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Proceeding of the LAVC

Latin American Veterinary Conference Oct. 16-19, 2009 – Lima, Peru

Reprinted in the IVIS website with the permission of the LAVC http://www.ivis.org/

LAVC 2009 Página 200

Dr. Michael Fry

COMMON LEUKOGRAM ABNORMALITIES IN DOGS AND CATS

ANEMIA – CLASSIFICATION, LABORATORY EVALUATION, MECHANISMS OF DISEASE

CYTOLOGY BASICS AND COMMON SKIN/SUBCUTANEOUS LESIONS

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Proceedings - Lima, Perú


ANEMIA CLASSIFICATION, LABORATORY EVALUATION,

MECHANISMS OF DISEASE

Anemia refers to subnormal circulating red blood cell mass, characterized by decreases in

one or more (usually all) of these complete blood count (CBC) parameters:

• Erythrocyte concentration • Hematocrit (or packed cell volume) • Hemoglobin concentration

Anemia causes clinical signs referable to decreased oxygen-carrying capacity (e.g., pallor of mucous membranes, lethargy, weakness, exercise intolerance) and may also cause other laboratory abnormalities secondary to tissue hypoxia (e.g., increased liver enzyme activities as a result of hypoxia-induced damage to hepatocytes). Anemia also causes decreased viscosity of the blood, and in marked cases frequently causes heart murmurs as a result of a

decrease in laminar blood flow.

Classification of anemia

Anemia

Non-regenerative Regenerative “Pre-regenerative”

Classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease, as discussed in more detail below (also

see Appendix A).

Impending

reticulocytosis

No

reticulocytosis Reticulocytosis




The hallmark of regenerative anemias (except in horses) is reticulocytosis – i.e., increased concentration of immature erythrocytes in circulation. Reticulocytosis indicates increased erythropoiesis – characterized by erythroid hyperplasia in the bone marrow, and release of erythrocytes into the circulation before they are fully mature (further back in the production

“pipeline”). Thus, reticulocytosis inidicates an appropriate compensatory response to anemia.

Normal reticulocyte counts are species-dependent. Here are some guidelines (ideally, the

reference values should be laboratory-specific):

Reticulocyte reference Reticulocytes �� with

Species values (#/uL) regenerative response?

Dogs & cats ≤ 80,000 Yes

Cattle None Yes

Horses None No

Absence of reticulocytosis indicates that erythropoiesis is being inhibited in some way. There are many potential underlying causes of non-regenerative anemia, as will be discussed

further.

The term “preregenerative” is sometimes used to describe anemia with a regenerative response that is impending, but not yet apparent on the CBC. Confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow

or emergence of a reticulocytosis on subsequent days.

Recall that the stimulus for increased erythropoiesis is increased secretion of erythropoietin (Epo) in response to hypoxemia. Although the action of Epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. One of the main effects of Epo is to expand the pool of early stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. In a case of acute blood loss or hemolysis, it typically takes 3 to 4 days until reticulocytosis is evident on the CBC, and several more days until the regenerative response peaks.




Qualitative v. quantitative assessment of regeneration

Reticulocytes differ from mature erythrocytes in size, content, and staining properties:

Relative Relative Contain Routine New Methylene Blue

RBC type Size [Hgb] rRNA Staining Staining

Mature Normal Normal No Pink Negative

(Normochromasia)

Reticulocyte Larger Lower Yes Purple Positive*

(Polychromasia)

*In cats, only count aggregate (not punctate) reticulocytes.

Polychromatophilic RBC Aggregate reticulocytes Punctate reticulocyte(cat)

The best qualitative indicator of a regenerative response (again, except in horses) is increased polychromasia – i.e., increased numbers of polychromatophilic RBCs. This is accomplished by microscopic examination of a routinely stained, well-made blood smear. Becoming




proficient at evaluating blood smears requires being able to distinguish normal morphology,

abnormal morphology, and artifact – and depends mostly on experience.

Accurately quantifying reticulocytes requires special staining techniques. This is done automatically by some hematology analyzers, and can also be performed manually using new methylene blue (NMB) or a similar stain (see Appendix B). In feline samples, punctate reticulotyes are not counted as reticulocytes, and they have the same appearance as mature

erythrocytes on routine blood smear examination.

