Lecture 3 - Hematology (Red Blood Cell Morphology, Hemacytometer, Refractometer)

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- Avian and reptilian RBCs are oval and nucleated

Avain/Reptilian RBC

- The avian and reptilian equivalent of the neutrophil.
- Heterophils in birds have segmented nuclei that are partly obscured by the large red-orange granules that pack the cytoplasm. Mature granules are fusiform and many are slightly refractile (shiny)

Heterophil

- Avian eosinophils are slightly smaller than heterophils and their granules are round and a slightly different shade of orange. The granules of eosinophils in psittacine birds are light blue.

Eosinophil (Avian/Reptilian)

Pathological changes are due to some disease-effect on the red blood cells, and as such, are very important

Pathological Changes

Artificial changes (artifacts) occur due to effects of preparation or the environment on the RBCs after or during sampling, and as such are not important (artifacts are not noted in the RBC assessment)

Artifactual Changes

A smear artifact is the presence of any changes on a blood smear which is not due to disease (pathologic) or physiologic. These changes are often due to the stain involved, age of the smear, or exposure of the sample of smear to water, debris, or chemicals
- Stain Precipitate Artifact
- Water Distortion Artifact

Smear Artifact

Echinocytes (crenated RBCs)
- Are spiculated RBCs
- The projections of the cell membrane may be sharp or blunt, are usually numerous and tend to be evenly spaced around the circumference.
- Usually represent an in vitro artifactive change, resulting from aging of the blood and/or exposure to excessive concentrations of EDTA as occurs when the sample tube is significantly underfilled. Can also be due to snake envenomization.

Name the cell type

Rouleaux Formation
- Occurs when RBCs form stacks or rolls.
- Common in normal horses and to a lesser extent normal cats.
- In other species, excessive Rouleaux is often due to either an artifact (resulting from not preparing the blood smear soon enough after placing the blood on the slide), or the presence of high concentrations of abnormal globulins or fibrinogen.

Name the cell type

Nucleated Red Blood Cells (nrbc, normoblast)
- Occurs when the demand for RBCs is so great that new red blood cells are released from the bone marrow prematurely.
- The cells generally have slightly basophilic cytoplasm and a dense, round nucleus (pyknotic nucleus).
- These are seen in anemic conditions

Name the cell type

Howelll Jolly Bodies
- Small remnants of a nucleus
- They represent "factory-defects", where the bone marrow just misses a few
- They are generally removed by the spleen and liver unless there is a spleen or liver problem

Name the cell type

Schistocytes
- Are RBC fragments that result from membrane damage encountered during passage through vessels.
- They occur in microangiopathic hemolytic anemia, severe burns, uremia, and hemolytic anemias caused by physical agents, as in disseminated intravascular coagulation (DIC)
- Sometimes referred to as "bite cells"

Name the cell type

Acanthocytes
- Are RBCs with irregularly spaced projections. These projections vary in width but usually contain a rounded end.
- They may be found in abetalipoproteinemia and certain liver disorders

Name the cell type

Target Cells (Codocytes)
- Have the appearance of a target with a bullseye
- Such cells are thin and have a disproportional increase in the surface membrane area to volume ratio. This increased ration may result from an increase in membrane surface area or a decrease in hemoglobin content.
- Morphologically, target cells have a central, hemoglobinized area surrounded by an area of pallor. The periphery of the cell contains a band of hemoglobin
- In mammals, target cells are usually an artifact of drying, however, the presence of excessive target cells within mammalian blood smears may indicate chronic disease including liver disease or iron deficiency, or following a splenectomy

Name the cell type

Hypochromasia
- Refers to erythrocytes that have a decreased density of the characteristic hemoglobin color OR by calcuation of the Mean Corpuscular Hemoglobin Concentration (MCHC).
- Is frequently observed in iron deficiency anemia as caused by chronic blood loss or parasitism.
- Stains lighter red and often have increased central pallor zones

Name the cell type

Spherocytes
- RBCs which have assumed the form of a sphere rather than the normal discoid shape. As a result, they appear on routine blood films as cells that are smaller and more dense than normal red cells of the species, and have a reduced or absent central pallor area.
- Because cats, horses, and cattle normally have red cells with little central pallor, recognition of spherocytes is more difficult in these species than in dogs, whose normal red cells have distinct pale centers.
- Large spherocytes (macrospherocytes) are seen in hemolytic anemia
- Small spherocytes (microspherocytes) are sometimes seen in severe burn cases

Name the cell type

Reticulocytes (Polychromatophilic Cells)
- In the presence of some anemias, the body increases the production of red blood cells (RBCs), and sends these cells into the bloodstream before they are mature
- Are characterized by a network of filaments and granules
- With Wright stain, reticulocytes stain a slight blue-purple
- With Supra-vital stains (New Methylene Blue) the filaments and granules show up clearly
- Reticulocytes normally make up 1% of the total RBC count, but may exceed levels of 4% when compensating for anemia and is an important diagnostic finding

