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.     

Limitations of the immediate spin crossmatch when used for detecting ABO incompatibility

In the eleventh edition of the AABB's Standards for Blood Banks and Transfusion Services the requirement for an antiglobulin crossmatch was deleted if no clinically significant unexpected antibodies are detected in recipient serum testing and if there is no history of detection of such antibodies. Test methods that demonstrate ABO incompatibility remain a requirement; however, the means to accomplish this may prove controversial. The immediate spin crossmatch has been used for the purpose of detecting ABO incompatibility by many workers. Nonetheless, limitations of this technique became apparent in tests between A2B donor red cells and group B patient sera. The results of 204 of 531 immediate spin crossmatches, between these two blood groups, were found to be negative.     

Role of the crossmatch in testing for serologic incompatibility

Nine unexpected antibodies of unquestioned clinical significance were detected when the major crossmatch was performed on 31,320 pretransfusion blood samples from 8969 patients whose screening test for unexpected antibodies was nonreactive. Three of the antibodies retrospectively were found to manifest a positive screening test. Another antibody was not detected by the antibody screening test due to an error in preparation of the screening red blood cells. The overriding importance of the major crossmatch is the assurance of ABO compatibility between donor blood and recipient. Therefore, while this study does not resolve whether the antiglobulin phase of the procedure might be considered optional, the major crossmatch should not be eliminated.     

The sensitivity and specificity of the immediate-spin crossmatch

The immediate-spin (IS) crossmatch is used to detect ABO incompatibility between donor red cells (RBCs) and the serum of the intended recipient. However, this test may be positive in the absence of ABO incompatibility (false positive) or it may be negative when ABO incompatibility exists (false negative). During a 25-month study, the rates of both false-positive and false-negative IS crossmatch results were evaluated, and the sensitivity and specificity of the IS crossmatch were determined. During the study period, 53,656 IS crossmatches were performed for patients without significant RBC antibodies. Fifty-five patients had positive IS crossmatches, and no false-negative reactions were found. In tests of 55 patients with positive IS crossmatches, 77 false-positive and 5 true-positive reactions were noted. The causes of the false-positive reactions were rouleaux (36 patients), cold-reactive antibodies (8 patients), a combination of rouleaux and cold-reactive antibodies (2 patients), fibrin clot (1 patient), and undetermined (3 patients). The sensitivity and specificity of the IS crossmatch were 100 and 99.86 percent, respectively. Laboratory personnel should be aware that the IS crossmatch may have false-positive or false-negative results, and they should develop written protocols to distinguish quickly between true-positive and false-positive reactions.     

Effect of delayed centrifugation or reading on the detection of ABO incompatibility by the immediate-spin crossmatch

The immediate-spin (IS) crossmatch is a method that detects ABO incompatibility. However, under certain circumstances, this test may show unwanted negative or weak results, even though the red cells (RBCs) and serum being tested are ABO incompatible with each other. The present study investigated the potential effect of delayed centrifugation or reading on the IS crossmatch test performance. When the centrifugation step of the IS crossmatch between group O sera and group A1 RBCs was delayed for 2 minutes, 5 of 200 crossmatches showed no agglutination with only trace (n = 2) or moderate (n = 3) hemolysis, and one crossmatch showed only weak, macroscopic agglutination, but moderate hemolysis. All six sera contained A antibodies that were lytic in vitro and, therefore, probably were capable of causing in vivo hemolysis of transfused A1 RBCs. Delaying the reading of the IS crossmatch test for 2 minutes had no apparent effect on test performance. These data demonstrate the importance of technologists' recognition of hemolysis as a positive result on IS crossmatches, especially if the performance of the centrifugation step of the test is delayed. Furthermore, the unwanted negative agglutination results were abolished by suspending the group A1 RBCs in saline containing EDTA. The authors' laboratory has modified its IS crossmatch procedure so that donor RBCs are routinely suspended in saline containing EDTA before testing. This procedural change should increase the safety of the IS crossmatch.     

Incompatible crossmatch following nonreactive antibody detection test. Frequency and cause

In order to determine the frequency and cause of an incompatible crossmatch found in specimens for which the antibody detection test was nonreactive, 261, 136 crossmatches performed on 116,278 patient samples from approximately 46,000 different patients were reviewed. One-hundred and one patients were identified whose sera were incompatible in at least one crossmatch following a nonreactive antibody detection test. The cause of the incompatibility was determined in 69 of these. Incompatibility was detected only after 37 degrees C incubation and/or after the antiglobulin phase in 36 (52%). Incompatibility was detected after an immediate-spin and/or incubation at room temperature in 33 (48%). Serologic evaluation did not identify the cause of the incompatibility in 16 of the 101 patients. In eight of these 16 the incompatibility was noted only after 37 degrees C incubation and/or after the antiglobulin phase. Detailed serologic evaluation was not performed on 16 of the 101 patients (all of whose sera were incompatible with only 1 donor unit). In 12 of these 16, the incompatibility was detected only after the antiglobulin phase.     