MCV & MCHC alterations with regenerative anemias

A strong regenerative response may result in macrocytosis (MCV above the reference limit) and hypochromasia (MCHC below the reference limit), because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. A macrocytic, hypochromic anemia is almost certainly regenerative. However, the large majority of regenerative anemias do not fit this pattern. In most cases of regenerative anemia, even though the concentration of reticulocytes is increased, they comprise only a relatively small proportion of all erythrocytes – and there are not enough of them in relation to the number of mature RBCs to push the MCV and MCHC values into the abnormal range. Here's a hypothetical example in which a normal dog has a hemolytic episode and its red cell mass drops by approximately one-half, and a week later there is evidence of a strong

regenerative response:

Patient Patient (1 week Reference

RBC parameter Unit (baseline) post-hemolysis) values

RBC x 106/uL 7.9 4.0 5.5 - 8.5 Hct % 54 29 45 - 60 Retics x 103/uL 50 400 < 80 MCV fL 68 72 62 - 75 MCHC g/dL 35.4 34.9 34.5 - 36.2




In this case, the regenerative response is associated with an increased MCV and a decreased

MCHC – but both values are still within reference limits.

Of course, whether or not the patient becomes macrocytic & hypochromic depends on many factors, including its baseline MCV and MCHC values, the severity of anemia, the magnitude of the regenerative response, and the size and hemoglobin concentration of the reticulocytes. Most of the time it doesn't happen.

nRBCs and regenerative anemias

Another finding that may accompany regeneration, in addition to reticulocytosis, is the presence of nucleated erythroid cells (nRBCs). However, the presence of circulating nRBCs is not in itself definitive evidence of an appropriate compensatory regenerative response to anemia, and in fact may signify dyserythropoiesis (e.g., because of lead poisoning or bone marrow disease) or splenic dysfunction. When nRBCs are present as part of an appropriate regenerative response to anemia, they should be in low numbers relative to the numbers of

reticulocytes.

Mechanisms of disease: regenerative anemias

Regenerative anemia almost always occurs due to hemorrhage or hemolysis. Some find it useful to remember these as the “2 Hs” or, alternatively, the “2 Ls” (loss or lysis).

In the case of hemorrhage, erythrocytes and the other components of blood escape from the vasculature. Hemorrhage may be acute or chronic, internal or external. Causes of hemorrhage include trauma, abnormal hemostasis, some forms of endo- or ectoparasitism, ulceration, and neoplasia.

In general, regenerative anemias do not occur because of a problem with erythropoiesis, but one that occurs after erythrocytes are made. However, it is important to note that chronic hemorrhage can deplete the body’s iron stores, leading to iron-deficiency anemia, which may be either regenerative or nonregenerative. A regenerative response may occur when the deficiency is resolving or temporarily compensated (e.g., when hemorrhage ceases, or when a patient suddenly gains access to increased dietary or parenteral iron). Nonregenerative

anemias and iron-deficiency anemia specifically, are discussed in more detail below.




Hemolysis may be intravascular—in which case erythrocytes release their contents, mostly hemoglobin, directly into the blood—or extravascular, in which case macrophages phagocytose erythrocytes, and little or no hemoglobin is released into the blood. Both forms (mostly extravascular hemolysis) occur as part of normal homeostasis, and involve pathways to conserve iron and other reusable components in hematopoiesis. However, some diseases are associated with increased destruction of erythrocytes by one, or both, of these

mechanisms.

Here are some clinical and laboratory abnormalities associated with hemolytic anemia:

Abnormality Cause(s)_____________________

Hyperbilirubinemia / icterus Hemolysis (intra- or extravascular)

Other causes (e.g., liver disease,

Cholestasis)

Heinz bodies Hemolysis (intra- or extravascular)

Eccentrocytosis Hemolysis (intra- or extravascular)

Increased MCH & MCHC Intravascular hemolysis

Artifactual hemolysis

Lipemia

Hemoglobinuria Intravascular hemolysis

Splenomegaly Extravascular hemolysis

Other causes (e.g., congestion,

neoplasia)

Spherocytosis Extravascular hemolysis

Autoagglutination Extravascular hemolysis

Mechanisms of disease: nonregenerative anemias

Nonregenerative anemias are characterized by a lack of reticulocytosis on the CBC (recall that reticulocytosis does not occur in horses even in the context of regeneration). Most often the absence of reticulocytosis is due to decreased production in the marrow (i.e., erythroid hypoplasia). The most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease.




