Erythrocytes, Leukocytes, and Platelets

Chapter 22 Erythrocytes, Leukocytes, and Platelets

ERYTHROCYTE DISORDERS

Anemia: Overview

Anemia is characterized by a reduction in the overall erythrocyte content, number of erythrocytes, or hemoglobin concentration. It is one of the most frequent hematologic abnormalities encountered in practice. Anemia is not a disease but rather the reflection of a disease state. Causes of anemia may be divided into three general categories: blood loss, hemolysis, and decreased erythrocyte production.

Determine the cause of the anemia before giving supportive treatment.

Principles of Diagnosis

Several tests can be used to document and characterize the anemia morphologically or etiologically.

Complete Blood Count (Table 22-1)

• Packed cell volume (PCV) is the most accepted and least expensive method of documenting anemia. Spin microhematocrit tubes 5 minutes at a rate of 12,000 to 15,000 g and measure the proportion of the concentrated cell population of the total plasma volume. Evaluate the plasma color after centrifugation. Alternatively, the hematocrit (Hct) can be calculated by multiplying the number of the erythrocytes in millions by the mean cell volume divided by 10. There may be 1 to 3 percentage units increased in PCV over Hct related to trapped plasma.

• Determine plasma protein or total solids by refractometer.

• Erythrocyte, or red blood cell (RBC), count usually is performed by an automated cell counter. This value is necessary for calculation of the erythrocyte indices.

• Hemoglobin (Hgb) concentration in grams per deciliter is determined most accurately by the cyanmethemoglobin method, using a colorimeter or spectrophotometer. Artifactual increases may occur with gross lipemia or an excessive number of Heinz bodies, both of which produce light interference.

• Mean cell or corpuscular volume (MCV) may be determined directly by the automated cell counters, or it may be calculated as follows: MCV (femtoliters) = PCV × 10 divided by RBC (10⁶/μl).

• MCV reflects erythrocyte size and may be categorized as macrocytic, suggesting increased red cell turnover; microcytic, suggesting defective cell growth; or normocytic, unchanging cell size. Note that Akita and Shiba dogs normally have microcytic erythrocytes, while some toy and miniature poodles, as well as greyhounds, may have macrocytic erythrocytes.

• Other erythrocyte indices such as mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) help classify the types of anemia into normochromic and hypochromic. Decreased levels reflect the presence of reticulocytes or abnormal hemoglobinization. Increased levels occur artifactually from hemolysis. MCH may be calculated by dividing Hgb by RBC (10⁶/μl) and multiplying this by 10, but it provides no additional value than MCHC, which is calculated by dividing Hgb by PCV and multiplying by 100.

• Blood smear evaluation involves quantitation of changes in size (anisocytosis), shape (poikilocytosis), and color (polychromasia). The presence of immature nucleated forms and basophilic stippling often signals abnormal erythropoiesis or defective circulation of erythrocyte precursors. Infectious parasites and Heinz bodies often require careful examination of the smear.

Table 22-1 HEMATOLOGY REFERENCE RANGES FOR DOGS AND CATS ^*

Parameter	Canine	Feline
PCV or Hct (%)	37–55	30–45
Hemoglobin (g/dl)	12–18	8–15
RBCs (×10⁶/μl)	5.5–8.5	5–10
MCV (fl)	60–75	40–55
MCHC (g/dl)	32–36	30–36
Reticulocytes (×10³/μl)	<80	<60 aggregate
Platelets (×10³/μl)	200–900	300–700
WBCs (×10³/μl)	6–17	6–18
Segmented neutrophils (×10³/μl)	3–12	3–12
Band neutrophils (/μl)	0–300	0–300
Lymphocytes (/μl)	1000–5000	1500–7000
Monocytes (/μ)	150–1350	50–850
Eosinophils (/μl)	100–1250	100–1500
Basophils (/μ)	<100	<100
Plasma protein (g/dl)	6.0–8.0	6.0–8.0
Fibrinogen (mg/dl)	200–400	150–300

Hct, hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume; RBCs, red blood cells; WBCs, white blood cells.

