Bone Marrow Collection

Bone marrow evaluation is an underutilized technique in veterinary medicine. Any veterinarian with the easily obtained proper supplies can collect bone marrow. For a diagnostic technique so full of potential rewards, the risks are minimal. As with any other diagnostic test, proper patient selection is important to avoid performing an unnecessary procedure.

Equipment and Supplies

Most hospitals will have, or can easily obtain, the supplies required to collect a bone marrow sample. These supplies include a bone marrow biopsy needle, chemical restraint, surgical scrub, sterile fenestrated surgical drape, slides, anticoagulant, a 12-mL syringe, a dozen glass microscope slides, sterile gloves, and a scalpel blade. If a core biopsy will be performed, tissue fixative such as 10% formalin will be required. Local anesthesia will be used for patients in which chemical restraint is contraindicated. Other supplies that may be useful, but are not required, include pipettes and microhematocrit tubes.

Several types of bone marrow needles are available (Figure 25-1). An 18-gauge needle is an appropriate size for collecting marrow from a cat. The Jamshidi and Rosenthal needles are made of stainless steel and can be heat sterilized. The Illinois needle contains plastic and requires gas or cold sterilization. The presence of a stylet in the needle keeps the lumen from becoming plugged with a core of cortical bone at the beginning of the procedure. The stylet must be completely in place until the actual sample is collected, or a frustrating obstruction of the needle will occur. For hospitals without a bone marrow needle, an 18-gauge venipuncture needle may be substituted. Because there is no stylet to keep the lumen open, an obstruction of the venipuncture needle is likely. This means a second needle will be needed, and dexterity will be required to find the hole in the cortical bone made by the first needle.

FIGURE 25-1 Bone marrow biopsy needles. Left to right, Jamshidi disposable core-aspiration biopsy needle (11-gauge, 4 inches), stylet for the Jamshidi biopsy needle, Rosenthal reusable core-aspiration biopsy needle (16-gauge, inches), stylet for the Rosenthal needle.

Bone marrow collection is a painful procedure. The struggling of an uncooperative and anxious patient makes collecting a diagnostic sample difficult. It may also be unethical to put a cat through unnecessary pain and anxiety when chemical restraint is available; trauma to assistants can also be avoided. If chemical restraint is not appropriate, a local anesthetic can be used to minimize the pain of passing the needle through the skin to the periosteum. Cortical bone itself has no pain receptors. Unfortunately, the endosteum cannot be anesthetized, and most of the pain of this procedure occurs when the endosteum is torn during sample collection.

Bone marrow clots readily when collected. The use of an anticoagulant is recommended so that there is no rush to process the sample after collection. A 2.5% solution of ethylenediaminetetraacetic acid (EDTA) can be made by injecting 0.35 mL of sterile saline into a 3-mL lavender-top EDTA blood collection tube. The contents are withdrawn and injected into a second EDTA tube.⁷¹ The resulting 0.5 mL volume is placed in a 12-mL syringe and should be adequate for preventing coagulation of the collected marrow sample.

Collection Sites

Bone marrow can be collected from one of three sites: the proximal humerus (Figure 25-2), the iliac crest, or the femur (Figure 25-3). The proximal humerus is easily accessible, has little overlying tissue, and offers a large surface for needle placement. The greater tubercle is palpated, and the needle inserted into the flat surface of the craniolateral humerus just distal to the tubercle. The needle is inserted in a craniomedial direction perpendicular to the long axis of the bone.

FIGURE 25-2 Bone marrow collection site from the proximal humerus.

(Redrawn from Grindem CB: Bone marrow biopsy and evaluation, Vet Clin North Am 19:674, 1989.)

FIGURE 25-3 Bone marrow collection sites from the iliac crest and the proximal femur.

(Redrawn from Grindem CB: Bone marrow biopsy and evaluation, Vet Clin North Am 19:673, 1989.)

The iliac crest may be wide enough only in large cats and is difficult to palpate in obese patients. The needle is directed ventrally and slightly medially into the most dorsally palpable portion of the ilium, where the bone is widest. Occasionally, the needle will come to rest against the opposing cortical wall. If aspirating a sample from the iliac crest is difficult, the clinician should withdraw the needle slightly before concluding that the procedure is a failure.

The proximal femur is an easily accessed site for collecting marrow from cats. The greater trochanter is palpated and the needle inserted into the trochanteric fossa medial to the trochanter. The needle is directed parallel to the long axis of the femur. A potential, but uncommon, complication with using this site is damage to the sciatic nerve running medial and caudal to the greater trochanter. There should be plenty of room, however, to obtain the sample while leaving the nerve untouched.

Cross-Match

A crossmatch test can identify compatibility or incompatibility between the blood donor and transfusion recipient. It tests for the presence of alloantibodies, induced or naturally occurring, for which blood typing does not test. At the present time, typing for the Mik erythrocyte antigen is not readily available. Other, unknown erythrocyte antigens may be present and cause blood incompatibility. Because of the potential presence of unknown alloantibodies in feline blood, a cross-match should be performed before any transfusion, even if both donor and recipient are blood typed and a prior cross-match has indicated compatibility.

