Suspected human intravenous immunoglobulin-induced acute haemolytic anaemia in a dog.

A 2-year-old mixed breed dog presented with a 1-year history of crust and erosion on the nasal planum. Because histopathological examination revealed ruptured intraepidermal pustules and superficial dermal inflammation, the dog was diagnosed with pemphigus foliaceus. Human intravenous immunoglobulin was administered in two consecutive doses of 0.5 g/kg/day due to poor therapeutic response to previous immunosuppressive therapy. From Day 3 after the first dose of human intravenous immunoglobulin, tachypnoea, pale mucous membrane, haemoglobinuria and haemoglobinemia were observed, thus confirming haemolytic anaemia. Other drug-induced haemolytic anaemias were excluded because no additional drugs had been administered before the haemolysis occurred. Immune-mediated haemolytic anaemia was also excluded because the direct antiglobulin test was negative. Two transfusions were performed, and haemolysis was not observed from Day 4 of haemolytic anaemia onset. In conclusion, human intravenous immunoglobulin-induced haemolytic anaemia should be considered in dogs that develop haemolysis following the administration of human intravenous immunoglobulin.

Comparison of serum versus EDTA plasma in canine major crossmatch reactions.

BACKGROUND: Protocols for crossmatch reactions vary in veterinary medicine, particularly regarding the use of recipient serum vs plasma. Sources suggest that major crossmatch results might differ when recipient plasma is used instead of serum, but there are conflicting reports as to the exact effects on the results. OBJECTIVES: The aim of this study was to determine the frequency and degree of discrepancy, if any, between canine major crossmatch reactions using serum versus EDTA plasma, performed via a standard tube method. METHODS: One hundred duplicate canine major crossmatch reactions were performed with both serum and EDTA plasma from 100 different "recipient" dogs against erythrocytes from a single, healthy "donor." Decreasing concentrations of a rabbit anti-dog erythrocyte antibody were added to generate strong positive, weak positive, and negative results for each crossmatch reaction. Crossmatch results were followed through the following phases: immediate spin, cold, warm, albumin, and Coombs. Semi-quantitative results were compared between reactions using serum vs EDTA plasma. RESULTS: Weak positive, major crossmatch reactions were significantly more likely to demonstrate stronger agglutination in EDTA plasma compared with serum in the immediate spin phase, cold phase, warm phase, and albumin phase (P < 0.001). There was no statistically significant difference between serum and EDTA plasma results in the Coombs' phase (P = 0.313). CONCLUSIONS: In this experimental setting, EDTA plasma and serum were both deemed acceptable for use in canine major crossmatch reactions. EDTA plasma might be preferable to detect weak agglutination with more sensitivity and reduce recipient blood volumes needed to complete the crossmatch reaction.

Effect of dilution of canine blood samples on the specificity of saline agglutination tests for immune-mediated hemolysis.

BACKGROUND: Saline agglutination tests (SATs) are widely recommended for diagnosis of immune-mediated hemolytic anemia in dogs, but there are frequent false-positive results. OBJECTIVES: Specificity of SATs will improve at higher saline-to-blood ratios. ANIMALS: One hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%. METHODS: Prospective diagnostic accuracy study. Immune-mediated hemolysis (IMH) was considered present if a gel direct antiglobulin test (DAT) was positive and there was clinical evidence of hemolysis (n = 9), absent if another mechanism for anemia was identified and the DAT was negative or there was no hemolysis (n = 138), and if IMH status was unclear, dogs were excluded (n = 3). Saline agglutination tests were prepared at 1 : 1, 4 : 1, 9 : 1, and 49 : 1 saline-to-blood ratios, and microscopic agglutination was considered a positive result. RESULTS: Specificity for IMH increased from 29% (95% confidence interval 20-38) at a 1 : 1 dilution to 97% (93-99) at a 49 : 1 dilution. Sensitivity was 88% (47-100) at 1 : 1 and 4 : 1 dilutions and 67% (30-93%) at 9 : 1 and 49 : 1 dilutions. Diagnostic accuracy increased from 33% (24-42) at 1 : 1 dilution to 95% (90-98) at 49 : 1 dilution. CONCLUSIONS AND CLINICAL IMPORTANCE: If performed using a 49 : 1 saline-to-blood ratio, SATs achieve high specificity for IMH. Based on a gold standard of positive DAT and evidence of hemolysis, lower saline-to-blood ratio results should not be used because false-positive results are common.