Source: Purdue University, Veterinary Clinical Pathology Laboratory.

* These values are only meant as a guide; individual laboratories may vary in their ranges, depending on instrumentation and regional differences in animal populations.

The most important part of the complete blood count (CBC) is an evaluation of the blood smear for morphologic abnormalities.

Reticulocyte Count

This is the most accurate method to evaluate regeneration. Perform reticulocyte counts when the PCV falls below 30% in dogs and 20% in cats.

• Incubate equal volumes of blood and 0.5% new methylene blue stain together for 15 to 20 minutes; then make a routine blood smear and count the number of aggregate and punctate forms per 1000 erythrocytes. Absolute numbers of reticulocytes per microliter are calculated by the following formula:

• Dogs normally have 0% to 1% reticulocytes (0–80,000/μl). In healthy cats, aggregated reticulocytes range from 0% to 0.4% (0-60,000/μl) and punctate reticulocytes are less than 5% (<500,000/μl). Aggregate reticulocytes represent the most active or recent form of regeneration in the dog and cat. Punctate reticulocytes in cats, which do not stain as polychromatophilic cells, represent regenerative attempts that occurred 2 to 4 weeks previously.

• By using the PCV instead of the erythrocyte count, a corrected reticulocyte percentage may be calculated as follows:

• Normal PCV is considered 45% for dogs and 35% for cats. A corrected percentage for a reticulocyte value greater than 1% indicates increased RBC regeneration. Once initiated at the bone marrow level, regeneration may take at least 3 days before reticulocytes appear in the circulation.

Fecal Examination and Urinalysis

These tests are performed to determine sources of blood loss and function of the kidney.

Biochemical Profile

Biochemical profiles may reveal organic disease that can affect the ability to regenerate erythrocytes.

Bone Marrow Evaluation

Bone marrow evaluation is necessary whenever regenerative attempts appear diminished. Hemolytic conditions do not warrant marrow examination unless the response is less than normally expected. Results always must be compared with a concurrent CBC to determine the current status of regeneration.

Perform bone marrow evaluation by obtaining both aspirate and core biopsies together because each provides different information.

• This is a sterile procedure that may be performed with only local anesthesia, sedation with reversible analgesia, or general anesthesia.

• Sites frequently used in dogs include the dorsal ilium, humerus (best for obese animals), and femur (small dogs or puppies). Sites frequently used in cats include the femur and dorsal ilium by a transilial or perpendicular approach.

Principles of Transfusion Therapy

Therapy depends on the etiology of the anemia. Rapid decreases in PCV warrant replacement of whole blood. However, slow daily decreases in PCV of 1% to 3% may not cause clinical signs of dyspnea or weakness. Whole blood transfusion is discussed here, and blood component therapy is discussed in Chapter 23.

Cross-matching

Perform cross-matching before transfusion. The procedure, which involves collection of erythrocytes and serum or plasma from the donor and the patient, is as follows:

• Wash erythrocytes and pellet 3 times in 0.9% saline.

• Make a 4% cell suspension by adding 4.8 ml of saline to 0.2 ml of cells.

• Measure compatibility between donor and patient optimally at three temperatures: 37°C, 20°C, and 4°C.

Perform cross-matching prior to all blood transfusions to prevent incompatibility reactions.

• Donors are incompatible if any agglutination or hemolysis occurs in the major cross-match. The types of cross-match are defined as follows:

• Major: 100 μl donor cells + 100 μl patient serum/plasma

• Minor: 100 μl patient cells + 100 μl donor serum/plasma

• Donor or patient controls: 100 μl of donor or patient cells to 100 μl of donor or patient serum/plasma, respectively

• Under emergency conditions, donors may be used if mild agglutination occurs in the minor cross-match.

Whole Blood Transfusion

• In dogs, this procedure is best performed using animals negative for the dog erythrocyte antigen DEA-1 and DEA-7 blood types.

