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Hematology & Transfusion Medicine Beth Davidow, DVM, DACVECC Seattle, Washington Approach to the Anemic Patient Physiology Anemia is defined as a deficiency of red blood cells or hemoglobin (Hb). Anemic animals are usually pale, may tire easily, and are often weak. These clinical signs of anemia are related to hemoglobin's (Hb) role in delivering oxygen to cells. Oxygen delivery (DO2) can be defined as: DO2 = Cardiac output * [(PaO2*0.003) + (SaO2*Hb*1.34)] Most red blood cells are produced in the bone marrow from precursors. Proerythroblasts differentiate to basophil erythroblasts (prorubricytes), then polychromatophil erythroblasts (rubicytes), then orthochromatic erythroblast [metarubicyte or nucleated red blood cells (nRBCs)], and finally to reticulocytes and red blood cells. During anemia, red blood cells can also be produced in the spleen and liver, a process known as extramedullary hematopoesis. Erythropoietin (EPO) is the main control hormone for RBC production. It is a 34.4 - kD glycoprotein hormone produced primarily by the peritubular interstitial cells in the renal cortex and to a smaller extent by the parenchymal liver cells. This hormone binds to receptors on erythroid progenitor cells promoting their proliferation and maturation and preventing their apoptosis. EPO gene expression is induced by hypoxia and decreased oxygen delivery. Decreases in PaO2 or RBC mass, or an increase in RBC oxygen affinity can cause increases in EPO production. In a healthy individual, acute hemorrhage leads to increased serum erythropoietin levels within minutes. Normal physiologic stimulation of EPO in humans starts when hemoglobin falls below 12g/dl. EPO production rises exponentially in response to falling hematocrit in normal individuals. Reticulocytes are usually first seen 3-4 days after erythropoietin stimulation and maturation of reticulocytes into mature RBCs usually takes an additional 1-3 days. EPO production is decreased by a high number of RBC precursors. In addition, inflammatory cytokines may interfere with EPO gene expression. In a study using human hepatocellular carcinoma cell lines which under hypoxic conditions produce EPO, it was shown that adding IL-1, TNF, and transforming growth factor - beta inhibited this production. EPO formation is also decreased by increased plasma viscosity, as can be seen in patients with inflammation. The normal life span of a red blood cell is 120 days. Aged and damaged red blood cells are removed in the spleen through phagocytosis. Iron is released from the Hb and carried back to the bone marrow. The porphyrin portion of Hb is converted to bilirubin, released into the blood and then secreted into the bile. Categories and differentials There are three major causes of anemia: 1) Loss of red blood cells 2) Destruction of red blood cells and 3) Lack of production. Anemia can also be categorized as regenerative or non-regenerative. A regenerative response is defined as production of new red blood cells that is appropriate given the degree of anemia. Regeneration can be identified on a complete blood count and differential as a high MCV (new RBCs are larger), polychromasia, anisocytosis, reticulocytes, and nRBCs. One method to determine whether this regeneration is adequate for the degree of anemia is to calculate the reticulocyte production index (RPI). Method:
Once anemia has been identified as from blood loss, then the cause of the blood loss must be identified. Trauma is usually the easiest to identify. Other causes include disorders of primary or secondary hemostasis, neoplasia, gastrointestinal ulcers, and gastrointestinal parasites. Destruction of red blood cells can occur for a number of reasons. Destruction may be secondary to blood borne parasitic diseases such as Babesia canis or Mycoplasma hemofelis or infections such as FeLV. Neoplasia can also cause increased destruction either by infiltration of the spleen (hemangiosarcoma) or as a paraneoplastic syndrome in which haptens on the red blood cells lead to increased phagocytosis. Toxins such as lead, zinc, or onions can also lead to hemolysis. Severe hypophosphatemia, seen clinically most often during treatment of diabetic ketoacidotic crises, can also lead to hemolysis. Pyruvate kinase deficiency, a rare genetic disorder, causes impaired energy generation in the red blood cell leading to hemolysis. Phosphofructokinase deficiency is another genetic disorder in which the red blood cells have increased fragility and rupture under alkalotic conditions. Primary immune mediated hemolytic anemia in which increased destruction is triggered by unknown factors is diagnosed when other causes have been ruled out. Anemia that is due to lack of production can occur for three main reasons. The first is that EPO is not being produced normally. This usually results from chronic renal disease. The second reason is that there is a decrease in the response to the EPO that is being produced. This is often seen in the anemia of chronic illness or the anemia of critical illness when inflammatory cytokines interfere both with EPO production and also with the action of EPO at the level of the bone marrow. The third reason is that the bone marrow itself can not respond to EPO. This can be due to neoplastic infiltration, immune mediated destruction of precursors, toxic insults, infection such as FELV or rickettsial diseases, or endocrine mediated impairment. Iron deficiency falls less neatly into the above categories. It is often due to chronic blood loss and can be regenerative initially. As iron stores are depleted, red cell production becomes ineffective and the anemia becomes non-regenerative. Diagnostic Approach A thorough history is the most important first diagnostic step. The time frame of clinical signs and the acuity of onset are important. Animals with a very acute onset of signs will not have immediate evidence of regeneration. Travel history is very important as some diseases have geographic ranges. It is important to ask about toxin exposure and to ask specifically about onions and zinc containing items such as pennies and diaper cream that owners might not know can be harmful. Associated clinical signs and previous medical history are also very important. The combination of vomiting, diarrhea and acute anemia is associated with zinc toxicity. A new onset of eating dirt, rocks, or other unusual items (pica) can be associated with slowly progressive anemias and is more often seen with bone marrow disorders or iron deficiency. Non-regenerative anemia is expected if we know the animal has chronic renal failure. It is important to note if the animal has a fever or not. Fever is associated with diseases that are infectious, immune-mediated, inflammatory, or neoplastic. The next step is measuring a PCV and total protein (TP). If an animal has a lower PCV than expected based on clinical signs, the anemia may be slowly progressive. If the animal has a higher than expected PCV based on clinical signs, the anemia has probably been very acute. The total protein can be helpful in differentiating causes of anemia. In most blood loss situations, the TP is decreased. In destructive anemia and lack of production anemia, the TP is usually normal. The exceptions to this rule are situations that cause a decrease in PCV and TP independently. One example would be protein losing nephropathy in which protein is lost in the urine but a low PCV could be related to impairment of EPO production from progressive kidney disease. Slide agglutination should be examined whenever a destructive anemia is suspected. A drop of blood is placed on a microscopic slide along with a drop of saline. If agglutination is seen, an immune-mediated destructive anemia is present. Agglutination can be differentiated from rouleaux microscopically. Rouleaux appears as stacks of coins while agglutination is more diffuse clumping of cells. A fresh blood smear should always be examined in house. In some cases, blood borne organisms such M.felis or B.canis will be seen. The presence of anisocytosis, polychromasia, and nucleated red blood cells indicates that the anemia may be regenerative. If spherocytes can be identified, a destructive anemia is present. Acanthocytes or spur cells are associated with splenic and liver diseases. FeLV and FIV should be tested in any cat with anemia prior to further diagnostics. These viruses should be tested for even in the face of vaccination and indoor only status. A complete blood count with reticulocyte count, chemistry panel and urinalysis should always be done when either the cause of the blood loss is not immediately evident or the animal has a suspected destructive or lack of production anemia. Coagulation panels should be done in any animal with unexplained blood loss. A regenerative response is expected with chronic blood loss anemia but in acute situations, the response may look non-regenerative as reticulocytes are usually not seen until 3-4 days after the start of the loss. The same is true in destructive anemias. A RPI greater than 3 is highly suggestive of a hemolytic process. Iron deficiency can occur in some animals with chronic blood loss and result in an insufficient regenerative response. The red blood cells in these animals are often hypochromic and microcytic. Elevations in total bilirubin often indicate a hemolytic process. However, animals with severe liver disease can have gastrointestinal bleeding so the total protein and other liver enzymes should be considered when differentiating the two. Radiographs and ultrasound are usually the next diagnostic steps. In animals with hemolytic disease, abdominal radiographs should be considered in addition to ultrasound as pennies in the stomach can be difficult to see on ultrasound. A bone marrow aspirate is indicated when a non-regenerative anemia can not be explained through the previous diagnostics. It is important to consider whether the anemia has presented long enough for regeneration to occur. Bone marrow aspirates are also indicated if: 1) There is pancytopenia 2) There are circulating abnormal cells that are either neoplastic or precursors that are seen "out of order" in the blood 3) Staging for lymphomas, mast cell tumors, and carcinomas or if the anemia is accompanied by 4) Unexplained hypercalcemia or severe hyperproteinemia. Cases will be discussed during the presentation to further illustrate the diagnostic process. References: Cope RB. Allium species poisoning in dogs and cats. Vet Med. August 2005: 562. Day M, Mackin A, Littlewood J. editors. Manual of Canine and Feline Haematology and Transfusion Medicine. Gloucester, UK: BSAVA, 2000. Harvey JW. Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses. Vet Clin Pathol 2006; 35: 144-56. Rogiers P, Zhang H, Leeman M, Nagler J, et al. Erythropoietin response is blunted in critically ill patients. Int Care Med 1997; 23: 159-162. Scharte M, Fink MP. Red blood cell physiology in critical illness. Crit Care Med 2003; 31(Suppl): S651-657 IMHA: What's New? Introduction Immune mediated hemolytic anemia (IMHA) is a devastating disease with one year mortality reaching 70% in some studies. IMHA is a type II hypersensitivity reaction that occurs when antibodies are formed against antigens on the surface of red blood cells. The RBC antigens may either be auto-antigens (primary IMHA) or drug, infection, toxin, or tumor induced (secondary IMHA). Mortality usually results not from anemia but from overwhelming infection secondary to use of immunosuppressive medications, acute renal failure from hemoglobin nephrosis, or most commonly, thromboembolism. Despite many veterinary publications on the disease, cause of the primary condition and best treatment protocols are still very much in question. In order to discuss ideal treatment protocols, one must start with an understanding of the immune system and classification of types of IMHA. In addition, one must understand how commonly used medications work. This presentation will review current knowledge and discuss evidence for treatment protocols. Review of Immunology Antigens are proteins that induce an immune reaction, usually the production of antibodies. In IMHA, these proteins can either be innate structures of red blood cells or acquired proteins. Innate proteins that are commonly attacked include red cell glycophorins, spectrin (a cytoskeletal protein), and CD233, which is a membrane anion exchange protein that is also a target used to remove aged cells. Acquired proteins include drugs that either attach to the surface of the red blood cells (such as penicillin) or alter the surface membrane properties (cephalosporins). Proteins can also be induced by infection with agents such as rickettsial organisms or by tumor antigens such as those produced by lymphoma. Antibodies, or immunoglobulins, are produced by B lymphocytes. Antibodies bind to antigens on the red blood cells and cause destruction by one of two mechanisms. When IgG is the primary immunoglobulin involved, macrophages bind their Fc receptors to the Fc portion of the antibody-antigen complex. This binding leads to phagocytosis and extravascular hemolysis, predominantly in the spleen and liver. When there is a large amount of IgG or if IgM is also involved, complement is activated and membrane permeability is increased. This leads to cell swelling and rupture and intravascular hemolysis occurs. Agglutination occurs when antibody levels are very high allowing antibodies to bind more than one RBC at a time. IMHA has been divided into classes of disease based on the immune process involved.
Immunosuppressive Agents The mainstay of treatment of IMHA is suppression of the immune system. Corticosteroids are the initial medication chosen. Corticosteroids cause lysis and induce movement of lymphocytes out of the bloodstream. Corticosteroids also increase production of the protein I?B-? in the nucleus of cells. This protein is an inhibitor of NF-?B, a transcription factor that when stimulated causes synthesis of many of the cytokines involved in inflammation. The effect of this inhibition is depressed antigen processing, phagocytosis, and chemotaxis by macrophages; and depressed proliferation, T cell responses, T cell-mediated cytotoxicity, and lymphokine production by lymphocytes. Response to corticosteroids is dependent on the dosage, type of steroid used, and timing of administration. However, the correct dosage and type of corticosteroid has not been fully identified in dogs and cats. Some authors have advocated that injectable dexamethasone may have a faster onset of action but a retrospective study of 105 dogs in Colorado showed no difference. An increase in hematocrit usually takes 3-7 days after the start of therapy. The author uses prednisone at 2mg/kg divided by mouth twice daily as the initial dose. Dexamethasone SP at 0.4 mg/kg IV is used if the animal is vomiting on admission and can not tolerate oral medication. Corticosteroids have many side effects including polyuria, polydipsia, polyphagia, tachypnea, muscle wasting, hypertension, poor wound healing, and increased susceptibility to infection. These side effects often become intolerable when these drugs are used at high doses for prolonged periods. Azathioprine has been recommended as the next immunosuppressive of choice in several veterinary studies. Studies from Virginia Tech and Cornell both showed increases in long term survival when azathioprine was used in addition to prednisone. Azathioprine is a cytotoxic drug that inhibits cell division of proliferating lymphocytes. It is a pro-drug with its metabolites causing the pharmacologic action. It is a purine analog which competes with purines in the synthesis of nucleic acids, thus inhibiting RNA and DNA synthesis and mitosis. Chromosome breaks may occur from incorporation into the nucleic acids and because coenzyme formation is also disrupted, cellular metabolism is impaired. Azathioprine also affects the production of macrophages but does not affect cytokine production. Because this drug works by slowing production of immune cells, its effect is delayed. Clinical response may take from 7 days to up to six weeks. Azathioprine is thought to improve long term mortality because it allows tapering of the corticosteroids, and thus a decrease in side effects. A dosage of 2mg/kg by mouth once daily is recommended to start. Azathioprine's main side effects include bone marrow suppression, gastrointestinal upset, poor hair growth, uncommon hepatotoxicity, and rare pancreatitis. In addition, in people, this drug has been shown to be carcinogenic when used long term. Due to the risk of these side effects, complete blood counts should be checked frequently while on this medication, and a complete chemistry panel is recommended 2-3 weeks after starting therapy. Testing for thiopurine methyltransferase activity is commonly performed in people before starting azathioprine. Thiopurine methyltransferase (TPMT) is an enzyme in red blood cells which produces inactive metabolites of azathioprine. People with a high level of this enzyme are less likely to have myelosuppression than those with low levels. A 2004 study of TPMT levels was performed in 299 healthy dogs and in 9 dogs receiving azathioprine. In this study, no dog was found to have a TPMT deficiency. In the dogs receiving azathioprine, neutrophil counts were lower in those dogs with less TPMT. However, when TPMT was measured in 6 clinical dogs with azathioprine induced myelosuppression, activity of the enzyme was intermediate to high in all, indicating that it is not the only factor involved in dogs. Cyclophosphamide has previously been advocated for use when no response is seen to prednisone alone. Cyclophosphamide is also a pro drug with its active metabolites responsible for the clinical action. The metabolites are alkylating agents that interfere with DNA replication, RNA transcription, and RNA replication. This action makes it toxic for rapidly dividing immunocompetant cells. It impairs T and B cell response and blocks mitogen and antigen-induced cell division and affects the production of IFN-?. Side effects of cyclophosphamide include myelosuppression, gastroenteritis, alopecia and sterile hemorrhagic cystitis. Several veterinary studies have shown no improvement in survival when added to the treatment protocol and in two retrospective studies, cyclophosphamide was associated with increased mortality. Its use is no longer recommended. Danazol is an androgen that has also been used in non-responding IMHA cases. It appears to reduce affinity of antibody with the Fc receptor of macrophages. A study was done in dogs comparing protocols with and without Danazol and no improvement in survival was found. Cyclosporine is a potent immunosuppressant that binds and blocks the intracellular transmitter calcineurin. This blockage inhibits early T cell activation and prevents production of IL-2 and IFN- ? by T cells. These actions lead to a decrease in cell-mediated immunity and in T cell-dependent B cell antibody production. Because this drug's action is specific for lymphocytes, myelosuppression does not occur. Neoral® is a micro emulsion preconcentrate that has improved absorption over Sandimmune®. Atopica® is similar to Neoral in its absorptive properties. However, absorption is still variable and trough blood levels should be monitored. In addition, because cyclosporine is metabolized in the liver, liver dysfunction or drugs that are also metabolized by the cytochrome P-450 system can dramatically affect blood levels. A trough whole blood level of 200-400 ng/ml is recommended and should be checked 48 hours after starting this medication. An initial dose of 5mg/kg PO BID of Neoral® is recommended. Side effects of this medication include anorexia, vomiting, and diarrhea. In addition, it enhances production of TGF-? and many promote tumor growth when administered long term.. There are two published reports in dogs of neoplasia occurring in animals on cyclosporine. Use of cyclosporine for treatment in IMHA dogs has been reported in two small abstracts (3 and 8 dogs only) and was examined retrospectively in 24 dogs in a larger IMHA study. A statistically significant effect could not be found. However, trough levels were not measured and sample sizes were small. Despite the lack of prospective studies, a combination protocol of prednisone, azathioprine, and cyclosporine has been advocated by some clinicians in severe IMHA cases. This protocol was recently described for canine renal transplant patients. In this study, no rejections were seen, but there was a high incidence of bacterial infections, indicating that it may be too much immunosuppression. Mycophenolate mofetil (MMF) is a pro-drug whose metabolite blocks guanine nucleotide synthesis. Both B and T cells are very dependent on this pathway for proliferation and functioning while other cells are not. A 2005 abstract at ECVIM reported use of MMF and prednisone in 8 dogs with IMHA. 7 dogs responded to treatment within one month. The eighth responded to cyclosporine and prednisone and all dogs were still alive at four months. A dosage of 14-17mg/kg by mouth was used in this study. MMF has also been successfully used in a dog with immune-mediated glomerular disease and in a dog with myasthenia gravis. Leflunomide has been mentioned as another possible immunosuppressive that is well tolerated. It is used in people with rheumatoid arthritis and is an inhibitor of pyrimidine biosynthesis. It is very expensive and there are only anecdotal reports of its use in dogs with IMHA. A dosage of 4mg/kg daily has been recommended with adjustment to reach a serum trough level of 20 ug/ml. Because the risk of thromboembolism is highest during rapid hemolysis and many owners are concerned with the cost of multiple transfusions during the initial hospitalization, ways to obtain rapid acting immunosuppression have been sought to control the immune system faster than can occur with glucocorticoids or other immunosuppressive drugs. Intravenous Human immunoglobulin (IVIG) has been used to suppress the immune system in a few studies. IVIG works by binding to the Fc recptors of macrophages so that they are not able to bind to antibody-antigen complexes on RBCs. In humans, IVIG was reported to work in 40% of IMHA cases in one study. IVIG was used in 5 dogs with non-regenerative immune mediated anemias and all 5 dogs had improvement in their anemias in 24-72 hours. IVIG was also used prospectively in 10 dogs who had failed to respond to prednisone and cyclophosphamide or azathioprine. 