Quantitation of D Sites on Selected ‘Weak D’ and ‘Partial D’ Red Cells

Measurements have been made of the number of available sites on 10 examples of red cells in which the only abnormality appeared to be a quantitative reduction in the expression of D (weak D cells); these estimates were carried out using three monoclonal anti-D antibodies, Fog-1, Brad-3 and Los-2. The values varied with the monoclonal antibody that was used and fell within the range of 170–1,870 sites/cell. A further 3 examples of weak D cells which had brought about immunisation following transfusion were found to have between 390 and 1,470 sites per red cell. The implications of the D site density on the immunogenicity of weak D cells are discussed. The number of sites on red cells with structurally abnormal D (partial D cells) were also estimated, using the antibody Fog-1. Four of the 5 examples of cells of category IVa (probable phenotype R₀r) were found to have a high expression of D (range 29,300–41,300), but the available D sites of categories D^Va, D^VIa, and D^VII were considerably reduced (<500, <500 and 2,400–7,500 sites/cell, respectively). As a working hypothesis, it is suggested that there are two types of genetic abnormality leading to an abnormal expression of D. First, a defect in genomic DNA leading only to a quantitative reduction in the number of available D sites; this genomic lesion should be termed ‘weak D’. Secondly, genomic defects leading to amino acid sequence abnormalities and structural change in the D polypeptide; these lesions should be collectively known as ‘partial D’.     

Observations of the Number of Available G (rhG,Rh 12) Antigen Sites on Red Cells1

Abstract. The number of available G antigen sites within the rhesus system were estimated using trace-labelled antibody. The results given as available antigen sites_ per cell were as follows: R₁R ₁ : 9,900-12,200; r'r': 8,200-9,700; ryr: 8,600; RNaN: 6,600; r‘^xr: 6,200; RB₁r: 6,200; R_ZR_Z: 5,400; Cdes: 4,800-5,100; R₂R ₂ : 3,600-5,800; R₀R ₀ : 4,500-5,300; R₂r 4,200; r“Gr: 1,700-3,600; RB₂r: 1,400-1,900. The G antigen is detected in optimal amount on C-positive samples. When D is present without C, the number of G sites is apparently dependent on the expression of all the D antigens within the recognized six categories of D antigens.     

Observations of the Number of Available c, D, and E Antigen Sites on Red Cells

Abstract. The number of available antigen sites within the Rh system were estimated using trace-labelled antibodies. The results were as follows, given as the number of sites per red cell: c-antigen: on cc cells, 70,000–85,000: on Cc, 37,000–53,000; D sites on cells of phenotype -D-: 110,000–202,000; E-antigen sites showed considerable heterogeneity depending on phenotype as well as source of anti-E, and estimates varied between 450 and 25,600; e-antigen: on eE cells, 13,400 and 14,500: on ee cells, 18,200 and 24,400.
The average equilibrium constants of the antibodies were: anti-E, 4 × 10⁸ 1/mol; anti-e, 2.5 × 10⁸ 1/mol; anti-c, 3.2 × 10⁷ and 5.6 × 10⁷ 1/mol.     

Demonstration of Seven Epitopes on the Rh Antigen D Using Human Monoclonal Anti-D Antibodies and Red Cells from D Categories

The agglutination patterns have been established for the reaction between 29 monoclonal antibodies with specificity for the Rh antigen D and red cells of D categories IIIa, IIIc, IVa, IVb, Va, Vc, VI and VII, which are known to lack certain epitopes on the D polypeptide. Six different agglutination patterns were recognized and interpreted to indicate the recognition of seven different epitopes. These epitopes are termed epD1 through to epD7. The separate existence of epD6 and epD7 is deduced from previous observations in inhibition studies using purified 125I-labelled antibodies; they cannot yet be distinguished in agglutination tests. The number of epitopes lacking from cells of each category varied between two and five. As all the antibodies agglutinated cells of categories IIIa, IIIc and VII and cells of categories II, IIIb and Vb were not available, it is probable that there are epitopes other than the seven presently recognized. Eighteen out of the 29 antibodies which were examined recognized epitopes epD6/7 and it is suggested either that antibodies recognizing these epitopes predominate in polyclonal anti-D sera, or that the lymphocytes producing these antibodies are preferentially selected during establishment of cell lines.     

Variant of Group B Lacking the B Antigen on the Red Cells

Abstract. A group B variant, lacking B antigen on the red cells, but secreting small amounts of B substance in the saliva is described. Family investigation showed both parents and three sibs to be normal B. Among six children no normal or abnormal B occurred. It is discussed whether the B variant is due to mutation of the B gene or to the effect of a recessive suppressor gene.     

