Warm Reactive Autoantibodies Clinical and Serologic Correlations

Coagulation and Transfusion Medicine / CHARACTERISTICS OF WARM AUTOANTIBODIES Warm Reactive Autoantibodies Clinical and Serologic Correlations Christine A. Wheeler, MD, * Loni Calhoun, MT(ASCP)SBB, and Douglas P. Blackall, MD Key Words: Autoantibodies; Hemolysis; RBCs Abstract Warm reactive autoantibodies are encountered relatively frequently in tertiary care hospitals. We studied 100 consecutive patients with warm autoantibodies to correlate their clinical and serologic features. Study patients (56 male, 44 female) had various diagnoses and a mean age of 53.5 years (range, 3-90 years). Autoimmune hemolysis was documented in 29 patients; 20 patients (69%) in this subset had diseases classically associated with warm autoimmune hemolytic anemia (hematologic and autoimmune disorders). All study patients demonstrated IgG on their RBCs (direct antiglobulin test [DAT] reactivity range, microscopic to 4+); 49 also demonstrated C3 (reactivity range, microscopic to 3+). The DAT for IgG was 2+ or more in 25 (86%) of 29 patients with hemolysis; the DAT for IgG was 1+ or less in 45 (63%) of 71 patients without hemolysis. In patients with hemolysis, 21 (72%) of 29 had a DAT reactive for C3. These findings may be useful in determining the clinical significance of warm autoantibodies and the extent to which patients should be followed up for hemolysis. Warm reactive autoantibodies are RBC-directed immune responses that are maximally reactive at 37 C. These antibodies react with the patients own RBCs and typically bind to all reagent or donor RBCs against which they are tested. However, they sometimes have specificity (absolute or relative) for a blood group antigen, most commonly a member of the Rh blood group system. Warm reactive autoantibodies are usually polyclonal IgG antibodies, although IgA or monomeric IgM antibodies are seen occasionally. Complement (C3) also may accompany these antibodies, as detected by the direct antiglobulin test (DAT). The clinical significance of warm reactive autoantibodies relates to their hemolytic potential and the interference that they can cause with routine pretransfusion compatibility testing (eg, the detection of blood group antibodies and the provision of crossmatch-compatible units of blood). 1 Warm reactive autoantibodies are encountered relatively frequently at our institution. During the years 2001 and 2002, warm autoantibodies constituted approximately 7% of all antibody identification evaluations. However, it was our impression that hemolytic anemia occurred in only a minority of patients with warm autoantibodies. This study was undertaken to correlate the clinical and serologic features of patients with warm autoantibodies and to determine whether clinical assessment practices could or should be improved. Materials and Methods The study patients were selected from a daily log of all patients who had antibody (alloantibody, autoantibody, or both) detected in their serum or plasma or on their RBCs. In 680 Am J Clin Pathol 2004;122:680-685 Downloaded 680 from https://academic.oup.com/ajcp/article-abstract/122/5/680/1759385

Coagulation and Transfusion Medicine / ORIGINAL ARTICLE this retrospective study, 100 consecutive patients with warm autoantibodies were evaluated. Patients with insufficient clinical or laboratory data to evaluate for the presence or absence of hemolysis due to warm autoantibodies were excluded. Age, sex, diagnosis, and serologic reactivity were recorded. EDTA-anticoagulated blood samples were used for indirect antiglobulin testing and the DAT. Serologic testing conformed with methods provided in the Technical Manual of the American Association of Blood Banks. 