• Although most cats have type A blood, it is best to cross-match blood, because as little as 1 ml of mismatched blood can cause a fatal transfusion reaction in cats.

• Estimated dosage is 10 to 20 ml/kg in dogs or cats, with a maximum of 40 ml in an adult cat. A more specific guideline for the amount of donor blood in anticoagulant (ml) is as follows: kg × 80 (dog) or 60 (cat) × (desired PCV − patient PCV) divided by donor PCV.

• When performing whole blood transfusion, use appropriate filters and administration sets to retain clotted blood or debris.

• Warm blood before administration.

• For the first 30 minutes, keep the initial rate of administration slow (0.25 ml/kg) to observe any incompatibility reactions (recommended rate of whole blood infusion in general is 10 ml/kg/hour). The rate of infusion depends on the hydration status of the patient.

• Evaluate the hematocrit before and after transfusion to document relative improvement and to screen for hemolysis as an indicator of incompatibility.

Blood Loss Anemia

Large volumes of blood must be lost before appreciable changes occur in the numbers of erythrocytes or PCV. The immediate loss of blood results in little or no change to the PCV owing to the concurrent loss of erythrocytes and plasma fluids. Over the course of several hours to days, redistribution of fluids occurs, resulting in a lowered PCV and plasma protein. Depending on the amount of blood lost and the time period over which it is lost, regenerative responses may range from moderate to extremely weak. Chronic blood loss causes decreased iron stores, resulting in decreased erythrocyte production.

Treatment

Administer cross-matched whole blood for acute hemorrhage when the PCV drops to 20% to 25% in the dog or cat. Also consider autotransfusion if hemorrhage has occurred recently in a body cavity and the blood is not contaminated with bacteria or neoplastic cells.

Hemolytic Anemia

Causes of hemolytic anemia include congenital abnormalities, immune-mediated destruction, infections, chemical or toxic agents, mechanical fragmentation, and hypophosphatemia. The net effect of hemolytic loss of erythrocytes is often a very strong to moderate regenerative response (Fig. 22-1). However, in some cases, the anemia may occur so rapidly that the animal may have too little time to mount a regenerative response by the time the condition is recognized.

Figure 22-1 Diagnostic approach to common regenerative anemias in dogs and cats.* See Figures 22-2 and 22-3. (APTT, activated partial thromboplastin time; FDP, fibrin degradation product; PT, prothrombin time.)

A period of 2 to 3 days is necessary for erythrocyte maturation and reticulocytosis once the stimulation from hypoxia or severe erythrocyte depletion has occurred.

• Depending on the severity or acuteness of the destruction and the conjugating ability of the liver, icterus may or may not be present.

• Plasma protein concentrations usually are normal.

Congenital Erythrocyte Abnormalities

Pyruvate Kinase Deficiency

Pyruvate kinase deficiency is an erythrocyte enzyme deficiency associated with an autosomal recessive inheritance. During anaerobic glycolysis, pyruvate kinase is necessary for the production of adenosine triphosphate (ATP), which erythrocytes need to maintain their integrity and viability.

• The condition has been documented in several dog breeds, including the basenji, beagle, West Highland white terrier, Cairn terrier, American Eskimo dog, miniature poodle, Chihuahua, and pug.

• It has been reported in Abyssinian, Somali, and domestic shorthaired cats.

Diagnosis

This disease is suggested clinically by the presence of an intense reticulocytosis (15-50%), along with frequent nucleated red cells in a young dog. Echinocytosis or erythrocytes with sharp projections unrelated to crenation may be seen in circulation.

• Polymerase chain reaction (PCR) based genetic tests are currently available for the basenji, West Highland white terrier, and Cairn terrier to detect affected and carrier animals.

• Definitive diagnosis for other breeds requires a special erythrocyte assay for the enzyme.

Phosphofructokinase Deficiency

Phosphofructokinase deficiency has been identified in English springer and American cocker spaniels as an autosomal recessive inheritance. This glycolytic enzyme deficiency causes a decrease in 2,3-diphosphoglycerate (2,3-DPG) concentration, resulting in an increase in intracellular pH. Erythrocytes from affected animals are especially fragile under alkaline conditions.