The major cross-match tests for alloantibodies in the plasma of the recipient that may hemolyze the donor’s red cells. The minor cross-match tests for alloantibodies in the plasma of the donor that may attack the recipient’s erythrocytes. Autoagglutination in the major cross-match predicts that antibodies in the recipient’s plasma will attack the donor’s red cells, likely eliciting a transfusion reaction. A minor cross-match incompatibility suggests that antibodies in the donor’s blood will attack the recipient’s red cells. Incompatible blood should not be used for transfusions.

A quick means of performing a major cross-match is to mix 2 drops of plasma from the recipient with 1 drop of anticoagulated blood from the donor on a slide at room temperature.³⁵ The opposite will be a minor cross-match. Development of macroscopic agglutination within a minute suggests the presence of anti-A alloantibodies in the plasma sample of the recipient (major cross-match) or donor (minor cross-match). In either case the blood is incompatible. Autoagglutination can make interpretation of the test difficult. Running a control test using saline instead of plasma may help with interpretation.

For hospitals performing frequent transfusions, a standardized gel agglutination test is available for in-hospital use. Although more time consuming than the previously described method, the gel test is less vulnerable to operator interpretation error. Because it is stable, the test result can be saved and reviewed at a later time if needed.¹¹⁸ Two commercially available products include the DiaMed-ID cross-match gel (DiaMed, Switzerland) and the Rapid Vet-H companion animal cross-match gel (DMS Laboratories, Flemington, New Jersey). More rigorous and time-consuming methods involving washing, centrifuging, and incubating samples have been published.^25,42

Slide Agglutination Test

A positive, properly performed slide agglutination test suggests the presence of antibody-coated erythrocytes and negates the need for performing a direct Coombs’ test. It is important to differentiate erythrocyte clumping caused by autoagglutination from that caused by rouleaux formation. These types of erythrocyte clumping are differentiated by washing the cells with saline, which will reliably break up clumps formed by rouleaux. A quick method of performing the test is to mix a drop of EDTA anticoagulated blood on a slide with 2 to 5 drops of 0.9% NaCl followed by a gross and microscopic examination of the sample.⁵⁴ The “stacked coins” appearance characteristic of rouleaux formation (Figure 25-4) will disperse, whereas the random or rosette clumping of autoagglutination will not (Figure 25-5). If the test is negative, a direct Coombs’ test should be requested. An important limitation to this test is its inability to separate primary from secondary immune-mediated disease.

FIGURE 25-4 The “stack of coins” arrangement associated with rouleaux formation.

(Courtesy Rick Cowell.)

FIGURE 25-5 Macroscopic agglutination is apparent on the slide. If clumping remains after proper washing, autoagglutination would be the conclusion.

(From Fry MM, McGavin MD: Bone marrow, blood cells, and lymphatic system. In McGavin MD, Zachary JF, editors: Pathologic basis of veterinary disease, ed 4, St Louis, 2007, Mosby.)

Quantitative Erythrocyte Parameters

Erythrocytes can be classified by their size and hemoglobin concentration on the basis of quantitative parameters such as the mean corpuscular volume (MCV, the average cell size), the red cell distribution width (RDW), and the mean corpuscular hemoglobin concentration (MCHC). Macrocytosis, normocytosis, and microcytosis refer to cell size above, within, or below the reference range, respectively. The RDW is derived from the cell numbers versus cell volume histogram (Figure 25-7); an increase in RDW indicates a greater than normal variation in cell size. The RDW may be artifactually affected by the overlap in size between feline platelets and red cells. Normochromia and hypochromia refer to MCHC within or below the reference range, respectively. Hemoglobin makes up approximately 33% of the volume of the cell. Erythrocytes cannot carry more hemoglobin in their cytoplasm than normal, so they cannot be hyperchromic. An increased MCHC is usually associated with hemolysis, either a result of disease or of improper venipuncture or sample handling. A change in any of these parameters requires a review of a blood smear for an explanation.¹²⁶

FIGURE 25-7 The erythrocyte volume distribution histogram. The mean cell volume is represented by the vertical bar. Increased variation in cell volume (anisocytosis) causes the curve to widen, increasing the red cell distribution width (RDW).

(From Weiser MG: Disorders of erythrocytes and erythropoiesis. In Sherding RG, editor: The cat: diseases and clinical management, ed 2, Philadelphia, 1994, Saunders.)

Qualitative Erythrocyte Parameters

Qualitative erythrocyte parameters are based on a blood smear evaluation. Increased variations in cell size, color, and shape are known as anisocytosis, polychromasia, and poikilocytosis, respectively. Anisocytosis is present if there is a combination of normal-size cells along with an appreciable number of larger or smaller cells. Anisocytosis may result in an increased RDW. The larger cells are often reticulocytes, although infection with the feline leukemia virus (FeLV) can result in larger cells without increased reticulocyte numbers. Polychromasia is usually due to the presence of increased numbers of aggregate reticulocytes and indicates regeneration.¹²⁶ Lack of polychromasia, however, does not rule out regeneration.¹⁸ Variations in cell shape may be artifactual or due to disease (Figure 25-8). Echinocytes are crenated red cells with uniform, often pointy, projections. They are usually artifacts but are important to recognize; when the projections are viewed end on, they may appear as small rings and mimic the ring form of hemoplasmosis (e.g., Mycoplasma haemofelis). Acanthocytes are similar to echinocytes but have fewer and more rounded projections. They are frequently present in cats with hepatic disease. Erythrocyte fragments, such as schistocytes or keratocytes, are the result of cell trauma. When there are many fragments, the presence of turbulent blood flow or microangiopathic disorders such as hemangiosarcoma or disseminated intravascular coagulation (DIC) should be considered. Iron deficiency may also cause increased fragmentation.¹⁸ Spherocytes are smaller cells that are the product of immune-mediated removal of antibody-coated parts of the erythrocyte membrane, after which the cell is reconfigured into a sphere. Because normal feline erythrocytes are small and lack central pallor, spherocytosis in this species is difficult or impossible to appreciate and identification is best left to an experienced veterinary hemocytologist.