Frequency of delayed hemolytic transfusion reactions following antibody screening and immediate-spin crossmatching

In view of the continuing controversy regarding the use of immediate-spin crossmatch procedures in preparing blood for transfusion to patients in whom unexpected clinically significant antibodies have not been found by antibody screening by the indirect antiglobulin test (IAT), a review of 8 years' experience with such a policy was conducted. In that period, 54,725 units of packed red cells or whole blood were transfused to 10,146 patients. Four clinically overt delayed hemolytic transfusion reactions and 18 clinically silent delayed serologic transfusion reactions were found. In 3 of the 22 patients, the offending antibody(ies) were detectable in the pretransfusion serum by an enzyme IAT, but none was detectable by routine saline IAT against either a three-cell screening panel or the transfused cells. Thus, the incorporation of saline indirect antiglobulin crossmatch would not have prevented the delayed reactions. It can be concluded that the use of a saline indirect antiglobulin crossmatch offers no significant advantage over the current policy of using only immediate-spin crossmatch for those patients whose pretransfusion serum gives negative results in a three-cell screen using a saline IAT.     

Acute extravascular hemolytic transfusion reaction due to anti-Kpa antibody missed by electronic crossmatch

BackgroundKpa antigen is a low incidence red blood cell antigen within the Kell system. Anti-Kpa alloantibody may be associated with acute and delayed hemolytic transfusion reactions.Case StudyWe report a case of a clinically significant acute extravascular hemolytic transfusion reaction mediated by previously unrecognized (and undetected) anti-Kpa alloantibody. This reaction occurred in a patient who met all criteria for electronic crossmatch, resulting in the transfusion of an incompatible red cell unit.ResultsPost-transfusion investigation showed the transfused red cell unit was crossmatch compatible at the immediate spin phase but was 3 + incompatible at the antiglobulin phase. No evidence of intravascular hemolysis was observed upon visual comparison of the pre- and post-transfusion peripheral blood plasma. Further testing showed the presence of anti-Kpa antibody. The clinical course of the patient included acute febrile and systemic reaction.ConclusionAcute extravascular hemolytic transfusion reaction may occur due to undetected anti-Kpa alloantibody. Various strategies for crossmatching are discussed in the context of antibodies to low incidence antigens.     

Electronic crossmatching

The crossmatch (XM) is an important part of routine pretransfusion testing. It is used to detect ABO incompatibility and other clinically significant antibodies. The XM has been modified many times and, in recent years, has been abbreviated. The 2 common types of XM currently being done are the immediate spin XM for antibody-negative patients and the antiglobulin XM for the rest. The antiglobulin phase of the XM is generally considered optional, unless a clinically significant antibody is present. Originally, the XM was intended to be a final check for ABO compatibility and for the detection of unexpected antibodies. Electronic crossmatching was first introduced at the University of Michigan Medical Center in 1992. This paper summarizes the basic principles, current guidelines, regulatory requirements, and some recommendations that may prove useful for the implementation of electronic crossmatching.     

Multiple transfusion fail to provoke antibodies against blood cell antigens in human infants

We conducted studies of both red cell (RBC) and leukocyte (WBC) antibody formation in infants following multiple transfusions given during the first weeks of life. Fifty-three infants received 683 RBC transfusions from 503 different donors, plus 62 platelet, 4 granulocyte, and 53 fresh-frozen plasma units during the first 4 months of life. Three hundred fifty serum samples were obtained before, during, and after the transfusions. None of the infants formed unexpected RBC antibodies when tested at 37 degrees C by a two-cell low-ionic-strength solution antibody screen that included an anti-globulin phase. Twenty posttransfusion serums were negative when tested at room temperature. Lymphocytotoxic and granulocytotoxic WBC antibodies were measured in posttransfusion serums from 13 infants, and none were found. Despite exposure to many RBC and WBC antigens, no infants produced alloantibodies against blood cell antigens. Thus, immunologically mediated transfusion reactions should be quite rare in young infants, and this study supports recommendations of the American Association of Blood Banks Standards to omit repeat RBC compatibility testing during the first 4 months of life in infants whose initial RBC antibody screens reveal no unexpected antibodies.     

