The Detection and Quantification of Rh(D) Antigen Sites on Human Leukocytes

Radiolabeled eluates of human anti-D were used to measure the capacityof leukocytes to bind the D antibody in cell suspensions prepared from 16normal and 3 leukemic bloods from Rh(D) donors. The contamination of theleukocyte suspensions by D positive red cells was measured and the contribution of D antigen sites by these cells was estimated. After correction wasmade for the D antibody bound by the contaminant red cells, no specific binding of D antibody by Rh(D) leukocytes could be detected. Three pairs ofRh(D) and rh(d) leukocyte dilution curves of I¹³¹-labeled anti-D uptakewere compared with the uptake by D positive and D negative red cell dilutions. No significant differences among the D negative erythrocyte, the rh(d)leukocyte and the Rh(D) leukocyte curves were obtained. The results werecollated with previous serologic evidence concerning the presence of ABOand Rh antigens on human leukocytes.

Submitted on April 16, 1963 Accepted on June 7, 1963     

Quantitative Serologic and Isotopic Studies on the Rho Variant--Du

1. Type D^u cells take up less than 10 per cent of the I-131 anti-D boundto D positive cells, so that the quantity of cell bound I-131 did not differ significantly from that bound nonspecifically to D negative cells.

2. Papain modification of D^u cells results in an almost threefold increasein the uptake of I-131 anti-D, whereas, D negative cells show no consistentincrease after such treatment. Enzyme modification, therefore, serves to clearlydelineate the two cell types.

3. The range of uptake of I-131 anti-D to enzyme treated D^u cells is fromapproximately 7 to 17 per cent of that by R₁R₂ cells. Most of these results wereobtained from studies on Mendelian D^u types. Studies on several bloods frommembers of a family having Rh genotypes typical of the "gene interaction" D^uconfiguration (C in transposition to D) revealed no significant reduction inI-131 anti-D uptake when compared with D positive cells.

4. Decreased antibody binding to D^u cells was confirmed by the serologicmethods used. Absorption of an anti-D serum and antiglobulin inhibitionwith sensitized D^u cells indicated that the quantity of D antigen was lessthan that observed with the isotopic technic. Elution studies did not show asmarked a reduction of D antigen on the D^u cell as the other serologic technicsindicated. Possible reasons for these differences have been discussed.

5. Although, from both the serologic and isotopic studies, the data indicate that the type D^u red cell is quantitatively deficient in D antigen, theyare inadequate to rule out other explanations for their reduced D antigenactivity. Irrespective of the mechanism(s) responsible, the effective bindingof anti-D by D^u cells is reduced several fold; this would appear to be theprimary cause of the weak second stage of red cell antigen-antibody reactionwhich characterizes type D^u blood.

Submitted on July 28, 1964 Accepted on October 8, 1964     

In Vivo Localization of Heterologous Anti-Erythrocyte Antibodies in the Rat Bone Marrow and Their Effect on the Proliferative Capacity of Immature Erythroid Cells

The in vivo localization of heterologous anti-erythrocyte antibodies in therat bone marrow was determined by the I¹³¹-labeled antibody technic. I¹³¹-labeled anti-erythrocyte antibodies localized specifically in the bone marrowindicating the presence of localizing antibody. Both the localizing antibodyand the incomplete antibody were thermostable, whereas hemolysins andhemagglutinins were thermolabile. Following an intravenous injection of antierythrocyte antibodies in rats, hemolysins and hemagglutinins were clearedrapidly from the plasma. The incomplete antibodies became attached to circulating red cells within 6 hours and red cell sensitization persisted for 1 week.The localizing antibody localized in the bone marrow within 30 minutes, leaving no activity in plasma.

The anti-erythrocyte antibodies markedly reduced the uptake of tritiatedthymidine by erythroblasts in vitro, demonstrating their inhibitory effect onthe proliferative capacity of erythroblasts.

