1. Week Four Hematology/CBC/Leukocytes

2. Hematology Values Provide direct cellular information
RBCs
WBCs
Platelets
Plasma protein concentration
Provide diagnostic information
Diagnosis of healthy and disease states
Monitoring response to therapy
The complete blood count (CBC) is one of the most commonly performed laboratory tests because it provides information about red blood cells (RBCs, or erythrocytes), white blood cells (WBCs, or leukocytes), platelets, and plasma protein concentration that can be helpful in determining health or disease and in monitoring response to therapy.

3. Complete Blood Count (CBC)
Blood samples for a CBC are collected in a tube containing an anticoagulant.
Several types of blood collection tubes and their intended uses are presented in Table 12-1, shown here. 

4. CBC Usual Values Provided
PCV—packed cell volume or hematocrit
RBC count
Hemoglobin concentration
MCV—mean cell volume
MCHC—mean corpuscular hemoglobin concentration
RDW—RBC distribution width
Platelet count
WBC count
WBC differential
The CBC typically includes the values shown here.

5. Complete Blood Count (CBC)
2-mL blood collection tube used most often
Do not overfill or underfill
Mixing blood: after collection
Gently invert tube several times
Inspect for small clots before processing
Mixing blood (for even distribution)
After collection
Before PCV count
Before blood smears
Before processing
If clots are present before processing, collect a new sample.

6. Direct Laser (CBC) Measurements
Usual CBC values, plus:
RBC hemoglobin content (CH)
Hemoglobin content of reticulocytes (CHr)
Corpuscular hemoglobin concentration mean (CHCM)
Hemoglobin concentration distribution width (HDW)
Mean cell volume of reticulocytes (MCVr)
Platelet volume distribution width (PDW)
Plateletcrit (PCT)
Newer instruments with direct laser measurement may include RBC hemoglobin content (CH), hemoglobin content of reticulocytes (CHr), corpuscular hemoglobin concentration mean (CHCM), hemoglobin concentration distribution width (HDW), mean cell volume of reticulocytes (MCVr), platelet volume distribution width (PDW), and plateletcrit (PCT).

7. CBC Additional Values Plasma protein concentration Blood smear
The CBC also may include determination of plasma protein concentration and, in many laboratories, examination of a blood smear to evaluate cell morphology and perform a manual differential WBC count.

8. Erythrocyte Mass Indication of oxygen-carrying capacity Evaluated by:
Packed cell volume (PCV)
Hematocrit (HCT)
Total RBC count
Hemoglobin concentration
PCV (or HCT), RBC count, and hemoglobin concentration are used to evaluate RBC mass, which is an indication of the oxygen-carrying capacity of blood. In veterinary medicine, the PCV is the most commonly used parameter to assess RBC mass because it can be measured manually.

9. PCV or Hematocrit (HCT)
PCV = % of RBCs in blood
Quick and inexpensive
Hold tube to grid; align bottom with zero
Challenges to correct measures of HCT
PCV is the percentage (%) of RBCs in a specific volume of blood. PCV is determined by filling a plain microhematocrit tube two thirds to three quarters full with EDTA anticoagulated blood, sealing one end with a specific kind of clay, and centrifuging the sample in a microhematocrit centrifuge for a specified length of time and speed according to the manufacturer’s recommendations.

10. PCV, cont’d
After centrifugation, three components of the blood are visible in the microhematocrit tube: a column of packed RBCs at the bottom, a buffy coat layer of WBCs and platelets just above the packed RBCs, and plasma at the top.  
Illustrated by Tim Vojt, Biomedical Media, The Chio State University College of Veterinary Medicine. Copyright The Ohio State University

11. PCV, cont’d
To measure the PCV, align the bottom of the RBC column with zero and the top of the plasma portion with 100, and determine where the top of the RBC column intersects the grid; this point indicates the PCV as a percentage (%).
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

12. RBC Count and Hemoglobin
RBC count = RBC × 1012/L or 106/mcg/L
Manual counts not recommended
Useful in calculating RBC indices; classifying anemia
Hemoglobin (g/dL)
RBC protein that carries O2 from lungs to tissues
Changes usually proportional to PCV (HCT) changes
Useful in determining RBC indices (anemia)
Manual RBC counts are not very accurate compared with RBC counts determined by automated instruments and are not recommended.
Because changes in RBC count usually are proportional to changes in PCV and HCT, most clinicians use PCV and HCT to evaluate RBC mass.
Hemoglobin concentration cannot be measured accurately by manual methods.