The utility of diagnostic tests for immune-mediated hemolytic anemia.

BACKGROUND: A definitive diagnosis of immune-mediated hemolytic anemia (IMHA) can be difficult to make. However, it is critical to differentiate IMHA from other causes of anemia due to the impact on prognosis and outcome for IMHA patients. Recently published American College of Veterinary Internal Medicine recommendations for the diagnosis of IMHA should be followed to concurrently confirm ongoing anemia, verify in vivo hemolysis, and detect anti-erythrocyte antibodies. The reliability of immunologic IMHA tests varies depending on which test is used and how it is performed. OBJECTIVES: Our aims were to determine which tests are currently used in veterinary medicine to diagnose IMHA and review the utility of assays that have historically been used to diagnose IMHA. METHODS: A short survey was designed to see which diagnostic tests for IMHA were currently being used by veterinary practices. The survey was distributed via list-serves to veterinarians and veterinary technologists. A literature review was performed to report the utility of diagnostic tests for the diagnosis of IMHA. RESULTS: Survey respondents indicated a variability in test protocols used to diagnose IMHA. Most respondents perform saline agglutination or Coombs' tests to detect anti-erythrocyte antibodies. Additional tests that can be used to support a diagnosis of IMHA are discussed in this review. CONCLUSIONS: A standardized diagnostic approach should be followed to differentiate IMHA from other causes of anemia. Test methodology can vary from one laboratory to another, and clinicians should be familiar with the procedures used by their laboratory.

Detection of naturally occurring alloantibody by an in-clinic antiglobulin-enhanced and standard crossmatch gel column test in non-transfused domestic shorthair cats.

BACKGROUND: Blood typing for the A and B antigens is essential and crossmatching testing is generally recommended before transfusing blood to cats. OBJECTIVE: To evaluate 2 crossmatch (XM) tests. ANIMALS: Forty-nine healthy domestic shorthair cats that had not received a blood transfusion. METHODS: Prospective study. Blood samples were typed for AB using immunochromatographic and flow cytometric techniques. A gel column (GC) and a feline antiglobulin-enhanced gel column (AGC) XM tests were used for crossmatching. RESULTS: The population included 34 type A, 13 B, and 2 AB cats, with concordant results (r = 1, P < .005) by flow cytometry and immunochromatographic strip kit. The plasma from type A cats had either no or weak anti-B alloantibodies. The plasma of 12 of 13 type B cats contained strong anti-A alloantibodies. For crossmatching, plasma to RBC pairings were prepared using the GC (n = 446) and AGC (n = 630) tests. Both methods showed compatibilities in 329 and incompatibilities in 102 pairings including all A-B mismatches. Additionally 15 pairings showed agglutination by the AGC but not GC method. Fourteen incompatibilities outside the expected A-B mismatches were only revealed by AGC. CONCLUSIONS AND CLINICAL IMPORTANCE: AB typing using immunochromatographic strip is as accurate as laboratory flow cytometry. The 2 XM methods had good agreement with additional incompatibilities being recognized by the AGC XM beyond A-B incompatibilities. In clinic, feline AB typing and sensitive XM test kits are available and recommended before each transfusion, although the clinical implications of incompatible XM test results and clinical benefits of such crossmatching have not been documented.

Prevalence of antinuclear and anti-erythrocyte antibodies in healthy cats.

BACKGROUND: Positive antinuclear antibody and direct antiglobulin tests support diagnoses such as systemic lupus erythematosus and immune-mediated anemia, respectively. Positive tests may occur in cats, but the prevalence of positive results in healthy cats is not well known. OBJECTIVE: The study's purpose was to determine prevalences of positive antinuclear antibody and direct antiglobulin tests in healthy cats. METHODS: Antinuclear antibody titers were measured by indirect immunofluorescence, and anti-erythrocyte antibodies were measured by the microtitration direct antiglobulin test at 37, 23, and 4°C in 61 client-owned and 28 facility-owned cats. Differences between the 2 groups were examined using chi-squared tests. RESULTS: For the antinuclear antibody tests, 70% of client-owned cats were negative, 10% had weak titers (1:40-1:80), and 20% had strong titers (1:160-1:320). Facility-owned cats had significantly fewer positive titers with 96% negative and one positive (1:8). For the antiglobulin test at 37°C, 93% of all cats were negative, 2 cats in each group were positive at low dilutions (1:2), and 2 client-owned cats were transiently positive at high dilutions (≥ 1:2048). At 23°C, 90% of all cats were negative, and 2 client-owned and 5 facility-owned cats were positive at low dilutions (1:2-1:8). At 4°C, 67% of client-owned cats had invalid results (negative control well agglutination), and 33% had negative results, while of facility-owned cats 14% had invalid results, 14% had agglutination at low dilutions, and 72% were negative. CONCLUSION: Healthy cats may have positive antinuclear antibody and direct antiglobulin tests, but the prevalence of strong reactions is low.