Submitted on February 3, 1964 Accepted on May 12, 1964     

HTLV-1 screening of blood donations: We are systematically missing opportunities

RH diversity among patients and donors contributes to Rh immunization despite serologic Rh-matched red cell transfusions. Anti-D can occur in D+ patients with RHD variants that encode partial D antigens. Anti-D has also been reported in patients with conventional RHD transfused primarily with units from Black donors who frequently have variant RHD. We report 48 anti-D in 690 D+ transfused individuals with sickle cell disease, categorized here as expressing conventional D, partial D or D antigen encoded by RHD*DAU0. Anti-D formed in a greater proportion of individuals with partial D, occurred after fewer D+ unit exposures, and remained detectable for longer than for those in the other categories. Among all anti-D, 13 had clinical or laboratory evidence of poor transfused red cell survival. Most individuals with anti-D were chronically transfused, including 32 with conventional RHD who required an average of 62 D− units/year following anti-D. Our findings suggest that patients with partial D may benefit from prophylactic D− or RH genotype-matched transfusions to prevent anti-D. Future studies should investigate whether RH genotype-matched transfusions can improve use of valuable donations from Black donors, reduce D immunization and minimize transfusion of D− units to D+ individuals with conventional RHD or DAU0 alleles.     

Hematologic and Biochemical Indices in the East African Baboon

Hematologic values for peripheral blood and marrow, serum proteins, B₁₂and folic acid levels are given for recently caught and captive baboons (P.anubis and P.sinocephalus) in Nairobi.

The hemaglobin levels were higher in captive than in wild animals. Therewas no change in the serum proteins unless the animals were on a riboflavindeficient diet.

Marrow activity as estimated by counting red cell precursors as a percentageof the total nucleated red cell population agreed with that of red cell uptakeof Fe⁵⁹ ferric chloride.

Hemosiderin was present in the marrow in all the animals.

Protein turnover studies using I¹³¹-labeled albumin with ion exchange resinsindicated that the protein catabolic rate and turnover was high. This may havebeen due to the selective destruction of the human albumin in the baboon.

Submitted on December 21, 1964 Accepted on February 1, 1965     

Weak D Antigen Testing Using the Groupamatic System

Reliable detection of the weak D (Du) antigen has been a problem for the Groupamatic typing equipment. Using several anti-D sera of known concentration and Du red cells, dose-response curves produced by the Groupamatic revealed variation in the ability to detect the Du antigen by commercial anti-D reagents. Variation in the reactivity of the Du antigen among Du-positive donors was noted. Studies were conducted to determine optimal conditions for the Groupamatic to detect the weakest Du cells. When these conditions were applied to the retesting, 28 cell samples previously found falsely negative by a number of blood centers were accurately detected.     

Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fcγ receptors

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti-D and IgG2 anti-A were isolated by affinity purification from an unusual anti-D serum (DEL) and anti-A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti-D in in vitro functional assays of the interaction between IgG-coated red cells (EA-IgG) and cells bearing IgG Fc receptors (FcγR). Dimeric IgG2 anti-D bound efficiently to cell lines transfected with FcγRIIa-H131, an allotypic form of FcγRIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, -D-phenotype red cells coated with IgG2 anti-D did not form rosettes with these cells, whereas EA-IgG2 anti-A and EA-IgG1 and EA-IgG3 anti-D effectively formed rosettes with these transfectants at the same sensitization level (100 000 molecules IgG/red cell). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, lysis of EA-IgG2 anti-A was mediated via FcγRIIa, whereas lysis of EA-IgG1 and EA-IgG3 anti-D was mediated via FcγRI or FcγRIII; EA-IgG2 anti-D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG–FcγR interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti-D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of FcγRIIa-H131 to the FcγR recognition site on the relatively inflexible IgG2 anti-D, but not to that of IgG1 or IgG3 anti-D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red-cell-bound IgG2 anti-D and IgG2 anti-A with cells bearing Fcγ receptors can occur.     