Name the cell type

Heinz Bodies
- Oxidation causes precipitation of hemoglobin, which may then coalesce to form intracellular inclusions called Heinz bodies (HB).
- A small number of non-pathologic HB can be found in blood of many healthy cats, but their presence is abnormal in other species.
- May be seen in Wright stained smears, especially if they are large and distort the red cell membrane, however, HB can be easily observed with supravital stains, such as new methylene blue.
- Extensive HB formation and anemia can be seen in cats with acetaminophen toxicity. Hemolytic anemia results because attachment to the membrane increase the rigidity of the red cells and render them susceptible to fragmentation and entrapment in the spleen where they are phagocytized

Name the cell type

Hemobaronellosis
- RBC parasite
- Haemobartonella felis is a rickettsial organism that adheres to cat RBCs, damaging the cell surface and leading to removal of infected erythrocytes from the circulation by phagocytes in the spleen and liver
- They appear as short chains of coccoid organisms on the periphery of RBCs
- Infection is often asymptomatic, and clinical disease may develop only when the cat suffers stress or is immunosuppressed, for example by feline leukaemia or feline immunodeficiency virus infections
- Dogs can be infected with H. canis, but this rarely causes clinical disease

Name this disease/parasite

Anaplasmosis
- Seen in cattle and other ruminants (A. marginale)
- Small dark cocci on the margin of RBCs (not in chains)
- Resembles Howell-Jolly bodies
- High numbers of RBCs (50%) affected early in infection

Name this disease/parasite

Cytauxzoonosis
- Cytauxzoon felis is a protozoal parasite that causes fatal disease in cats. The disease has been reported in Missouri and Oklahoma; cytauxzoonosis is an extremely unlikely diagnosis in cats that have not lived in endemic areas.
- The cat is a dead-end host, with the bobcat being the primary host and transmission being mediated by Dermacentor tick
- The parasite has a tissue phase, in which macrophages throughout the body are distended with organisms, as well as a red cell phase
- Infection of red cells results in hemolytic anemia

Name this disease/parasite

Babesiosis
- Common in cattle (B. bigemina, B. bovis)
- Known as Texas fever, redwater fever, cattle tick fever
- Less common in horse (B. equi, B. caballi)
- Tear drop shaped frequently seen in pairs

Name this disease/parasite

- The most common in domestic animals is the microfilaria of the heartworm Dirofiliaria immitus

Blood parasite

- Determining whether the number of cells in la given blood sample has increased or decreased is an important diagnostic indicator
- With RBCs, the count is generally a Total RBC count
- With WBCs, the count is generally the Total WBC count (the total number of WBC is a sample of blood), and an Absolute Differential Count (the totals of the different kinds of WBCs)
- Cells in a blood sample may be counted by automated, electronic cell counting machines, or by the manual method

Cell Counting

- Counts by measuring the cells ability to alter electric resistance as they pass through a small aperture
- Size proportional to degree of impedance

Impedance Counter
(Electronic Cell Counter)

- Counts by detecting light diffraction of a cell as it passes through a light beam

Darkfield Illumination Counter

- A laser-based technology in which cells are counted as they travel in suspension one by one past a sensing point

Flow cytometry

- Hemocytometer: this instrument is a special microscope slide on which precise grids have been etched within a counting chamber designed to hold an exact volume of diluted blood sample.
- The blood cell determination involves counting cells in several squares of the grid and obtaining an average number. That number is multiplied by a factor that compensates for the amount of dilution.
- The final result expresses the number of red blood cells per cubic millimeter of the original blood sample
- The hemocytometer is filled by using a special diluting system called Unopette
er squac

Unopette Cell Counting System
(Manual Cell Counting)

- Observe the hemocytometer under 40x magnification
-Count the cells in the proper square cells by cell. When it comes to cells touching a line, *only count cells touching the left and top walls of the square when moving left to right, and the right and top walls of the square when moving right to left

Hemocytometer Counting Chamber

# cells counted x # squares x chamber depth x dilution = erythrocyte count (in cu mm)

Constants:
Chamber depth is 10
Dilution is 200

Erythrocyte calculation

# cells counted + 10% of cells counted x dilution = leukocyte count (in cu mm)

Dilution is 100

Leukocyte calculation

- When counting WBCs using the hemocytometer, nucleated RBCs are included in the count (if present in high numbers), throwing off the real count
- To correct for these nRBCs, the percentage of nRBCs is determined by reviewing the blood smear, then use the following formula:
% nRBC = (# nRBCs/ 100 RBCs)

corrected WBC = WBC count - (WBC count x % nRBC)

Correcting the WBC Count for Nucleated RBCs (nRBCs)

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