A proposal for compatibility testing incorporating the manual hexadimethrine bromide (Polybrene) test

In November 1984, the Standards Committee of the American Association of Blood Banks changed the requirements for pretransfusion testing by making the performance of an antiglobulin crossmatch optional when the antibody screening test is negative. The crossmatch would be necessary only to confirm ABO compatibility. Many will welcome this change; others will persist in their current methods. This article presents data supporting the use of the manual hexadimethrine bromide (Polybrene) test, a 1-minute room temperature procedure, as a crossmatch technique when the antibody screening test is negative. The manual Polybrene test (MPT) is an effective method for detecting ABO incompatibility. Forty-seven randomly selected serums gave expected results with A1, A2, and B red cells. Only 66 percent of 84 group B sera were serologically incompatible with A2B red cells by MPT, but the same results (69% positive) were observed using a 5-minute low-ionic-strength solution (LISS) room temperature technique. As only 37 percent of these crossmatches were incompatible using a LISS immediate spin (IS) method, the reliability of an IS method is questioned. An MPT crossmatch provides added security in that most unexpected blood group antibodies are demonstrable by this method. Of 106 serums tested which contained antibodies, 83 reacted. We believe that the MPT provides a rapid and sensitive test that, accompanied by a carefully performed antibody screening test, meets the requirements of Standards and will provide for safe red cell transfusion without the need for an antiglobulin crossmatch.     

Comparison of a modified manual hexadimethrine bromide (Polybrene) and a low-ionic-strength solution antibody detection technique

Manual hexadimethrine bromide (Polybrene) tests (Polybrene in low-ionic medium) were used in parallel with manual low-ionic-strength solution (LISS) procedures for the routine testing of patient samples referred to a general hospital blood bank. Of 5646 consecutive sera tested, 5167 (91.5%) did not react with either technique; 320 sera (5.7%) reacted in both methods. The Polybrene technique detected 63 antibodies which did not react in the LISS methods. One hundred sera did not react in the Polybrene test, but did react in the LISS methods. Sera showing discrepant results between the two methods were further tested in a reference laboratory. Polybrene tests appeared to be better in avoiding reactions due to clinically nonsignificant antibodies. The LISS methods, however, appeared to be more sensitive in detecting antibodies of potential clinical significance.     

The manual hexadimethrine bromide (Polybrene) test. Effects of serum proteins and practical applications

Serum proteins affect the performance of the manual hexadimethrine bromide (Polybrene) test. Red cells suspended in the low-ionic medium adhere to the surface of test tubes when centrifuged. This adherence is prevented by serum components apoprotein B, fibronectin, von Willebrand factor, or serum diluted less than 75-fold, but not by serum albumin or immunoglobulin fractions. A high concentration of serum reduces the sensitivity of the test and destabilizes antibody-dependent aggregation. Disaggregation time (DT), defined as the time required for antibody-dependent aggregates to disperse, was used as an indicator of agglutination stability. DT was often shortened when antibodies were in native serums, but could be prolonged if serums were diluted with 0.85 percent saline. Other factors that influenced DT included antibody concentration, temperature, specificity, and sources of antibodies within specificities. When antibodies were present in a mixture, they produced various DTs related to individual specificities. Determination of these differences allowed the successful identification of multiple antibodies. The procedure, called differential disaggregation time, was also used for typing red cells coated by autoantibodies. Additional modifications improved the performance of the antiglobulin phase.     

Red cell phenotyping using hexadimethrine bromide (Polybrene) in a microplate system

To provide rapidly phenotyped units of blood, we adapted the hexadimethrine bromide (Polybrene) technique to microplate technology. Pilot samples from 282 donor units were phenotyped for antigens in the Rh, Kidd, Kell, Duffy, and Ss systems with a standard tube-testing method and a Polybrene-microplate (P-MP) technique. Diluted antisera and a 1 percent red cell suspension were used to give P-MP reactions that were accurate and easy to interpret. One microplate, containing 96 tests, was prepared and read within 15 minutes for P-MP tests yielding direct agglutination (Rh, Jkb, Fya, Fyb), or within 19 minutes for P-MP tests requiring an antiglobulin phase (Jka, K, S, s). No false-positive results were found. No false-negative reactions were found in typing for Rh, K, S, s, or Jka antigens. Phenotyping for Jkb and Duffy antigens gave a false-negative rate of less than 0.015. Considerable savings in reagents were obtained through microplate miniaturization and through the enhancement of apparent antibody avidity in the Polybrene/low ionic medium which permitted dilution of reagent antisera. The P-MP technique affords a rapid, accurate, simple, and inexpensive means of phenotyping large numbers of donor units.     

