Search

A micro enzyme-linked immunosorbent assay (ELISA), using lyophilized stroma as carrier of red blood cell antigens, which stays stable longer than usual, using intact erythrocytes, was developed for determination of blood-group antibodies in the ABO, Kell and Lewis-systems. Stroma being fixed on microtiter plates was incubated with antisera and peroxidase-conjugated anti-human globulin. The transformation of the substrate added was determined photometrically. A binding of antibodies to the stroma could be demonstrated up to an antibody dilution of 1:1024 for the ABO-system, of 1:512 for the Kell-system and of 1:64 for the Lewis-system. By standardization of this method the quantitative determination of antibodies becomes possible without being restricted by the limited stability of intact erythrocytes.

Current textbooks for transfusion medicine state that anti-A and/or anti-B (anti-A/B) agglutination titers--and thus the respective antibody concentrations--reach their maximum in individuals 5 to 10 years old and then gradually decline with the increasing age of the individual. This statement is largely based on a study by Thomsen and Kettel that dates to 1929. In the present article, ABO antibodies in sera of 175 healthy persons aged 61 to 97 years, as well as sera of 170 newborn infants and children aged 0 to 17 years, were analyzed. Microhemagglutination tests were performed with all sera and complemented by ABO enzyme-linked immunosorbent assays to measure the immunoglobulin class (IgM, IgG, and IgA) of the anti-A/B. As in a previous study using sera of persons aged 20 to 67 years, individual differences exceeded age-related changes for all variables. Median values of IgG and IgA anti-A/B were elevated in elderly persons of blood group O, whereas no significant changes were observed in other variables. In particular, the decrease in agglutination titers with the increasing age of the individuals was far less pronounced than previously described; even in sera of persons aged 90 to 97 years, median agglutination titers of 128 were found. Results in the sera of children confirm previously reported data that agglutination titers and IgM anti-A/B reached adult levels at the age of 5 to 10 years.

The presence of residual donor red cells in transfused patients impairs the ability to obtain a reliable phenotype for subsequent allogeneic blood transfusions. Post transfusion phenotyping is particularly important in transfused patients developing multiple or weak blood group antibodies. However, separation of autologous and transfused red cells from recently transfused patients is necessary for the proper identification of any suspected red cell alloantibody or autoantibody. The standard technique for the separation of autologous from transfused red cells involves microhematocrit centrifugation with phytate ester solution, a laborious and time consuming method. We have examined the effectiveness of a simplified microhematocrit technique, omitting the use of esters, to separate autologous and transfused cells by performing Rhesus (Rh) and Kell (K) blood group phenotyping, both manually and by automation, pre and post transfusion. The Rh phenotype and K type were recorded on pre- and post-transfusion peripheral blood samples from 100 newly transfused operating theatre patients, both manually and by automation using the DiaMed Classic ID-Gel Station and Diaclon ’Rh subgroups+K’ gel cards. On post transfusion blood samples, separation of autologous from transfused cells was performed using the Haematospin 1300, based on the difference in the specific gravity between reticulocytes and mature red cells where the autologous cells concentrate in the top layer of the microcapillary tube. This separation was performed on blood samples taken three days post transfusion, based upon the normal reticulocyte maturation time of three days. On all samples tested, there was 100% correlation between manual and automated ID-GEL Station results. On Rh phenotyping of post transfusion blood samples prior to cell separation, 46 of the 100 patients had a mixed field (Mf) reaction. Repeat Rh phenotyping of day three post transfusion samples, following separation of autologous cells using the microhematocrit centrifugation technique, showed complete correlation with pre transfusion Rh phenotyping results in all but two cases, where a Mf reaction persisted. These two cases had had massive blood transfusions (40 and 36 units of red cells respectively) compared to the amount of red cells (mean 3.3 units, range 1–15) transfused to the other 98 patients. One week after initial Rh phenotyping, allowing sufficient time for autologous reticulocyte recovery in these two patients, repeat Rh phenotyping of separated autologous cells correlated with that of the pre transfusion results. 6 of the 100 patients were K positive on pre transfusion testing. Repeat testing of post transfusion blood samples prior to cell separation showed a Mf reaction for the K antigen in two of these 6 cases, suggesting transfusion of K antigen negative red cell units. Once cell separation was complete, both patients were confirmed as K positive. In conclusion, the simplified microhematocrit centrifugation technique and automated ID-Gel station are reliable, sensitive and less hazardous in providing an accurate Rh phenotype and K type on patient blood samples taken three days post transfusion and could be easily performed in the standard blood transfusion laboratory. The only limitation to this technique is an inability to separate autologous and transfused cells from patients who have undergone massive transfusions within a short space of time.