Pre- and Post-Transfusion Alloimmunization in Dogs Characterized by 2 Antiglobulin-Enhanced Cross-match Tests.

BACKGROUND: When dogs are transfused, blood compatibility testing varies widely but may include dog erythrocyte antigen (DEA) 1 typing and rarely cross-matching. OBJECTIVES: Prospective study to examine naturally occurring alloantibodies against red blood cells (RBCs) and alloimmunization by transfusion using 2 antiglobulin-enhanced cross-match tests. ANIMALS: Eighty client-owned anemic, 72 donor, and 7 control dogs. METHODS: All dogs were typed for DEA 1 and some also for DEA 4 and DEA 7. Major cross-match tests with canine antiglobulin-enhanced immunochromatographic strip and gel columns were performed 26-129 days post-transfusion (median, 39 days); some dogs had an additional early evaluation 11-22 days post-transfusion (median, 16 days). Plasma from alloimmunized recipients was cross-matched against RBCs from 34 donor and control dogs. RESULTS: The 2 cross-match methods gave entirely concordant results. All 126 pretransfusion cross-match results for the 80 anemic recipients were compatible, but 54 dogs died or were lost to follow up. Among the 26 recipients with follow-up, 1 dog accidently received DEA 1-mismatched blood and became cross-match-incompatible post-transfusion. Eleven of the 25 DEA 1-matched recipients (44%) became incompatible against other RBC antigens. No naturally occurring anti-DEA 7 alloantibodies were detected in DEA 7- dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: The antiglobulin-enhanced immunochromatographic strip cross-match and laboratory gel column techniques identified no naturally occurring alloantibodies against RBC antigens, but a high degree of post-transfusion alloimmunization in dogs. Cross-matching is warranted in any dog that has been previously transfused independent of initial DEA 1 typing and cross-matching results before the first transfusion event.

Increased erythrocytic osmotic fragility in anemic domestic shorthair and purebred cats.

OBJECTIVES: Increased erythrocytic osmotic fragility and splenomegaly have been reported in anemic Abyssinian and Somali cats. Here we report on this condition in anemic domestic shorthair cats and two other breeds, and describe common features of the clinicopathological profiles, management and outcomes. METHODS: Anemic cats, other than Abyssinians and Somalis, were included. The erythrocytic osmotic fragility test was performed, known causes of anemia were excluded, the illness was followed and medical records were reviewed. RESULTS: Twelve neutered cats were first found to be anemic between 0.5 and 9.0 years of age. Pallor, lethargy, inappetence, pica, weight loss and splenomegaly were commonly observed. A moderate-to-severe macrocytic and hypochromic anemia with variable regeneration was noted. Infectious disease screening, direct Coombs' and pyruvate kinase DNA mutation test results were negative. Freshly drawn blood did not appear hemolysed but became progressively lysed during storage at 4°C. The sigmoid osmotic fragility curves were moderately to severely right shifted, indicating erythrocytic fragility at 20°C. Cross-correction studies indicated an intrinsic red cell effect rather than plasma effect. Most cats were treated with immunosuppressive doses of prednisolone and doxycycline, with variable responses. Five cats with recurrent or persistent anemia responded well to splenectomy. However, two had occasional recurrence of severe anemia: one was found to be Bartonella vinsonii-positive during one episode and responded to azithromycin and prednisolone, while the other cat had two episodes of severe anemia of unknown cause. Finally, six cats were euthanized within 1 month and 7 years after initial presentation. Histopathology of six spleens revealed mainly congestion and extramedullary hematopoiesis. CONCLUSIONS AND RELEVANCE: Similarly to Abyssinian and Somali cats, domestic shorthair and cats of other breeds can also develop severe erythrocytic osmotic fragility with anemia and splenomegaly, which should be considered as a differential diagnosis in anemic cats.

