Antiglobulin Techniques Research Articles

A prospective study of the incidence of red cell allo-immunisation following transfusion.

To establish the incidence and timing of red cell allo-immunisation following transfusion, pretransfusion and serial post-transfusion samples were screened for allo-antibodies in a total of 452 patients who had undergone elective surgery. Antibody screening was performed by 2-stage papain, manual polybrene and indirect antiglobulin techniques (IAT). Red cell allo-antibodies were found in 42 patients and 38 of these (8.4% overall) demonstrated antibodies only after transfusion; 76% of them had Rh specificity. This rate of red cell allo-immunisation is higher than what would be expected if transfused patients were tested only once post-transfusion, as has been the case in several previous studies. For the type of patients studied, this finding may not be of clinical relevance at present because most patients undergoing elective surgery do not require further transfusion in their lifetime. However, this is changing with the longer life expectancy of the population and the increased probability of repeat surgery. Twenty-two (58%) of the antibodies were initially detected by the 2-stage papain and/or polybrene techniques, when the IAT was negative, although later 19 became positive by IAT; this added sensitivity of techniques other than the IAT, to detect early allo-immunisation may be relevant in pretransfusion testing to prevent haemolytic transfusion reactions in patients requiring repeated transfusions.

Two cases of “hrB‐like” autoantibodies appearing as alloantibodies

Two cases are described in which autoantibodies mimicked alloantibodies. The direct antiglobulin test (DAT) on the red cells (RBCs) from both patients was negative when routine manual hexadimethrine bromide (Polybrene) and enzyme-linked antiglobulin techniques were used. The RBCs also did not react on direct bromelin and direct Polybrene tests. However, an "hrB-like" antibody was eluted from the RBCs of both patients. The sera from these patients reacted with all e+ hrB+ RBCs but not with e+ hrB-, e-, or their own RBCs. The antibody in the serum of one patient was not adsorbed by R2R2 RBCs. Serologic tests initially suggested (by direct testing and adsorption studies) that the serum antibodies were alloantibodies rather than autoantibodies. RBCs taken from one patient, 8 months after her sample was first referred to our laboratory, reacted with a serum sample from her first admission. An RBC sample taken from the other patient, initially typed e+ and hrB- but 1 month later typed e+ and hrB+ by using the same anti-hrB sera, was used to test the earlier samples.

A survey of platelet serology in UK laboratories (1987): An assessment of the efficacy of using chloroquine-treated platelets to distinguish between platelet-specific and anti-HLA antibodies

This survey was designed to determine the ability of participants to distinguish between platelet specific and anti-HLA antibodies. Four well characterized reference reagents and 10 unknown samples were sent to 29 laboratories affiliated to the UK Platelet and Granulocyte Serology Working Group and to three overseas reference laboratories. Results were obtained from 27 Working Group laboratories and from 2 reference laboratories. Overall, the results indicate that the use of chloroquine-treated platelets alone, without an independent test for anti-HLA antibodies, was insufficiently reliable to elucidate mixtures of platelet specific and anti-HLA antibodies. Laboratories using chloroquine-treated platelets were no more successful in elucidating the antibody composition of sera containing platelet-specific and anti-HLA antibodies than those laboratories which did not use this technique. The results may indicate a lack of experience with the chloroquine technique and suggest that further studies into the performance of the technique are required. The survey also provided insights into other areas of platelet serology: (1) There was a decrease in the incidence of false-positive results compared to earlier UK surveys. [2) All laboratories detected potent platelet-specific antibodies but weakly reactive antibodies were less readily detected. (3) Laboratories using fluorescent antiglobulin techniques recorded fewer errors than laboratories using ELISA techniques. (4) Laboratories using paraformaldehyde (PFA)-treated platelets in fluorescent antiglobulin techniques did not perform better than laboratories using untreated platelets.

Solid‐phase antiglobulin test for screening and identification of red cell antibodies

A newly developed automatically readable solid-phase antiglobulin test is described. The solid-phase test in comparison with conventional antiglobulin techniques for screening and identification of red cell antibodies showed agreement in 2346 (98.7%) of samples tested. The increased sensitivity of the solid-phase antiglobulin test is demonstrated by examination of serial twofold dilutions of antibodies.