FIGURE 25-8 Some common terms and synonyms are given beneath a drawing of selected morphologic changes in red blood cells. These illustrations are of the cell as it appears on a Wright-stained smear, except for reticulocytes and Heinz bodies, which are preferentially stained with new methylene blue. A canine erythrocyte is included for comparison purposes.

(From Weiss D, Tvedten H: The complete blood cell count and bone marrow examination: general comments and selected techniques. In Willard MD, Tvedten H, editors: Small animal diagnosis by laboratory methods, ed 4, St Louis, 2004, Saunders.)

Microagglutination and rouleaux formation may be visible on blood smears. Agglutination appears as a random, disorganized clumping of cells not dispersed by the addition of saline. True autoagglutination indicates an immune-mediated disease affecting the erythrocyte. Rouleaux formation looks similar to a stack of coins (see Figure 25-4) and will disperse after the addition of saline (see Figure 25-5). Circulating monocytes may phagocytose antibody-covered red cells; this is called erythrophagocytosis. Although not observed very often, erythrophagocytosis also suggests the presence of immune-mediated red cell damage. Heinz bodies are areas of oxidatively denatured hemoglobin within the cell (discussed later). The altered hemoglobin is pushed off to one side and often seen as a projection off the surface of the cell membrane. Heinz bodies stain dark with new methylene blue stain, somewhat clear with Diff-Quik, and the same as the cytoplasm with Wright’s stain¹⁸ (Figures 25-9 and 25-10). Howell–Jolly bodies are intracytoplasmic remnants of nuclear material found in erythrocytes that may mimic red cell parasites. Blood smear examination is an essential part of a CBC, particularly when it comes to evaluating the erythron. There is no other way to identify the morphologic changes in red cells that can yield clues to the etiology of erythrocyte disease. Without a blood smear evaluation, a CBC is incomplete.

FIGURE 25-9 Heinz bodies appear as dark-stained structures in this new methylene blue–stained smear of feline blood.

(Courtesy Rick Cowell.)

FIGURE 25-10 Heinz bodies stain pale on a Wright-stained feline blood smear. They may be seen projecting from some erythrocytes, whereas others appear as pale areas in the erythrocyte, as shown by the arrows.

(Courtesy Rick Cowell.)

Blood Types

There are three well-known, clinically important blood groups in cats: A, B, and AB. Another potentially important group called Mik has recently been identified.¹²⁴ The blood groups are genetically determined erythrocyte surface antigens. The A-allele is dominant over the b-allele so that cats with genotypes A/A and A/b will be type A, whereas only the homozygous b/b will have the type B phenotype. A third allele, Ab, occurs rarely and is said to be recessive to the A-allele and dominant to the b-allele, although controversy exists regarding the exact inheritance. A and B antigens are produced on the same red cell only in cats with the genotypes Ab/b or Ab/Ab.³⁵ A more in-depth discussion of feline blood types has recently been published.⁷ Blood typing can be performed by a diagnostic laboratory using various methods or in the hospital with a card typing system (DMS RapidVet-H [feline], DMS Laboratories). If the card-typing system is used, type AB and type B results should be confirmed by a referral laboratory because some cross-reactions have been known to occur.¹⁰⁴ A recently introduced option for patient-side blood typing is the gel column agglutination test (DiaMed-Vet feline typing gel, DiaMed, Switzerland). This test is easier to interpret than the card method, although it requires a specially designed centrifuge that may be cost prohibitive in some settings.¹¹⁸ An evaluation of various blood typing methods for the cat concluded that the gel column test is reliable compared with the gold standard, the Penn tube assay.¹⁰⁴

Genetic blood typing using buccal mucosal swabs is available from certain labs and may allow breeders to identify heterozygous type A cats (A/b). Breeding two of these cats together would be expected to produce 25% type B kittens (b/b) and 50% heterozygous type A kittens (A/b). However, genetic typing cannot distinguish between type A and type AB.

Understanding feline blood groups is important because, unlike other mammals, cats produce naturally occurring antibodies, called alloantibodies, against the erythrocyte antigens not present on their own cells. The kitten produces these alloantibodies at approximately 2 to 3 months of age, as a result of exposure to antigens on plants, bacteria, or protozoa that are structurally similar to red cell antigens. No alloantibodies are produced against antigens that are similar to self-antigens, and no previous exposure to blood products (transfusions) is necessary to produce the alloantibodies.