The computer crossmatch: a safe alternative to the serological crossmatch

The crossmatch has evolved from including a wide range of techniques through a test purely to eliminate ABO incompatibility (immediate spin) to computer crossmatching in which no serological testing is carried out and validation ensures the correct ABO/RhD type blood is issued. The crossmatch was always considered to be the most important feature of the compatibility test and in particular the antiglobulin phase; however, there are potential risks associated with serological and computer crossmatching including technical and procedural errors. The use of immediate spin and computer crossmatch change the emphasis for safety of the compatibility test from the crossmatch to the antibody screen. UK guidelines have now been published describing the features necessary for the introduction of computer crossmatching. Computer crossmatching is used by many institutions in various countries. It is considered safe practice and brings benefits to the laboratory and the patient. Compatibility testing is only one element of the blood transfusion procedure; the others are equally as important and include correct patient identification at the time of collection of the blood sample and at the administration of the blood transfusion.     

The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited

Direct antiglobulin tests (DATs) using anti-IgG were performed on 65,049 blood samples from prospective transfusion recipients; 3570 tests (5.49%) were positive. Using criteria published previously (primarily excluding patients not transfused within the preceding 14 days), 778 samples from other than neonatal patients were selected for further evaluation. Eluates that did not react were obtained on 518 (66.6%) of these samples. Warm-reactive autoantibodies were apparent in 192 eluates, while 16 contained drug-related antibodies, anti-A or anti-B from prior transfusion with ABO mismatched blood components, or anti-D passively acquired from immune serum globulin. Fifty-two eluates contained alloantibodies; however, in only six of these cases did the corresponding serum lack unexpected alloantibodies, as determined by routine pretransfusion studies. Three additional weakly reactive clinically significant alloantibodies were detected solely through additional serum tests performed on DAT-positive samples. On the basis of these findings, the DAT had a low predictive value when used to detect the early manifestations of an immune response to recently transfused red cells. Elimination of the autocontrol from routine pretransfusion testing, therefore, carries minimal risk to patients yet will undoubtedly contribute to the containment of health care costs. Moreover, the risk is lower than that associated with the elimination of the antiglobulin crossmatch.     

Crossmatch procedures used in organ transplantation

Several lymphocyte crossmatch procedures used in clinical histocompatibility laboratories, including complement dependent (CDC, AHG-CDC) and complement independent (flow cytometric, chromium release) techniques, are used to assess the likelihood of allograft rejection due to preformed donor specific antibody. Crossmatch assays can be extremely sensitive and detect very low levels of donor reactive antibody present in the potential recipient. Since positive crossmatches are usually associated with allograft rejection, a positive crossmatch is generally a contraindication to organ transplantation. Recent data, however, suggests that not all positive crossmatches lead to graft rejection, particularly those due to autoantibodies. This underscores the need to critically evaluate any positive crossmatch to determine if the antibodies involved in the reaction are relevant to allograft rejection.     

A fully automated blood typing system for hospital transfusion services. ABS2000 Study Group

BACKGROUND: The results of routine blood bank testing by a fully automated blood typing system (ABS2000) were compared with those obtained by standard manual methods in six hospital transfusion services. STUDY DESIGN AND METHODS: The ABS2000 system uses microtiter plates for determining ABO and D types, solid-phase red cell adherence (SPRCA) assays for antibody detection, and modified SPRCA plates for IgG crossmatches. The transfusion services used their standard manual test tube methods. RESULTS: Of 3779 donors' samples tested for ABO types (red cell typings only), 3.0 percent could not be interpreted by the ABS2000 system's neural network, because of clots, hemolysis, or lipemic samples. The results for ABO types were concordant for 99.8 percent of the remaining samples. Of 3779 donors' samples tested for D types, the results were concordant for 98.7 percent. Of 7580 patients' samples tested for ABO types (red cell and plasma typings), 5.8 percent could not be interpreted by the ABS2000 system. There was 100-percent concordance of ABO typing results for the remaining 7140 samples. There was 99. 7-percent concordance of results for patients' D types. The results of 96.7 percent of antibody detection tests and 98.8 percent of crossmatches were concordant. Neither method failed to detect a serologically incompatible crossmatch that was associated with a specific, clinically significant alloantibody. The ABS2000 system performed 45 confirmatory donor ABO and D types in 115 minutes, 22 antibody detection tests in 116 minutes, 16 patients' ABO/D types in 149 minutes, and 40 crossmatches in 140 minutes. CONCLUSION: The ABS2000 blood typing system automates routine blood bank tests with accuracy comparable to that of hospital transfusion services' standard manual methods.     

Pretransfusion blood group serology. Limited value of the antiglobulin phase of the crossmatch when a careful screening test for unexpected antibodies is performed

The limited value of the antiglobulin phase of the cross match when a careful antibody screening is performed was demonstrated by analyzing 73,407 compatibility tests for 23,857 patients. By the blood grouping and screening of these patients, 178 cases were detected with unexpected blood group antibodies that had not been previously observed. Unexpected antibodies were detected in an additional 13 patients by the saline phase of the cross-match. In addition, the antiglobulin phase disclosed only one patient with a very weak anti-Lea and two patients whose sera gave doubtful reactions, possibly representing antibodies, but too weak to be identified.     

