Hematology A branch of medicine that deals with the morphology of blood and blood-forming tissues, and with their

physiology and pathology.

Complete Blood Count (CBC) The complete blood count (CBC), consists of

A. white blood cell count.

B. red blood cell count.

C. hemoglobin.

D. hematocrit.

E. white blood cell differential.

Also included are the red blood cell indices, which indicate the relative and absolute hemoglobin content

and size of the average red blood cell. When performing the differential, the white blood cells are

identified and categorized, all cells are examined for abnormalities, and the platelets are reviewed for

number and morphologic features.

Additives in Collection Tubes ANTIGLYCOLYTIC AGENT

This substance inhibits the use of glucose by blood cells. Such inhibition may be necessary if testing for

glucose level is delayed. Examples of antiglycolytic agents are sodium fluoride and lithium iodoaceate.

Tube containing sodium fluoride alone will yield serum. Potassium oxalate (an anticoagulant) is

combined with sodium fluoride or potassium ethylene-diaminetetra-acetic acid (EDTA) to yield plasma

for more rapid testing.

ANTICOAGULANT

This substance prevents blood from clotting. The mechanism by which clotting is prevented varies with

the anticoagulant; for example, EDTA, citrate and oxalate bind with calcium, whereas heparin prevents

the conversion of prothrombin to thrombin. Examples of anticoagulants are EDTA, sodium citrate, and

lithium heparin. Tubes must be inverted several times to ensure proper mixing after collection, according

to manufacturer’s instructions.

CLOT ACTIVATOR

This substance helps initiate or enhance the clotting mechanism. Clot activators include glass or silica

particles that provide increased surface area for platelet activation and a clotting factor such as thrombin

GENERAL PRINCIPLES: Haematology 1. What is erythropoietin, where is it produced, and what are its principal effects?

Erythropoietin is a glycoprotein hormone produced by the peritubular capillary endothelium of the kidney

in response to reduced oxygen tension (hypoxia). Erythropoietin’s principal site of action is the bone

marrow.

2. How is iron, either dietary or recycled, transported through the body?

Iron is transported in the blood by transferrin, a β-globulin (serum protein), from the site of absorption in

the intestine or from macrophage stores to the marrow and tissues. Iron can then be incorporated into

hemoglobin during heme synthesis.

3. How are aged or damaged erythrocytes normally removed from circulation?

Aged or damaged erythrocytes have changes in either their cell membranes or cytosolic enzymes that

allow them to be recognized by macrophages within the spleen and liver. These erythrocytes are

subsequently phagocytosed by the macrophages and removed from circulation.

Additionally, a small percentage of “old” or abnormal erythrocytes are removed from circulation through

intravascular hemolysis.

What is methemoglobin, and how is it normally metabolized to hemoglobin? Methemoglobin is structurally identical to hemoglobin except that the iron moiety of the heme group is in the

ferric (3+) state instead of the ferrous (2+) state. Therefore, methemoglobin is oxidized hemoglobin that is no longer capable of binding oxygen. A small percentage of hemoglobin is oxidized to methemoglobin in healthy animals. Methemoglobin is continuously changed back to hemoglobin by an enzyme found within erythrocytes known as methemoglobin reductase.

What are reticulocytes, and how do you identify them? Reticulocytes are immature, anucleate, circulating erythrocytes that contain residual cytoplasmic ribonucleic acid

(RNA), mitochondria, and organelles (also known as reticulum) that are visible when stained with new methylene blue. Reticulocytes appear bluish on a Romanowsky stain (Wright’s, Diff-Quik) and are referred to as polychromatophilic cells

Approximately how long does it take for reticulocytes to be produced and released

from the bone marrow? It takes approximately 2 to 3 days for reticulocytes to be produced and released from the bone marrow in

response to anemia. It takes 5 to 7 days for reticulocytes to reach a maximum response.

What are nucleated erythrocytes (nRBCs)? Nucleated erythrocytes are RBC precursors that have not yet expelled their nuclei. Nucleated RBCs include

metarubricytes, rubricytes, prorubricytes, and rubriblasts. Usually, nRBCs refer to metarubricytes

When are increased numbers of nRBCs expected in peripheral blood? Nucleated erythrocytes are normally present in the bone marrow. Increased numbers of nRBCs can be released

from the bone marrow with both strongly regenerative anemias and increased erythropoietin secretion. This condition is known as appropriate metarubricytosis. Appropriate metarubricytosis is accompanied by reticulocytosis.

Define inappropriate metarubricytosis and identify associated conditions. Inappropriate metarubricytosis occurs when metarubricytes are released from the bone marrow, often when

there is an alteration of the blood–bone marrow barrier. Reticulocytosis is not associated with inappropriate metarubricytosis.

Inappropriate metarubricytosis has been associated with several conditions, including lead toxicosis, myelodysplasia, erythroid leukemia, splenic disease/neoplasia, and endotoxemia.

ERYTHROCYTE MORPHOLOGY What are Howell-Jolly bodies, and when do they usually occur?

Howell-Jolly bodies are remnant portions of nuclear material that are found within the cytoplasm of

erythrocytes. These bodies are basophilic and spherical. Howell-Jolly bodies are usually observed with

accelerated erythrocyte production or after splenectomy.

What are Heinz bodies, and what causes them? Heinz bodies are precipitates of denatured hemoglobin attached to the erythrocyte membrane. These bodies

occur following oxidation of hemoglobin. Heinz bodies are found in low numbers in health in certain

species (e.g., cat) or may be associated with hemolytic anemias

How are Howell-Jolly and Heinz bodies distinguished morphologically? Although both structures are round, Howell-Jolly bodies are basophilic and located deeper within the

cytoplasm than Heinz bodies, which protrude from the red blood cell (RBC) surface.