Knowledge of this system is important in the prevention of transfusion reactions and neonatal isoerythrolysis. Cats with type B blood have anti-A antibodies with strong hemolytic potential. Even a small volume of type A or AB blood administered to a type B cat can cause potentially life-threatening hemolysis within minutes of the transfusion.³⁶ Hemolysis of type B blood administered to a type A cat will result in a reduced life span of the transfused erythrocytes, but severe reactions are uncommon.⁴² Ingestion of colostrum from a type B queen by a type A newborn results in absorption of anti-A alloantibodies and subsequent rapid hemolysis of the kitten’s erythrocytes. This is known as neonatal isoerythrolysis and occurs only when type A or AB kittens are born to a type B queen.

The distribution of blood types varies by geographic region and breed (Tables 25-1 and 25-2). Type A is the most common type among cats. There is, however, a geographic variation in the number of type B domestic shorthair cats. More than 10% of the domestic shorthair cats in Australia, France, Greece, India, Italy, Japan, Turkey, and some regions of England are type B. Distribution of blood types among pedigreed breeds does not vary as much by location because of the international exchange of breeding cats. More than 30% of British Shorthair cats, Cornish and Devon Rex cats, and Turkish Angora or Vans have type B blood. In contrast, Siamese and related breeds are almost exclusively type A. Ragdoll cats appear to be unique with regard to blood types. Approximately 3% of Ragdoll cats are discordant when genotyping is compared to serology, necessitating further investigation in this breed.⁷ The AB blood type is very rare, and the frequency of the Mik blood type is unknown. The presence of erythrocyte antigens in addition to the A and B groups may explain why transfusion compatibility is not guaranteed by blood typing; cross-matching is recommended before any transfusion.¹²⁴ Breeding queens, along with blood donors and, if possible, blood recipients, should be blood typed.

TABLE 25-2 Worldwide Frequencies of Blood Types A, B, and AB in Different Breeds

Clinical Evaluation of Cats with Anemia

Anemia is defined as a decrease in the number of circulating red blood cells, the PCV, or the hemoglobin concentration. Because anemia is a sign of disease, making appropriate therapeutic decisions depends on identifying the underlying etiology. As with any disease, the first, most important steps include taking a thorough history and performing a detailed physical examination. The signs associated with anemia are often nonspecific. They are the result of decreased oxygen-carrying capacity of the blood, decreased blood volume, or the underlying disease. The severity of the signs is related to the rate of onset of the disease and the severity of the anemia. Most anemic cats are presented for evaluation of weakness, lethargy, or anorexia. Bleeding may or may not be obvious, depending on its location. The owner should be asked about previous illnesses in addition to the duration and course of the present illness. Exposure to drugs or toxins, such as acetaminophen or onions, as well as the outdoors is important to ascertain. Cats that go outside have a greater risk of trauma and increased exposure to other cats and thus infectious diseases such as retroviral infections. Outdoor cats are also more likely to be exposed to fleas or ticks, possible vectors for important infectious causes of anemia. Discolored urine from hemoglobinuria must be distinguished from hematuria. The geographic location of the cat and its travel history may provide clues as to the cause of the disease. The blood type of a neonate’s parents may be critical information if an ill day-old kitten exhibits signs of neonatal isoerythrolysis. Other signs, such as polyuria and polydipsia, can indicate the presence of chronic diseases. Gastrointestinal signs may lead to the consideration of chronic blood loss or inflammatory disease. Recent surgery or trauma may result in blood loss anemia.

Mucous membrane pallor is a common physical finding. If hemolysis is present, the mucous membrane color may be icteric. Decreased peripheral perfusion from causes such as shock or congestive heart failure may also cause pallor, whereas hepatic failure can result in icterus. Evidence of volume contraction, such as tacky mucous membranes or prolonged skin tenting, may be present. Remember that older cats lack skin elasticity and may have a prolonged skin tent even if well hydrated. A moderate decrease in erythrocyte numbers leads to decreased blood viscosity and tissue hypoxia. A soft murmur may be present because turbulent blood flow is directly related to decreased blood viscosity. Hypoxia leads to vasodilation, resulting in an increased heart rate in an attempt to increase cardiac output and oxygen delivery to the tissues. Tachypnea is also a common finding. Fleas or ticks may be found during a detailed examination of the skin, particularly in young animals. Fever may be present in cats with infectious causes of anemia, and splenomegaly is a common finding in cats with hemolysis of any etiology. Small kidneys may be appreciated in a cat with chronic renal disease. Any abdominal mass should be noted for further evaluation. Petechial or ecchymotic hemorrhages indicate bleeding from hemostatic disorders, whereas bleeding wounds may be evidence of recent trauma. Discolored urine may stain the perineum of a longhaired white cat. The severity of the clinical signs shown by anemic cats is more often related to the chronicity than degree of anemia. Chronic anemia allows the cat to adapt physiologically and behaviorally to decreased tissue oxygenation, whereas acute anemia does not allow this adaptation to take place.