Anti-A and anti-B blood group antibodies in the cynomolgus monkey sera were serologically characterized. 2-Mercaptoethanol (2-ME) inactivated these antibodies of both group-A and group-B sera. Furthermore, soluble human ABH substances neutralized these antibody activities. However, these activities were not inhibited by anti-human IgG or IgA serum but by anti-human IgM serum. By gel filtration on Sephacryl S-300, the anti-A and anti-B antibodies reacting readily in saline media were detected in the fractions corresponding to the IgM antibodies. Thus, anti-A and anti-B antibodies in group-B and group-A sera were judged to be mainly of IgM. In contrast to group-A and group-B sera, group-O serum contained IgG antibodies of high titers besides IgM antibodies. Neither 2-ME nor anti-human IgM serum affected these antibody activities. On the basis of the results of the inhibition test using anti-human immunoglobulin sera as well as of gel filtration, group-O sera were divided into the following two categories, the one having only IgG reacting well in the Coombs method and the one having both IgM and IgG.

A two-channel Auto Analyzer II system (AAII) was tested for routine screening of blood group antibodies in 8, 980 sera during past 18 months. A low ionic strength polybrene method (LISP) was employed to detect IgG antibody.Manual conventional method was tested in parallel and compared with this method. AAII was not outstanding with respected to detection of some naturally occurring antibodies such as anti-Lewis, anti-P1. For the purpose of preferential detection of antibodies in the P and Lewis systems, we used commercial panel red cells instead of pooled red cells made in our laboratory.On the contrary, weak immune antibodies missed in the manual method were well detected by AAII. These results suggest that this system is ideally suitable to the detection of IgG antibodies which appears early after transfusion.we consider that the automated LISP method is very useful in a laboratory engaged in the detection and identification of red cell antibodies.

Blood banks face significant problems supplying blood units compatible with erythrocyte antigens other than ABO and rhesus D. In this study the number of such units was evaluated between 1973 and 1982, and it was found that 1.5% of the total number of blood units were compatible with other antigens than ABO and rhesus D. Based on data of the number of units requested for each such antigen and the frequency of the antigen in the European population, a logistical system providing sufficient numbers of ‘antibody-compatible’ blood units is proposed.

P blood group system antibodies are encountered frequently in clinical transfusion practice. The authors describe an improved method for removing P antibodies from patient specimens without dilution. Solid- phase synthetic P antigens were used to neutralize serums containing P blood group system antibodies; 26 alloantibodies with specificity other than P were not inhibited. The synthetic P antigens are also used to characterize the heterogeneous immunochemical fine specificity of antibodies to P1 and P + P1 + Pk.

Immunologic injury is an important contributing factor in failure of aortic valve allografts. The etiology of this immune reaction is unknown, but blood group antibodies have been postulated. In 46 patients, ABO and rhesus blood group compatibility bore no relationship between the success or failure or aortic valve allotransplantation. Therefore, circulating blood group antibodies do not contribute to valve failure, and recipients do not have to receive aortic valve allografts matched for ABO or rhesus compatibility.