First report on seroepidemiology of Toxoplasma gondii infection in pigs in Central Ethiopia.

BACKGROUND: Toxoplasma gondii is one of the most widely prevalent cyst forming Apicomplexan parasites with significant impact on animal production particularly in sheep, goats and pigs. The objectives of this cross-sectional study were to estimate the seroprevalence and to assess risk factors of Toxoplasma gondii infection in pigs. A systematic random sampling technique was used to collect 402 blood samples from pigs in Central Ethiopia. Direct Agglutination Test (DAT) was used to test sera. A questionnaire survey was made to assess potential risk factors and knowledge of farm attendants about toxoplasmosis. RESULTS: An overall seroprevalence of 32.1% [95% confidence interval (CI): 27.6%-36.9%] was found. Multivariable logistic regression analysis showed that extensively managed pigs (39.7%) are nearly twice (adjusted odds ratio [aOR]:=1.91, 95% CI: 1.01, 3.63) at higher risk of acquiring toxoplasmosis than intensively managed pigs (30.5%). Pigs supplied with feed containing animal byproducts had nearly four times (OR = 3.84, 95% CI: 2.01, 7.36) higher risk of acquiring T. gondii infection. Most of the farm attendants had little knowledge of health risks due to cats, neither to human nor to animals. Absence of rodent control, high neonatal mortality and history of abortion were found among herds of the studied pig farms. CONCLUSIONS: T. gondii infections in pigs are wide spread. Extensive management systems and pig feed types containing animal byproducts are independent predictors of T. gondii seropositivity. The high seroprevalence suggests that pigs might serve as an important source of T. gondii infection for people. This is the first report of seroepidemiology of T. gondii infection in pigs in Ethiopia. Further studies are warranted for designing appropriate prevention and control strategies.

Comparison of 4 direct Coombs' test methods with polyclonal antiglobulins in anemic and nonanemic dogs for in-clinic or laboratory use.

BACKGROUND: Difficulties with the direct antiglobulin test (DAT) and its apparent lack of sensitivity and specificity for immune-mediated hemolytic anemia (IMHA) in dogs have raised skepticism regarding its diagnostic value. OBJECTIVE: To compare different DATs and other hematologic parameters in dogs. ANIMALS: Anticoagulated blood samples from 59 nonanemic and 46 anemic dogs (± IMHA) from a research colony and veterinary clinics. METHODS: Prospective observational study: Immunochromatographic strip, gel microcolumn, and capillary techniques were compared with standard microtiter DAT using 2 polyvalent antiglobulins. Spherocytosis, autoagglutination, osmotic fragility, and clinical data were assessed. RESULTS: Blood samples from all 59 nonanemic dogs were DAT-. Among 46 anemic dogs, 33 were suspected of IMHA, but only 20 were DAT+. Old and new DAT methods yielded comparable and consistent results even after storage of chilled blood samples for 1 week. Spherocytosis and autoagglutination (that did not persist after washing) were noted in 15 and 16 DAT+ dogs, respectively. The other 26 anemic dogs, including 21 previously transfused dogs and 4 with autoagglutination, tested DAT- by the other methods. Osmotic fragility was increased in 70% (19/27) of anemic and all 15 DAT+ dogs tested. Limited follow-up testing revealed DAT+ results for 3-70 days. CONCLUSIONS AND CLINICAL IMPORTANCE: The novel strip and capillary DAT methods are promising adjunct in-clinic tools. Despite prior immunosuppressive treatment and presence of autoagglutination, the DAT was positive in anemic dogs with IMHA. Transfusion did not cause false DAT+ results. Our results support DAT as a cornerstone in the diagnosis of canine IMHA.

Coombs' testing and its diagnostic significance in dogs and cats.

The Coombs' test can detect both immunoglobulin and complement on the surface of RBCs, and as such can be of value as an aid in the diagnosis of IMHA. Techniques that may improve sensitivity include use of monovalent reagents, increased dilutions of antiglobulin to avoid a prozone effect, and testing at 4°C. These techniques are not without controversy, and positive tests should always be interpreted in the presence of other clinical and hematologic evidence for IMHA. Alternate techniques, such as flow cytometry, can improve detection of RBC-bound immunoglobulin, but require a flow cytometer and further standardization between laboratories.