1 Antibody detection and identification were performed by gel testing according to the manufacturer s specifications (Ortho-Clinical Diagnostics, Raritan, NJ). Autologous or allogeneic adsorption was performed, as needed, to detect underlying alloantibody and to confirm autoantibody reactivity. DATs were performed in the following situations: positive autocontrol in antibody identification studies, suspicion of hemolysis, or request by a clinician. DATs were performed by test tube methods using a licensed, commercial, polyspecific antiglobulin reagent (Ortho-Clinical Diagnostics). Monospecific antiglobulin reagents for IgG and C3 (Ortho-Clinical Diagnostics) were used to test samples with positive DAT results. In some cases, antibody was eluted to help establish the presence of a warm autoantibody or to evaluate for the presence of an alloantibody on RBCs. Elutions were performed using a commercially available acid elution kit (Elu- Kit II, Gamma Biologicals, Houston, TX). The eluates were tested using the gel-igg method with a last wash saline control. The presence of a warm autoantibody was established by pan-reactivity of an acid eluate prepared from patient RBCs or by pan-reactivity of patient plasma or serum tested against a panel of reagent RBCs. Clinical and laboratory data were reviewed for all patients using a computer database. The patients were placed into 1 of 8 disease categories based on their predominant diagnosis: autoimmune disorder, hematologic malignancy, other hematologic disorder, nonhematologic malignancy, end-stage renal or liver disease, idiopathic autoimmune hemolytic anemia, cardiac disease, or other disease. Clinical records, transfusion requirements, and the following laboratory parameters were used as a basis for determining whether an individual patient was likely to be experiencing hemolysis: hematocrit, less than 37.4% (<0.37); hemoglobin, less than 12.3 g/dl (<123 g/l); reticulocyte count, more than 2.06% (>0.021) or an absolute count of more than 107.2 10 3 /µl (>107.2 10 9 /L); total bilirubin concentration, more than 1.5 mg/dl (>26 µmol/l; majority unconjugated); lactate dehydrogenase level, more than 220 U/L (>220 U/L); and haptoglobin, less than 6 mg/dl (<0.06 g/l). Responses to transfusion also were reviewed for patients who experienced hemolysis due to warm autoantibodies. A full response to transfusion of 1 U of RBCs was considered to be a hematocrit increase of 2% to 3% (0.02-0.03) or an increase of 1 g/dl (10 g/l) in the hemoglobin concentration. Other therapies (eg, steroids, intravenous immune globulin, and splenectomy) also were reviewed. Results There were 56 male and 44 female study patients. Their ages ranged from 3 to 90 years (mean age, 53.5 years). Table 1 shows the disease categories of all patients with warm autoantibodies, the number of patients with warm autoantibodies who experienced hemolysis due to these antibodies, and the number of patients with warm autoantibodies who did not experience hemolysis. Autoimmune disorders included systemic lupus erythematosus (6), rheumatoid arthritis (1), polyglandular autoimmune disorder (1), Graves disease (1), ankylosing spondylitis (1), ulcerative colitis (4), autoimmune liver disease (2), antiphospholipid syndrome (2), and an unspecified category (3). Hematologic malignant neoplasms included lymphoma (7), mycosis fungoides (1), leukemia (6), and Castleman disease (1). Other hematologic disorders included idiopathic thrombocytopenic purpura (3), congenital dyserythropoietic anemia (1), myelodysplastic syndrome (4), lymphadenopathy (1), and sickle cell disease (2). Nonhematologic malignant neoplasms (1 or 2 cases of each entity) included malignant mesothelioma, adenocarcinoma, non small cell carcinoma (lung), transitional cell carcinoma, hepatocellular carcinoma, and malignant melanoma. Other diseases (1 or 2 cases of each entity) were subarachnoid hemorrhage with cerebral aneurysm, moyamoya disease, cerebrovascular accident, chronic lung disease, abdominal aortic aneurysm, gastrointestinal bleeding, hepatitis or liver disease, and interstitial cystitis. One patient who was given methyldopa and then developed warm autoantibodies, without hemolysis, is included in the other diseases category. Table 1 Disease Categories in Patients With and Without Autoimmune Hemolysis * Disease Category Hemolysis No Hemolysis Autoimmune disorder (n = 21) 9 (43) 12 (57) Cardiac disease (n = 19) 1 (5) 18 (95) Hematologic malignancy (n = 15) 8 (53) 7 (47) End-stage renal or liver disease (n = 12) 3 (25) 9 (75) Other diseases (n = 12) 3 (25) 9 (75) Other hematologic disorders (n = 11) 3 (27) 8 (73) Nonhematologic malignancy (n = 8) 0 (0) 8 (100) Idiopathic hemolysis (n = 2) 2 (100) 0 (0) Total (n = 100) 29 (29) 71 (71) * Data are given as number (percentage). Includes 1 patient who also experienced a delayed hemolytic transfusion reaction. Downloaded from https://academic.oup.com/ajcp/article-abstract/122/5/680/1759385 Am J Clin Pathol 2004;122:680-685 681 681 681