The key mediator of anemia of inflammation is the hormone hepcidin, an acute phase protein synthesized mainly in the liver and which was first identified as an antimicrobial peptide. Hepcidin expression increases with inflammation, infection, or iron overload, and decreases with anemia or hypoxia. The bioactive form is a 25-amino acid peptide that exerts its effects by binding to the cell surface iron efflux protein, ferroportin, and inducing its internalization and degradation. The effect of this interaction is to inhibit both absorption of dietary iron

from intestinal epithelium and export of iron from macrophages and hepatocytes.

Iron overload

↑ expression

of hepcidin

Hepatocyte

Inflammation

EnterocyteMacrophage

...

....

..

.

.....

.

Ferroportin

Iron

. .. ..

. ....

Hepcidin binds to ferroportin and causes its internalization and degradation, thus inhibiting efflux of iron into the plasma.

Anemia of inflammation almost certainly involves factors besides decreased iron availability. For example, experimentally induced sterile inflammation in cats resulted in a shortened erythrocyte life span, suggesting anemia of inflammation is also a function of accelerated

erythrocyte destruction.

True (or absolute) iron deficiency has long been recognized as a cause of anemia. Iron deficiency in domestic animals occurs most commonly because of chronic blood loss, and thus loss of iron-rich hemoglobin, and much less frequently due to nutritional deficiency. Although iron deficiency often results in nonregenerative anemia, it is not always so, especially if caused by chronic hemorrhage and when nutritional iron is not a limiting factor. There are many underlying conditions that involve hemorrhage-induced iron deficiency anemia, including primary or secondary gastrointestinal disease (e.g., hookworms, neoplasia, ulceration) and marked ectoparasitism.




The classic hematologic picture with iron deficiency is microcytic (subnormal MCV), hypochromic (subnormal MCHC) anemia. Microcytosis typically develops before hypochromasia. In severe cases, microcytosis and hypochromasia may be discernible on review of a blood smear as RBCs that are abnormally small, or paler staining because of their subnormal hemoglobin concentration, respectively. However, microscopic examination is not

a reliable means of detection, especially in the case of mild abnormalities.

Other causes of decreased erythropoiesis include the following:

• Decreased hormonal stimulation (e.g., � Epo production secondary to chronic renal failure; hypothyroidism)

• Infection of erythropoietic cells (e.g., FeLV) • Toxic insult to the bone marrow – likely to result in decreased hematopoiesis in

general • Other disease involving the bone marrow (e.g., fibrosis, neoplasia) – likely to result in

decreased hematopoiesis in general • Immune-mediated destruction of erythroid precursors – see below • Malnutrition • Inherited conditions

It is important to point out that nonregenerative anemia is not always caused by decreased erythropoiesis. For instance, immune-mediated hemolytic anemia (IMHA) is typically strongly regenerative, but there are also nonregenerative forms of IMHA. Bone marrow findings in dogs with severe nonregenerative IMHA range from a complete absence of erythropoiesis, known as pure red cell aplasia, to erythroid hyperplasia in a majority of patients in one study. The latter situation is an example of ineffective hematopoiesis (in this case, ineffective erythropoiesis), in which cells are being produced at normal or increased levels but are destroyed, presumably because of an immune-mediated mechanism, before they enter the

circulation.

Appendix A: Anemia Classification & Mechanisms of Disease

Regenerative Non-regenerative

CBC hallmarks Reticulocytosis & Absence of reticulocytosis

polychromasia (except horses)

+/- � MCV, � MCHC +/- � MCV, � MCHC (iron def.*)




Causes Hemorrhage Hemolysis

Trauma Extravascular Anemia of inflammation

Hemostasis defect Immune-mediated Iron sequestration

Neoplasia Hemoparasitism � RBC life span

GI ulceration Toxicity

Parasitism Intravascular Altered humoral signaling

Immune-mediated � Epo

Hemoparasitism Endocrinopathies

Toxicity

Enzymatic Ineffective erythropoiesis

Hypophosphatemia Immune-mediated dz

Feline leukemia virus

Myelodysplastic syndrome

Primary production problem

Bone marrow toxicity

Myelophthisis

Pure red cell aplasia

Pancytopenic disorders

Iron deficiency* Iron deficiency*

Notes Evaluate in concert with history, Evaluate in concert with history,

physical exam, other lab data physical exam, other lab data




Fluid shift occurs within hours of Bone marrow evaluation may

acute hemorrhage be indicated

Regeneration takes apx. 3-4 days to *Iron deficiency anemia may be

become evident in blood, apx. 7-10 regenerative or non-regenerative

days to reach maximum response

Appendix B: Manual method of counting reticulocytes

• Mix anticoagulated whole blood and NMB (1:1)