A microcapillary method utilizing phthalate esters or an ultracentrifuge method are both capable of separating autologous from homologous erythrocytes in polytransfused patients. The microcapillary technique which is readily adaptable to blood bank laboratories provides a previously unavailable method for defining blood group antigen typings in transfused patients. Such typings are of vital importance in the laboratory evaluation of transfused patients with multiple or weak blood group antibodies.

The addition to a 15-channel blood-grouping machine of two singlechannel antibody screening systems, based on the principles described by Marsh and Lalezari has produced a modified machine capable of detecting most clinically significant irregular blood group antibodies with a sensitivity equal to normal manual screening tests.

Red cell ghosts prepared by conventional methods were mixed with an equal volume of 0.6% γ globulin and converted to an insoluble “slurry” with glutaraldehyde. A 50% suspension of the washed “slurry” in phosphate buffered saline using 0.1% NaN3 as preservative was used for preparing hemoglobin-free eluates of anti-D and other blood group antibodies suitable for radiolabeling; it remained active during storage at room temperature for longer than 3 months. The procedure can be used for preparing immunoadsor-bents containing red cell stroma coupled to other substances.

It has been shown that red cells sensitized with anti-Rh antibody are agglutinated by anti-γ-globulin sera, regardless of the "order" of Rh antibody activity. Red cells sensitized with anti-Lewis antibody, on the other hand, are agglutinated by anti-sera to non-γ-globulin substances. Of six other blood group antibodies studied, five provided γ-globulin red cell coating materials and one, an anti-Kidd, a non-γ-globulin material. The participation of complement, or complement-like, substances was shown in some instances. Various pitfalls in the technics employed in absorbing Coombs sera so as to render them immunochemically specific are discussed.

We have always experienced difficulties in the past in treating cells sensitized with blood group antibody with the first component of complement so that they became strongly active following such treatment. Such cells, prepared as described by other workers (Harboe, Muller-Eberhard, Fudenberg, Polley and Mollison, 1963) have always proved to be insufficiently active for our experiments. We therefore explored a number of alternative methods, and found that a pellet obtained by ultracentrifugation of human serum (Naff, Pensky and Lepow, 1964) could be used for this purpose. The object of this paper is to describe the treatment of cells sensitized with blood group antibodies with this pellet and to describe the reactions of the cells so prepared.

The present work is concerned with further investigations on a swine iso-immune antiserum in which three different populations of blood group antibodies have been detected. Two of these antibody populations, anti Ea, and anti Ee, are described previously (Andresen and Wroblewski, 1959). A third antiserum fraction, containing anti Gb, has been isolated. The results from population studies on 148 random pigs of Danish Landrace breed from commercial herds made it likely that the two factors, Ga and Gb, are contrasting characters in a closed genetic system. This theory was supported by a gene frequency analysis on combined material of 127 random pigs from breeding centers and the 148 random pigs from commercial herds. Also details of investigations on another iso-immune antiserum which contained anti Ea, anti Fa, anti Ka, and a fourth population of antibodies to be called anti Hb are presented. Population studies on 127 random pigs suggested a serological association between Ha and Hb. The possibility of detecting a genetical association was investigated. The results from blood typing limited family material are concordant with the theory that Ha and Hb are produced by allelic genes in a multiple allelic system. Afsnit A omhandler fortsatte undersogelser af et iso-immun antiserum, hvori der er pavist tre forskellige blodtypeantistoffer. To af disse, anti Ea og anti Ee, er beskrevet tidligere (Andresen & Wroblewski, 1959). Et blodtypereagens indeholdende det tredje af de naevnte antistoffer, anti Gb, er blevet fremstillet. Blodtypebestemmelse af 148 tilfaeldigt udvalgte slagterisvin samt 127 tilfaeldigt udvalgte soer og orner fra svineavlscentre viste, at G systemet ma opfattes som et lukket genetisk system, der omfatter to allele gener Ga og Gb. Undersogelser af et andet iso-immun antiserum er beskrevet i afsnit B. Dette antiserum viste sig at indeholde antistoffer overfor blodtypefaktorerne Ea, Fa og Ka samt et fjerde antistof, anti Hb. Resultatet af blodtypebestemmelse af de 127 orner og soer fra avlscentre tydede pa et serologisk slaegtsskab mellem blodtypefaktorerne Ha og Hb. Dette muliggjorde tillige en genetisk sammenhaeng, og resultatet af de pafolgende familieundersogelser underbygger den teori, at det genetiske grundlag for faktorerne Ha og Hb er to allele gener i et blodtypesystem, der omfatter mindst tre alleler, Ha, Hb og H−.