• Intravascular hemolysis may occur during hyperventilation-induced alkalemia, resulting in periodic bouts of hemoglobinuria and sometimes bilirubinuria.

• A constant state of hypoxia is produced by the low levels of 2,3-DPG, with subsequent increased oxygen binding to hemoglobin.

• The result is persistent reticulocytosis (10-30%) even though the PCV is normal or mildly decreased, except during hemolytic episodes.

Clinical Signs.

The clinical signs may involve lethargy, pale or icteric mucous membranes, mild hepatosplenomegaly, myopathy, and fever. Dogs may have a normal life span if properly managed.

Diagnosis.

The enzyme deficiency is diagnosed by specialized erythrocyte assays in homozygous and heterozygous animals. A PCR-based test is available to screen for affected and carrier animals of any age.

Hereditary Stomatocytosis

Hereditary stomatocytosis is an autosomal recessive trait in Alaskan malamutes associated with chondrodysplasia. The defect may involve abnormalities of the cell membrane or of ion transport, leading to increased water content and a larger cell size (96 fl). Morphologically, some erythrocytes appear to have a slit-like or mouth-like area of central pallor.

• Dwarfism is recognized clinically in affected Alaskan malamutes, and there is a mild hemolytic anemia and slight reticulocytosis (2%). Erythrocyte numbers are reduced, but the PCV remains normal because of enlarged erythrocytes.

• Other breeds associated with congenital stomatocytosis include Drentse Patrijshonds and miniature and giant schnauzers.

Hereditary Non-spherocytic Hemolytic Anemia

Hereditary non-spherocytic hemolytic anemia is recognized in two breeds: poodles and beagles. In both breeds, erythrocytes are morphologically normal and enzyme activities are not affected.

Poodles.

Poodles develop an autosomal dominant condition that resembles pyruvate kinase deficiency, but decreases in this enzyme are not demonstrable. Affected animals have a persistent macrocytic hypochromic anemia (PCV of 13-31%) with moderate reticulocytosis. It is clinically evident by 1 year of age and fatal by 3 years of age. Myelofibrosis, osteosclerosis, and hemosiderosis are present at death.

Beagles.

In beagles, the condition presents with chronic anemia and moderate reticulocytosis. The mode of inheritance is likely autosomal recessive. The disease is less severe in beagles than in poodles, and the disorder can remain subclinical for up to 4 years in affected dogs. The short erythrocyte life span in beagles may be caused by an unidentified membrane abnormality.

Immune-Mediated Hemolytic Anemia

See the chapter on systemic immune-mediated diseases (Chapter 24).

Infectious Causes of Hemolysis

Hemoplasmosis

Hemoplasmosis or hemotrophic mycoplasmosis, previously known as hemobartonellosis, is caused by a mycoplasma organism that infects the erythrocytes of dogs (Mycoplasma haemocanis) and cats (M. haemofelis and “Candidatus M. haemominutum”). It can be transmitted by ticks and fleas or by queens to their newborn kittens in the absence of blood-sucking arthropods. Splenectomy (especially in the dog), immunosuppression caused by glucocorticoid therapy, or stress in cats with latent M. haemofelis infection predispose to the development of clinical disease in the animal infected by the organism.

Clinical Signs.

Clinical signs include those generally observed with anemia. Weight loss may occur if the anemia develops slowly, whereas acute, severe anemia produces sudden depression and icterus. Splenomegaly often is noted.

Diagnosis

Laboratory findings generally reflect a strong regenerative response in the CBC. The absence of marked anisocytosis or polychromasia in confirmed cases of feline hemoplasmosis may suggest peracute infections with too little time to respond or concurrent infection with feline leukemia virus (FeLV) or feline immunodeficiency virus (FIV).

• Thin smears, made directly without anticoagulant, assist in finding the epicellular organisms. Blood films should be well stained without precipitate to minimize any confusion in identifying the parasite.