5 dogs had a clinically meaningful improvement after treatment. 5 of the 7 dogs who died within 12 months had evidence of thromboembolism at necropsy, leading some concern that IVIG may increase hypercoagulability. IVIG was also examined in 13 dogs with IMHA. 11 dogs had an increase in PCV of 4% within 4 days. However, in a more recent blinded, randomized clinical trial, the addition of IVIG versus placebo to treatment with steroids had no impact on initial response or hospitalization time. Because this is a human product, reactions are likely with repeated doses. Dosages from 0.5 g/kg to 1.5 g/kg are reported. A recent article in Experimental Hematology presented a novel way to obtain rapid immunosuppression. Liposomal clodronate (LC) is a compound that is preferentially phagocytized by macrophages. Once ingested, the liposome breaks open, releases the clodronate and the macrophage is destroyed. By injecting a large quantity of LC, splenic and hepatic macrophages can be depleted leading to quick acting immunosuppression without myelosuppression. In addition, the LC mediated macrophage death does not lead to cytokine release so inflammation is not increased. In this study, LC was tested in both healthy dogs and in 7 dogs with severe IMHA. The drug appeared safe with only mild side effects of diarrhea seen. 2 of 7 dogs had dramatic decreases in RBC clearance within 24 hours of treatment and the group of dogs had statistically improved survival over a historical control group. Because this drug targets macrophages, it will not be useful in those dogs with IgM mediated complement activation. Splenectomy Splenectomy is often performed in people with IMHA who do not respond to corticosteroids. About 50% of people will have an initial complete response after splenectomy. However, there are common relapses thought to be due to increased activity of hepatic macrophages. About 3% of patients develop postsplenectomy sepsis syndrome, which is fatal in approximately half of the cases. A 2003 ACVIM abstract reported on use of early splenectomy in dogs with IMHA. In this small study, 12 dogs had a splenectomy within 48 hours of presentation and were compared to 8 dogs who did not. All dogs were also treated with prednisone and azathioprine. Dogs who were splenectomized had a faster return to a normal hematocrit and a longer term survival. A 2009 retrospective looking at splenectomy in dogs with IMHA showed 9 out of 10 dogs were still alive at 30 days. Splenectomy is only expected to be useful in cases where extravascular hemolysis predominates. Venous thrombosis and PTE are two of the most common causes of mortality in dogs with IMHA. Dogs with IMHA have been found to have high fibrinogen, high soluble fibrin and high D-dimer levels. In addition, these dogs often have low anti-thrombin (AT) activity. A recent study showed that dogs with IMHA also have an increase in the proportion of activated platelets as measured by an increase in P selectin expression. Drugs that have been used or discussed to decrease the incident of thromboembolism in IMHA patients include unfractionated heparin, low molecular weight heparin, aspirin, and more recently, anti-platelet drugs such as Clopidogrel (Plavix®). Unfractionated heparin is a glycoprotein that binds to antithrombin catalyzing its inhibition of Xa and IXa and allowing inactivation of thrombin. Studies in people indicate that best anti-thrombotic effect with the least risk of bleeding occurs when the heparin level is 0.3-0.7 I/ml or the APTT is increased 1.5 times baseline. Unfractionated heparin has very variable absorption and pharmokinetics and thus patients need to be monitored carefully while on this drug. Several studies in dogs with IMHA have addressed dosing and effectiveness of unfractionated heparin. A recent prospective study of 18 dogs with IMHA at Purdue revealed that even aggressive dosing of 300 U/kg every 6 hours was inadequate to achieve therapeutic anti-Xa activity. 6 dogs that died in this study had necropsies and 3 of the 6 had evidence of thrombosis. In a retrospective study from Texas A & M, dogs with IMHA were divided into those who had received heparin and those who had not. The dogs who had received heparin were less likely to survive to discharge. In a final study of dogs with hypercoagulable diseases, antithrombin levels were low in all 9 dogs with IMHA. In this study, antithrombin levels decreased in those dogs who received heparin or a combination of heparin and fresh frozen plasma. Low molecular weight heparins are shorter, more uniform size glycosaminoglycan chains that bind to antithrombin. Their smaller size reduces binding to thrombin and to other proteins and cells so that the pharmokinetics of this drug are more predictable. The risk of bleeding is less than with unfractionated heparin. In people, these drugs can be given once daily. However, pharmokinetic studies in dogs show that Enoxaparin® must be given at 0.8 mg/kg q 6 hrs to maintain anti-Xa activity in the target range of 0.5-2.0 U/ml. In addition, as mentioned above, because antithrombin is decreased in many dogs with IMHA, heparins of any type may not be the best choice for thromboembolism prevention. The finding that dogs with IMHA have circulating activated platelets indicates that anti-platelet drugs may be a better way to address thromboembolism. Aspirin works by blocking production of thromboxane A, which is an inducer of platelet aggregation. A dose of 0.5 mg/kg PO SID -BID has been shown to reduce thromboxane A. A recent study from Cornell University retrospectively looked at 151 cases of IMHA. In that study, 0.5 mg/kg PO SID aspirin was used along with prednisone and azathioprine. This protocol was compared to using prednisone, azathioprine, and heparin and both short and long term survival was much improved with aspirin. Newer anti-platelet medications might be even more efficacious, especially if used in combination with aspirin in the initial treatment. Clopidogrel (Plavix®) acts through an active metabolite (metabolized in the liver) that inhibits the platelet release reaction and activation of the platelet fibrinogen receptor by binding to a specific platelet ADO receptor. This drug has been studied in cats but information in dogs is limited to IV dosing in experimental models of thrombosis. Abciximab is an antagonist of the platelet GP IIb/IIIa receptor. This drug has also not been used clinically but in canine experimental models of thrombosis, doses of 0.2-0.8 mg/kg resulted in thrombus dissolution and almost complete inhibition of platelet aggregation. Antibiotics Because a major cause of secondary IMHA is rickettsial diseases, doxycycline is recommended in the initial treatment protocol in areas where these diseases are prevalent. Although secondary infections are a common cause of morbidity and mortality in dogs with IMHA, antibiotics such as cephalosporins and penicillins are also associated with RBC antigen formation and should be avoided when possible. In addition, many antibiotics have possible interactions with cyclosporine so blood levels should be rechecked if an antibiotic is added to the treatment regime. Antibiotics are thought necessary by some due to the leukocytosis often seen in these cases. However, leukocytosis is often a leukemoid response of the bone marrow to the anemia and not indicative of infection. Steroids can also cause leukocytosis. Finally, the leukocytosis can also be indicative of hypoxic tissue injuries. Suspected infections should be confirmed with cultures and antibiotic selection should be based on sensitivity results when at all possible. Hemoglobin and blood products Hypoxia is also a cause of end organ injury in dogs with IMHA. In a necropsy study of dogs who died of IMHA, ischemic necrosis within the liver, kidney, heart, lung, and spleen were often identified. Some of the lesions were associated with thromboembolism but some were related to anemic hypoxia. This finding suggests that maintenance of oxygen delivery through transfusion is an important aspect of successful treatment. However, there may be some risks with transfusion in dogs with IMHA. Raising the hematocrit may decrease the stimulus for erythopoiesis by the bone marrow. Some studies have suggested that transfusion may worsen the hemolysis which may in turn increase the risk of thromboembolism. However, transfusion has not been associated with a worse prognosis in studies that have looked at this variable. Because cross matching and typing is often difficult or impossible if the patient is auto-agglutinating, blood from universal donors is recommended (ideally DEA 1.1, 1.2, 3, 5, and 7 negative). Although Packed RBCs are commonly recommended, there has been some examination of whether whole blood or the addition of plasma could be helpful. Several studies have shown that dogs with IMHA meet some of the criteria of DIC and also have reduced levels of natural anti-coagulants such at AT. Plasma administration could be hypothesized to help prevent thromboembolism by providing some of these natural anticoagulants. A study was done in 13 dogs with IMHA who were given fresh frozen plasma along with heparin within 12 hours of admission. AT levels were measured before and 24 hours later and there was no change. 