2. Are the characteristics of blood-group antibodies in vitro exactly related to their effects in vivo ? For example, if an antibody binds complement in vitro does it bring about the removal of red cells in a different way from that of an antibody which does not bind complement ? 3. Is there a close relationship between the potency of an antibody, judged by tests in vitro, and the speed and extent of the red-cell destruction which it causes in vivo ?

Summary. Antomated antibody screening has been performed on donor bloods for six months, using a filter paper model Antoanalyzer and a specially built automatic pooler for an antiglobulin technie. This combined system screens donor blood for antibodies which react by either enzymes or anti-human globulin methods. The method appears to be fast and sensitive and to be adaptable to the procedures at a large blood center.

Resume Plusieurs observations d'accidents hemolytiques lies a l'iso-immunisation (anti c, E, Kell, Fya, S, Jka) sont signalees dans ce travail. L'analyse des faits cliniques observes montre que, quel que soit le type serologique d'anticorps, le risque d'anurie est important si le malade est transfuse au cours d'une intervention chirurgicale. Le collapsus est alors un element de gravite. Le diagnostic serologique peut etre rendu difficile du fait que l'anticorps responsable est masque pendant plusieurs jours. Un controle serologique post-transfusionnel est souhaitable chez les sujets particulierement aptes a l'iso-immunisation. L'apparition d'agglutinines antileucocytes permet parfois de deceler cette aptitude, selectionnant ainsi certains malades qui seront alors soumis a des controles serologiques comportant toute la precision technique souhaitable. Summary A number of hemolytic transfusion reactions due to anti-c, anti-E, anti-K, anti-Fya, anti-S, and anti-Jka are reported. The clinical data show that, independently of the specificity of the antibody concerned, the risk of anuria is greater if the patient is transfused during a surgical operation. Collapse increases the severity of the reaction. Serological diagnosis may be rendered difficult by the disappearance of the responsible antibody for several days. It is advisable to examine a post-transfusion sample of serum in patients with a particular aptitude for making antibodies. Such patients can sometimes be spotted by the finding of anti-leucocytic antibodies in their serum, and this should be thoroughly examined for blood group antibodies using a battery of varied techniques. Zusammenfassung Eine Reihe von hamolytischen Transfusionsreaktionen, die auf Isoimmunisierungen gegen die Antigene c, E, K, Fya, S und Jka beruhen, wird beschrieben. Diese Beobachtungen zeigen, das unabhangig von der Antikorperspezifitat bei inkompatiblen Transfusionen, die wahrend chirurgischen Eingriffen durchgefuhrt werden, eine erhebliche Anuriegefahr besteht. Der Kollaps stellt bei solchen Patienten eine schwere Komplikation dar. Die serologische Abklarung solcher Falle ist haufig erschwert, da oft wahrend Tagen keine freien Isoantikorper nachgewiesen werden konnen. Bei Patienten mit einem gesteigerten Antikorperbildungsvermogen ist es zweckmasig, post-transfusionelle serologische Kontrolluntersuchungen durchzufuhren. Das Auftreten von Leukozytenagglutininen ist ein wertvolles Indiz fur ein gesteigertes Antikorperbildungsvermogen. Durch den Nachweis solcher Antikorper gelingt es, diejenigen Patienten zu erfassen, bei welchen eine eingehende posttransfusionelle serologische Kontrolle besonders angezeigt erscheint.