Wheeler et al / CHARACTERISTICS OF WARM AUTOANTIBODIES Autoimmune hemolysis occurred in 29 patients. Three of these patients experienced hemolysis at an outside institution but no longer had evidence of hemolysis when evaluated at our institution. One patient died as a direct result of autoimmune hemolysis, which was intravascular. Three patients experienced hemolysis not thought to be attributable to their warm reactive autoantibodies: 1 had passenger lymphocyte syndrome after allogeneic stem cell transplantation, 1 had hemolysis secondary to aortic valve stenosis, and 1 had systemic lupus erythematosus and Kasabach-Merritt syndrome with chronic, low-level disseminated intravascular coagulation. One patient with systemic lupus erythematosus had a warm reactive autoantibody and evidence of a delayed hemolytic transfusion reaction due to anti-jk a (as evidenced by rapid emergence of the alloantibody shortly after transfusion). Based on persistent laboratory evidence of hemolysis and continued transfusion requirements, it seemed that autoimmune hemolysis was also present. Of note, 20 (69%) of 29 patients with warm autoantibodies who had autoimmune hemolysis had diseases classically associated with warm autoimmune hemolytic anemia: autoimmune disorders (including idiopathic thrombocytopenic purpura) and hematologic malignant neoplasms. The DAT was reactive for IgG in all patients with warm autoantibodies and ranged in strength from microscopically reactive to 4+. In Table 2, DAT results (for IgG) are correlated with the presence or absence of autoimmune hemolysis, and the predictive value of individual DAT reaction strengths is shown. The DAT was 2+ or greater in strength in 25 (86%) of the 29 patients with warm autoantibodies who experienced autoimmune hemolysis, and the predictive value of a strongly reactive DAT ( 2+) was 49%. The DAT was 1+ or weaker in strength in 45 (63%) of 71 patients who did not experience hemolysis. The relationship between the presence or absence of hemolysis and the DAT strength was highly statistically significant by χ 2 analysis (P <.005). The DAT for C3 was reactive in 49 patients with warm autoantibodies and ranged in strength from microscopically reactive to 3+. Of these patients, 21 (43%) experienced hemolysis, and 28 (57%) were unaffected. However, closer evaluation of the 29 patients who experienced hemolysis revealed that 21 (72%) had a positive DAT for C3. In Table 3, DAT results (for C3) are correlated with the presence or absence of autoimmune hemolysis, and the predictive value of individual DAT reaction strengths is shown. The relationship between hemolysis and the presence of complement was highly statistically significant (P <.005). Autoantibody only was detected on the RBCs of 30 patients but also was detected in the serum or plasma of the other 70 patients. In 12 patients, the autoantibody mimicked the specificity of an alloantibody as follows: anti-e, 6; anti-e, 2; anti-c and anti-e, 1; anti-d, 1; anti-f, 1; and anti-jk b, 1. In 34 patients, autoadsorptions were performed; in 30 patients, alloadsorptions were performed. Alloantibodies were detected in 53 patients. Of these, 24 had single specificities, and 29 had multiple antibodies (range, 2-7). The specificity of the alloantibodies detected by blood group system is shown in Table 4. Cold agglutinins (all reactive at room temperature or lower) also were present in 8 patients. Of the 29 patients who had autoimmune hemolytic anemia, 23 received transfusions of RBCs. Of these, 16 (70%) had at least a partial response to transfusion. In 7 patients, the responses to some or all of the transfusions could not be determined because hemoglobin and hematocrit values were not Table 2 DAT Results (for IgG) Correlated With Hemolysis DAT No. of Patients No. of Patients Predictive Value * Strength With Hemolysis Without Hemolysis (%) m+ 2 14 13 1+ 2 31 6 2+ 13 16 45 3+ 7 9 44 4+ 5 1 83 Total 29 71 DAT, direct antiglobulin test; m+, microscopically reactive. * Likelihood that a patient with a given DAT result will experience hemolysis, calculated as follows: predictive value = TP/(TP + FP), where TP indicates patients with a positive DAT result who experience hemolysis and FP patients with the same positive DAT