Also, Heinz bodies on Wright’s stain are the same color as the erythrocyte’s cytoplasm because they are

derived from hemoglobin.

What is the cause of basophilic stippling in erythrocytes, and when does it occur? Basophilic stippling results from minute aggregations of residual ribonucleic acid (RNA) that collect within

the cytoplasm of the erythrocyte and is an indicator of RBC immaturity.

Stippling is readily observed with either Wright’s or Diff-Quik staining. Basophilic stippling is occasionally

observed in anemic cats and indicates a regenerative response. Basophilic stippling with polychromasia and

anemia may be an appropriate response to the anemia.

Define anisocytosis and identify associated conditions. Anisocytosis is variation in cell size. Although mild anisocytosis can be observed in erythrocytes from

healthy dogs and cats, more pronounced anisocytosis is associated with avariety of conditions. These include

regenerative anemias, some hemolytic anemias, Heinz-body anemia, and breed-associated variations in RBC

size

What are macrocytes and microcytes? Macrocytes are larger erythrocytes of normal shape.

Microcytes are abnormally small erythrocytes.

What are spherocytes? Spherocytes are small, spherical erythrocytes that lack central pallor. Spherocytes are more easily observed

in the dog; dogs normally have erythrocytes that are slightly larger than other domestic animals, and dog

erythrocytes have well-defined central pallor. Rare spherocytes may be seen in circulation before being

removed by the liver or spleen, since immunoglobulin G (IgG) increases on the surface of “old”

erythrocytes. This is the process by which old erythrocytes are removed from circulation

How are spherocytes formed? Spherocytes are formed when macrophages within the liver and spleen remove a portion of a damaged

erythrocyte’s cell membrane. This generally occurs after the erythrocyte’s surface has been coated with

either antibody or complement. Because a portion of the membrane is removed, the resulting RBC has

reduced cell membrane surface area relative to cytoplasmic volume. Therefore, spherocytes appear as

smaller, dense cells that lack central pallor.

Spherocytes are typically associated with what disease? Spherocytes are associated with immune-mediated hemolytic anemia (IMHA). Spherocytes also occur as

part of a natural aging process, but then are seen only in low numbers in circulation.

When are increased numbers of nRBCs expected in peripheral blood? Nucleated erythrocytes are normally present in the bone marrow. Increased numbers of nRBCs can be

released from the bone marrow with both strongly regenerative anemias and increased erythropoietin

secretion. This condition is known as appropriate metarubricytosis. Appropriate metarubricytosis is

accompanied by reticulocytosis.

Define inappropriate metarubricytosis and identify associated conditions. Inappropriate metarubricytosis occurs when metarubricytes are released from the bone marrow, often

when there is an alteration of the blood–bone marrow barrier. Reticulocytosis is not associated with

inappropriate metarubricytosis.

Inappropriate metarubricytosis has been associated with several conditions, including lead toxicosis,

myelodysplasia, erythroid leukemia, splenic disease/neoplasia, and endotoxemia.

How are Howell-Jolly and Heinz bodies distinguished morphologically? Although both structures are round, Howell-Jolly bodies are basophilic and located deeper within the cytoplasm

than Heinz bodies, which protrude from the red blood cell (RBC) surface.

Also, Heinz bodies on Wright’s stain are the same color as the erythrocyte’s cytoplasm because they are derived from hemoglobin.

How are schistocytes formed? Schistocytes are formed secondary to mechanical trauma in the vasculature. Physical trauma to the erythrocyte

membrane as it passes through fibrin deposits within vessel walls, as seen with disseminated intravascular coagulation (DIC), can result in schistocyte formation.

What diseases have schistocytes been associated with in small animals?

Schistocytes have been associated with a number of fibrin-forming conditions, including

hemangiosarcoma, vasculitis, congestive heart failure, DIC, myelofibrosis, microangiopathic

anemia, and glomerulonephritis.

What are acanthocytes, and with what diseases are they associated? Acanthocytes are erythrocytes with several unevenly spaced, irregular projections or spicules that form

secondary to an altered membrane lipid and cholesterol content

Do dogs have circulating reticulocytes in health? Low numbers of reticulocytes are present in the blood of healthy dogs (<1%).

27. What type of reticulocyte is found in the peripheral blood of dogs? Canine reticulocytes are aggregate reticulocytes, in which larger aggregates of reticulum are found within the

cytoplasm (see Figure 1-1).

28. Identify and describe the two types of reticulocytes found in the cat, indicating

which

type is most useful in the determination of a bone marrow response to anemia.

a. Punctate reticulocytes. These reticulocytes have small, punctate clumps of residual RNA and are more mature than aggregate reticulocytes .Also, they remain in circulation for several weeks. Punctate reticulocytes are not evaluated as often in reticulocyte counts because they persist so long in circulation, and they are not considered a good index of the current bone marrow response.

b. Aggregate reticulocytes. These reticulocytes are similar in appearance to those in the dog .Aggregate reticulocytes may exist in low numbers in the circulation of healthy cats (< 0.4%) and are the type of reticulocyte evaluated in the determination of a bone marrow response.

High reticulocyte levels could indicate: acute bleeding

chronic blood loss hemolytic anemia kidney disease Erythroblastosis fetalis, also called hemolytic disease (a potentially fatal blood disorder in a fetus or newborn) Low reticulocyte levels could indicate: iron deficiency anemia folic acid deficiency

aplastic anemia

radiation therapy

cirrhosis of the liver kidney disease

vitamin B12 deficiency bone marrow failure (caused by drug toxicity, an infection, or cancer)