Bovine immune-mediated hemolytic anemia: 13 cases (November 2008-August 2009).

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) occurs in cattle; however, there are few reported cases. OBJECTIVE: The aim of this study was to investigate the prevalence of IMHA in cattle with anemia, describe the associated clinical and laboratory findings, including osmotic fragility, and identify potential causative infectious agents or drugs. METHODS: This study included 42 anemic cattle (HCT < 27.5%) comprising 31 females and 11 bulls with a mean age of 3.5 years referred to the University of Tehran Veterinary Teaching Hospital during a 10-month period. CBCs, saline osmotic fragility tests, direct Coombs' tests, and biochemical profiles were performed, and blood smears were evaluated for spherocytosis, parasites, and microscopic agglutination. Five clinically healthy cattle were used as controls for testing osmotic fragility of RBCs. RESULTS: The Coombs' test was positive in 13/42 (30%) cattle; 5 had no evidence of concurrent disease or history of drug administration, and 8 had underlying or concurrent diseases, positivity for BLV, or exposure to drugs. The HCT (mean ± SE) of Coombs'-positive cattle (16 ± 1.7%) was significantly lower than that of Coombs'-negative animals (21 ± 0.8%). Hematologic and biochemical findings in cattle with IMHA included anisocytosis (2), polychromasia (2), basophilic stippling (2), spherocytosis (2), hyperfibrinogenemia (5), left-shifted neutrophilia (3), and hyperbilirubinemia (8). RBCs from Coombs'-positive anemic cattle were more fragile than those from Coombs'-negative anemic cattle. Four osmotically different populations of RBCs were detected in cattle with IMHA, whereas RBC populations were homogeneous in the Coombs'-negative anemic cattle and in normal cattle. CONCLUSION: IMHA was identified in a significant proportion of anemic cattle. Idiopathic IMHA and IMHA secondary to infectious diseases and administration of certain drugs occur in cattle.

Production of an anticanine globulin (polyspecific) reagent for laboratory investigation.

OBJECTIVES: Canine allo- or autoantibodies are clinically important, but attachment of these immunoglobulin G (IgG) antibodies does not produce observable haemagglutination. Antibody to canine globulins is required to demonstrate sensitisation of red blood cells. Commercial reagents are available, but these often differ in sensitivity and specificity. Rabbit anticanine globulins (polyspecific) were produced for use in canine blood compatibility testing and in the investigation of immune-mediated haemolytic anaemia. METHODS: Canine sera was pooled, IgG was purified and subsequently used to immunise rabbits. A rising titre of anticanine IgG was demonstrated by indirect enzyme-linked immunosorbent assay. Rabbit anticanine complement was isolated and investigated by agglutination of complement-coated canine red blood cells. Both antibodies were purified and checked for crossreactivity before being combined to polyspecific anticanine globulins. The obtained reagent was used to indicate sensitised canine red blood cells and free antibodies in three different types of clinical samples, including blood for compatibility testing and that for investigation of immune-mediated haemolytic anaemia and screening for post-transfusion alloantibodies and was also compared to commercial Coombs' reagent. RESULTS: The product provided results in accordance with those from commercial Coombs' reagent. The sensitivity for canine crossmatching was 100% and specificity for diagnosing immune-mediated haemolytic anaemia was 87%. CLINICAL SIGNIFICANCE: This product is helpful for canine crossmatching purposes and in the investigation of immune-mediated haemolytic anaemia.

Coombs', haemoplasma and retrovirus testing in feline anaemia.

OBJECTIVE: To investigate the associations between Coombs' testing, haemoplasma and retroviral infections, and feline anaemia. METHODS: Haematology, Coombs' testing (including assessment of persistent autoagglutination) and selected infection testing (haemoplasma, feline leukaemia virus/feline immunodeficiency virus provirus) were performed in blood samples collected from 60 anaemic and 60 non-anaemic cats. RESULTS: No association between infection and anaemia or Coombs' positivity existed. Anaemic cats (21.7%) were significantly more likely than non-anaemic cats (0%) to have cold autoagglutination (P<0.0001), but significance (set at

Pattern of Coombs' test reactivity has diagnostic significance in dogs with immune-mediated haemolytic anaemia.