Summary Extracts from roe of female brown trout, rainbow trout, seatrout and salmon are found to give reactions of saline agglutinating anti-B with human red cells. Some trout roe also contain saline agglutinating anti-A and/or anti-AB.

Summary So-called naturally-occurring antibodies seem, as a rule, to be composed of 19 S globulin even when they behave as haemolysins (anti-P + P1 or as partially incomplete antibodies (anti-Le1). 19 S naturally-occurring antibodies may be accompanied by 7 S antibody with the same specificity which has possibly been produced as an immune response; an example of anti-Lea consisting partly of 7S and partly of 19S globulin is described. Immune incomplete antibodies seem normally to be composed of 7S gamma globulin but it appears that they may be composed partly of 19 S globulin; in the present paper two antibodies of this kind, one anti-D and one anti-K, are described. Zusammenfassung Die sogenannten naturlichen Antikorper scheinen in der Regel aus 19 S-Globulin zu bestehen. Dies gilt selbst dann, wenn diese Antikorper den Charakter von Hamolysinen (Anti-P+P1) oder partiell inkompletten Antikorpern (Anti-Lea) aufweisen. Neben den naturlichen 19 S-Antikorpern kommen 7 S-Antikorper mit derselben Spezifitat vor, die wahrscheinlich auf Immunisierungen zuruckzufuhren sind. Als diesbezugliches Beispiel wird ein teils aus 7 S-, teils aus 19 S-Globulin bestehendes Anti-Lea-Serum beschrieben. Die inkompletten Immunantikorper bestehen vorwiegend aus 7 S-γ-Globulin; es scheint aber, das sie teils auch aus 19 S-Globulin bestehen konnen. In der vorliegenden Arbeit werden zwei solche Antikorper, ein Anti-D und ein Anti-K beschrieben. Resume Dans la regie les anticorps dits «naturels» semblent etre composes de globulines 19 S, malgre le fait que certains d'entre eux se comportent comme des hemolysines (anti-P + P1) ou comme des anticorps partiellement incomplets (anti-Lea). Les anti-corps naturels 19 S peuvent etre associes a des anticorps 7 S presentant la meme specificite et dont la presence peut etre le resultat d'une reponse immunitaire; les auteurs decrivent un exemple d'anti-Lea compose en partie de 7 S et en partie de 19 S. Les anticorps immuns incomplets semblent etre composes normalement de gamma-globulines 7 S, mais il semble qu'ils peuvent etre composes en partie de 19 S; dans cet article, les auteurs decrivent deux anticorps de cette espece: un anti-D et un anti-K.

Summary Solutions of colloidal silica react with human erythrocytes and, depending on their method of preparation, show the phenomena of agglutination, zoning, blocking, enhanced agglutination with papaintreated cells, and an effect similar to the Coombs test. Cells coated with “incomplete colloid” appear to be capable of taking up an additional coat of serum protein. These effects are similar to those found with blood group antibodies, and some of the effects have also been found with extracts of certain seeds. The varied properties of different preparations of colloidal silica may cause difficulty when this material is accidentally present in serological reagents. Resume Les solutions colloildales de silicates reagissent avec les hematies humaines; selon la methode de preparation on obtient les phenomenes suivants: agglutination, phenomene de zone, bloquage, agglutination intensifiee d'hematies traitees a la papaine, enfin un effet semblable a la sensibilisation dans le test de Coombs: il semble que les hematies qui ont deja fixe du «collolde incomplet» peuvent en outre fixer des proteines seriques. Ces effets sont semblables a ceux que produisent les anticorps, et quelques uns d'entre eux sont egalement produits par certains extraits de graines. II ressort de ces diverses proprietes des preparations collordales de silicate que lorsqu'il y a une contamination accidentelle des tests serologiques par ces substances, cela peut causer des difficultes considerables. Zusammenfassung Menschliche Erythrozyten werden durch kolloidale Kieselsaurelosungen in verschiedener Weise beeinflust. Je nach Art der Herstellung dieser Losungen beobachtet man Agglutinationen, Zonen- und Hemmungsphanomene, antiglobulinahnliche Effekte sowie eine verstarkte Agglutinabilitat von papainbehandelten Zellen. Kieselsaurekolloid beladene Erythrozyten vermogen zusatzlich Serumproteine zu binden. Die Wirkung der Kieselsaurekolloide gleicht derjenigen von blut-gruppenspezifischen Antikorpern und Pflanzenagglutininen. Verunreinigung von serologischen Reagenzien mit Kieselsaurekolloiden fuhrt unter Umstanden zu Fehlergebnissen.

