgb x 10 = ______________ pg (picograms)

RBC
b. picogram = one millionth of a gram
c. Normal: 27 - 32 pg
 2. MCHC = mean corpuscular Hgb concentration - expressed as
percentage

a. MCHC = Hgb x 100 = _______________%

Hct
b. Normal: 32 - 36%

3. Polychromatophilia - caused by reticulocytes (also called polychromasia, diffuse basophilia)

RED BLOOD CELL MORPHOLOGY TERMINOLOGY

SIZE

Normocytic Normal Size

Microcytic Smaller Than Normal
Macrocytic Larger Than Normal
Anisocytosis Cells of Various Sizes Present

HEMOGLOBIN CONTENT :

Normochromic Normal Color & Hemoglobinization

Hypochromic Pale Color & Lack of Adequate Hemoglobin

SHAPE

Poikilocyte Abnormally Shaped Red Cell

Ovalocyte Oval Red Cell
Elliptocyte same as Ovalocyte
Sickle Cell or Dense Elliptocyte seen with Hgb S and Hemoglobinopathy
Tear Drop Pear Shaped Red Cell
Schistocyte Cellular Fragment
Keratocyte Red Cell Shaped Like a Military Helmet "Helmet Cell"

Spiculated Red Cells

Echinocyte or Crenated Red Cell (regularly spiculated; usually reversible)
Acanthocyte (irregularly spiculated; irreversible)
Spherocyte Round Red Cells, not biconcave
Stomatocyte Red Cell with Mouth Shaped area of Central Pallor
Target Cell Area of Central Pallor with Dense Center like "bullseye"
(RBC Morphology, continued)

RED CELL INCLUSIONS

Basophilic stippling usually RNA or Hemoglobin residue

Fine
Coarse or Punctate
Howell - Jolly Body Fragment of Nucleus
Siderocyte Granules (Pappenheimer Bodies) Iron Granules

RED CELL AGE

Polychromatophilic Red Cell "Multi-colored" young RBC, reticulocyte

Shift or Stress Erythrocyte Nucleated Red Cell in Peripheral Blood
Basophilic Red Cell Same as Reticulocyte
Megaloblast Large Immature RBC with N/C asynchrony

RED CELL DISTRIBUTION ON SMEAR

Agglutination clumps of RBC's

Rouleaux RBC's aligned in "stack of coins" fashion end to
end, NOT clumped

ARTIFACTS

"Drying" and Anticoagulant changes

RED BLOOD CELL MORPHOLOGIC ARTIFACTS

SPICULATED (CRENATED) CELLS

Contaminants (moisture, grease, dirt, etc.)

I.V. running
Excessive amount of anticoagulant to specimen
Old blood - long standing
Warm environment (room temperature) may hasten changes

If spiculated RBC's are a true feature, they are usually uniformly distributed and do not affect most of the
red cells. However, it is advisable to repeat on a fresh sample.

TARGET CELLS

Very easy artifact to make (dehydration environment) May be caused by contamination with dirt or
alcohol on slide, finger, needle or tube. Usually artifactual target cells will be unevenly distributed
---present in some areas of the smear and absent in others. True target cells are usually evenly distributed
throughout the smear.
DRYING (MOISTURE) ARTIFACTS:

Pseudo-Hypochromia Pale central area has a sharp demarcation rather than gradual transition in color
intensity. Sometimes drying artifact appears as vacuoles or small bubbles which are refractile areas in
the RBC's that will appear to change in size (grow larger or smaller) as the focus in changed-this type is
probably more stain-related than preparation related. Crenated (Moth-eaten) periphery sometimes present.
CAUSED BY:
Humidity
Moist slides
Anemia (increased amounts of plasma to red blood cells)
Pernicious anemia and folate deficiency with
low hematocrits frequently demonstrate this.)
Under-fixation.
Water in alcohol fixation (hygroscopic absolute methyl
alcohol in bottle absorbs water vapor if not
kept tightly closed).
Excess buffer to stain.
Thick smears (and thick area of smear)
ELIMINATE BY:
Avoid Humidity
Air conditioning
Smears made in humid-free lab
Desiccator or Dehydrating Set-up
Calcium Chloride crystals
Smear in desiccator for one hour
Examine red cells in thin area of smear
Is MCHC Compatible?

FRAGMENTS

Blotting smear with filter paper or towel.

