Erythropoiesis
Erythrocytes are produced in the bone marrow, largely under the influence of erythropoietin (EPO), with increased erythropoiesis occurring in response to blood loss or hemolysis.
Horses are unique in that equine erythrocytes generally remain in the bone marrow until fully mature before they are released into circulation. As a result, peripheral indicators of regeneration used in other species such as polychromasia and reticulocytosis are typically absent or unreliable in the horse. Detection of relatively low numbers of reticulocytes in the peripheral blood of anemic horses, however, has been described using an automated hematology analyzer, which may be a more sensitive means of detecting these cells than the manual methods used previously.1-5Laboratory Evaluation of Erythrocytes
General reference intervals are presented for common erythrocyte parameters in large animals (Table 24.1); however, laboratory- or instrument-specific reference intervals should be used whenever possible. A complete discussion of age- and breed-related influences on erythrocyte parameters is found elsewhere.6-8 What follows is a brief review of common laboratory evaluators of the erythron.
Hematocrit, Packed Cell Volume, Hemoglobin Concentration, and RBC Count
Hematocrit (HCT), packed cell volume (PCV), hemoglobin (Hgb) concentration, and red blood cell (RBC) count are all ways of quantifying the erythrocytes in peripheral blood, and these measures tend to increase (erythrocytosis) or decrease (anemia) together. HCT and PCV are similar in that they describe the percentage volume of whole blood occupied by erythrocytes. The HCT is a calculated value and is a function of the mean cell volume and RBC count, whereas the PCV is determined by centrifugation of whole blood in a microhematocrit tube. The Hgb concentration and RBC count are measured values generated by hematology analyzers.
Mean Cell Volume
The mean cell volume (MCV) is directly measured by most hematology analyzers and represents the average erythrocyte volume. An increased MCV can be the result of a regenerative anemia in the ruminant and occasionally in the horse. Decreased MCV is seen with iron-deficiency anemia (also copper) and can also be seen in healthy calves and foals.3,4,7,8
Mean Cell Hemoglobin Concentration and Mean Cell Hemoglobin
The mean cell hemoglobin concentration (MCHC) is the cellular Hgb concentration per average erythrocyte and is calculated from the Hgb concentration and HCT. A decreased MCHC can be seen in ruminants with regenerative anemias, but this would not be expected in the horse. Both horses and ruminants can have decreased MCHC as the result of iron- deficiency anemia. A low MCHC has also been reported in
■ TABLE 24.1
Reference Intervals for Ruminants and Horses
| Cattlea | Sheepb | Goatsb | Horses (Hot-Blooded Breeds)b | Horses (Cold-Blooded Breeds)b | |
| PCV/HCT (%) | 22-33 | 27-45 | 22-38 | 32-53 | 24-44 |
| Erythrocytes (?106∕μL) | 5.1-7.6 | 9-15 | 8-18 | 6.8-12.9 | 5.5-9.5 |
| Hemoglobin (g/dL) | 8.5-12.2 | 9-15 | 8-12 | 11-19 | 8-14 |
| MCV (fL) | 38-50 | 28-40 | 16-25 | 37-58.5 | — |
| MCHC (g/dL) | 36-39 | 31-34 | 30-36 | 31-38.6 | — |
| Reticulocytes | — | 0 | 0 | — | — |
aFrom George JW Snipes J, Lane M: Comparison of bovine hematology reference intervals from 1957-2006.
Vet Clin Pathol 39:138, 2010. bFrom Jain NC: Schalm,s veterinary hematology, ed 4, Philadelphia, 1986, Lea & Febiger.PCV, Packed cell volume; HCT, hematocrit; MCV, mean cell volume; MCHC, mean cell hemoglobin concentration.
calves (from these organisms in some cases.