13. RBC Indices MCV: MCHC: MCH: Indicator of average RBC size
Average amount of hemoglobin in a specific volume of blood
MCH:
Average amount of hemoglobin in each RBC
MCV is used as an indicator of the average size of RBCs and is reported in femtoliters.
The analyzer measures the volume of each RBC counted and determines the mean volume for the RBC population.

14. MCV Manual Determination
MCV also can be calculated from a manually determined PCV and RBC count, but manual RBC counts are not accurate.

15. MCV, cont’d Normocytic RBC = normal MCV Macrocytic RBC = increased MCV
Microcytic RBC = decreased MCV
RBCs with normal MCV are called normocytic, RBCs with an increased MCV are called macrocytic, and RBCs with a decreased MCV are called microcytic.

16. MCHC Mean corpuscular hemoglobin concentration
Average amount of hemoglobin in a specific volume of blood
Calculated from hemoglobin concentration and HCT as determined by automated instrument
Challenges in measuring
RBCs with a normal MCHC are called normochromic, and RBCs with a decreased MCHC are called hypochromic.
RBCs often are normochromic in health and in nonregenerative anemia. Hypochromic RBCs occur in marked regenerative anemia and in some animals with severe iron deficiency.

17. MCH Mean corpuscular hemoglobin
Average amount of hemoglobin in each RBC
Reporting in picograms
Calculated from hemoglobin concentration and RBC count
Not clinically useful
Mean corpuscular hemoglobin (MCH), the average amount of hemoglobin in each RBC, is reported in picograms.
MCH is calculated from the hemoglobin concentration and the RBC count as determined by the automated instrument, but MCH is not used clinically and may not be reported as part of a CBC.

18. White Blood Cell (WBC) Count
Total WBC count
Hemocytometer
Automated hematology analyzers
Sources of errors
WBC estimate
Buffy coat
Automated hematology most accurate
WBCs can be counted manually or by automated instruments, or they can be estimated from the buffy coat layer or blood smear evaluation.
For manual WBC counts, a hemocytometer and commercially available reagents are used.

19. Manual Determination of WBC
An improved Neubauer hemocytometer is shown on the left, and the counting chamber is shown on the right. 
WBCs are counted in the four large corner squares of the grid, and the number of WBCs is multiplied by 50 to determine WBCs/µL.
The pink circle indicates a corner square, which can be visualized through the 10× objective.  
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

20. Preparing a Blood Smear
Microscopic evaluation of a blood smear is an important component of a CBC because some hematologic abnormalities are recognized only on the blood smear.
Preparation of high-quality blood smears is a technical skill that requires practice. Blood smears should be made with new, clean microscope slides using one of several methods.
Most commonly, a small drop of blood is placed close to one end of a microscope slide that is held stationary on a flat surface, and a second “spreader” slide is used to push the drop forward in a smooth, even motion, resulting in a blood smear that is evenly distributed across the stationary slide.
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

21. Blood Smears Dry quickly to prevent RBC artifacts
Stain within 48 hours
Store at room temperature
Ship separately from biopsy specimens exposed to formalin
Blood smears should be dried quickly before staining to prevent RBC artifacts.
Slides should be labeled at one end with the date and the animal’s name or identification number and ideally should be stained within several hours. A lapse longer than 48 hours may result in inadequate staining.
Unstained smears being sent to a reference laboratory should be stored at room temperature, not in a refrigerator or freezer because water condensation will damage the cells.
Unstained smears should be stored away from formalin fumes and shipped separately from surgical biopsy specimens that have been placed in formalin because formalin vapors inhibit optimal staining.