When presented with a pale cat, the first diagnostic step is to measure the PCV and total plasma protein concentration. If the PCV is normal, the veterinarian should look for other causes of pallor. If the PCV is low, the next step is to determine if the anemia is regenerative or nonregenerative (Figure 25-11). The single, best indicator of regeneration is an increase in the absolute aggregate reticulocyte count.¹²⁶ The severity of anemia should not be assessed until any volume deficits have been corrected. A CBC with a platelet and aggregate reticulocyte count and a blood smear examination will provide evidence of regeneration as long as sufficient time has elapsed since the initial insult. If the protein content is low, acute bleeding should be suspected. Additional tests to consider include a slide agglutination test, a direct Coombs’ test, testing for retroviral infection, and a polymerase chain reaction (PCR) test for hemotrophic Mycoplasma. Other tests to consider include thoracic and abdominal radiography and abdominal ultrasonography, a serum biochemical profile, urinalysis, and coagulation profile. If the anemia is nonregenerative, evaluation of the bone marrow may be required to make an etiologic diagnosis. An attempt should be made to biopsy any masses identified during the evaluation. Examination and sampling of the gastrointestinal mucosa may be required to diagnose causes of blood loss from this system. To differentiate anemia of inflammatory disease from iron deficiency anemia, serum iron, ferritin, and transferrin (total iron-binding capacity) will have to be measured. By following a logical, ordered diagnostic approach to anemia, the veterinarian can often make an etiologic diagnosis, allowing specific therapy to be instituted.

FIGURE 25-11 An algorithm that may be useful in evaluating a cat with anemia. Clinical judgment should be used when following any algorithm because an individual cat may not follow the rules. Further testing should be performed as needed.

Basic Feline Transfusion Medicine

The indications for the use of blood products are many and include hemorrhage, anemia, hemostatic defects, and hypoproteinemia.⁴² Many blood products are available or may be prepared, although most veterinary hospitals have hospital cats to use as needed for whole blood donation.

Many blood products are available and have specific uses. Fresh, whole blood contains erythrocytes, platelets, clotting factors, and serum proteins. Storage of whole blood results in the loss of platelets in 2 to 4 hours and clotting factors V and VIII within 24 hours of collection.⁴² Packed red cells maintain the oxygen-carrying capacity of whole blood in a smaller volume. This product may be used when volume expansion is unwanted, such as anemic cats with heart disease. Fresh frozen plasma contains albumin and all the clotting factors and is used in cats hemorrhaging from coagulation disorders such as liver failure, DIC, or anticoagulant rodenticide toxicity. The use of plasma products to treat hypoalbuminemia is beneficial only in the short-term because transfused albumin rapidly equilibrates with the extravascular space.²⁵ The addition of synthetic colloids may prolong the oncotic effects of a plasma transfusion in these cats.⁴² Platelet-rich plasma is indicated for cats bleeding from platelet deficiency or dysfunction. Sources for products other than fresh whole blood include a local emergency or referral hospital or a regional veterinary blood bank. Oxyglobin (Biopure, Cambridge, Mass.) is a bovine hemoglobin product containing 130 g/L of hemoglobin that has been licensed for use in dogs. Because no cell membranes are present, there is no antigenicity and the product can be used when compatible blood is unavailable. However, availability of the product has been erratic, and at the time of this writing, it is not available.

Donor cats should be healthy larger cats with a PCV above 35% and fully vaccinated.⁴² Donors should be blood typed before blood is collected. No abnormal morphologic cell types should be present, and platelet numbers should be in the reference range. The American College of Veterinary Internal Medicine (ACVIM) consensus statement on screening blood donors recommends testing donor cats for M. haemofelis; Candidatus Mycoplasma haemominutum; FeLV antigen; feline immunodeficiency virus (FIV) antibody; and, possibly, Bartonella infections.¹²² Cats that test positive for FIV antibody should be excluded even if vaccinated against the disease because development of reliable tests to discriminate between antibodies present from a natural infection versus vaccination has been difficult. Heartworm disease cannot be transmitted by blood donation, insofar as the larvae require passage through the mosquito to become infective. Screening for cytauxzoonosis is unnecessary because most cats with the disease are ill. Toxoplasmosis and feline infectious peritonitis have not been documented to be transmitted by transfusion.¹²² Donor cats should be kept indoors to reduce the risk of exposure to infectious diseases. Healthy cats can donate 10% to 20% of their total blood volume without adverse effects. A cat’s total blood volume is approximately 66 mL/kg. For example, approximately 50 mL of blood (10 mL/kg) can be collected from a 5-kg cat no more often than every 4 to 6 weeks. Subcutaneous fluids should be administered at 2 to 3 times the volume of donated blood. Collection of more than 70 mL of blood from a 5-kg cat can lead to hypovolemia, and the volume should be replaced with intravenous fluids. Many donors resent sitting still long enough to have this volume of blood removed and may require sedation or general anesthesia.

For the treatment of anemia, there are no established levels of PCV below which a cat requires a blood transfusion. The decision to transfuse is instead based on the condition of the patient and assessment of the potential benefits weighed against the risks. Indications that an anemic cat may require a transfusion include respiratory distress, weak pulses, or severe weakness.¹²⁶ Both the donor and recipient should be blood typed. Even if the blood types are known, cross-matching should be performed before blood administration to prevent incompatible transfusions caused by untested or unknown erythrocyte antigens such as Mik. The half-life of appropriately matched red blood cells in the cat is 29 to 39 days, but for mismatched transfusions the half-life may be a matter of hours. When type B blood is transfused to a type A cat, the life span of transfused red blood cells is only 2 days. When type A blood is transfused to a type B cat, in addition to a potentially severe and fatal reaction, the life span of the transfused cells is only a few hours. A cross-match should be performed again if more than 4 days have elapsed since the last transfusion from the same donor or if another donor is used.