A New Method for Detection of Red Blood Cell Antibodies

A new method for the detection of red blood cell antibodies has been developed. Polybrene®, a positively charged polymer, was utilized to produce agglutination of red blood cells. This agglutination could be reversed by the addition of hyper tonic salt solution. However, red blood cells remained agglutinated in the presence of antibodies. This principle was applied to antibody detection automated by AutoAnalyzer^rG. The method has proved to be highly sensitive and has a wide spectrum of usefulness for the detection of both "complete" and "incomplete" antibodies.     

Comparison of a manual hexadimethrine bromide-antiglobulin test with saline- and albumin-antiglobulin tests for pretransfusion testing

A prospective double-blind study compared a manual hexadimethrine bromide (Polybrene) antiglobulin antibody detection test (P-AHG) and crossmatch with the albumin-antiglobulin antibody detection test and saline-antiglobulin crossmatch routinely used in our laboratory. A total of 10,084 pretransfusion blood samples from approximately 6000 patients were tested. The P-AHG method detected 153 of 157 alloantibodies for which antigen-negative, crossmatch-compatible blood is routinely provided. All four antibodies not detected were anti-K. The routine techniques detected 147 of the 157 alloantibodies. The P-AHG method detected only 36 percent of the alloantibodies for which crossmatch-compatible blood is routinely provided without determination of the antigen status of the donor unit's red cells (e.g., anti-Lea), whereas the routine method detected 91 percent of such antibodies. Eighty-six percent of the 189 alloantibodies detected by the Polybrene technique were found before the addition of antiglobulin. The manual Polybrene test is a rapid and sensitive technique; it may be used without an antiglobulin phase as a routine crossmatch procedure when accompanied by a sensitive antibody detection test that includes antiglobulin and an additional test to ensure ABO compatibility.     

Is a room-temperature crossmatch necessary for the detection of ABO errors?

The detection of anti-A and anti-B isohemagglutinins by low-ionic- strength saline tests at 37 degrees C and by the indirect antiglobulin technique, without an "immediate'spin" or room-temperature phase, has been studied. Using such a procedure, all but one of 2746 patient blood samples reacted in accordance with ABO type when tested against A2 and B red cells. However, the discrepant sample also was nonreactive when tested by "immediate-spin" technique against saline-suspended A2 red cells. Our findings indicate that compatibility tests performed at 37 degrees C in low-ionic-strength saline are as sensitive as "immediate- spin" tests with saline-suspended red cells for the detection of ABO errors. Performing serologic tests for unexpected alloantibodies and donor-recipient compatibility without an "immediate-spin" or room- temperature phase abbreviates pretransfusion testing and reduces the detection of clinically insignificant alloantibodies solely reactive at room temperature.     

Radiolabeled red cell viability. II. 99mTc and 111In for measuring the viability of heterologous red cells in vivo

The authors developed a double in vivo crossmatch method using 2 to 3 ml of potential donor blood labeled with either 400 microCi of 99mTc or 30 microCi of 111In-oxine. Data are presented for 19 crossmatches on nine patients, using one blood specimen labeled with 99mTc or two specimens, one labeled with 99mTc and the other with 111In. Normal values are given for standardization purposes. This method appears to have advantages over earlier in vivo crossmatch techniques using 51Cr-labeled RBCs. These advantages include the rapidity with which the in vivo crossmatch may be repeated, the ready availability of 99mTc and 111In-oxine, and the lower radiation absorbed doses with the shorter-lived radionuclides.     

全自动血型分析仪应用于献血者血型筛查

目的探讨并评价全自动血型分析仪应用于献血者血型筛查和盐水不规则抗体检测。方法采用全自动血型分析仪(全自动法)对25 554例献血者标本作ABO及RhD血型鉴定、盐水不规则抗体初筛,并与加样仪加样手工比色法(半自动法)作比对实验。ABO正反定型不一致而无法定型、O细胞凝集、RhD阴性的标本送血型红细胞参比实验室鉴定。结果全自动法与半自动法比较,ABO、RhD阴性血型1次准确定型率:99.93%(25 535/25 554)vs99.95%(25 542/25 554)(P>0.05);O细胞凝集阳性率:0.18%(46/25 554)vs0.10%(26/25 554)(P<0.05),经参比实验室确认,盐水不规则抗体分别为43、24例(0.17%vs0.09%,P<0.05);正反定型不符:17例(0.06%)vs10例(0.04%),参比实验室确认,经参比实验室确认2种方法共同拥有亚型5例,亚型漏检各2例(0.01%),余为正常血型(10/17vs3/10,P>0.05)。结论全自动血型分析仪作ABO、RhD血型筛查的技术相关性好、重复性好;除了全自动化、操作规范化、标准化等优点外,全自动血型分析仪更易发现盐水不规则抗体。