OBJECTIVES: To investigate the clinical significance of the pattern of Coombs' test reactivity in dogs with immune-mediated haemolytic anaemia. METHODS: Sixty-five anaemic dogs with a positive Coombs' test were included. Coombs' testing was performed at 4 and 37 degrees C with polyvalent canine Coombs' reagent and antisera specific for each of canine immunoglobulin G, immunoglobulin M and complement factor C3. The impact of performing testing with only polyvalent antiserum at 37 degrees C was assessed. Chi-squared tests were used to compare Coombs' test reactivity in dogs with primary immune-mediated haemolytic anaemia (group A) and in dogs with concurrent/underlying disease (group B). Following Bonferroni correction, significance was set at P < or = 0.003. RESULTS: Eleven dogs would have been regarded as Coombs' negative had they been tested with polyvalent antiserum at 37 degrees C alone. Group A dogs were significantly more likely to be positive with polyvalent antiserum and/or anti-dog immunoglobulin G at 4 and/or 37 degrees C (P < or = 0.001) and tended to be less likely to be positive with anti-dog immunoglobulin M at 4 degrees C (P=0.040). CLINICAL SIGNIFICANCE: Testing of anaemic dogs with polyvalent Coombs' reagent at 37 degrees C was less sensitive than testing with monovalent reagents at 4 and 37 degrees C. The pattern of Coombs' test reactivity differed significantly between dogs with primary immune-mediated haemolytic anaemia and those with concurrent/underlying disease.

Canine immune-mediated hemolytic anemia: pathophysiology, clinical signs, and diagnosis.

Immune-mediated hemolytic anemia (IMHA) is a common type of anemia in dogs and cats that results from a type II hypersensitivity reaction. The disease is most common in middle-aged female dogs, especially American cocker spaniels. The common clinical signs are associated with severe anemia and the resultant inflammatory response. There is no pathognomonic test for IMHA, but the following are suggestive of it: the presence of hemolytic anemia in a young adult or middle-aged dog of a predisposed breed, autoagglutination and/or spherocytosis, positive results from a direct antiglobulin (Coombs') test, elimination of any other underlying cause of anemia, and an appropriate response to immunosuppressive therapy.

Performance of 2 microtiter canine Coombs' tests.

BACKGROUND: The Coombs' test, which detects immunoglobulin or complement on RBC surfaces, has long been the standard for laboratory confirmation of immune-mediated hemolytic anemia (IMHA), a common cause of hemolytic anemia in the dog. This test, however, suffers from relatively low sensitivity. Optimization of test sensitivity would lead to fewer discrepancies between laboratory results and clinical diagnoses, and in some cases institution of appropriate therapy in a timely manner. OBJECTIVES: The objectives of this study were to 1) characterize the sensitivity and specificity of 2 canine Coombs' tests for the detection of IMHA, 2) document the efficacy of using multiple antisera dilutions beyond what is directed by manufacturers, and 3) evaluate the necessity of monovalent antisera in the test protocol. METHODS: Sixty-five canine whole-blood samples submitted for Coombs' testing were evaluated. Patients were classified as IMHA positive or negative based on a set of predetermined criteria. IMHA classification was compared to Coombs' test results from 2 different Coombs' tests adapted to a microtiter-plate format. One Coombs' test (VMRD Coombs' test) utilized a single polyvalent antiserum (VMRD, Inc, Pullman, WA, USA), while a second Coombs' test (University of Minnesota [U of MN] Coombs' test) used both polyvalent and monovalent antisera. RESULTS: Sensitivity and specificity were 61% and 100% for the VMRD Coombs' test, and 82% and 95% for the U of MN Coombs' test. The use of multiple antisera dilutions resulted in 6 additional Coombs' positive test results. All positive Coombs' test results were positive by polyvalent antisera. CONCLUSIONS: When used in a microtiter-plate format, the U of MN Coombs' test was a more sensitive test for the detection of IMHA in canine patients when compared to the VMRD Coombs' test. The use of multiple antisera dilutions increased test sensitivity. Sensitivity, however, was not increased by the use of monovalent antisera in the Coombs' test protocol.