result who do not experience hemolysis. Table 3 DAT Results (for C3) Correlated With Hemolysis DAT No. of Patients No. of Patients Predictive Value * Strength With Hemolysis Without Hemolysis (%) 0 8 43 16 m+ 6 15 29 1+ 8 12 40 2+ 6 1 86 3+ 1 0 100 4+ 0 0 0 Total 29 71 DAT, direct antiglobulin test; m+, microscopically reactive. * Likelihood that a patient with a given DAT result will experience hemolysis, calculated as follows: predictive value = TP/(TP + FP), where TP indicates patients with a positive DAT result who experience hemolysis and FP patients with the same positive DAT result who do not experience hemolysis. Table 4 Patients With Alloantibodies by Blood Group Blood Group No. of Patients * Rh 40 Kell 21 Kidd 5 Duffy 6 MNS 7 Lutheran 2 High-titer, low avidity 6 * A total of 53 patients had alloantibodies, some multiple. 682 Am J Clin Pathol 2004;122:680-685 Downloaded 682 from https://academic.oup.com/ajcp/article-abstract/122/5/680/1759385

Coagulation and Transfusion Medicine / ORIGINAL ARTICLE available or transfusions were given elsewhere. Twenty-five patients received other therapy for their hemolytic anemia, including steroids, intravenous immune globulin, rituximab (anti-cd20 monoclonal antibody), 6-mercaptopurine, chlorambucil, cyclosporine, vincristine, methotrexate, mycophenolate, pentostatin, thalidomide, and splenectomy. Discussion During the last 15 years, the medical literature has contained little new information focused on correlating the clinical significance of warm reactive autoantibodies with associated serologic findings. This is an important concern because this time frame has borne witness to a variety of novel immune modulating agents and therapies, a consequence of which might be autoantibody development. When RBC autoantibodies are detected, it would be useful to be able to predict the biologic behavior of these antibodies to plan therapy and provide appropriate patient follow-up. A better understanding of the serologic features of warm reactive autoantibodies might prove helpful in this regard. In the present study, only 29% of the patients with warm autoantibodies actually experienced a hemolytic anemia. However, 20 (69%) of 29 patients with hemolysis had diseases classically associated with warm autoimmune hemolytic anemia: autoimmune disorders, including idiopathic thrombocytopenic purpura, and hematologic malignant neoplasms. A few studies of patients with a specific disease entity who also had warm autoantibodies address the issue of the proportion of patients who experienced hemolysis. In one study of patients with carcinoma and warm reactive autoantibodies (not associated with cold agglutinins), 49 of 99 patients experienced autoimmune hemolytic anemia. 2 In a second study, 46 patients with myelodysplastic syndrome had erythrocyte autoantibodies (warm, mixed warm and cold, or cold); 15 of these patients had autoimmune hemolysis, with 7 patients experiencing hemolysis secondary to warm autoantibodies alone. 3 In another study, 14 pediatric patients with sickle cell disease developed warm autoantibodies after receiving multiple erythrocyte transfusions; 4 of these patients experienced autoimmune hemolysis. 4 Issitt et al 5 describe 3 groups of a total of 150 subjects with warm autoantibodies: 87 had autoimmune hemolytic anemia, 33 had taken α-methyldopa but most did not experience hemolysis, and 30 were hematologically healthy without evidence of hemolysis (25 healthy blood donors and 5 additional patients without autoimmune disorders). All patients in our study had warm reactive autoantibodies and a DAT reactive for IgG. Of these patients, 49% also had a DAT reactive for complement. Several research reports in the medical literature describe the nature of DAT results in patients with warm autoimmune hemolytic anemia. 