• Incubate for 15 minutes

• Make a blood smear from the mixture

• Review the smear microscopically and count the % of reticulocytes

o In cats, punctate reticulocytes should not be counted

o The more cells counted, the more accurate the estimate (ideally, count 1,000 cells)

• Multiply the reticulocyte % by the RBC count to calculate the reticulocyte concentration:

reticulocytes (#/uL) = reticulocyte % x RBC count (#/uL)

o The RBC count can be determined either by an automated instrument or using a hemacytometer

The disadvantage of expressing the reticulocyte count as a percentage is that the value needs to be corrected for the magnitude of the patient’s anemia:




corrected retic % = uncorrected retic % x (patient Hct ÷ average normal Hct)

Here’s hypothetical data to illustrate the point – the reticulocyte percentage (uncorrected) is

the same in both examples:

Example: Example: Reference

RBC parameter Regenerative Non-regenerative values

RBC (x 106/uL) 4.8 2.4 7.5 – 11.7

Hct (%) 20 10 34 - 48

Reticulocytes

concentration (x 103/uL) 144 (��) 72 (not ��) < 80

% (uncorrected) 3 3

% (corrected to Hct = 40%) 1.5 0.75




Common Leukogram Abnormalities

We will limit our discussion of leukogram abnormalities to some of the most common

patterns seen in dogs and cats:

Categorized by cell type Categorized by process

Neutrophils Inflammation

Mature neutrophilia

Left shift Glucocorticoid-mediated (“stress leukogram”)

Toxic change

Epinephrine-mediated (“physiologic leukocytosis”)

Lymphocytes

Lymphopenia Immunologic response to antigenic stimulation

Lymphocytosis

Reactive lymphocytes

Of course, there are many other leukogram abnormalities ….

First, a little bit of background:

Overview of leukocyte production and function

Granulocytes (neutrophils, eosinophils, basophils) and monocytes have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages); parasiticidal activity and participation in allergic reactions (eosinophils and basophils); and antigen processing and presentation, and cytokine




production (macrophages). Many molecules influence production of these cell types, including cytokines such as granulocyte and granulocyte-monocyte colony- stimulating factors (G-CSF and GM-CSF, respectively) and interleukins (such as IL-3 and IL-6). Inflammatory mediators

stimulate production of cytokines promoting granulopoiesis and monocytopoiesis.

The earliest granulocytic or monocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division to produce 16 to 32 progeny cells. The normal transit time from myeloblast to mature neutrophil is approximately 5 days. In addition, a reserve of neutrophils is maintained in the bone marrow. The size of this so-called storage pool is species-dependent. Neutrophils normally are the predominant leukocyte type in blood of most domestic species. Neutrophils are only in circulation for a short time (less than 12 hours).

T lymphocytes and B lymphocytes are the key effectors of cell-mediated and humoral immunity, respectively. T cells originate in the bone marrow and migrate to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. B-cell development occurs in two phases, first an antigen-independent phase in the bone marrow and ileal Peyer’s patches (the site of B-cell development in ruminants), then an antigen-dependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosal associated lymphoid tissue or MALT).

Trafficking of lymphocytes occurs under the direction of chemokine (chemoattractant cytokine) signals. Once they have migrated to the peripheral lymphoid tissue, lymphocytes may undergo clonal expansion in response to antigenic stimulation. Unlike other hematopoietic cells, which are unidirectional in the blood vessels, lymphocytes travel in both blood and lymphatic vessels, continually recirculating between the two systems. In most species, the majority of lymphocytes in blood circulation are T lymphocytes.

What is normal?

Veterinary laboratories typically provide species-specific hematology reference values based on data from a representative population of clinically normal adults. However, it is important to remember that what is normal may vary not only between species, but also as a function of

other factors, including:

• Age • Breed




• Environment/husbandry • Geographic location • Differences in laboratory methodology.



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