6 of the 10 dogs who died had evidence of thromboembolism at necropsy. A study by Rozanski, et al did show that AT levels in dogs with IMHA were maintained but not increased when plasma, but not heparin, was administered. It is possible that in dogs with IMHA and evidence of DIC that a much higher dose of plasma than that traditionally used would be needed to provide enough AT to see a benefit. Polymerized hemoglobin products (Oxyglobin®) have been used in IMHA patients to provide oxygen carrying capacity without red cell membranes that might worsen the immune response. In one retrospective study from Texas A & M, all dogs who received Oxyglobin® died but this product was only used in the most severe cases. There are no prospective studies examining whether this product does provide a benefit over blood transfusion. Conclusion Despite a large number of veterinary publications on IMHA, much is still not known about the disease. Based on available evidence, a protocol of prednisone, azathioprine, low-dose aspirin and the possible addition of IVIG in severe cases is recommended. Transfusion with red blood cells is recommended as needed to maintain oxygen delivery. Cyclosporine, Mycophenolate mofetil, Liposomal clodronate, and newer anti-platelet drugs should be studied further for efficacy in treatment of this disease. Texts Cohn L. Acute hemolytic disorders. In: Silverstein DC, Hopper K, eds. Small Animal Critical Care Medicine. 1st ed. Canada: Saunders, 2009. Gregory CR. Immunosuppressive Agents. In Current Vet Therapy XIII ed. Bonagura, JD. WB Saunders 2000, pp.509-513. Hardman JG, Limbird LE ed. Goodman and Gilman's The Pharmacological Basis of Therapeutics. 10th ed. New York: McGraw-Hill, 2001. King KE, Ness PM. Treatment of autoimmune hemolytic anemia. Seminars in Hematology 2005; 42:131-136. Miller E. CVT Update: Diagnosis and Treatment of Immune-Mediated Hemolytic Anemia. 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Efficacy of intravenous immunoglobulin in the treatment of autoimmune hemolytic anemia: results in 73 patients. Am J Hematol 1993;44:237-242. Gladwin MT, Crawford JH, Patel RP. The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radic Biol Med. 2004 Mar 15; 36(6):707-17. Grundy SA, Barton C. Influence of drug treatment on survival of dogs with immune-mediated hemolytic anemia: 88 cases (1989-1999). JAVMA 2001; 218: 543-546. Haneberg B, Matre R, Winsnes R, et al. Acute hemolytic anemia related to diphteria-pertussis-tetanus vaccination. Acta Paediatr Scand 1978; 67: 345-350. Ho WK, Martinelli A, Duggan JC. Severe immune haemolysis after standard doses of penicillins. Clin Lab J Haematology 2004; 26: 153-156. Horgan JE, Roberts BK, Schermerhorn T. Splenectomy as an adjunctive treatment for dogs with immune-mediated hemolytic anemia: ten cases (2003-2006). JVECCS 2009; 19: 254-261. Jackson, ML, Kruth SA. Immune-mediated hemolytic anemia and thrombocytopenia in the dog: a retrospective study of 55 cases diagnosed from 1969 through 1983 at the Western College of Veterinary Medicine. Can Vet J 1985; 26: 245-250. Jacobs, RM, Murtaugh RJ, Crocker DB. Use of microtiter Coombs' test for study of age, gender, and breed distributions in immunohemolytic anemia of the dog. JAVMA 1984; 185: 66-69. Kellerman DL, Bruyette DS. Intravenous human immunoglobulin for the treatment of immune-mediated hemolytic anemia in 13 dogs. JVIM 1997; 11:327-332. Klag AR, Giger U, Shofer FS. Idiopathic immune-mediated hemolytic anemia in dogs: 42 cases (1986-1990). JAVMA 1993; 202: 783-788. Klein, MK, Dow SW, Rosychuk RAW. Pulmonary thromboembolism associated with immune-mediated hemolytic anemia in dogs: Ten cases (1982-1987). JAVMA 1989; 195:246-250. Mason N, Duval D, Shofer FS, Giger U. Cyclophosphamide exerts no beneficial effect over prednisone alone in the initial treatment of acute immune-mediated hemolytic anemia in dogs: a randomized controlled clinical trial. JVIM 2003; 17: 206-212. Mathes M, Jordan M, Dow S. Evaluation of liposomal clodronate in experimental spontaneous autoimmune hemolytic anemia in dogs. Experimental Hematology 2006; 34: 1393-1402. McManus, PM, Craig LE. Correlation between leukocytosis and necropsy findings in dogs with immune-mediated hemolytic anemia: 34 cases (1994-1999). JAVMA 2001; 218: 1308-1313. Miller SA, Hohenhaus AE, Hale AS. Case-control study of blood type, breed, sex and bacteremia in dogs with immune-mediated hemolytic anemia. JAVMA 2004; 224: 232-235. Piek CJ, Junius G, Dekeer A, Schrauwen E, et al. Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs. JVIM 2008; 22:366-373. Reimer ME, Troy GC, Warnick LD. Immune-mediated hemolytic anemia: 70 cases (1988-1996). JAAHA 1999; 35: 384-391. Robbins RL, Wallace SS, Brunner CJ, et al. Immune-mediated haemolytic disease after penicillin therapy in a horse. Equine Vet J 1993; 25: 462-464 Rodriguez DB, Mackin A, Easley R, Boyle CR, et al. Relationship between red blood cell thiopurine methyltransferase activity and myelotoxicity in dogs receiving azathioprine. JVIM 2004; 18(3):339-45. Scott-Moncrieff JC, Reagan WJ, Snyder PW, Glickman LT. Intravenous administration of human immune globulin in dogs with immune-mediated hemolytic anemia. JAVMA 1997; 210: 1623-1627. Scott-Moncrieff JC, Treadwell NG, Brooks MB. Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia. JAAHA 2001; 37: 220-227. Scott-Moncrieff JC, Reagan WJ, Glickman LT, DeNicola DB, Harrington D. Treatment of nonregenerative anemia with human-?-globulin in dogs. JAVMA 1995;206:1895-1900. Weinkle TK, Center SA, Randolph JF, Warner KL, Barr SC, Erb HN. Evaluation of prognostic factors, survival rates, and treatment protocols for immune-mediated hemolytic anemia in dogs: 151 cases (1993-2002). JAVMA 2005; 226: 1869-1879. Weiss DJ, Brazzel JL. Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia. JVIM 2006; 682-686. Whelan MF, O'Toole TE, Chan DL, et al. Use of human immunoglobulin in addition to glucocorticoids for the initial treatment of dogs with immune-mediated hemolytic anemia. JVECCS 2009; 19: 158-164. Proceeding Articles Husbands B, et al. Prednisone and Cyclosporine vs. Prednisone Alone for Treatment of Canine Immune Mediated Hemolytic Anemia (IMHA) ACVIM Proceedings2004 Fryer JS, McMichael MA, Slater MR. Effect of heparin use on survival to discharge of dogs with immune-mediated hemolytic anemia. ACVIM Proceedings 2005, 829. Lunford K, Mackin A, et al. Pharmacokinetics of the biological effect of subcutaneous enoxaparin in dogs. ACVIM Proceedings 2005. Nielsen L, Niessen S, Ramsay M, Ramsay IK. The Use of Mycophenolate Mofetil in Eight Dogs with Idiopathic Immune-Mediated Haemolytic Anaemia. ECVIM Congress 2005 Proceedings. Whelan MF, Rozanski EA, et al. Use of the Canine Hemolytic Anemia Objective Score (Chaos) to Predict Survival in Dogs with Immune-Mediated Hemolytic Anemia. ACVIM Proceedings 2006 Uses for Plasma Products and Albumin: Evidence and Experience The indication for plasma products has become more debated in recent years and overall use is declining in both human and veterinary medicine. The times in which plasma transfusions are often given include: 1) Actively bleeding with documented coagulation factor defects 2) Severe pancreatitis for alpha macro globulin supplementation 3) Prior to invasive procedures in animals with documented changes in coagulation parameters 3) Suspected SIRS or DIC for general supplementation of coagulants and anti-coagulants and 4) Hypoalbuminemia. Despite these uses, the only time in which plasma has been conclusively proven to be effective is in actively bleeding patients with documented coagulation factor defects. When deciding to use a plasma product in a patient, it is important to look at: 1) Which product is most appropriate, 2) is a plasma product conclusively indicated, and 3) possible risks of transfusion versus potential advantages. Plasma products There are several different types of plasma products available that contain different components. Fresh frozen plasma has been frozen within 8 hours of collection of the blood. It contains all the clotting factors, anticoagulants, fibrinogen, albumin, and alpha macro globulin and is considered fresh frozen for one year. Coagulation factors are defined as either labile or non-labile, based on their activity with storage. Labile clotting factors include V and VIII. Non-labile include the vitamin K dependent factors II, VII, IX, and X. Frozen plasma is created when either the plasma was frozen greater than 8 hours after collection of the blood or the plasma was initially fresh frozen but is now over 12 months old. The labile clotting factors are not active in frozen plasma but the vitamin K dependent factors are so this product can still be used to treat anticoagulant rodenticide poisoning or as a source of albumin. Cryoprecipitate is made when fresh frozen plasma is slowly thawed to the slush stage and then centrifuged. The supernatant is removed and the remaining slush/sediment is a concentrated form of factor VIII, von Willebrand's factor, and fibrinogen. The cryoprecipitate can then be refrozen and is good for 12 months from the date of the initial blood draw. Cryoprecipitate is the preferred treatment for active bleeding in dogs with hemophilia A or von Willebrand's disease (vWD). It is also preferred for prophylactic use during elective surgery in animals with known vWD or hemophilia A. Cryo-poor plasma is the supernatant that is removed in the process of making cryoprecipitate. This plasma is still a source of the non-labile clotting factors II, VII, IX, and X and thus can also be used to treat anticoagulant rodenticide poisoning. It is also a source of albumin. Albumin can be extracted from plasma to make a concentrated product. Concentrated human serum albumin has been used in both canine and feline patients with extremely low albumin levels. Although several papers have shown relative safety with this product in sick patients, 2 healthy dogs died when given human serum albumin in a research trial due to severe type III hypersensitivity reactions. In addition, a follow up study showed that even sick patients do develop significant antibodies to human albumin after administration. A new canine albumin product has recently been released on the market (ABRI, Michigan). Each bottle contains 5 grams of albumin. An initial safety study performed by ABRI showed no signs of reactions in 6 dogs who received the product weekly for four weeks. Indications for Use Use of the correct plasma product in an actively bleeding patient with documented coagulopathy has been proven to be beneficial. It is important in these situations to use enough of the correct product. 