• Cats often have coccoid or ring shapes, whereas dogs mostly have linear chain forms.

• A positive Coombs test result may be associated with feline hemoplasmosis, presumably because of erythrocyte membrane alteration.

• PCR-based assays are available for the diagnosis of hemoplasma infections in cats.

Babesiosis

Babesiosis is a tick-borne protozoal disease affecting erythrocytes of dogs (Babesia canis, B. gibsoni). B. canis is common in kennel conditions and is associated especially with greyhounds. B. gibsoni has been associated with pit bull and American Staffordshire terriers. Babesia felis has not been reported in North American cats but is reported in Africa and southern Asia. Concurrent infections with other tick-borne agents such as Ehrlichia canis are common.

Clinical Signs.

Clinical signs in babesiosis range from an acute hemolytic crisis to an unapparent subclinical form. Signs of the acute disease include splenomegaly, icterus, anemia, thrombocytopenia, hemoglobinuria, and fever.

Leptospirosis

Leptospira serovars can cause kidney disease, liver disease, hemolysis, icterus, and coagulopathies. Toxins released from proliferating organisms interfere with cellular metabolism and damage cells. The clinical findings, diagnosis, and treatment are discussed in Chapter 19.

Chemical or Toxic Injury of Erythrocytes

Oxidant-Induced (Heinz Body) Anemia

Heinz body anemia is produced by oxidant agents that cause precipitation of hemoglobin. Oxidative injury to the iron moiety of hemoglobin (methemoglobinemia) is discussed in a separate section later in this chapter. Accelerated red cell destruction involves intravascular fragmentation or extravascular phagocytosis by the spleen.

• Eccentrocytosis is another erythrocyte change associated with oxidative damage resulting in hemolysis, which occurs from fusion of the erythrocyte membrane, producing a thin, colorless portion or hemighost on one side of the cell and a dense, hemoglobin portion on the opposite side. This has been associated with onion and garlic ingestion in dogs.

Zinc Toxicity

Zinc toxicity can produce intravascular hemolysis in dogs. Sources of the zinc include ingestion of galvanized wire or kennel cage nuts and pennies produced since 1983.

• Diagnosis involves the presence of a regenerative anemia and proof of zinc exposure. Spherocytosis and Heinz bodies have been noted in some cases on blood smear evaluation.

• Radiograph the abdomen to identify metallic foreign bodies in the GI tract and remove the source of zinc via surgery or endoscopy.

Mechanical Fragmentation of Erythrocytes

Disseminated Intravascular Coagulopathy

Disseminated intravascular coagulopathy may cause a microangiopathic hemolytic anemia because of fibrin deposition that result from damage to small blood vessels. Mechanical fragmentation of erythrocytes appears in blood smears as schizocytes or spherocytes.

• Etiology involves a variety of conditions, such as neoplasia, infections, necrosis, toxins, and immune complex formation. These can lead to massive tissue destruction and activation of clotting pathways.

• Diagnosis is based on consumption of platelets (thrombocytopenia), reductions in clotting factors (prolonged prothrombin time [PT], activated partial thromboplastin time [APTT], hypofibrinogenemia), and increased amounts of fibrin degradation products (see Chapter 23).

• Treatment is aimed at the inciting condition (see Chapter 23).

Hypophosphatemia

Cases of feline diabetes mellitus and hepatic lipidosis have been associated with hypophosphatemia-induced hemolysis. Heinz body formation may play a role in the development of hemolytic anemia in these cases.

Nonregenerative Anemia

Nonregenerative anemia generally is related to direct toxicity of erythroid precursors in the bone marrow or secondary suppression of erythropoiesis (Fig. 22-2). Often neutrophils and platelets also are affected (Fig. 22-3).

• Reduction in erythrocytes, leukocytes, and platelets in circulation is termed pancytopenia.

• The term aplastic anemia is used to describe bone marrow failure involving the three cell lines that leads to peripheral pancytopenia. The bone marrow in this condition is severely hypoplastic, with total or near complete replacement of hematopoietic elements by adipose tissue.