5-11 These studies reported the DAT to be reactive for IgG alone in 21% to 56% of patients. The DAT was reactive for both IgG and C3 in 31% to 64% of patients, and the DAT was reactive for C3 alone in 0% to 45% of patients. Some of these studies also demonstrated the DAT to be reactive for IgA, without IgG or C3. In the study by Issitt et al, 5 all 150 subjects had warm autoantibodies and a DAT reactive for IgG; 91 had a DAT reactive for IgG alone; 49 for IgG and complement; 5 for IgG, IgM, and complement; 2 for IgG, IgA, IgM, and complement; 2 for IgG and IgA; and 1 for IgG, IgA, and complement. In the present study, 25 (86%) of the 29 patients with warm autoantibodies who experienced hemolysis had a DAT for IgG that was 2+ or greater in reaction strength. Of 71 patients with warm autoantibodies who did not experience hemolysis, 45 (63%) had a DAT for IgG that was 1+ or weaker. The medical literature addresses the strength of reactivity of the DAT in patients with warm autoimmune hemolytic anemia but does not focus on individuals with warm autoantibodies without hemolysis. Issitt and Anstee 11 commented that in the majority of cases of warm autoimmune hemolytic anemia, the DAT usually is strongly reactive. In one study of 23 patients with warm autoimmune hemolytic anemia due to IgG autoantibodies, 21 patients had a DAT that was 4+ and 2 patients had a DAT that was 3+. 12 In another study, 19 (61%) of 31 patients with warm autoimmune hemolytic anemia had a DAT for IgG of 2+ or greater. 13 A study evaluating erythrocyte autoantibody behavior in the monocyte monolayer assay revealed that the level of RBC sensitization with IgG was an important positive predictor of in vivo hemolysis. 14 Furthermore, in patients with warm reactive autoantibodies and positive monocyte monolayer assay results, testing indicated that this type of autoantibody was more likely to be associated with phagocytosis than simple RBC adherence (identified more commonly in patients with cold and mixedtype RBC autoantibodies). Thus, it seems that in patients with warm reactive autoantibodies, there is an important correlation between DAT reaction strength and the probability of hemolysis. However, when heterogeneous groups of individuals with positive DAT results have been evaluated for hemolysis, there is not a good correlation between DAT reaction strength and the likelihood of hemolysis. 15 In our study, there also was a correlation between autoimmune hemolysis and the presence of complement on RBCs. In addition, the strength of reactivity of the DAT for complement also was important. Although the relationship between the C3 DAT and hemolysis is not as well studied as that of the IgG DAT hemolysis relationship, there is evidence that the presence and amount of C3 is an important predictor of hemolysis and might act synergistically with IgG. 14 In contrast, several studies have shown that approximately 8% of hospitalized patients had a positive DAT with no evidence of hemolysis. 7,9,16-18 The positive DAT results usually were weakly reactive, 1+ or less, and Downloaded from https://academic.oup.com/ajcp/article-abstract/122/5/680/1759385 Am J Clin Pathol 2004;122:680-685 683 683 683

Wheeler et al / CHARACTERISTICS OF WARM AUTOANTIBODIES 80% or more of these positive DAT results represented the presence of complement without immunoglobulin on the RBCs. As Issitt and Anstee 11 describe, it follows that the remaining 20% of these hospitalized patients (with a positive DAT) and no hemolysis must have IgG (with or without complement) on their RBCs. Thus, between 1% and 2% of hospitalized patients have a weakly reactive DAT for IgG but do not experience hemolysis. These same studies also demonstrated that 1% of hospitalized patients without hemolytic anemia had a DAT reactive for complement at a strength of 2+ to 4+. In the series by Issitt et al, 5 in most of the 30 hematologically healthy subjects with positive DAT results (25 healthy donors and 5 patients with diseases other than autoimmune disorders), the strength of the DAT was 3+ to 4+: 21 subjects had a DAT reactive for IgG alone; 7 had a DAT reactive for IgG and complement; 1 had a DAT reactive for IgG and IgA; and 1 had a DAT reactive for IgG, IgM, and complement. In our series, autoantibodies were identified in the serum or plasma of 70% of patients, with 30% of patients having autoantibodies detected on their RBCs alone. These data are comparable to other data in the medical literature. In the study of 150 subjects with warm autoantibodies by Issitt et al, 5 62% of patients had autoantibodies in their serum samples (78% of patients with warm autoimmune hemolytic anemia, 52% of subjects receiving α-methyldopa, and 27% of the hematologically healthy subjects). Petz and Garratty 7 identified warm autoantibodies in serum samples of 57% of patients with warm