Comparison of measurement of fat absorption using I131- and C14-labeled fats

Summary 1. The relative validity of C¹⁴-labeled fat and I¹³¹-labeled fat in measuring fat absorption was checked in subjects with Neomycin-induced mal-absorption syndrome.2. From these studies it would appear that C¹⁴ produces a better measurement of fat absorption than I¹³¹.The technical assistance of E. S. Hercker is appreciated.     

Quantitative Hemagglutination Studies in the Rh Blood Group System. II. A Study of the D (Rho) Agglutinogen

The reactivity of Rh positive red cells with saline anti-D sera has beeninvestigated by means of quantitative hemagglutination methods. The inhibitory effect of C on the D antigen has been confirmed and the possibility ofinhibition by this antigen in the cis position suggested. It is also suggested thatthe e antigen has suppressive effect on D. The presence of companion antigensresults in a -D-> R₂ > R_o > R₁ progression of decreasing agglutinability.Within families differences in the agglutination behavior between homozygousand heterozygous D positive cells were found. The heterogeneity of thisantigen was confirmed.

Accepted on November 20, 1960     

Development of Non-Rh Antibodies in Volunteers Stimulated for the Production of Hyperimmune Anti-D

At the North London Blood Transfusion Centre, red cells from accredited Rh-D-positive donors, matched for all antigens capable of inducing clinically significant antibodies, are used to stimulate immune plasma donors to achieve higher anti-D levels. Despite such careful matching, antibody to the relatively non-immunogenic M antigen developed in 3 out of 20 NN donors (15%) stimulated with M-positive cells. In general, good responders to the Rh antigen D are good responders to other red cell antigens; our report exemplifies the importance of using fully matched accredited red cells for immune stimulation and the need to perform thorough antibody screening after each stimulation.     

A summary of atypical antibodies,rare genotypes,and ABO hemolytic disease encountered in a one year survey

1. Among 13,000 selected blood specimens studied in 1954, there were 1882which contained atypical antibodies, mainly anti-D.

2. Of the 177 sera with atypical antibodies other than anti-D, 85 containedanti-c, anti-E or anti-K and 18 sera contained anti-Le^a.

3. There were 52 cases of intragroup hemolytic disease, more than half ofwhich were due to anti-c.

4. Intragroup isoimmunization with production of antibodies other thananti-D was detected in 47 cases as incompatibility in the crossmatching. In 31additional cases failure to detect incompatibility resulted in transfusion reactions.

5. There were 30 examples of very rare Rh genotypes due to the presence ofrare chromosomes dCe (r'), dcE (r'), DCE (R^z), and the rarest of all, dCE (R^y).All told, there were 7 bloods which contained chromosome dCE (r^y) includingone—the first of its kind—which was homozygous.

6. It is recommended that rare genotypes be registered on a world-wide basisunder the auspices of a suitable agency and that these bloods be made availableas test cells or as compatible donors.

7. Sixty-two cases of ABO hemolytic disease are reviewed briefly. In all butfour the mothers were in group O. Of the 55 group O mothers tested, all but 5showed enhancement of the specific antibody in the indirect Coombs reaction.

Submitted on April 9, 1956 Accepted on June 25, 1956     

The Rh chromosome frequencies in England

The results are reported of testing 1073 English bloods with the Rh antibodiesanti-C, anti-C^w, anti-c, anti-D, anti-E and anti-e. The results of another series of927 bloods, already published, are here reproduced. The total of 2000 bloods hasbeen used by Fisher to estimate, by his method of maximum likelihood, the Rhchromosome frequencies in England. The estimates are: CDe 40.75 per cent, cde38.86 per cent, cDE 14.11 per cent, cDe 2.57 percent, C^wDe 1.29 per cent, cdE 1.19per cent, Cde 0.98 per cent, and CDE 0.24 per cent.

A brief account is given of the three pairs of alternative antigens shown byFisher to be the basis of the Rh blood groups. Fisher’s interpretation must now beconsidered as established beyond doubt. A possible genetic basis of these relatedantigens is discussed.