緊急時における血液製剤の供給 都立墨東病院における供給状況

The prescribed form about releasing of blood products in urgent situations had not been established in our hospital. In 1986, however, agreement for emergent transfusion was decided in hospital transfusion committee and the manual was made about releasing of blood products in urgent situations. The outline of the manual contents is following. In this manual, the degree of uregent is composed of three classes that is, class A, B, and C respectively according to extent of urgency. Blood products are transported to doctors within ten minute as soon as blood grouping tests and crossmatching test by saline technique are completed in a case of class A. Blood products are issued at twenty minute after finishing crossmatching tests by saline and enzyme (bromelain) techniques in class B or are provided at thirty five minute after completing crossmatching tests by saline, enzyme and antiglobulin techniques in class C.Then, we analyzed the annual state of transfusion according to this manual at the emergency center in Bokuto hospital. As the result of findings, ordered number as class A had about one third of total urgent orders. The cases of multiple trauma due to traffic accidents which were most frequent occupied 43.2% of total cases in class A. Ordered number as class B was under 10%, and as class C was over 60% respectively. In class B, cases of gastrointestinal bleeding which was most frequent had 43.2% of total cases belonging to this class. In class C, although the request of blood products prepared for urgent operation was most frequent, actually transfused cases were about one third of total cases in this class.From investigation in this time, it was noticed that the situation called for uregent transfusion was various but there was no case to that transfusion could not be waited for ten minute at least.

Eighteen Months' Experience with a Manual Polybrene Crossmatch in a Large Hospital Transfusion Laboratory

For 18 months in this laboratory the manual polybrene technique (MP) has been used as the only crossmatching procedure preceded or accompanied by an antibody screen comprising two-stage papain and LISS antiglobulin techniques. There were 17,161 requests representing 43,006 blood units crossmatched and 20,841 units transfused. Non-specific reactivity with the MP required use of an antiglobulin crossmatch in approximately 0.2% of patient samples. Heparin in excess of 20 IU/ml reduced polybrene aggregation of red cells necessitating an antiglobulin crossmatch for 2 patients. Of 288 antibodies detected 20 reacted exclusively by MP compared with 18 by the papain procedure. The data supported the use of MP as an alternative to enzymes in antibody screening protocols. The polybrene technique was found to be a superior abbreviated crossmatch compared with the immediate spin technique and was applicable to all patients including those with known antibodies.

Discrimination between innate and cytophilic immunoglobulin on human peripheral blood lymphocytes: Analysis by the direct antiglobulin rosette-forming reaction

Bovine red blood cells linked to polyclonal or monoclonal anti-immunoglobulin antibodies are used in the direct antiglobulin rosetting reaction to detect surface-Ig on human lymphocytes. The sensitivity of this test is markedly increased by pretreating the red cells with trypsin. Enzyme-treated red cells, coupled to anti-human Fab or anti-light chain antibodies, react not only with innate Ig on B lymphocytes but also with smaller amounts of passively adsorbed, cytophilic Ig on up to 25% of freshly prepared peripheral blood (non-B) lymphocytes. In contrast, trypsinized red cells carrying anti-Ig isotype-specific antibodies react exclusively with B cell surface-Ig. Cytophilic Ig is abnormally firmly bound to lymphocytes separated on Ficoll-Hypaque at 20°C or below, and is released very slowly during 3 h or more at 37°C in vitro. Lymphocytes are free of detectable cytophilic Ig when isolated on Ficoll-Hypaque at 37°C, and very little Ig is retained by non-B cells in suspensions purified on Percoll which, unlike Ficoll, does not increase Ig binding affinity. These lymphocyte separation procedures are recommended as a preliminary to B cell assays by sensitive antiglobulin techniques.

An Immunohematologic Complication of Isoniazid

The characteristics of an immunohematologic reaction to isoniazid are described with emphasis on the mechanism of development of the positive antiglobulin test. In contrast to previous findings, the antibody is a firmly bound IgG complement-binding antibody, reacting best at 37 degrees C by antiglobulin techniques and only with isoniazid presensitized cells. A method for preparing isoniazid-sensitized cells is described.

Modulation and regrowth of allotype on normal rabbit peripheral blood lymphocytes: Allelic inclusion of b4 and b6 in single cells

Allelic inclusion at the b-locus by heterozygous peripheral blood rabbit lymphocytes was demonstrated by using the mixed antiglobulin techniques (6, 7, 19). Heterozygous cells (64, 6) were treated with monospecific antiallotype reagents at 4 °C and warmed at 37 °C. The removal of surface allotype determinants was studied and, both the b4-and b6-specificities co-modulated after sensitization with either anti- b4 or anti- b6. Experiments were undertaken in which cells stripped of one or both allotypes by antiallotype induced modulation were cultured overnight. Allotype was then regenerated. Double expression of allotype by cells before antiallotype treatment was recovered following overnight regrowth at levels equal to those seen before treatment. Such was not the case when b44 and b66 cells were cultured together. These results indicate that normal heterozygous peripheral blood lymphocytes may express both b-locus alleles and that these determinants are in some manner physically associated with one another in the cell membrane.