Wiping off oil with tissue or gauze. Especially enhanced under following conditions
1. Poor or inadequate fixation
2. Wetting smear with Xylene and wiping with tissue.

MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR

Thin area-Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will
be found in other (Good) areas of smear.

Thick area - Rouleaux, which is normal in such areas.

Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a
"stack of coins" fashion..
Red Blood Cell Morphology

A normal red blood cell should be approximately the same size as a normal lymphocyte nucleus or 2
normal sized red blood cells should fit side by side across a normal sized poly (not a hypersegmented
poly).

1+ 2+ 3+ 4+
1-6 per oil imm. field 7-10 per OIF 11-20 per OIF > 20 per OIF

1. Target cells 14. Microcytosis

2. Stomatocytes 15. Macrocytosis
3. Schistocytes 16. Hypochromia
4. Burr cells 17. Anisocytosis
5. Sickle cells 18. Poikilocytosis
6. Ovalocytes
7. Acanthocytes
8. Spherocytes
9. Stippled red blood cells
10. Howell-Jolly bodies
11. Hemoglobin C crystals
12. Pappenheimer bodies
13. Polychromasia

Where possible use macrocytic and microcytic, rather than simply anisocytosis alone, when describing
red cell morphology.

Use specific cell morphology when possible, rather than simply reporting poikilocytosis.

When red cells are normocytic, normochromic, report out as NORMAL. When abnormal morphology
has been noted, DO NOT indicate normal on the report form.

EXAMPLE: 7-10 microcytic RBC's/OIF is reported out as: 2+ microcytosis

ABNORMAL DIFFERENTIALS

1. 200 Cell diff:

a. WBC > 15.0 (>20.0 for babies under 1 month and labor unit)
b. Three or more basophils seen.

2. If more than five immature WBC's are seen (or any blasts) let someone else diff slide and average
results.

3. Correct WBC for NRBC's if you seen ten or more nrbc's/100 WBC.

4. Always indicate number of cells counted on diff.

5. If any cell type is extremely elevated (such as bands, monos, or eos > 20) indicate that you are aware
of the abnormality by circling or checking on the card next to the results.
Examination of the Peripheral Blood Smear: Common Findings Associated with Disease

DISEASE FINDINGS ON THE BLOOD SMEAR

Elliptocytosis Elliptocytes &/or ovalocytes

Hereditary Spherocytosis Spherocytes; polychromatophilia

Acquired hemolytic anemia,Spherocytosis; polychromatophilia;

compensated erythrocyte agglutination

Thalassemia trait Microcytes; poikilocytes, target cells; hypochromia, basophilic

stippling

Hemoglobin C Disease Target cells; microspherocytes

Lead Poisoning Basophilic stippling, coarse

Sideroblastic Anemia Two RBC populations; siderotic granules

Incipient Pernicious Anemia or Hypersegmented neutrophils; macrocytes (oval); poikilocytosis

(fragments)
Folic Acid Deficiency

Consumptive Coagulopathy (DIC) Schistocytes (fragments); reduced platelets

Mechanical Hemolysis Schistocytes (fragments)

Cold Agglutinin Disease Agglutination

Multiple Myeloma Rouleaux formation; agglutination

Acute Leukemia Blast cells

(Early or "Leukopenic")

Metastatic Tumor Leuko-Erythroblastic picture (Immature neutrophils & NRBC)

Severe Infection Relative increase in neutrophils; increased band forms; toxic

granulation; Dohle bodies

Sepsis Bacteria &/or vacuoles in neutrophils & monocytes

May-Hegglin Anomaly Megathrombocytes; Dohle bodies

Agranulocytosis Decreased neutrophils; increased monocytes; relative lymphocytosis

Allergic Reactions Eosinophilia

Parasitic Infestation Eosinophilia

Chronic Lymphocytic Leukemia Relative lymphocytosis; smudge cells

(Early)

Infectious Mononucleosis Reactive-stimulated or transformed lymphocytes

Viral Infections Reactive lymphocytes; reticular (basophilic) lymphocytes; plasma

cells

Lipid Storage Disorder Vacuoles in lymphocytes

Mucopolysaccharide Disorder Granules in lymphocytes

Hurler's Disease Inclusions in leukocytes

Malaria Parasites in the erythrocytes

Histoplasmosis, Loa Loa, etc. Fungus, Parasite, etc.

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Wednesday, October 13, 2010