Blood collected for immediate use can be anticoagulated with heparin. Heparin has no preservative properties, and heparinized blood should be used within 8 hours.⁴² If blood is to be stored for a longer period, citrated anticoagulants should be used. The required blood volume can be collected into a large syringe. If stored blood is used, it should be warmed to room temperature. Blood is administered through a filter connected to a dedicated intravenous line or a line without the presence of calcium-containing fluids. The transfusion is usually administered using gravity flow, although an infusion pump can be used if the manufacturer has stated that it can be used for this purpose. Bacterial contamination is a potential risk, and aseptic techniques for blood collection should be followed. Blood products can be administered intravenously or intraosseously in small patients.

For severely anemic cats the goal of transfusing whole blood is to ameliorate life-threatening decreases in PCV. This can be accomplished by attempting to raise the PCV to over 20%.⁴² The volume of whole blood required to increase the PCV to a desired level can be calculated by using the following formula:

The volume to be delivered is equal to 70 times the recipient cat’s weight in kilograms times the difference between the desired and the patient’s PCV divided by the donor’s PCV. Administer 2 to 3 mL of typed and cross-matched blood over 5 minutes and watch for evidence of adverse reaction, such as an increased body temperature, increased heart or respiratory rate, or a prolongation of the capillary refill time. Whole blood can be administered at 10 mL/kg/hour in a normovolemic cat or 2 to 4 mL/kg/hour in a cat with heart disease. The recipient should be constantly monitored for increased heart and respiratory rate, fever, and any signs of adverse reaction (e.g., vomiting) until the transfusion is complete. The transfusion should be completed within 4 hours to avoid bacterial contamination and the PCV measured 1 to 2 hours after completion.

For cats with incompatible cross-matches or in cases when blood products are unavailable, Oxyglobin (Biopure, Cambridge, Mass.) may be administered at a dose of 5 to 15 mL/kg at a rate of 5 mL/kg/h. Because the product contains no red cells, the hemoglobin concentration is measured to assess the effectiveness of the treatment. Oxyglobin has high colloidal properties, and cats are prone to volume overload with its use. Adverse reactions in cats included vomiting, pulmonary edema, and pleural effusion. In one study of cats receiving Oxyglobin, 20% of cats developing respiratory signs required furosemide or supplemental oxygen during or after treatment. Most of these cats had preexisting heart disease.³² The lower dose should be used cautiously in cats with cardiac disease. The oxygen-carrying effects of Oxyglobin last up to 3 days in circulation.⁴²

Adverse effects of blood transfusions can be immunologic reactions to incompatible blood or nonimmune events and may occur within 1 or 2 hours after the transfusion begins. Occasionally, they may be seen up to 48 hours later.⁴² In a study of 126 cats that received blood transfusions, 11 cats (8.7%) suffered acute reactions.⁵³ Multiple red blood cell transfusions (either whole blood or packed red blood cells) are also well tolerated in cats and may be critical for survival of some severely ill patients.⁹⁸ Immune-mediated reactions can include hemolysis, allergic reactions, fever, or graft-versus-host reactions. Bacterial contamination of the blood product, hemolysis, hypocalcemia (from citrate toxicity), hypothermia, hyperammonemia, and volume overload are examples of nonimmune adverse reactions. In either case, the life span of the transfused erythrocytes may be shortened. Some reactions are severe enough to cause death. Despite the best efforts to prevent them, transfusion reactions may still occur. Depending on the severity, therapy may include glucocorticoids, epinephrine, crystalloid intravenous fluids, and discontinuation of the transfusion. Fever is usually mild, requiring no treatment. Furosemide should be administered if volume overload occurs. To prevent hypothermia, the blood product can be warmed to no more than 37° C. If the reaction is relatively mild, the transfusion can be restarted at a slower rate. Cross-matching blood is the best means of preventing immune-mediated transfusion reactions even if the blood type is known for both cats. It is also imperative that blood be collected and administered as aseptically as possible and that cats receiving blood products be carefully monitored.

Regenerative (Responsive) Anemia

Immune-Mediated Hemolysis

Immune-mediated hemolytic anemia (IMHA) occurs when an immune response is directed against antigens on erythrocytes, leading to their removal by the mononuclear phagocyte system of the spleen (extravascular hemolysis) or complement-mediated lysis (intravascular hemolysis). If the immune-mediated event is associated with another disease, it is a secondary IMHA. Infectious or inflammatory disease may lead to secondary IMHA, as might neoplasia or drug administration. When no underlying cause can be discerned, it is termed primary IMHA.