Plasma exchange in autoimmune hemolytic anemia (AIHA)

Plasma exchange therapy in autoimmune hemolytic anemia (AIHA) was used in four patients (two with warm hemolytic anemia and two with cold hemolytic anemia). The size of each plasma exchange approximated 1 plasma volume; three consecutive daily exchanges removed 80–90% of the immunoglobulins—immunoglobulin G (IgG) and immunoglobulin M (IgM)—. complement (C₃, C₄), and reduced antibody titers. Transfusion requirements dramatically decreased after plasma exchange in each case. In two patients, red blood cell (RBC) survival studies were performed to more accurately assess the effect of plasma exchange therapy, since steroid and/or immunosuppressive therapy was given concomitantly. In one case of cold AIHA, homologous ⁵¹Cr-RBC were injected 4 days prior to plasma exchange and repeat injection (same donor) following completion of plasma exchange. The survival curve prior to plasma exchange therapy had a T 1/2 = 7.8 days (r = ?0.988) and after plasma exchange therapy had a T 1/2 = 20.4 days (r = ?0.925). RBC survival studies using homologous ⁵¹Cr-RBC were also performed in a patient with warm AIHA. The survival curve before plasma exchange had a T 1/2 = 2 days (r = ?0.95), and postplasma exchange a T 1/2 = 1.8 days (r = ?0.91). Plasma exchange therapy seems to have a beneficial effect in cold rather than warm autoimmune hemolytic anemia.     

自身免疫性溶血性贫血的微柱凝集技术诊断应用

目的　探讨微柱凝集体技术在诊断自身免疫性溶血性贫血 (AIHA)中的应用。方法　采用BioVue系统微柱凝集技术对 2 6例AIHA患者进行检测 ,同时用传统的试管法抗人球蛋白试验作比较。结果　统计学上两种方法对AIHA检出率无显著性差别 ,但微柱凝集技术能检出试管法所不能检出的阴性血样 ,比试管法检测凝集强度高 1 ～ 2 。结论　BioVue系统微柱凝集技术能快速准确检测出AIHA ,具有更高的灵敏度 ,是直观可靠的方法。     

自身免疫性溶血性贫血的微柱凝集技术诊断应用

目的探讨微柱凝集体技术在诊断自身免疫性溶血性贫血(AIHA)中的应用.方法采用BioVue系统微柱凝集技术对26例AIHA患者进行检测,同时用传统的试管法抗人球蛋白试验作比较.结果统计学上两种方法对AIHA检出率无显著性差别,但微柱凝集技术能检出试管法所不能检出的阴性血样,比试管法检测凝集强度高1+～2+.结论BiVue系统微柱凝集技术能快速准确检测出AIHA,具有更高的灵敏度,是直观可靠的方法.     

pH-dependent anti-U in autoimmune hemolytic anemia

Auto anti-U of unusual serologic characteristics is described in a Caucasian patient with autoimmune hemolytic anemia associated with chronic lymphocytic leukemia. The direct antiglobulin test was strongly positive (3+) with broad spectrum and anticomplement reagents. Auto anti-U was demonstrated only when the serum was acidified to pH 6.5 and anticomplement reagent was used in the antiglobulin test. Multiple transfusions of U positive red blood cells had clinically decreased survival manifested by jaundice and return of hemoglobin to pretransfusion levels within days. The autoantibody reacted weakly with U positive cord red blood cells interpreted as mosaicism of the U antigen possibly on a developmental basis.     

Anti-Jka, -C, and -E in a single patient, initially demonstrable only by the manual hexadimethrine bromide (Polybrene) test, with incompatibilities confirmed by 51Cr-labeled red cell studies

Published reports have confirmed the superior sensitivity of the manual hexadimethrine bromide (Polybrene) test (MPT) for demonstrating many alloantibodies in vitro; however, the clinical significance of alloantibodies demonstrable exclusively by MPT has not been shown conclusively. A patient with macroglobulinemia experienced chills, fever, hemoglobinemia, and hemoglobinuria following the transfusion of 1 unit of red cells (RBCs) shown to be compatible by the low-ionic-strength antiglobulin (LIS-AG) method. Serologic investigation was negative. Intravascular hemolysis occurred with a second "compatible" unit. Serologic studies were again negative by LIS-AG and ficin-AG methods, but revealed anti-Jka by MPT. Both donors were Jk(a+b-), and 51Cr studies of the second donor's RBCs revealed a t1/2 of less than 30 minutes, with marked intravascular hemolysis. A LIS-AG-compatible Jk(a-) unit was transfused uneventfully, but with no rise in hematocrit. MPT next revealed anti-C; subsequent 51Cr studies with the Jk(a-), Cc donor's RBCs showed a 51Cr t1/2 of 100 minutes with slight intravascular lysis. Four transfusions of Jk(a-), C- blood were uneventful, but 5 days later the patient's hemoglobin declined. The following day, anti-E was demonstrable exclusively by MPT. 51Cr-labeled Jk(a-), C-, E- RBCs had normal 24-hour survival. The patient's hemoglobin rose to 11 g per dl following transfusions of Jk(a-), C-, E- RBCs, and he was discharged. In vitro studies employing the patient's purified IgM paraprotein revealed no interference with alloantibody binding or detection.     

