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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.

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.

A review is given of the ante- and peri-natal immunohaematological problems arising when ‘non-Rhesus’ irregular red-cell antibodies, especially those reacting with ‘public’ antigens, are encountered in pregnancy, and a case of anti-Ve in pregnancy is reported. The antibody did not cause haemolytic disease of the newborn. The direct Coombs test was negative, and no maternal antibody was present in the infant's serum. The results of the serological and immunological investigations are discussed in relation to the condition of the newborn infant, and the difficulty in assessing whether an infant is likely to be affected when such antibodies are present in pregnancy is stressed.

The properties of four albumin-agglutinating sera demonstrating a spectrum of reactivities are reported. All cause pan- and auto-agglutination in albumin. Direct and indirect reacting caprylate-dependent agglutinins are described, as are two sera with undefined specificity not related to the presence of caprylate stabilizer in the albumin. Immunoelectrophoretic evidence was obtained to support the concept of albumin-immunoglobulin complex formation in the genesis of albumin agglutination. Most albumin agglutinins react only with albumin stabilized by short-chain fatty acids; however, other specificities may be represented. Albumin agglutinins should be clearly identified in the blood bank to prevent confusion with blood group antibodies.

IN a previous report1 we showed that ganglioside, a class of complex sphingolipids isolated from brain tissue, inhibited several blood group antibodies, namely, anti-A, anti-M and anti-Rh0 (D). Since then2 we have found that anti-I and anti-O also were inhibited by gangliosides although anti-H was not. It has recently been found that anti-H (lectin) was less easily inhibited by gangliosida than human anti-H. In an extensive study of the association between ABH (O) and I, Gold3 found a parallel between the agglutinability of cells by anti-O, anti-A1, and anti-I. Schmidt and McGinniss4 demonstrated that the serological similarity between anti-O and anti-I sera does not extend to anti-H reagents and suggested establishing this blood group system as the OI rather than the HI system.

Ten commercial “broad-spectrum” antiglobulin sera, which had satisfied NIH standards, were evaluated and compared with six antiglobulin sera of varying specificities prepared in the authors' laboratories. The majority of the commercial antisera had adequate anti-IgG, although four were definitely weaker and failed to detect weak IgG antibodies. Most of the commercial antiglobulin sera had inadequate anticomplement, eight failing to react with cells strongly sensitized with complement, from patients with hemolytic anemia. Most of the commercial antiglobulin sera reacted poorly with cells weakly or moderately sensitized with complement-binding blood group antibodies, such as anti-Lewis. Evidence that adequate anticomplement activity should be present in routinely employed antiglobulin sera is discussed.

Pregnant cows can be successfully immunized with different proteins and subsequently deliver large volumes of potent antisera in their colostrum. An anti-human immunoglobulin has been prepared in this way which is excellently suited for the demonstration of incomplete blood group antibodies of the IgG class. At a dilution of 1:50 it was equivalent to a commercial antiglobulin serum of high quality. Anti-complementary antibodies were also present but at a considerably lower concentration than the antibodies against IgC.

THE PRESENT case was deemed worthy of report for three reasons: (1) the occurrence of acquired hemolytic anemia subsequent to transfusion incompatibility in a patient with acute disseminated lupus erythematosus; (2) the occurrence and demonstration of blood group antibodies, anti-C, anti-A 1 , and a third incompletely identified atypical hemagglutinin, subsequent to multiple whole blood transfusions; (3) the use of exchange transfusion as a therapeutic agent during the most acute phase of intravascular hemolysis. REPORT OF CASE The patient, a 15-year-old white girl, was admitted to the hospital for the first time on July 9, 1949, with complaints of generalized weakness and fatigability and of migratory joint swellings. Her first difficulties began seven months prior to admission with a brief episode of low-grade fever and widespread joint pains which subsided until six weeks before admission, when fever and alternate warm painful swellings of the finger, elbow, and knee points again developed.

Summary From the known effect of pH, temperature, antigen and salt concentration on the reaction between an example of anti-D and red cells, it has been found possible to suggest optimum conditions for the detection of other antibodies by the antiglobulin test. By lowering the salt concentration of the suspending medium the rate of association of antibody and red cells is considerably increased, so that the incubation time can be reduced from 1 hour to 5 minutes. Performance of the antiglobulin test under these conditions has been found to enhance the titre of antibodies against the following antigens: A, c. D, e, E, M, K, Lea, Fya, Jka. Using 131I-labelled anti-D it was estimated that approximately 500 molecules of blood group antibody on the surface of each red cell would give a just detectable agglutination reaction with antiglobulin serum under standard conditions. Resume Connaissant l'effet du pH, de la temperature, de la concentration en antigene et en sel sur la reaction entre l'anti-D et les erythrocytes, il nous a semble possible de trouver les conditions optimales permettant de detecter d'autres anticorps au moyen du test a l'anti-globuline. En diminuant la concentration en sel du milieu de suspension, le taux d'association de l'anticorps avec les erythrocytes est considerablement augmente, si bien que le temps d'incubation peut etre reduit de 60 minutes a 5 minutes. La pratique du test a l'anti-globuline dans ces conditions a demontre une augmentation du titre de l'anticorps contre les antigenes suivants: A, c, D, e, E, M, K, Lea, Duffya, Jka. Lorsqu'on utilise de l'anti-D marque au 131I, on a pu estimer que, approximativement, 500 molecules d'anticorps de groupe sanguin fixes a Ia surface de chaque erythrocyte donnent une reaction d'agglutination aisement decelable avec le scrum anti-globuline dans des conditions standard. Zusammenfassung Anhand der bekannten Beeinflussung der Reaktion eines Anti-D-Antikurpers mit Erythrozyten durch das pH, die Temperatur, die Antigen- und Salzkonzentration wurden die optimalen Bedingungen fur den Nachweis anderer Antikurper mittels des Antiglobulintestes ermittelt. Durch Herabsetzung der Salzkonzentration der Aufschwemmungsflussigkeit gelang es die Geschwindigkeit der Antikurperbindung an die Erythrozyten derart zu beschleunigen, das die Inkubationszeit von 1 Stunde auf 5 Minuten herabgesetzt werden konnte. Bei Innehaltung dieser Bedingungen gelang es im Antiglobulintest den Titer der Antikurper gegen folgende Antigene zu erhuhen: A, c, D, e, E, M, K, Lea, Fya, Jka. Mittels 131I-markierten Anti-D-Antikurpern gelang es zu zeigen, das unter Standardbedingungen 500 Antikurpermolekule pro Erythrozyt eben ausreichen, um im Antiglobulintest eine positive Agglutination auszulusen.