Studies on the Immune Response to Penicillin and Cephalothin in Humans

Abstract This study evaluated the critical factors in the titration of penicillin and cephalosporin antibodies using hemagglutination techniques. Emphasis was placed on the development of sensitive and reproducible methods for further investigational work as well as the development of a simple and practical method suitable for use by clinical laboratories with only occasional need for performing such tests. Conditions for optimal sensitization of erythrocytes with the antibiotics were evaluated including concentration of the antibiotics; pH, temperature, and time of sensitization; comparison of red cells from different donors; and the effects of varying conditions of storage and age of the red cells. Optimal sensitization of red cells by penicillin are obtained when 1 ml of fresh erythrocytes is incubated with 1,000,000 units Kbenzylpenicillin G in 15 ml Trimethylamine (TMA)-buffer, pH 10.0, at room temperature for 2 hr. Similarly, for preparation of cephalothin-sensitized red cells, 1 ml of fresh erythrocytes is incubated with 400 mg Na-cephalothin in 10 ml TMA-buffered saline (pH 10.0) at 37°C for 2 hr. Red cells sensitized at pH 10.0 with penicillin gave titers 30 times higher than those sensitized at pH 7.3, but only marginal differences were noted with cephalothin. Several hemagglutination systems were evaluated, including standard blood-banking methods such as saline, albumin and antiglobulin techniques. These were compared with agglutination in a diluent containing dextran, normal rabbit serum, and Tris-buffered saline (Dex-NRS-TBS). The indirect antiglobulin (Coombs') test using antibiotic-sensitized cells is optimal for clinical purposes. However, if numerous penicillin and cephalothin antibody titrations are to be performed for investigational work, direct agglutination in Dex-NRS-TBS is generally more practical.

Results of an antibody screening programme at an antenatal clinic: relevance to haemolytic disease of the newborn and blood transfusion reaction

Results of a routine antenatal antibody screening programme from March 1967 to March 1970 were presented. 5,354 sera from 4,724 pregnancies were screened. The indirect antiglobulin and saline techniques at 37° on separate selected commercially available screening cells were used throughout. All patients were screened at their first visit and Rh negative patients again at approximately 28 and 36 wk. gestation. Antibodies were identified with commercial cell panels. Eighty sera contained detectable antibodies (1.7%). In 44 pregnancies (41 patients) the sera contained anti-D or anti-C + D and in 36 there were other antibodies. In 12 of these (2 anti-C, 2 anti-E, 1 anti-c, 6 anti-Kell (5 patients) and 1 anti-Fya) the antibody was capable of causing haemolytic disease of the newborn but only 2 babies from this group were affected and only one (an anti-Kell) required exchange transfusion. The remaining non-rhesus antibodies could have caused transfusion reactions or cross-matching difficulties (17 anti-Lewis and 7 unidentified antibodies). Of the 44 pregnancies in which anti-D or anti-E + D were found, 12 were negative, 8 were positive at the initial antibody screen and 24 were known from previous pregnancies. Some of the 12 developing detectable antibodies during the pregnancy produced quite severe haemolytic disease. There was a decreasing rate of appearance of new examples of anti-D in each year of the programme and only one of the 20 cases developing anti-D for the first time had had anti-D gamma globulin with her last pregnancy. The yield of antibodies relevant to haemolytic disease was small but the screening of all antenatal patients for antibodies was felt to be worthwhile for the anticipation of cross-matching difficulties and for its effect in improving standards.

Anti-Nuclear Antibodies in Mice

Summary Indirect immunofluorescent antiglobulin techniques were utilized to examine the incidence of autoimmune anti-nuclear antibodies in aging retired-breeder and virgin A/J, aging retired-breeder DBA/1J, and aging virgin (A/JxDBA/1J)F1 hybrid mice. The incidence of anti-DNP antibody in aging retired-breeder A/J animals increased almost linearly to approximately 90% by 23 months of age. The greater incidence in 8-month-old retired-breeder females than in both retired-breeder males and virgin females of the same age disappeared as more animals in each group began to produce anti-DNP antibody during aging. Anti-DNA antibody was also produced spontaneously during aging, but only in very old animals. Aging DBA/1J (retired-breeders) and AJDF1 hybrids never developed either anti-DNP or DNA antibodies. The injection of calf thymus DNP or DNA, combined with complete Freund's adjuvant, did not induce 4 ½-month-old virgin A/J mice to produce anti-DNP or DNA antibodies. Similar treatments of 8-month-old retired-breeder A/J mice which lacked anti-DNP antibody prior to injection resulted in the production of antibody reactive with both calf and rabbit thymus DNP and autochthonous nuclei (LE cell formation). Antibody with specificity for DNA could not be detected in these sera. The injection of BSA or complete Freund's adjuvant alone also resulted in anti-DNP antibody production. In contrast, DBA/1J and (A/JxDBA/1J)F1 hybrids given similar injections did not produce anti-DNP or DNA antibodies.