Blood group antibodies tend to occur in a small group of sensitive people. Thus, in Rh-negatives, those who produce anti-Rh are more likely than others to form still other antibodies. In the sensitive group production of anti-Rh may be a forerunner of other antibody production. In a study of sera from 2,851 antenatal patients it was found that 13.9% of women who had formed anti-Rh also formed a non-Rh-system immune antibody. In women with no anti-Rh only 0.1% formed an immune antibody.

Summary 1 Complement fixing blood group antibodies are described. 2 A description is given of some of the reactions of cells sensitized with antibody in the absence of complement; the action of complement on cells is also described. 3 The effect of altering various conditions in the indirect anti-globulin test, when employed for the detection of complementfixing blood group antibodies, on the strength of the result of this test is described. Resume 1 Les anticorps de groupe sanguin qui fixent le complement. 2 Quelques-unes des reactions que presentent des erythrocytes sensibilises par des anticorps en l'absence de complement et l'action du complement sur les erythrocytes. 3 L'effet produit sur la qualite des resultats du test indirect a l'anti-globuline utilise pour la detection des anticorps de groupe sanguin fixant le complement lorsqu'on modifie de manieres diverses les conditions d'execution du test. Zusammenfassung 1 Es werden komplementbindende Blutgruppenantikorper be-schrieben. 2 Das Verhalten sensibilisierter Erythrozyten in Abwesenheit von Komplement sowie die Wirkung des Komplementes auf solche Zellen wird beschrieben. 3 Die Reaktionskinetik des indirekten Antiglobulintestes beim Nachweis von komplementbindenden Blutgruppenantikorpern wurde studiert und die optimalen Bedingungen dieser Reaktion heraus-gearbeitet.

Three patients are described who produced a variety of blood group antibodies following exposure to foreign red cells. These findings are considered in relation to the theory that a small group of individuals possess an ability, possibly genetically determined, to readily form antibodies and the theory that the first antibody formed may augment the production of other antibodies.

A statistical study of the incidence and specificity of multiple irregular blood group antibodies shows predominance of those within the Rh-Hr, Kell, and Duffy systems. When multiple antibodies included those in the Lewis, Lutheran, MNS, P, or Kidd systems, the other antibodies were usually in the Rh-Hr, Kell, and Duffy systems. The following categories of donors: 1a Group O Rh-neg. (dce/dce) K-neg. Fya-neg. 1b Group O Rh-neg. (dce/dce) K-neg. Fyb-neg. 2a Group O Rh-pos. (R1R1-DCe) K-neg. Fya-neg. 2b Group O Rh-pos. (R1R1-DCe/DCe) K-neg. Fyb-neg. 3a Group O Rh-pos. (R2R2-DcE7DcE) K-neg. Fya-neg. 3b Group O Rh-pos. (R2R2-DcE/DcE) K-neg. Fyb-neg. are further subtyped for Lea, Leb, P, Jka, Jkb, M, N, S, s, and Lua. Erythrocytes from these donors, frozen in glycerol, with Anti-A, and Anti-B antibodies removed during deglycerolization simplifies the problem of finding compatible blood for patients with multiple antibodies.