Dysregulation of the immune system results in a loss of self-tolerance. Antibodies may be formed against erythrocyte antigens (type II hypersensitivity), against a non-erythrocyte antigen attached to the red cell surface (type III hypersensitivity), or an antibody may be produced against an unassociated antigen that is similar to an erythrocyte antigen. Alloantibodies present in transfused blood or in colostrum from a type B queen ingested by a type A kitten may lead to immune-mediated hemolysis. Some erythrocyte antigens are hidden and exposed to the immune system only after the cell membrane has been damaged. New antigens that cross-react with red cell antigens or attach to the red cell membrane may be released into circulation by infection or inflammation.³⁴

The antibodies involved in the immune process are usually IgG, although IgM can be present alone or with IgG. Macrophages of the mononuclear phagocyte system have receptors for the Fc portion of the IgG antibody but not for IgM. Fc receptors are proteins on the surfaces of cells such as macrophages and neutrophils that contribute to the protective functions of the immune system. Fc receptors bind to the Fc portion of antibodies attached to pathogens or infected cells and stimulate the activity of phagocytic or cytotoxic cells. The antibody-coated cells are removed after antibody binds to the receptor on the macrophage, mostly in the red pulp of the spleen. The result is extravascular hemolysis. Complete phagocytosis may not occur, and only a portion of the membrane may be removed. The cell’s volume-to-surface-area ratio is reduced, and the cell becomes spherical. Spherocytes are less able to deform, making passage through the spleen more difficult. Because of the nonsinusoidal nature of the feline spleen (see the section on splenic diseases later in this chapter), less cell deformability is required for cells to pass through, and decreased numbers of spherocytes are trapped and destroyed than in dogs. Splenic macrophages also have receptors for complement. If a sufficient quantity of IgG antibodies coat the cell membrane, complement may also bind to the erythrocyte. The presence of complement on the cell membrane increases the efficiency of erythrocyte removal. If a sufficient number of the antibodies are IgM, complement-mediated lysis can occur, resulting in intravascular hemolysis. However, none of the 19 cats with primary IMHA in one study had intravascular hemolysis despite the presence of IgM in 8 of the cats.⁵⁷

Cats with IMHA, whether primary or secondary, will exhibit signs related to the anemia. These can include anorexia, lethargy, weakness, or respiratory difficulties. Additional signs as a result of the underlying disease may be present in cats with secondary IMHA. Most of the cats with primary IMHA are young adults. In the previously mentioned report of 19 cats with primary disease, six were younger than 2 years old; the median age for all 19 cats was 2 years.⁵⁷ Eleven of the cats were male, and eight female. Cats with secondary disease will have a signalment related to the underlying disease. Physical changes that might be present include pale or icteric mucous membranes, tachycardia, tachypnea, or splenomegaly as a consequence of increased processing of damaged erythrocytes. Tachypnea and tachycardia are attempts at compensating for the decreased oxygen-carrying capacity of the anemic patient; pulmonary thromboembolism, so common in dogs with IMHA, is rare in cats.⁵⁷ Splenomegaly occurs in many nonimmune disorders causing hemolysis as the organ attempts to deal with the increased numbers of damaged red cells. Body temperature is likely to be normal unless the patient is moribund, in which case hypothermia may be noted. A systolic murmur may be heard during auscultation of the thorax.

Diagnosis of IMHA can be frustrating. There are many mechanisms causing hemolysis that do not involve the immune system. Distinguishing primary from secondary IMHA is important because therapy may be different. With aggressive diagnostic investigation, an underlying cause is often found. Two sources state that primary IMHA is rare in cats.^34,75 However, Kohn and coworkers⁵⁷ found that of 23 anemic cats with a positive Coombs’ test or persistent erythrocyte agglutination, an underlying cause was identified in only four patients after an extensive workup. Put another way, 19 of the 23 Coombs’-positive cats had primary IMHA.

To make a diagnosis of primary IMHA, other causes of hemolysis must be eliminated. A CBC with reticulocyte and platelet counts, serum biochemical profile, and urinalysis should be performed. The cat’s retroviral status should be ascertained and a PCR test for M. haemofelis DNA should be run. Thoracic radiography and an abdominal ultrasound examination should be performed to rule out the presence of potential bronchial infections, or thoracic or abdominal masses. A bone marrow evaluation may be useful when a regenerative response to the anemia is equivocal. Specific immunodiagnostic tests such as a slide agglutination test and a direct Coombs’ test should be performed.

If the hemolysis is severe enough and the anemia is not peracute, there should be evidence of regeneration in the CBC report. Increased numbers of aggregate reticulocytes should be present. Polychromasia and rubricytosis (nucleated red blood cells) may be present. The hallmark of IMHA in dogs, spherocytosis, is unlikely to be identified. The PCV may be surprisingly low for the condition of the patient; cats seem to tolerate a lower PCV than dogs.⁵⁷ The lower the PCV, the higher the aggregate reticulocyte count should be. If the reticulocyte count is not appropriate for the degree of anemia, it may be nonregenerative. In this case an immune response directed at erythrocyte precursors in the bone marrow might be considered. Another difference from dogs is the lack of leukocytosis or neutrophilia with left shift in cats with primary IMHA. In the aforementioned study by Kohn and coworkers⁵⁷ 17 of the 19 cats with primary IMHA had a white cell count within the reference range. The platelet count should be within the reference range, too. Evans syndrome, a combination of immune-mediated damage to both erythrocytes and platelets, is rare in cats. Although evidence of DIC is common in dogs with IMHA, it is uncommon in cats. Before convicting a cat with anemia of also having thrombocytopenia on the basis of an automated cell count, a blood smear should be examined to determine whether platelet clumping is present. The smear examination will also allow identification of intraerythrocytic parasites or morphologic changes in the erythrocyte that may suggest causes of anemia other than IMHA.