22. Blood Smear Evaluation
Three regions
Counting area: between body and feathered edge
Accurate evaluation of blood smears requires a systematic procedure for examination and expertise in identifying normal cells, morphologic abnormalities, and artifacts.
A blood smear consists of three regions: body, counting area, and feathered edge.
Illustration by Tim Vojt, Biomedical Media, The Ohio State University College of Veterinary Medicine. Copyright The Ohio State University

23. Agglutination in Blood Smear
Rouleaux formation
RBCs in chains that look like stacks of coins
Agglutination
Irregular RBC clumps
Excess antibody binding
IHA in dogs
In the body of the smear, RBCs can be evaluated for rouleaux formation or agglutination.
Rouleaux formation refers to RBCs in chains that resemble a stack of coins.
In horses and cats, rouleaux formation is common, whereas in dogs, rouleaux formation may be an indication of inflammation (increased plasma proteins), or it may be an artifact of smear preparation.
Agglutination refers to irregular, variably-sized clumps of RBCs that form because of excess antibodies bound to the surface of RBCs.

24. WBC Differentiation Marked species variation
Differential WBC count: Identify and enumerate at least 100 leukocytes in counting area of blood smear
Percentage of each cell type × total WBC (in mcg/L of blood)
Classifications
Segmented neutrophils, band neutrophils, eosinophils, lymphocytes, monocytes
Determination of the numbers of each type of WBC can be helpful in establishing a list of differential diagnoses.
A differential WBC count is performed by identifying and enumerating a minimum of 100 leukocytes consecutively encountered in the counting area of the blood smear.
At least 200 cells should be counted if the WBC count is increased.

25. Neutrophil Morphology
Segmented neutrophil
Nucleus
Cytoplasmic granules
Heterophils
Band neutrophil
Metamyelocyte
Figure 12-14A shows a segmented neutrophil (arrowhead), three bands (short arrows), and a metamyelocyte (long arrow).
The bands have C- or U-shaped nuclei. The metamyelocyte has an indented nucleus.

26. Neutrophil Morphology
Left shift
Increase in circulating neutrophils
Regenerative
Indicates release of less mature cells to meet demand
Degenerative
Band neutrophils outnumber segmented; poor prognosis
Toxic changes
Intense neutrophil production
Shortened maturation time
Mild, moderate, or marked
Rarely, formation of giant neutrophils occurs.
Toxic changes can be subjectively reported as mild, moderate, or marked, depending on the percentages of cells affected and the severity of the change.

27. Eosinophil Circulate in low numbers in health
Mediate hypersensitivity reactions
Protect against some parasites
All nucleated cells are eosinophils.

28. Eosinophils, Cont'd
Eosinophilia = increased numbers (allergic/defensive reaction to parasites)
Eosinopenia = decreased numbers (response to corticosteroids)
Marked species variation
There are three segmented neutrophils and an eosinophil in this blood smear from a horse.
In horses, eosinophils have numerous large granules, so they are easy to identify.

29. Basophil Morphology Very low numbers in healthy smears
Basophilia = increased numbers (allergic/defensive reaction to parasites)
Morphology varies with species
There is a basophil on the left and an eosinophil above a neutrophil on the right in this blood smear from a dog.
Basophils in dogs have a few dark granules in the cytoplasm.
Basophils appear larger than neutrophils, and the nuclei appear twisted with chromatin that is slightly less condensed than in neutrophil and eosinophil nuclei.

30. Lymphocytes Predominant circulating cell in cattle, sheep, goats
Usually smaller than neutrophils
Round nucleus almost fills cytoplasm
Lymphocytosis
Antigenic stimulation
Lymphoid neoplasia
Acute lymphoid leukemia (ALL)
Chronic lymphocytic leukemia (CLL)
Figure 12-14G (top) shows a normal lymphocyte in upper portion and a reactive lymphocyte in the lower portion of blood smear from a cat.
Figure 12-14I (bottom) shows neoplastic lymphocytes in ALL in a dog.

31. Monocytes Relatively low numbers Similar in most species
Larger than neutrophils
Abundant gray-blue cytoplasm
Monocytosis: inflammataion or hemolysis
Reactive: chemotherapy or inflammation
Figure 12-14K shows two monocytes in equine blood smear.
Shows typical abundant basophilic cytoplasm with clear vacuoles.
Nuclei are indented or irregular.

32. QUESTIONS?