Thirty-six teeth have been surgically transplanted; of these twenty-seven were homografts and nine were autografts. Both clinical and radiographic evaluations suggest successful acceptance of these teeth by hosts, even though some of the specimens are going on the fourth year. Histologic examination of one of the homotransplants (surgically removed en bloc) suggests some replacement of tooth structure by viable bone, but the overwhelming mass of the specimen has retained its dental identity. Where there was a difference in blood group antigens between donor and recipient, three of the transplanted hosts manifested an increase in blood group antibodies suggesting the possibility of an immune response. Further and more varied studies are anticipated.

Concentration of serum proteins, including blood group antibodies and complement components, is achieved rapidly and economically by dialysing the serum against dry Carbowax 20-M, a polyethylene glycol polymer with a molecular weight of 20,000. Such concentrates require no fluid or electrolyte readjustment for red cell testing and may be used for the identification of weak antibodies, the conversion of rare but unreliable antisera into useful laboratory reagents, and the preparation of serum specimens with high complement activity.

The standard prophylactic dose of anti-Rh immunoglobulin used in the United States is 300 microgram. For use after spontaneous or induced abortion and diagnostic amniocentesis, this dose represents immunological "overkill". A 50 microgram dose has been effectively used for these patients in other countries but the specific products used were not equivalent to the products available in the USA. No evaluation of this 50 microgram dose has been conducted in the USA. This is a pilot study of 315 patients given a 50 microgram dose or a 300 microgram dose of anti-Rh immunoglobulin after first trimester abortion. No patient in either treatment group developed atypical blood group antibodies within 6 months. This finding confirms that 50 microgram of the American product is protective after first trimester abortion.

This report documents a mild case of hemolytic disease of the newborn (HDN) associated with anti-Jk3. The Filipino mother had previously had six children none of whom had been affected by HDN. She had been transfused at the time of her second pregnancy. Anti-Jkb and anti-Jk3 were detected in the maternal serum at the time of her seventh delivery. No prenatal serologic tests for blood group antibodies had been performed. The cord blood was found to have a positive direct antiglobulin test and anti-Jkb plus anti-Jk3 were eluted. The infant was treated with phototherapy.

The investigation of a failure of proficiency showed that certain saline solutions are inappropriate for use in blood group serology tests. In particular, it was found that solutions of unexpectedly low pH and/or those autoclaved and stored in plastic containers could severely compromise the sensitivity of the antiglobulin test when used as wash solutions. The observed loss of sensitivity ranged from a reduction in titration score to a complete failure in the detection of clinically significant blood group antibodies. It is suggested that careful consideration should be given to the source, pH, and storage container of saline solutions intended for use in serological tests and that improved standardization and sensitivity could be achieved by using phosphate-buffered saline pH 7.0 to 7.2 for all such purposes. It is recommended that unbuffered saline solutions of pH less than 6.0 should not be used for serological testing.

When the ionic strength of blood samples is reduced, blood group antibodies bind to red blood cells that lack the corresponding antigen. Upon normalization of the ionic strength, the antibodies elute into the supernatant fluid. We compared, in semiquantitative terms, this nonspecific and reversible binding of various blood group antibodies. Studies with anti-CD antibodies showed that the nonspecific binding increased when the ionic strength was progressively lowered. In 1/10 normal ionic strength, practically all of a high-titer anti-CD antibody bound to Rh(D)-negative RBC (phenotype ce). The majority of the other antibodies studied (anti-C, -c, -Fya, -Fyb, and -S) behaved similarly in 1/10 normal ionic strength, but the binding of some (anti-E, -s, and -Jka) was, at times, less complete. Most examples of anti-K antibodies, however, demonstrated very poor nonspecific binding to K-negative RBC in low ionic strength (LIS). The authors propose that the augmentation of specific blood group antibody reactions in LIS is the direct result of this nonspecific binding, and that the failure of these conditions to support the nonspecific binding of anti-K antibodies might explain the lack of augmentation (and, at times, even the impairment) of the reactions of anti-K with its antigen.