Note: ACKNOWLEDGMENTSWe are deeply indebted to Professor Fisher for many reasons, but we should particularly like toacknowledge his kindness in allowing us to publish the results of his calculations of the chromosomefrequencies.For the antisera used in the investigations we are indebted to the following: Doctors E. F. Aubert,Sheila Callender, D. S. Dick, R. J. Drummond, Mr. I. Dunsford, Doctors A. J. McCall, Brenda Morrison,J. Murray, E. Wordley and R. A. Zeitlin.We also thank Dr. H. F. Brewer of the London Red Cross Blood Donor Service, and Dr. J. F. Loutit ofthe National Transfusion Service, for providing us with large numbers of blood samples.We also wish to acknowledge the assistance of Dr. Marjory N. McFarlane in the earlier part of thisinvestigation.

     

Failure to Demonstrate Dosage of U Antigen

Abstract. Tests in which 11 examples of anti-U were used in titration studies against the red blood cells of 9 obligate Uu heterozygotes, from 4 unrelated families, and random Negro and Caucasian donors (many of whom were of the presumptive UU genotype) have failed to demonstrate any dosage of the U antigen.     

Differential binding of IgG anti-D and IgG autoantibodies to reticulocytes and red blood cells

125I IgG anti-D binding to reticulocytes obtained by density fractionation is reduced relative to that bound to all other red cell (RBC) fractions. Maximum D antigen reactivity occurs following reticulocyte maturation with no detectable change in D reactivity of mature RBC throughout their life span. Reticulocytes have in the range of about 60% of the content of mature RBC. Previously reported increased anti-D agglutinability and binding to old RBC it is not due to an intrinsic increase in D antigen with age, but results from an 'apparent' decrease in anti-D binding to young RBC fractions due to reticulocyte enrichment. IgG RBC autoantibodies obtained by elution from the RBC of eight Coombs-positive blood donors, probably associated with alpha-methyldopa (alpha-MD) administration, showed decreased binding to reticulocytes as determined by 125I protein A (PA). Reticulocytes bound about 70% of the IgG bound to mature RBC, indicating that the membrane antigenic determinant defined by these autoantibodies was incompletely expressed in the reticulocyte. This difference in IgG autoantibody binding between reticulocytes and mature RBC is similar to the decreased D antigen content of reticulocytes and consistent with an autoantibody determinant associated with the Rh complex. Direct testing of density fractionated Coombs-positive RBC in four out of five patients with autoimmune haemolytic anaemia (AIHA) showed reduced quantities of IgG on reticulocytes. The distribution of IgG between reticulocytes and mature RBC may be useful in serologically characterizing patients with AIHA and in identifying subpopulations of patients with this disorder.     

H, A and A1 Reactivity in Two Polynesian Groups

Abstract. Polynesian O bloods from Samoans and Tokelau Islanders have the same mean H reactivity as Caucasian, and their A, bloods the same A₁ content. However, Polynesian A₁ bloods have significantly greater H reactivity than Caucasian, and the Tokelau Tslanders also showed enhanced A and I^T. It is suggested that the Polynesian AHI molecular conformation must differ from the Caucasian pattern, providing an arrangement of antigen sites more favourable to some antigen-antibody reactions.     

Serological Characteristics of Partial D Antigen Category VI in 8 Unrelated Blood Donors

Classification of subjects with a partial D antigen is traditionally performed with immune anti-D sera. The development of monoclonal antibodies enables a fine analysis to be made of the specificity of the epitopes that are present or missing in these cases. A systematic search in a Caucasian donor population of 17,500 revealed 8 unrelated male individuals (frequency 0.05%) with a red cell phenotype characteristic of partial D category VI, but without anti-D in their serum. The relation to the 'classic' partial D category VI was investigated and is discussed, as is the observed serological heterogeneity of the partial D category VI group. Clinical consequences for the prevention of immunization of these subjects are mentioned.     

Evidence for the in Vivo Localization of Heterologous Anti-Leukocyte and Anti-Bone Marrow Antibodies in the Rat Bone Marrow

1. The intravenous injection of I¹³¹-labeled heterologous anti-leukocyte and anti-bone marrow antibodies into rats resulted in a high specific in vivolocalization in the bone marrow, indicating the presence of localizing anti-body.