15-20 ml/kg of fresh frozen plasma is needed to provide adequate coagulation factors to reverse coagulopathies from vWD and hemophilia A in dogs. The use of adequate plasma has also been proven to be critical in preventing the dilutional coagulopathy that occurs with massive red blood cell transfusion. A recent study of 214 civilian trauma patients showed that 30 day survival was increased when maintaining a 1:1 ratio of plasma to red blood cell transfusion in massively transfused patients. Hypoalbuminemia has been another indication for plasma or albumin transfusion. Low albumin levels have been correlated with increased mortality in both humans and animals. Albumin makes up 70-80% of colloid oncotic pressure and is very important as a carrier molecule for medications, and for the removal of bacterial toxins and free radicals. It is an important buffer and low albumin levels can lead to acidosis. Furthermore, it is important for microvascular integrity and in reducing platelet aggregation. Despite the numerous theoretical benefits, exogenous albumin supplementation has not been conclusively proven to improve survival. Several large meta-analyses examined human trials of albumin supplementation and could find no benefit. The SAFE trial looked at the use of albumin versus crystalloids alone for resuscitation in 6997 patients and could find no difference between the groups in days of hospitalization, ventilator free days or in 28 day mortality. There was a trend toward worse outcome with use of albumin in trauma patients. A recent abstract compared use of hydroxyethyl starch and human serum albumin in 74 dogs with albumin levels less than 2 g./dl. Although albumin levels were both lower initially in the human serum albumin group and higher later on, there was no difference in mortality between the two groups. However, in subgroup analysis of the SAFE trial of severe septic patients there was a trend toward improved mortality. In addition, a prospective study looking at use of human serum albumin in patients with ARDS did show an improvement in oxygenation and perfusion parameters. If plasma is going to be used to supplement albumin, it should be noted that 22.5ml/kg are needed to raise the serum albumin 0.5 g/dl if there are no ongoing losses. Concentrated albumin may be more effective in animals with severely low albumins and concurrent peripheral edema. When using a concentrated albumin, the following equation can be used to calculate the amount of albumin needed. Albumin deficit (grams) = 10 x (alb. desired (g/dl) - patient's current alb. (g/dl)) x wt (kg) x 0.3 Plasma use has been recommended to replace alpha macroglobulins and maintain albumin levels in patients with pancreatitis. It has been documented that survival is decreased in patients with low alpha macroglobulin levels. In a human study, no benefit was seen compared to control when giving plasma to patients with pancreatitis, even though an increase in alpha macro globulin level could be shown. This paper has been cited as a reason plasma should not be used in veterinary patients with pancreatitis. However, the control group for this paper was patients receiving colloid support in the form of albumin. Thus it is not known whether the colloid support of plasma may still be useful for patients with pancreatitis. In addition, the causes and course of canine and human pancreatitis are different so care should be taken when using evidence from one species to make recommendations for the other. A recent retrospective study looked at the use of plasma in dogs with pancreatitis. No improvement in mortality could be seen with plasma administration. However, albumin levels were not reported pre or post treatment for either group. Risks of plasma products Transfusion reactions due occur to plasma products and can either be acute or delayed. These reactions can be as severe to those to red blood cell products. Type specific plasma products should be used, accurate transfusion records should be kept, and animals should be monitored closely for reactions while these products are being administered. Concentrated albumin products cause significant oncotic pull. This can be very useful in animals that have severe peripheral edema or in animals who are severely hypotensive. However, if a concentrated albumin product is given to an animal who is normovolemic, volume overload with resultant pulmonary edema is a risk. Blood pressure should be monitored while administering these products and the animal should be watched for signs of volume overload. References: Day M, Mackin A, Littlewood J. editors. Manual of Canine and Feline Haematology and Transfusion Medicine. Gloucester, UK: BSAVA, 2000. Francis AH, Martin LG, Haldorson GJ, et al. Adverse reactions suggestive of type III hypersensitivity in six healthy dogs given human albumin. JAVMA 2007; 230:873-9. Leese T, Thomas WM, Holliday M. A Multicentre Controlled Clinical Trial of High Volume Fresh Frozen Plasma Therapy in Prognostically Severe Pancreatitis. Annals of the Royal College of Surgeons of England. 1991; 73:207-214. Malone DL, Hess JR, Fingerhut A. Massive transfusion practices around the globe and a suggestion for a common massive transfusion protocol. J Trauma 2006; 60: S91-6. Martin LG, Luther TY, Alperin DC, Gay JM, Hines SA. Serum antibodies against human albumin in critically ill and healthy dogs. JAVMA 2008; 232: 1004-1010. Petrides M, Stack G, Cooling L, Maes LY. Practical Guide to Transfusion Medicine. 2nd edition. Bethesda, MD: AABB press, 2007. Shaz BH, Dente CJ, Nicholas J, MacLeod JB. Increased number of coagulation products in relationship to red blood cell products transfused improves mortality in trauma patients. Transfusion 2010; 50: 493-500. Smith CL, Ramsay NB, Parr AM, et al. Evaluation of a Novel Canine Albumin Solution in Normal Beagles. IVECCS Proceedings 2009. Stokol T, Parry B. Efficacy of fresh-frozen plasma and cryoprecipitate in dogs with von Willebrand's disease or hemophilia A. JVIM 1998;12:84-92. Torrente C, Bayarri K, Bosch L, Segura D. Comparative Evaluation of the Hydroxyethyl Starch and Human Serum Albumin Infusions in Critically Ill Hypoalbuminemic Dogs: A Retrospective Study. IVECCS Proceedings 2009. Trow AV, Rozanski EA, deLaforcade A, Chan DL. Evaluation of use of human albumin in critically ill dogs: 73 cases (2003-2006). JAVMA 2008; 233: 607-612. Weatherton, LK and Streeter EM. Evaluation of fresh frozen plasma administration in dogs with pancreatitis: 77 cases (1995-2005). JVECCS 2009; 19: 617-622. Indications for Platelet Transfusion Thrombocytopenia is seen commonly in emergency veterinary patients but platelet transfusions are used infrequently. There are limited publications in veterinary medicine about triggers for prophylaxis or therapeutic efficacy of platelet products in actively bleeding patients. Both the limited publications and infrequent use of these products are due largely to the complexity of processing and storing platelets for immediate use in all but the largest veterinary institutions. Recent advances in platelet storage may make platelet transfusion more readily accessible to veterinarians. Platelet Physiology Platelets were first noted to be a true blood component, rather than just slide debris, by Alfred Donne in 1842 and their role in coagulation was first proposed in the 1860s.1 These cytoplasmic fragments originate from megakaryocytes located primarily in the bone marrow, but also in the lung, spleen, liver and kidney. Thousands of platelets are formed from branching and separation of megakaryocytes. This fragmentation process and the discoid structure of the platelets are driven by microtubules. Platelets bind to exposed von Willebrand's factor and collagen in damaged endothelium. This binding is mediated by GP Ib-IX-V and GPVI complexes. Collagen binding causes intracellular signaling, leading to activation of the platelets with resultant platelet shape changes, changes in platelet receptor affinity and release of active molecules from the dense and alpha granules. Platelets aggregate to form the primary hemostatic plug through interactions of the activated GP11b-111a receptors with fibrinogen. Activated platelets also accelerate procoagulant activity and bind coagulation proteins to keep the reactions localized to the site of the injury.2 Average platelet survival in dogs is approximately 5-7 days while it is 8-10 days in people. In non disease states, platelets either become senescent and are removed by the spleen or are used in ongoing endothelial maintenance. Kinetic studies in people show that a set number of platelets are needed for endothelial maintenance resulting in higher percentages that are needed in thrombocytopenic conditions. Thus, average platelet survival is correlated with the degree of thrombocytopenia.3 Causes of Thrombocytopenia and Thrombocytopathia Thrombocytopenia can be caused by decreased production, accelerated removal, increased consumption, loss or dilution. The most common cause of thrombocytopenia in dogs is immune-mediated thrombocytopenia.3,4 Other causes include infectious diseases such as Ehrlichiosis, bone marrow insults, neoplasia, disseminated intravascular coagulation (DIC), and blood loss. Therapy with certain drugs can also result in thrombocytopenia. This can be a dose related toxicity or can be an idiosynchatic reaction. Azathioprine and chloramphenicol can lead to thrombocytopenia through a dose related affect on the bone marrow. In people, heparin can cause thrombocytopenia. This thrombocytopenia results from the development of antibodies against platelet factor 4 - heparin complexes. These antibodies lead to inappropriate platelet activation and consumption. It is most commonly seen in people who have received heparin previously. Drug therapy with potentiated sulfonamides can also result in an immune related thrombocytopenia in both dogs and people.5 In the inherited macrothrombocytopenia of Cavalier King Charles Spaniels, a genetic mutation in the beta-tubulin protein of the microtubules is known to be the cause.6 Cavaliers affected with this condition can have platelet counts as low as 50,000/ul but often have no signs of bleeding. It is important to distinguish this condition from immune causes of platelet destruction as these animals do not require treatment. In thrombocytopathic conditions, platelet numbers are normal but function is impaired. Acquired loss of platelet function is associated with medications and certain disease states. Non-steroidal anti-inflammatory drugs cause inhibition of the COX-1 enzyme, leading to decreased thromboxane generation and decreased platelet aggregation. Animals with both hepatic disease and uremia can have platelet function issues. The decreased function in hepatic disease thought to be due to changes in platelet phospholipids while uremia appears to impair platelet function by decreasing adhesion to the endothelium.7 Several hereditary thrombocytopathias have been identified in dogs and cats.8 Glanzmann thrombasthenia is a genetic disease in which the GP 11b-111a receptor is decreased on missing. This disease has been documented in Great Pyrenees and Otterhounds. In Basset Hound thrombopathia, platelets do not aggregate normally due to a defect in calcium exchange. The gene has been identified and is autosomal recessive. Similar calcium exchange genetic defects have been found in 2 Eskimo Spitz and in Landseer-ECT. A selected defect of ADP content of granules was noted in a family of Cocker