自身免疫性溶血性贫血的诊断

自身免疫性溶血性贫血(autoimmune hemolytic anemia，AIHA)是一种获得性溶血性疾病，患由于免疫功能紊乱产生抗自身红细胞的抗体．与红细胞表面的抗原结合，或激活补体使红细胞加速破坏而致溶血性贫血。国外报道本病约占溶血性疾病患总数的1／3，最低年发病率为1／80000，国内AIHA亦不少见。该病以青壮年为多，女性多于男性。鉴于AIHA是临床上较常见、难根治的贫血．     

IgA-mediated autoimmune hemolytic anemia in an infant

Autoimmune Hemolytic anemia (AIHA) a relatively uncommon form of hemolytic anemia in children, occurs due to the premature destruction of red blood cells caused by presence of autoantibodies directed against antigens on RBCs. Warm reactive AIHA is the most common form due to IgG isotype of immunoglobulin class binding to autologous RBCs at 37⁰C and confirmed with a positive DAT screening. We present a case of DAT-negative primary warm AIHA in an infant due to IgA antibody. A 10 month old male infant presented with dark colored urine and irritability for past two months, with associated history of fever, diarrhea and vomiting. He had received one red cell transfusion 10 days prior. On physical examination he had pallor with tachycardia without splenomegaly. On investigation his hemoglobin was 5.8 g/dl, WBC 25.9 × 10³/mm³ and normal platelets counts. Peripheral blood smear had spherocytes and biochemical values showed high bilirubin and LDH. Immunohematological work up revelaed polyspecific DAT was negative but monospecific DAT screening showed strong (4+) positivity for IgA and a weak IgG positivity. The patient was diagnosed as IgA-mediated Warm AIHA and was started on prednisolone at 2 mg/kg/day following which hemoglobin improved over the next 2 months. After 2 weeks, prednisolone was tapered and stopped by the end of 3 months. Patients with clinical and laboratory evidence of acute hemolysis, an additional screening for IgA antibody may be done even in cases where poly-specific DAT is negative. Early detection helps in avoiding further investigations and provide efficient management.     

Anti-Jka autoimmune hemolytic anemia in an infant

Anti-Jka was identified in the serum and on the red cells of an 8-month-old infant with anemia, splenomegaly, and symptoms of an upper respiratory infection. The anemia improved after transfusion and treatment with prednisone.     

A transgenic model of autoimmune hemolytic anemia

We made double transgenic mice bearing immunoglobulin heavy and light chain genes encoding an autoantibody against the mouse erythrocyte by the cross of C57BL/6 mice carrying the transgene for each chain of the immunoglobulin. Although no obvious disorders were found in the single-chain transgenic mice, severely anemic symptoms were found in some of the double transgenic mice, in which most B cells express, at least on their surface, the autoantibody reactive to self-antigens on the erythrocyte. Individual double-transgenic mice showed a wide variation of phenotypes between severe anemia and no symptoms. Both deletion and anergy of autoreactive B cells were seen in each individual mouse, but their relative contribution to self-tolerance was variable and not directly related to the severity of anemia or the amount of the autoantibody produced. This transgenic system provides a good autoimmune disease model for exploring its onset mechanism, and means of its treatment and prevention.     

难治性自身免疫性溶血性贫血一例

患者:男,19岁,因反复头昏、乏力、黄疸2年余,再发1个月而入院。患者2年前出现头昏、乏力、皮肤巩膜黄染、尿色加深。曾就诊于当地医院,查体:脾脏左肋下3cm,血常规:Hb 84g/L,RBC 2.67×10~(12)/L,WBC 7.17×10~9/L,N:0.78,L:0.16,M:0.03,PLT 154×10~9/L,网织红细胞0.39。尿常规:隐血阳性( ),余正常。肝功能:总胆红素40.04μmmol/L,直接胆红素13.50 μmmol/L,ALT、AST 等正常。肝、胆、脾的B 超检查提示脾脏肿大。直接抗球蛋白试验(DAT):IgG 阳性( ),C3阳性( ),Hams 试验阴性。骨髓象:有核细胞增生明显活跃,红系占58%,以中晚幼红增生为主,易见核分裂、变     

Hemolytic warm IgM autoagglutinins in autoimmune hemolytic anemia

The authors report a patient with fulminant autoimmune hemolytic anemia due to a rare warm IgM autoagglutinin more reactive at 37 degrees C than at lower temperatures and secondary to systemic lupus erythematosis. The patient's clinical course and the serologic and immunochemical characteristics of the antibody are described, including the possibility that transfusions of small amounts of incompatible red cells may have contributed to the hemolysis. The consequences of using the initial serologic test results as the basis for therapy are discussed.