autoimmune hemolytic anemia. Of the patients studied in the present series, 53% had alloantibodies in addition to their warm reactive autoantibodies. The alloantibodies were defined as such by demonstrating that patients lacked the corresponding blood group antigens on their own RBCs. However, because more demanding adsorption and elution studies were not performed, we cannot exclude the possibility that some of these antibodies actually were mimicking autoantibodies. A review of the literature shows differing incidences of accompanying alloantibodies. Laine and Beattie 19 detected serum alloantibodies in 37.6% of 109 serum samples containing unbound warm reactive autoantibodies. James et al 20 identified alloantibodies in 31.7% of such serum samples. Morel et al 21 identified alloantibodies in 40% of serum samples studied. However, Wallhermfechtel et al 22 demonstrated alloantibodies in only 15.2% of serum samples, and Issitt et al, 23 in a study of 138 patients, identified alloantibodies in only 23%. One explanation offered by Mollison et al 24 and Issitt et al 23 for the higher incidence of alloantibodies in some studies is that the primary or exclusive use of allogeneic adsorption may leave behind alloantibodies that really are autoantibodies with mimicking specificities. In our study, allogeneic adsorptions were performed in 30 cases; in only 8 patients were underlying alloantibodies detected. Therefore, the use of allogeneic adsorption is unlikely to account for the high percentage of alloantibodies in this series. Instead, this may relate to the heavily transfused nature of our study patients. Alternatively, by selecting patients for study with demonstrable warm autoantibodies, we also may have selected for patients with alloantibodies. There is a recognized relationship between the development of RBC alloantibodies and the later development of autoantibodies. 25-27 Our study demonstrates that the majority of patients with serologically detectable warm reactive autoantibodies do not experience hemolysis, that the vast majority of patients who experience hemolysis have primary clinical conditions classically associated with autoimmune hemolysis, and that the strength of a patient s DAT (for IgG and C3) correlates with the presence or absence of hemolysis. These findings may help in determining the hemolytic potential of warm reactive autoantibodies and the subsequent follow-up required to monitor patients for evidence of ongoing hemolysis. From the Department of Pathology and Laboratory Medicine, University of California, Los Angeles. Address reprint requests to Dr Blackall: Dept of Pathology, Arkansas Children s Hospital, Mail Slot 820, 800 Marshall St, Little Rock, AR 72202. Acknowledgments: We thank the technical staff of the UCLA blood bank for excellence in performing the serologic studies described in this article, and the Fenwal Division, Baxter Healthcare, Deerfield, IL, for continued support of the American Association of Blood Banks Fenwal Scholarship Awards for transfusion medicine fellows. * Dr Wheeler was a 2003 recipient of an American Association of Blood Banks Fenwal Scholarship award. This article resulted from work recognized by the fellowship award program. References 1. Brecher ME. AABB Technical Manual. 14th ed. Bethesda, MD: American Association of Blood Banks; 2002. 2. Sokol RJ, Booker DJ, Stamps R. Erythrocytes, autoantibodies, autoimmune hemolysis and carcinoma. J Clin Pathol. 1994;47:340-343. 3. Sokol RJ, Hewitt S, Booker DJ. Erythrocyte autoantibodies, autoimmune haemolysis and myelodysplastic syndromes. J Clin Pathol. 1989;42:1088-1091. 4. Castellino SM, Combs MR, Zimmerman SA, et al. Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance. Br J Haematol. 1999;104:189-198. 5. Issitt PD, Pavone BG, Goldfinger D, et al. Anti-Wr b and other autoantibodies responsible for positive direct antiglobulin tests in 150 individuals. Br J Haematol. 1976;34:5-18. 6. Worlledge SM, Blajchman MA. The autoimmune haemolytic anaemias. Br J Haematol. 1972;23(suppl):61-69. 7. Petz LD, Garratty G. Acquired Immune Hemolytic Anemias. New York, NY: Churchill Livingstone; 1980:193, 209. 684 Am J Clin Pathol 2004;122:680-685 Downloaded 684 from https://academic.oup.com/ajcp/article-abstract/122/5/680/1759385

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