Spaniels. A family of German Shephards was found to have a defect in which platelets did not express procoagulant activity. These dogs have signs more suggestive of a coagulopathy than a primary hemostatic defect but have normal coagulation screening tests and normal platelet function tests making diagnosis a challenge. At Guelph in 2001, 581 transfusions were given, but only 40 were for platelet support. All of the platelet support transfusions were in dogs, 50% for ITP and the rest for a combination of leukemias, aplastic anemias, DIC, splenic neoplasia, chemotherapy induced thrombocytopenia and thrombocytopenia associated with vitamin K antagonism toxicity.3 In a recently completed multicenter trial looking at lyophilized platelets, 43 dogs received platelet transfusions. Of these, 36 had ITP or Evans syndrome, 3 had underlying neoplasia, 2 were in DIC, 1 had Ehrlichiosis, and 1 had Basset Thrombocytopathia. Indications for Platelet transfusions and dosing In humans, platelets are recommended for prophylaxis in any patient with a count less than 10,000/ul and in patients who need an invasive procedure with counts less then 50,000/ul. Platelets are recommended therapeutically in any actively bleeding patient with a count less than 20,000/ul. Platelets are also recommended in patients with drug or hereditary impairment of platelet function that need an invasive procedure.9, 10 However, ITP is considered a unique situation due to the rapid clearance of any administered platelets. In addition, platelets in ITP are often younger and hyperfunctional so that bleeding may not occur until counts are extremely low. Platelet transfusions are usually not recommended for prophylaxis in this disease.10 The risk of bleeding with thrombocytopenia is affected by the degree of anemia. A higher packed cell volume reduces the risk of bleeding through several mechanisms. RBCs scavenge nitric oxide which leads to increases in platelet activity. In addition, a higher hematocrit pushes platelets toward the endothelium and reduces sheer stress. Lastly, RBCs are known to contain mediators that increase platelet activity.11 In cases of moderate thrombocytopenia and concurrent anemia, the risk of bleeding may be lessened with packed RBC transfusion alone. However, it has been recommended to increase the ratio of platelet and plasma transfusions to packed red blood cells in situations of massive blood loss. Two human retrospective studies of severe trauma have shown increased survival when these ratios were kept close to 1:1. 12, 13 Lyophilized platelet products may also be used as hemostatic agents even in cases where platelet numbers and function are normal. In a recent study, 20 swine were subjected to a grade III liver injury. They were then given either a placebo or lyophilized platelets. Two hours later, the liver was repaired. There was 80% survival in the group that received the lyophilized platelets while only 20% survival in the group receiving placebo. One pig did have thrombi in other locations on necropsy indicating the need for more study before the products are used in clinical patients for this indication.14 The risk of bleeding from thrombocytopenia must be weighed against the risk of transfusion reaction especially when prophylactic platelet transfusions are considered. The reported rate of febrile reactions in people is 38% while 2% have a severe adverse reaction. In addition, alloimmunization does occur in both people and dogs which can lead to platelet transfusion refractoriness. This alloimmunization can occur with as little as 3 transfusions in dogs.4 Dosing recommendations have been made by calculating the expected rise in platelet count. The expected one hour platelet increment in dogs can be calculated as: Total number platelets transfused x 0.51/blood volume, where blood volume is 85ml/kg. The standard fresh platelet concentrate dose of 1 U per 10 kg was derived to aim for a 40,000/ul increase in platelet count.3 Some investigators have looked at whether the interval between transfusions could be extended by using a higher dose initially. A meta-analysis in humans did show an increase in the interval between transfusions when a higher dose was used initially.15 Currently Available Products Fresh whole blood is the product most veterinarians use when platelets are needed. A 500 ml unit of fresh whole blood obtained from a canine donor is estimated to contain 7 X 1010 platelets. A dose of 10ml/kg of whole blood would be expected to raise the platelet count about 10,000/ul.3 The advantage of fresh whole blood is that no platelets are lost during separation. In addition, the platelets are less activated than in platelets obtained via centrifugation for concentrate.16 Fresh whole blood at room temperature is considered safe for use for 4-8 hours. Refrigeration of human platelets, either in whole blood or in platelet concentrate, rapidly leads to platelet aggregation and activation. In addition, refrigerated platelet survival is half that of platelets held at room temperature. This effect is due to clustering of the vWF receptors in response to temperature leading to increased clearance by hepatic macrophages.17 No studies have been done of canine refrigerated platelets to confirm if a similar response occurs.4 Fresh platelet concentrate has traditionally been made when initially processing whole blood. Whole blood is spun using a "soft" spin which separates the platelets into the plasma component. The plasma is expressed into a separate bag. This plasma is then known as platelet rich plasma (PRP). The PRP is then spun again to create a platelet concentrate (PC) and the plasma is removed and stored as fresh frozen plasma. One unit of PC is the amount made from one unit (500 ml) of whole blood but will contain a lower amount of platelets. A recent study showed a maximum in vivo platelet recovery of 80%.18 The dose is normally calculated as 1 unit/ 10 kg. Fresh platelets must be stored in gas soluble bags at room temperature to remain active. They must also be constantly agitated and thus are kept on continuous rockers. Bacterial contamination is a concern at room temperature and storage is limited to 5 days.10 Fresh platelet concentrate can also be made through plateletpheresis. In this process, blood is taken from the donor, split into components using an apheresis machine, the desired component is retained, and the rest is returned to the donor. The process is more time consuming for the donor but many more platelets can be collected and sterility of the product is maximized. Dog donors have been trained to remain still for successful apheresis. An apheresis donation will produce a platelet concentrate containing 1-4 x 1011 platelets, 4-6 times the amount in a centrifuged platelet concentrate.3 In addition, there is neglible WBC and RBC contamination. Fresh platelets collected in this manner must also be kept constantly agitated at room temperature. Because of the higher sterility during collection, they can be stored for up to 7 days.10 Frozen platelet concentrate is made by stabilizing apheresed platelets with 6% DMSO or with 2% DMSO and Thrombosol. In original work done by Valeri, canine platelet recovery after freezing with 6% DMSO at -80 F was shown to be 70% with a ½ life of 2 days versus 3.5 days for fresh platelets.19 The platelets were shown to be effective in halting active bleeding in thrombocytopenic dogs. A more recent study comparing 6% DMSO to 2%DMSO and Thrombosol showed only 49% and 44% platelet recovery respectively. Platelet half-life was confirmed to be about 2 days.18 The frozen product must be thawed at room temperature. Lyophilized platelets are currently in canine clinical trials through Animal Blood Resources International. Platelets are stabilized using a mild aldehyde crosslinking of membrane proteins and lipids. This process then allows for lyophilization and reconstitution with preservation of platelet structure and function. The lyophilized platelets can be stored for up to 24 months in the refrigerator, and are reconstituted with saline immediately prior to use. Research studies on these platelets have shown that they bind to collagen, von Willebrand factors and damaged endothelium. Receptors are activated normally and bind fibrinogen. Importantly, these platelets also retain the ability to increase procoagulant activity.20 In a study of dogs on cardiac bypass, infusion of the lyophilized platelet product led to improvement in venous bleeding time that was most pronounced at 20-30 minutes after infusion.21 A prospective, multicenter trial was recently completed to study the use of a lyophilized platelet product (LYO) in dogs with thrombocytopenia. Dogs who met the entry criteria were randomized and received either lyophilized platelets or fresh platelet concentrate. Fresh platelet concentrate was sent to each hospital weekly to be on hand as needed for the study. Physical exam, hematologic, and coagulation status data was collected at baseline, one hour and 24 hours after transfusion. 34 dogs met the criteria for the study: 21 dogs were given LYO and 13 dogs were given fresh platelets. An additional 7 patients received LYO and 2 patients received fresh platelet concentrate either during or after the study as a compassionate use. . In total, 28 dogs had received lyophilized platelets at the time of this publication. Of the dogs receiving the fresh platelets, 13% were noted to have possible transfusion reactions within one hour of the transfusion - one with facial uticaria and one with panting. Of the dogs receiving lyophilized platelets, 7% had possible transfusion reactions - one with a 2 degree F rise in temperature but no other signs and one with tachycardia and pale gums noted one hour after the infusion that improved with further packed RBC administration. Although no significant difference in bleeding score or platelet count could be measured in dogs receiving lyophilized platelets, in at least 2 cases, active bleeding was observed to stop immediately after the transfusion of the LYO. The LYO was found to be easy to use and administer. An additional finding of the study was that keeping 2 units of aphresed fresh platelet concentrate on hand at all times at each of the 5 institutions was economically unfeasible on a long term basis. References:
Should I have In-house Blood Donors? Selection and Testing Requirements It is extremely valuable to have blood available when a patient presents with an acute transfusion need. Purchased blood products can be expensive and it is difficult to keep correct quantities on hand. In addition, there are times when fresh whole blood is the best product for the patient. Correct evaluation and testing of in house blood donors is crucial to protect the safety of the donor and the recipient. When choosing blood donors for an in house program, physical characteristics, behavioral traits, blood type and infectious disease screening are all important. Physical exam and behavior Dogs: The best blood donors are between the ages of one and seven weigh over 25 kg (55 lbs). It is recommended that dogs not give more than 15-20 ml/kg of blood at one time and the standard collection bag holds 450ml. Dogs which are eager to meet new people, who are easy to exam and who are willing to lie still for 10-15 minutes are preferred. Dogs can be sedated for blood donation but care must be taken with medication choice to avoid hypotension during the draw and over sedation when the animal goes home. Cats: The best blood donors are between the ages of one and seven. Cats should weigh over 3.5 kg. This is to allow collection of 10-15ml/kg or approximately 60 ml from a 4 kg animal. Cats should be indoor only and live with cats that are also indoor only to minimize risk of infectious disease. It is best to have cats who allow thorough unsedated physical exams and small volume blood draws for testing. Unfortunately, the majority of cats will not allow unsedated blood donation. The best choice of sedation and anesthetic protocols for cats is widely debated. Blood typing Dogs: The DEA 1 system is the most important as it is the most antigenic. It has several subtypes that are allelic (DEA 1.1, 1.2, and 1.3). Dogs who are DEA 1.1 negative will not react to DEA 1.1 positive blood the first time it is given as they do not have natural antibodies to this allele. This is why it was originally thought that the first transfusion was always safe. However, the second time a DEA 1.1 negative dog is given 1.1 positive blood, they will have a severe hemolytic reaction. It has been shown that natural antibodies are present in some dogs for DEA 3, 5, and 7 and in occasional dogs for less common antigens (i.e. DAL in some Dalmatians). These natural antibodies can result in transfusion reactions. There are several available in house typing kits available for dogs. However, the kits can only test for DEA 1.1 status. In order to identify dogs that are true universal donors (DEA 1.1, 3, 5, and 7 negative, 4 positive), typing must be done through a commercial laboratory. The most common in house typing kits are the DMS card test (DMS Laboratories, NJ) and the Alvedia cartridge test (Alvedia, France). In a recent study, 84% agreement was noted between methodologies but both false negatives and false positives were found with the card test and rare false negatives were seen with the cartridge Cats: Cats are either Type A, Type B, or Type AB. It was originally reported that 97% of cats in the United States were type A and that only 0.3% of cats in the northeast part of the United States were type B. However, a 2005 study from the AMC had a 6% incidence of the B blood type. The B blood type is more common in certain exotic breeds of cats, including Devon Rex (41%), British Shorthair (36%), Cornish Rex (31%), Exotic Shorthair (27%), and Scottish Fold (19%) but is also seen in domestic breed cats. Type AB has been reported to occur in less than 1% of the general cat population. However, this incidence may be higher as newer methodologies seem better at identifying these cats. Type A cats may have weak anti-B alloantibodies which can cause shortened RBC survival if a B donor is used. Type B cats, however, have very strong anti-A antibodies and can have a fatal reaction from as little as 1ml of transfused Type A blood. Type AB cats have no alloantibodies against either type A or B blood in their sera. They should receive either AB or A blood if they need a transfusion. Other blood groups may be present as incompatible cross match results can be seen with Type A cats given repeated Type A blood In house typing of healthy cats is more reliable than in dogs. Both the DMS card test and Alvedia cartridge test are fairly accurate, although neither were 100% in a recent study. It is recommended that B and AB cats be confirmed by an outside laboratory. General health All donors should be up to date on vaccines and free of flea and ticks. PCV should be over 40% in dogs and over 35% in cats. A complete blood count, chemistry panel and fecal are recommended yearly for active blood donors. Although these are usually normal in young, healthy dogs with no clinical problems, an occasional change will be found that indicates an emerging problem. Infectious Disease Screening In human medicine, individual bags of blood are screened for infectious diseases. Only a limited number of diseases are screened due to cost concerns and test availability. Careful interview of donors is used to minimize risks of other diseases. In the veterinary field, it is cost prohibitive in a small scale setting to test each individual unit. Therefore, a combination of careful interview and blood screening of the donor is used to minimize the risk of infectious disease transmission. Screened diseases meet the following criteria:
http://www3.interscience.wiley.com/cgi-bin/fulltext/119821263/PDFSTART?CRETRY=1&SRETRY=0 The recommendations discussed which diseases to test for and which test methodologies to use. Since publication, PCR (polymerase chain reaction) assays have become more available for these diseases of concern. PCR assays when positive (and run correctly) indicate that organism has been identified in the blood stream and active infection is present. False negatives are possible if the organism is present only in very small amounts as only a small sample of blood is examined. Several labs now offer blood donor screening PCR panels. It is important when choosing a panel to evaluate whether the diseases included are correct for your geographic area. In addition, it is important to question the lab on the specificity and sensitivity of their tests as this can vary between laboratories. Dogs: Babesia canis and Babesia gibsoni are transmitted by ticks and have been documented to pass in blood transfusions. Testing can be by antibody titer, or ideally by PCR. Leishmania can also be transmitted via blood transfusion. It is common in foxhounds but is found in other breeds only in certain parts of the country so testing should be conditional. Ehrlichia spp and Anaplasma spp are also vector borne and can pass in blood transfusions. E.canis and A. phagocytophilum can be tested via a beside ELISA but other Ehrlichia spp may not be identified by this test. PCR may be a more accurate way to make sure less common ehrlichias are not being carried by the donor. M. haemocanis and Candidatus M. haematoparvum are also erythrocyte parasites that can cause hemolytic anemia in immunosuppressed or splenectomized dogs. PCR testing is recommended for these organisms. Brucellosis is a bacterial disease that has been documented to pass in human transfusions but not shown directly to be transmitted in canine transfusions. However, bacteremia in dogs can be longstanding. Any dog with a history of breeding should be screened for brucellosis antibodies by the rapid slide agglutination test (RSAT). Dogs that are currently breeding will need to be screened on a more regular basis. Chagas disease, or American Trypanosomiasis, is caused by a blood borne protozoan but has not been proven to be transmitted via blood transfusion. It is only found in Texas and the southwestern region and testing should be considered in dogs from this region Bartonella vinsonni (berkhoffi) is an intraerythrocytic organism that is thought to be tick borne. It has been suspected to have caused endocarditis and other fever syndromes in some dogs. It is not known if it is transmitted in blood transfusions but can be transmitted by intravenous infusion. This is an emerging disease and testing should be considered. Heartworm disease does not meet the criteria for testing as microfilaria from an infected donor transfused into a recipient will not cause heartworm disease. However, for the safety of the donor, screening for heartworm disease is recommended in endemic areas. Rocky Mountain Spotted Fever and Lyme's disease are not recommended for screening. RMSF does not have a carrier state and Lyme's disease has not been shown to be transmitted via blood transfusion. Cats: FeLV and FIV ELISA testing should be performed initially and for FeLV again one month later prior to use as a blood donor. PCR testing should be done for Mycoplasma hemofelis and Mycoplasma haemominutum, which used to be classified as Hemobartonella felis. It is debated whether cats used as blood donors should be tested for Bartonella spp. via PCR. The seroprevalence in cats is high, especially in areas where fleas are common so this test might exclude a number of donors. However, bartonella has been associated with uveitis and other fever diseases in cats. Testing for Cytauxzoonosis, Ehrlichia spp, Anaplasma spp and Neorickettsia spp. should be based on geographic area. FIP and toxoplasmosis are not recommended for testing as they have not been documented to be transmitted via blood transfusion. Von Willebrand's Factor (vWF) If donors will be used to provide plasma or cryoprecipitate for dogs with vWD, it is crucial that they have adequate vWF. Although bruising may occur from the initial bloodwork screening draw in potential donors with very low vWF, levels can be quite low with no clinical signs. Actual factor testing is recommended with donors excluded for use of plasma with levels < 70%. References: Blais MC, Berman L, Oakley DA, et al. Canine Dal blood type: A red cell antigen lacking in some Dalmations. JVIM 2007; 21:281-6. Day M, Mackin A, Littlewood J. editors. Manual of Canine and Feline Haematology and Transfusion Medicine. Gloucester, UK: BSAVA, 2000. Klaser DA, Reine NJ, Holenhaus A. Red blood cell transfusions in cats: 126 cases. JAVMA 2005; 226(6): 920-3 Owens SD, Oakley DA, Marryott K, et al. Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. JAVMA 2001; 219:1076-83. Seth M, Winzelberg S, Jackson KV, et al. Comparison of Gel Column, Card and Cartridge Techniques for DEA 1.1 Blood Typing of Dogs. ACVIM Proceedings, 2008 (abstract). Wardrop KJM, Reine N, Birkenheuer A, Hohenhaus A, et al. Canine and Feline Blood Donor Screening for Infectious Disease. JVIM 2005;19:135-142. Transfusion Reactions Although transfusions are life saving, it should be remembered that life threatening reactions are a risk of giving blood products. Reactions are possible to all blood products, including plasma, cryoprecipitate, and platelets. Reactions can either be acute (occurring within 48 hours of start of transfusion) or delayed. These reactions can be further classified as immunologic or non-immunologic. Immunologic Reactions Immunologic reactions are those that occur due to an antibody-antigen reaction. Hemolytic reactions are type II hypersensitivity reactions involving recipient IgG or IgM. These recipient antibodies bind to antigens on donor red blood cells and lead to destruction of cells. An antigen antibody reaction involving IgM will activate complement and can lead to a reaction within 5 minutes of the start of a transfusion. Signs include intravascular hemolysis, systemic hypotension, fever, tachycardia or bradycardia, vomiting, defecation, weakness, dyspnea and even cardiac arrest. In dogs, these hemolytic reactions usually occur when a DEA 1.1 negative dog receives a second transfusion from a DEA 1.1 positive dog. In cats, these reactions can occur during a first transfusion if a B cat is given type A cells. If an acute hemolytic reaction is suspected, the transfusion should be stopped and an investigation started. The identity and blood type of both the recipient and blood donor should be confirmed. History on the recipient should be confirmed by asking whether there is any chance the animal had a previous unknown transfusion. Blood samples should be obtained from both the recipient and from the donor bag. These samples should be used to re-type, consider cross match, possible coombs testing, and blood culture as a septic reaction can mimic an acute hemolytic reaction. Supportive care with IV fluids should be started and oxygen should be supplemented if the animal is dyspneic. Antigen-antibody reactions that don't activate complement can lead to increased red blood cell phagocytosis with extravascular hemolysis. Signs of this type of reaction are less severe and may include jaundice, fever, or an unexplained decrease in the PCV after transfusion. IV fluids are important if hemolysis is noted to protect the kidneys from the effects of hemoglobinuria. Use of glucocorticoids for hemolytic transfusion reactions is controversial and these drugs are usually not used in human cases. Although glucocorticoids can minimize some of the inflammatory response they do not acutely suppress the production of IgG or IgM antibodies. Allergic reactions, which are type I hypersensitivity reactions, also occur. These reactions are mediated by preformed IgE or IgG antibodies bound to mast cells and basophils. Binding of these antibodies to allergens such as anticoagulants or plasma proteins causes mast cell and basophil activation with release of histamine, leukotrienes and other vasoactive substances. Anaphylactoid reactions that are IgA mediated have been reported in IgA deficient people. Signs of this type of reaction may be pruritus, erythema, and uticaria within 45 minutes of the start of a transfusion. More severe signs could include vomiting, dyspnea, and non-cardiogenic pulmonary edema. Treatment for a mild reaction would be the administration of diphenhydramine at 2mg/kg IM and slowing of the transfusion. In more severe reactions, the transfusion should be stopped, diphenhydramine given and epinephrine 1:1000 - 0.01-0.02mg/kg IM or IV should be considered. A short acting steroid can be considered to help with inflammation but it should be remembered that steroids do not prevent the binding of IgE to mast cells. Non-hemolytic Febrile Reaction are one of the most common types of transfusion reaction seen. They are a temperature increase of 1-2 C within 1-2 hours of a transfusion. They are usually due to antibody reactions against donor leukocyte or platelet antigens. These type of reactions are greatly reduced when leukoreduction filters are used prior to storage of blood. Leukoreduction filters do work with canine blood products but the cost has prevented widespread use in veterinary medicine. It is important to watch animals carefully as a fever can also be an early sign of a more septic or hemolytic reaction. The transfusion should be slowed and a non-steroidal anti-inflammatory could be considered if the animal is clinical for the fever and no other type of reaction is suspected. Transfusion Related Lung Injury (TRALI) is one of the most serious complications in human transfusions. It was originally defined as the development of bilateral pulmonary edema, fever, and hypotension 1-6 hours after transfusion of a plasma containing blood product. More recently, it has been recognized that TRALI can also be delayed, especially in critical ill patients and manifest 6-72 hours after a transfusion. It is due to a leukocyte antigen reaction that leads to leukocyte aggregates in pulmonary circulation and the development of a high protein pulmonary effusion. It must be differentiated from cardiogenic pulmonary edema or volume overload. In people, it has been noted that the incidence of reaction can be decreased by not using women as plasma donors who had have multiple pregnancies as these pregnancies increase the incidence of neutrophils antibodies in their blood. Treatment is supportive with oxygen and ventilation. Diuretics are not effective due to the nature of the effusion. There are no published veterinary case reports. Thrombocytopenia does occur in people occasionally about one week after transfusion. This is usually non-clinical. The incidence is unknown in dogs but has been reported once. Several studies have also reported immunosuppression after transfusion possibly due to down-regulation of the immune system from immunoregulatory compounds in plasma. This syndrome has been termed TRIM (transfusion related immunomodulation). An increase organ transplant survival but also an increase in the incidence of infections after transfusion has been reported. Non-Immunologic Reactions The most important reaction to remember is circulatory overload. Blood products are natural colloids and can cause a dramatic increase in volume. Signs can include peripheral edema or respiratory compromise. Jugular distension is often visible if this is occurring. The transfusion should be slowed or stopped and the use of diuretics considered. In severe cases, phlebotomy is useful. Sepsis can occur if contaminated blood is administered. Signs of sepsis include fever, hypotension, low blood glucose and could even progress to DIC. It is very important that blood that is stored is collected with aseptic technique, blood is not left at room temperature for over 4-6 hours, and that open units, even those in the refrigerator, are discarded after 24 hours. Because it can be very difficult to differentiate an acute hemolytic reaction from a septic reaction in the early stages, samples should be obtained of both the blood unit and from the recipient for culture, type, cross-match and Coombs. Urine can also be obtained from the recipient for culture. A gram stain of any remaining blood product may also help identify the presence of bacteria and type. Antibiotics and other supportive care should be initiated if a septic reaction is suspected. Infectious Disease Transmission is one of the biggest risks of blood transfusion. The transmission of leishmania, hemobartonella, and babesia through transfusion has been reported in animals. Accurate transfusion records are crucial so that if an unexpected disease arises in a recipient, the blood can be traced back to the donor and appropriate testing done. It is strongly recommended that blood donors only be used when thoroughly screened for infectious diseases of importance. Citrate toxicity occurs with the rapid infusion of products that contain CPD or ACD. Citrate is used as an anticoagulant in blood because of its binding of calcium. Rapid administration can result in hypocalcemia. Signs include face rubbing, vomiting, tremors, tetany, and cardiac abnormalities. Citrate toxicity is more common in animals who are very small, who have liver problems, or who are cold. Treatment includes stopping the transfusion and administering 10% calcium gluconate at 0.5-1.5 ml/kg IV slowly while carefully monitoring heart rate or EKG. The transfusion can then be restarted at a slower rate. Hemolysis can occur within a blood unit due to improper blood handling, usually either freezing or overheating. When the hemolyzed product is administered, the recipient can develop hemoglobinuria and hemoglobinemia. The recipient is usually not harmed by this but it is important to differentiate from an immune-mediated hemolytic reaction. Examining a sample of the blood product for hemolysis is useful in making this determination. Hyperammonemia can occur in patients with liver disease who are given stored red blood cell products. Ammonia levels increase as blood is stored. Signs are similar to hepatic encephalopathy. In patients with severe liver disease, the freshest red blood cell products should be used for transfusion. Hypophosphatemia, hyperkalemia, and acidemia are also all potential complications from use of large quantities of stored blood. Hypothermia can develop if unwarmed products are given, especially to small or young animals. Coagulopathy can develop if massive transfusions of red cell products are given without concurrent administration of platelets and plasma. This is a dilutional coagulopathy. Due to the risks of transfusions, their use should always be closely examined. Animals should be monitored carefully during a transfusion and careful records should be maintained. References: Bracker KE, Drellich S. Transfusion Reactions. Compendium July 2005; 500-512. Giger U, Gelens CJ, Callan MB, Oakley DA. An acute hemolytic transfusion reaction caused by dog erythrocyte antigen 1.1 incompatibility in a previously sensitized dog. JAVMA 1995; 206: 1358-1362. Harrell K, Parrow J, Kristensen A. Canine transfusion reactions. Part I. Causes and Consequences. Compendium 1997; 19: 181-190. Harrell K, Parrow J, Kristensen A. Canine transfusion reactions. Part II. Prevention and Treatment. Compendium 1997; 19: 193-200. Hohenhaus A, Drusin LM, Garvey MS. Serratia marcescens contamination of feline whole blood in a hospital blood bank. JAVMA 1997; 210:794-798. Katja J, Melzer K, Wardrop J, Hale AS, et al. A hemolytic transfusion reaction due to DEA 4 alloantibodies in a dog. JVIM 2003; 17: 931-933. Lee, Justine. Transfusion Related Acute Lung Injury. IVECCS Proceedings 2009. Marik PE, Corwin HL. Acute lung injury after blood transfusion: expanding the definition. CCM 2008; 36: 3080-3084. Owens SD, Oakley DA, Marryott K, et al. Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. JAVMA 2001; 219:1076-83. Tocci LJ, Ewing PJ. Increasing patient safety in veterinary transfusion medicine: an overview of pretransfusion testing. JVECCS 2009; 19: 66-73. Waddell LS, Holt DE, Hughes D, Giger U. The effect of storage on ammonia concentration in canine packed red blood cells. JVECCS 2001; 11:23-26. Wardrop KJM, Reine N, Birkenheuer A, Hohenhaus A, et al. Canine and Feline Blood Donor Screening for Infectious Disease. JVIM 2005;19:135-142 Appendix A Cross Match Procedure
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