Evaluating a Blood Film, WBC Morphology, WBC Blood Parasites and Morphologic Abnormalities of Blood Cells Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Manual Determination of WBC We don’t use this anymore in practice to determine a WBC count since there are CBC analyzers 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

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

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.

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

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.

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.

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.

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.

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

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.

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.

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.

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.

Morphologic Abnormalities in WBCs Nuclear hyposegmentation Pelger-Huet anomaly Congenital defect Hyposegmentation of all granulocyte nuclei Nuclear hyposegmentation in neutrophils (N) and eosinophils (E) from a dog with Pelger-Huet anomaly (Wright stain) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Morphologic Abnormalities in WBCs (cont.) Nuclear hypersegmentation Very common Nuclei with 5 or more lobes Can be due to aging neutrophils either in vivo or in vitro (prolonged storage) Common in poodles with macrocytosis Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Morphologic Abnormalities in WBCs (cont.) Toxic change Common disease-induced cytoplasm changes in neutrophils Associated with inflammation, infection, drug toxicity Dogs – bacterial infection Cats – common in not severely ill cats Cytoplasmic basophilia, Döhle bodies, toxic granulation, giantism Caused by decreased length of time in the neutrophil maturation process with bone marrow Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Toxic Neutrophil Which neutrophil is toxic in this photo? Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Atypical Lymphocytes Atypical lymphocytes have basophilic cytoplasm and cleave nuclei Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Reactive Lymphocytes a.k.a. immunocytes Increase basophilic cytoplasm More abundant cytoplasm Sometimes a large, convoluted nuclei Usually caused by antigenic stimulation Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Smudge Cells a.k.a. basket cells Degenerative leukocytes that have ruptured Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Smudge Cells (cont.) Karyolysis – degenerative change to the nucleus by dissolution of the nuclear membrane Septic exudates Pyknosis – condensing of the nucleus as the cell dies Karyorrhexis – fragmentation of the nucleus after cell death Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.