There are no pathognomic changes for IMHA in the biochemical profile. Anemia may cause hepatic centrilobular hypoxia, hepatocyte injury, and subsequent increases in serum alanine transferase (ALT) activity. Hyperbilirubinemia and hyperproteinemia may be present. Volume contraction may be reflected by azotemia, which is likely prerenal in cats with primary IMHA. Other changes may be present if there is an underlying disease (secondary IMHA). Any thoracic or abdominal masses should be biopsied. An airway wash with cytology and culture might be attempted in cats with peribronchial thickening.

The direct Coombs’ test detects the presence of antibodies or complement on the red cell surface. A positive test is consistent with, but not necessarily diagnostic for, IMHA. However, a diagnosis of IMHA should include a positive direct Coombs’ test.³⁴ False-negative tests are unlikely. In the study by Kohn and coworkers, 78 cats with anemia had a direct Coombs’ test performed and 55 were negative, all of which had nonimmune etiologies identified as causing the anemia; an additional 14 cats without anemia were also direct Coombs’ test negative.⁵⁷ The direct Coombs’ test may become negative after a patient with IMHA enters remission, although a few days of immunosuppressive therapy is unlikely to cause a negative test.³⁴ A limitation of the test is the inability to differentiate between primary and secondary IMHA. A properly performed slide agglutination test may detect anti-erythrocyte IgM or large quantities of anti-erythrocyte IgG coating the erythrocytes. Autoagglutination must be distinguished from rouleaux formation by proper washing or dilution of the blood on the slide. A direct Coombs’ test is unnecessary if the slide agglutination test is positive because they both test for anti-erythrocyte antibodies; autoagglutination is considered indicative of IMHA.⁵⁷ Autoagglutination may artifactually increase the MCV because clumps of cells are counted as one.

Therapy for IMHA depends on the cause and severity of the anemia and must be tailored to the individual. Removal of an underlying cause or trigger will help bring secondary IMHA under control. If an infection, such as M. haemofelis, is thought to be contributing to the disorder, use of appropriate antibiotics is required. Surgical drainage of any fight-wound abscesses or removal of potentially neoplastic masses may be necessary. Removal of nonessential drugs, particularly those known to induce an immune response, may eliminate a potential trigger for the immune-mediated process.

Supportive measures should not be forgotten. Volume expansion in a severely ill cat will improve organ perfusion. Concerns regarding exacerbating hypoxia by decreasing the PCV with intravenous fluids are unwarranted. Although the PCV may decrease, the total amount of hemoglobin in the body does not. However, rehydration will reveal the true severity of the anemia. Depending on the cat’s condition and PCV, a blood transfusion may be required. A major cross-match before collecting and administering blood is imperative, even if the blood type of the donor and recipient is known. Unfortunately, autoagglutination may make interpretation of the cross-match difficult. Alternatively, a hemoglobin-containing solution, Oxyglobin, may be used to improve oxygen-carrying capacity. Stressful situations while in hospital, such as frequent handling or exposure to barking dogs, should be minimized in severely ill cats.

Reduction of the immune-mediated destruction of erythrocytes is the goal of drug therapy. The optimal drug protocol will decrease phagocytosis of antibody or complement coated red cells, reduce complement activation, and eliminate the production of anti-erythrocyte antibodies (Table 25-3). Glucocorticoids are the initial drug of choice. These drugs are both antiinflammatory and immunosuppressive, although higher doses are required to accomplish the latter. Oral prednisone is the most commonly used glucocorticoid, but it requires conversion by the liver to the active form, prednisolone.¹⁰⁰ There is some evidence that intestinal absorption or hepatic conversion of prednisone to prednisolone may be poor in cats,¹¹⁹ thus prednisolone is thought by some to be a better initial choice in this species. The pharmacologic effects are due to interference with cellular communication and interaction among cells of the immune system. Glucocorticoids also inhibit production of cytokines used to amplify the immune response.⁷⁵ Decreased production of IL-2 leads to decreased T_h1 helper cell proliferation and cytotoxicity.²⁷ Glucocorticoids stimulate maturation of T-suppressor cells and inhibit antibody-dependent cytotoxicity by natural killer cells,²⁷ resulting in inhibition of the cellular arm of the immune system. They are beneficial in reducing binding of the Fc component of the attached IgG to the Fc receptors on splenic macrophages. In addition, they may decrease antibody binding to the red cell membrane and complement activation.³⁴ There are few direct effects on B-lymphocytes, and therefore there is little effect on antibody production.^27,90 Cats have fewer and less sensitive cytoplasmic glucocorticoid receptors than dogs.¹⁶ This may explain why cats typically have less pronounced side effects; for instance, polyuria and polydipsia and steroid hepatopathy are not typical side effects of glucocorticoid use in cats.²⁷ Cats receiving immunosuppressive doses of glucocorticoids may have difficulty eliminating infections on their own, and some infections may be inapparent because the inflammation associated with the infection may be blunted by the antiinflammatory effect of the glucocorticoid.

TABLE 25-3 Immunosuppressive Drugs

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