A case of delayed hemolytic transfusion reaction in a 43-year-old patient is presented. Pretransfusion tests revealed no antibody, but tests performed when the reaction occurred demonstrated four. Ten months and 15 months later, when the patient's serum was again tested, the antibodies were undetectable. The significance of such antibodies is discussed.

Two hundred four examples of blood group antibodies, reactive only by the indirect antiglobulin test, were examined by the two-layer albumin capillary (TLAC) technique. Fifty-nine per cent of the examples tested were reactive by this technique. TLAC reactivity appeared to be related to antibody specificity, but a positive association between TLAC reactivity and IgG subclass composition was not confirmed.

Publisher Summary This chapter discusses practical procedures for ABO grouping, Rh grouping, and compatibility testing. Hemagglutination tests are usually carried out using either a test tube or a slide (tile) agglutination technique. Where possible, the tube technique should be used in preference to the slide technique, particularly when ABO grouping, as the slide method will often prove inadequate for detecting those blood group antibodies with a poor avidity time and the weaker red cell antigens and subgroups. Where a reagent has been specifically standardized for use by a slide or tile technique, the method recommended for its use should be strictly followed; and where time permits, a confirmatory tube test is advisable. Practice in handling and controlling a Pasteur pipette should be carried out using saline before attempting to perform any serological tests. Poor control of the pipette will lead to drops of serum or cells going into the wrong tubes. It is necessary for the interpretation and scoring of agglutination results to be as similar as possible between individuals, so that when one is reading the results of another person's work, a clear understanding of the strength of reactions is obtained.

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.

An improved method of acid elution is presented that simplifies the neutralization step by a sequential titration of small aliquots of base (Tris) with concomitant pH determinations. This technique is further adapted for use with small volumes of red cell starting material as might be found in certain obstetrical and pediatric situations. It is also useful in the determination of pH optima for select blood group antibodies.

By slowly lowering the surface tension of the aqueous medium through the admixture of 47.5% dimethyl sulfoxide (DMSO) and 0.1% bovine serum albumin (BSA) and by raising the pH to 9, complete elution of A, D, and K antibodies from sensitized erythrocytes (RBC) could be achieved. The eluates comprising the antibodies were subjected to dialysis to remove the DMSO and to neutralize the pH, and to ultrafiltration to remove the excess water. Recovery of a sizeable proportion of the eluted RBC (of blood groups A and K) proved possible by slow and careful removal of the DMSO with phosphate-buffered saline containing 2% BSA.

Antiglobulin sera, from nine different manufacturers, have been tested over a two-year period for their ability to detect different nonagglutinating, IgG and IgM blood group antibodies and for the presence of undesirable antibodies that cause the agglutination of nonglobulin coated red blood cells. There are not so many differences in anti-IgG levels among the sera as there are differences in anticomplement levels. Over the two-year period, there have been, in general, increases in the amounts of anti-IgM antibodies in the sera tested. Several of the sera, apparently prepared by dilution of raw rabbit serum and not by adsorption, contain antibodies that cause the agglutination of red blood cells not coated with IgG, IgM, or any of the components of complement.

The hospital blood bank plays a key role in the diagnosis of acute transfusion reactions involving red cell incompatibility. This paper discusses the interpretation of the serological tests performed by the laboratory. Because red cell incompatible transfusion reactions occur so infrequently it is difficult to accumulate practical experience in their laboratory presentation and this paper describes several of the pitfalls that may be encountered when laboratory findings deviate from classical descriptions. These include the absence of a positive direct antiglobulin test (Coombs) or an incompatible crossmatch, the absence of any apparent discrepancy between the pre- and posttransfusion specimens in cases of ABO incompatibility, the differentiation of auto-immune haemolytic anaemia from delayed transfusion reactions and the assessment of the clinical significance of any blood group antibodies that may be detected.