• In contrast, crowding from abnormal cellular infiltrates can suppress hematopoiesis, producing myelophthisis.

Figure 22-2 Diagnostic approach to common nonregenerative anemias in dogs and cats when only the erythrocyte line is involved. (FeLV, feline leukemia virus; MDS, myelodysplastic syndrome; M/E, myeloid-to-erythroid ratio; PCR, polymerase chain reaction; TIBC, total iron-binding capacity.)

Figure 22-3 Diagnostic approach to common nonregenerative anemias in dogs and cats when multiple cytopenias are present. (AML, acute myeloid leukemia; CML, chronic myeloid leukemia; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; MDS, myelodysplastic syndrome; PCR, polymerase chain reaction; RMSF, Rocky Mountain spotted fever).

Chronic, nonregenerative anemias may not require blood transfusion until the PCV falls below 15% in the dog and 12% in the cat.

Causes of nonregenerative anemia include infectious agents, nutritional disturbances, organic disease, endocrine abnormalities, toxic agents, myelophthisis, irradiation, and immune-mediated destruction.

Infectious Agents

Feline Leukemia Virus

FeLV-associated anemia is usually normochromic and normocytic to macrocytic. Nonregenerative anemia results from bone marrow suppression caused by both primary infection of hematopoietic stem cells and infection of the supporting stromal cells.

• Severe red cell hypoplasia is most common and is associated with depletion and arrest of maturation of erythroid precursors in the bone marrow.

• Approximately 10% of the cases are hemolytic with regeneration related to immune destruction of erythrocytes.

• One type of virus-induced anemia is associated with erythrocyte macrocytosis and is thought to be related to a defect in mitosis during erythrocyte maturation.

Diagnosis.

Diagnosis is based on positive enzyme-linked immunosorbent assay (ELISA) or IFA test results (see Chapter 8). Bone marrow aspirate smears or core biopsy sections indicate decreased cellularity with increased fat infiltration when the infection is not associated with a proliferative leukemia.

Treatment.

Treatment varies depending on concurrent conditions such as neoplasia, hemoplasmosis, FIV, or feline infectious peritonitis (FIP). Supportive care, in the form of blood transfusions (see “Principles of Transfusion Therapy”), antibiotics, appetite stimulants, and anabolic steroids, is often necessary (see Chapter 8 for additional information on FeLV).

Other Viruses

Other viruses, such as FIP, FIV, feline panleukopenia virus, canine parvovirus, and canine distemper virus, have been associated with suppression of erythropoiesis. The anemia is often mild to moderate and nonresponsive. Other cell lines, such as granulocytes or lymphocytes, are affected more severely. Diagnosis usually is determined by exposure history, clinical signs, virologic or serologic tests, characteristic histopathologic lesions, and identification of viral particles or inclusions within affected cells. In canine distemper virus, erythrocytes or leukocytes may contain one or more pale blue spots in the cytoplasm with Romanowsky-type stains. However, these inclusions stain a deep purple color with aqueous-based Wright stains (e.g., Diff-Quik).

Ehrlichiosis and Anaplasmosis

Ehrlichia spp. and Anaplasma spp. are tick-transmitted rickettsial infections that produce chronic polysystemic disease in dogs and cats associated with hyperglobulinemia, thrombocytopenia, mild to moderate non-regenerative anemia, and sometimes leukopenia or pancytopenia in the later stages. The epidemiology, clinical manifestations, diagnosis, and treatment of these infections are described in Chapter 17.

• Intracytoplasmic morulae are found infrequently in mononuclear cells (E. canis, Ehrlichia chaffeensis) or granulocytes (Ehrlichia ewingii, Anaplasma phagocytophilum), during the acute stage of the disease. Bone marrow, at this time, is often normocellular to hypercellular with increased numbers of megakaryocytes and plasma cells.

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Aug 27, 2016 | Posted by admin in SMALL ANIMAL | Comments Off

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