2. No in vivo localization in lungs was demonstrated, in possible contrastto the earlier concept of pulmonary emboli of clumped leukocytes sensitizedwith antibodies in experimental immunoleukopenia.

3. Heterologous anti-leukocyte and anti-bone marrow antibodies injectedintravenously disappeared from plasma and fixed to peripheral leukocytesand bone marrow cells within 1 hour.

Submitted on June 25, 1962 Accepted on December 24, 1962     

Blood group D antigen content of nucleated red cell precursors.

The D antigen content of nucleated red cell precursors in human bone marrow was estimated using autoradiography and 125I-anti-D. D antigen first appeared in the pronormoblast, and the quantity of antigen progressively increased during red cell maturation. Maximal anti-D binding occurred on mature red blood cells. Pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, and orthochromatic normoblasts, respectively, had approximately 1/4, 1/2, 2/3, and 3/4 the quantity of antigen found on mature red cells. None of the other cell types were found in bone marrow labeled with anti-D.     

Plasma infusions in immunologic deficiency states: metabolic and therapeutic studies

Serial immunologic measurements were used to study the metabolic behavior of the immune globulins (_G-, _M- and _A-globulins) in patients with agammaglobulinemia after plasmapheresis and plasma infusion and in newborninfants after exchange transfusion. These studies were supplemented by metabolic and distribution studies of I¹³¹-labeled _A-globulin (isolated from serumor breast milk) and I¹³¹-labeled _G-globulin in normal and agammaglobulinemic subjects. The therapeutic benefit of periodic plasma infusions in patientswith agammaglobulinemia and the Aldrich syndrome was also assessed.

In the agammaglobulinemic patients, the mean half-lives of _G-, _M- and_A-globulin were 32, 9.6 and 5.9 days, respectively. In the transfused infants,the mean half-lives of _M- and _A-globulins were 7.4 and 4.3 days, respectively.Agreement existed between simultaneously determined immunologic and radioactive survival times, except when I¹³¹-labeled _A-globulin isolated fromserum was used; this preparation had a shorter half-life than the _A-globulinof infused plasma, probably as a result of denaturation during the isolationprocedure. Studies on 2 normal and 3 agammaglobulinemic subjects showedthat 65 to 85 per cent of breast milk I¹³¹-labeled _A-globulin was distributedwithin the tissues. I¹³¹-labeled _A-globulin was not demonstrable in the breastmilk of 2 lactating women or in the saliva of 2 normal subjects. No _A-globulincould be demonstrated in the saliva of an agammaglobulinemic patient afterplasma infusion which raised the serum _A-globulin concentration to 50mg./100 ml.

The use of plasma instead of commercial -globulin for the therapy of immunologic deficiency states has several advantages. Plasma contains all threeimmune globulins, provides greater quantities of _G-globulin than can begiven by intramuscular injections, and is more acceptable to the patient. Because of the risk of serum hepatitis, this mode of therapy in the routine management of agammaglobulinemia is endorsed only if special precautions aretaken. A therapeutic trial of plasma infusions in 2 patients with the Aldrichsyndrome gave promising results.

Submitted on January 7, 1966 Accepted on April 25, 1966     

The Use of Radioactive Antiglobulin for the Detection of Erythrocyte Sensitization

Red cell sensitization by either agglutinating or incomplete antibodies wasdetected with an I¹³¹ labeled rabbit antihuman globulin serum (RAG). Nonspecific absorption of RAG by red cells was reduced to a minimum by theaddition of 6 per cent bovine albumin. The reactions between RAG and thesensitized erythrocytes were typical of antigen-antibody reactions and thesensitivity of the test was found to be greater than the standard Coombs test.Quantitative studies of the degree of erythrocyte sensitization by isoimmuneor autoimmune antibodies were possible with this technic.

Submitted on March 7, 1962 Accepted on April 27, 1962