NUCLEATED ERYTHROCYTES. Nucleated erythrocytes (nRBCs) are not normally seen in the peripheral blood of healthy horses or ruminants. In ruminants, small numbers associated with regenerative anemias can sometimes be present but should be accompanied by a significant degree of polychromasia. Bone marrow damage (e.g., toxic damage, hypoxia, neoplasia) can also result in circulating nRBCs. The presence of nRBCs and basophilic stippling in the absence of significant anemia should prompt consideration of lead toxicity (more common in ruminants).
BASOPHILIC STIPPLING. Basophilic stippling is seen as multiple small basophilic inclusions within erythrocytes and is the result of staining of ribosomal aggregates. Basophilic stippling is often seen in association with regenerative anemias in ruminants. As described previously, it can also be seen in cases of lead toxicity.
HEINZ BODIES. Heinz bodies form in erythrocytes as a result of oxidative damage that leads to precipitation of Hgb. They are recognized on Wright-stained blood smears as rounded, pale pink staining structures that protrude from the edge of erythrocytes. Heinz bodies can be confirmed by visualizing them with use of a new methylene blue stain that stains Heinz bodies as dark, rounded structures associated with the erythrocyte membrane. Ingestion of oxidants such as wilted red maple leaves, onions, and garlic; phenothiazine; Brassica spp.; and copper toxicity can result in Heinz body formation in large animals. Heinz body formation causes affected erythrocytes to be more susceptible to both intravascular and extravascular hemolysis.
ECCENTROCYTES. Eccentrocytes also form as a result of oxidative damage.
In the case of eccentrocytes, oxidative damage results in partial fusion of the erythrocyte membrane causing displacement of Hgb contents to one side of the erythrocyte. Oxidants listed as causes of Heinz bodies can also result in eccentrocyte formation. Eccentrocytes have also been reported in horses with glucose-6-phosphate dehydrogenase deficiency and flavin adenine dinucleotide deficiency. Both are rare erythrocyte enzyme deficiencies that result in the affected animal's erythrocytes being more susceptible to oxidative damage by endogenous or exogenous sources.10ROULEAUX. Rouleaux formation is seen as linear stacking of erythrocytes on a peripheral blood smear. Rouleaux formation is common in healthy horses. A consequence of rouleaux formation is that equine blood samples tend to have a relatively high erythrocyte sedimentation rate, and as a result whole blood samples should be thoroughly mixed before evaluation. Rouleaux formation is uncommon in healthy ruminants but can be seen with increased plasma proteins such as fibrinogen and immunoglobulins. Increases in these plasma proteins can also enhance rouleaux formation in horses. In any species, care should be taken to differentiate rouleaux from erythrocyte agglutination.
AGGLUTINATION. Agglutination is the result of surface - associated antibody on erythrocytes causing clumping. When viewed microscopically on a peripheral blood smear, aggregation produces clumps of erythrocytes that are described as having the appearance of “clusters of grapes.” Agglutination is significant in that it is indicative of immune-mediated hemolytic anemia (IMHA). In some cases, it may be difficult to differentiate prominent rouleaux from agglutination. A saline dilution test can be done to confirm true agglutination. This test is accomplished by diluting whole blood with saline and evaluating a wet preparation. Rouleaux will disperse with dilution, whereas true agglutination will persist. Generally a 1 : 2 to 1 : 4 dilution will disperse rouleaux; however, occasionally higher dilutions are needed. Agglutination confirmed by a saline dilution test is generally considered to preclude the need for Coombs testing. Agglutination in the horse has also been described as a result of treatment with unfractionated heparin.11
Erythrocyte Parasites
A variety of erythrocyte parasites can be detected by review of peripheral blood smears. Examples include Mycoplasma spp., Anaplasma spp., Theileria spp., and Babesia spp.12
Coombs Test
The Coombs test or direct antiglobulin test is used to detect erythrocyte surface-associated antibody. Positive Coombs test results support a diagnosis of IMHA (including neonatal isoerythrolysis). A negative Coombs test result, however, does not rule out IMHA because this test is known to have a relatively low sensitivity (i.e., many false negatives).