Observations on the Number of Available C Antigen Sites on Red Cells

The number of available C (rh', Rh 2) antigen sites within the Rh system was estimated using trace-labelled antibody. The results, as available sites per red cell, were as follows: CDe/CDe: 45,700-56,400; Cde/Cde: 42,200; CDe/cDE: 25,500-39,700; CwDe/cde: 21,500-40, 000; Cde/cde: 31,100; CDE/cDE: 8,500-9,800; Cde/cde: 7,200. The average equilibrium constant of the anti-C was 0.5 X 10(7) M-1.     

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     

Testing Efficacy of Anti-D Sera by a Panel of Donor Red Cells with Weak Reacting D Antigen and with Partial D Antigens

In 1987 the definition of an Rh-negative donor in the Netherlands was changed from C-, E- as well as Du-negative to D-negative only. The use of 2 different strong anti-D sera without an antiglobulin phase (Du test) was considered sufficient to reveal the clinically important D antigen. In applying this policy, we identified 32 donors in 13,500 consecutive blood donations whose indirect antiglobulin test (IAT) (Du)-positive red cells gave negative reactions with at least 2 out of 11 anti-D sera and whose cells might therefore be typed as Rh(-D-)-negative in routine investigations. IgG anti-D used by a one-stage bromelain technique and anti-D with modified IgG appeared to be relatively insensitive in detecting Du in this study. Polyclonal anti-D in an enhancement medium and monoclonal anti-D scored better, although differences exist between the products of various manufacturers. It is suggested that if IAT (Du) testing is omitted, only anti-D sera with a high index of detectability of cells expressing weak D antigens should be accepted.     

Observations on Subdivisions of the Rh Antigen D

The interactions between cells and sera of certain people thought to have D in their red cells and anti-D in their serum are recorded. The cells fall into six categories: in one (Rh^{a c d} in the classification of Wiener and Unger ) the abnormality seems to be attributable to the antigen G rather than to D.
Two further examples are in a different class for their « anti-D >> can be absorbed by dd cells.

Résumé

Les auteurs rapportent sur les interactions entre cellules et sérum observées chez certaines personnes qui possèdent à première we l'anti-D dam leur sérum et du D sur leurs érythrocytes. Ces érythrocytes peuvent faire partie de six catégories: dans l'une d'entre elles (Rh^{a c d}, dans la classification de Wiener et Unger ) l'anomalie sernble être d'avantage attribuable à l'antigène G qu'à l'antigène D. Deux autres exemples se trouvent dans une autre catégorie parce que leur « anti-D >> est absorbable par des érythrocytes dd.

Zusammenfassung

Bei Individuen, bei denen angeblich D-Antigen in den Erythrozyten und Anti-D-Antikörper im Serum festgestellt worden waren, werden die gegenseitigen Beziehungen zwischen den Zellen und den Serumantikörpern untersucht. Die Zellen lassen sich in 6 Gruppen unterteilen: bei einer Gruppe (Rh^{a c d} in der Klassifikation von Wiener und Unger ) betrifft die Ariomalie eher das Antigen G als D. Zwei weitere Beispiele gehören in eine weitere Gruppe, da deren « Anti-D >> durch dd-Zellen absorbiert werden konnte.     

Direct Agglutination of Weak D Red Cells by Tetramolecular Complexes Containing Monoclonal IgG Anti-D

The capacity of blood group antibodies to agglutinate red cells suspended in saline is largely dependent on the antibody isotype. The immunological cross-linking of IgG antibodies has previously been described as a means to increase the reactivity of IgG in many situations. We have prepared anti-D-containing complexes by blending a human IgG anti-D monoclonal antibody (mAb) and a murine anti-human IgG mAb. In standard red cell serology assays, the anti-D complexes exhibited a very high avidity and could agglutinate weak D-positive red cells in direct saline testing. These results indicate that potent saline hemagglutinating reagents of RhD and eventually of other blood group specificities can be prepared from IgG mAbs.     

Phagocytosis by Human Monocytes of Red Cells Coated with Rh Antibodies

Les monocytes humains démontrent la propriété de digérer les érythrocytes revêtus d'anticorps anti-Rh (anti-D, anti-C, anti-c ou anti-E) lorsque les érythrocytes et les leucocytes sont colligés par centrifugation.
On n'a pas observé de chémotaxisme, et, la phagocytose par les monocytes est indépendante de facteurs sériques autres que l'anticorps. L'érythrophagocytose (par d'autres types de cellules blanches) ne se produit pas dans ces conditions.

Zusammenfassung

Menschliche Monozyten vermochten Erythrozyten, die mit Rh-Antikörper (Anti-D, Anti-C, Anti-c, Anti-E) beladen worden waren, in sich aufzunehmen, sofern die roten und weißen Zellen durch Zentrifugation aneinandergelagert worden waren.
Chemotaxis wurde nicht beobachtet. Die Phagozytose durch die Monozyten erwies sich unabhängig, von Serumfaktoren abgesehen, von den Antikörpern. Ery-throphagozytose durch andere Typen weißer Zellen wurde bei diesen Versuchs-anordnungen nicht beobachtet.     

Flow Cytometry: A Tool in Immunohematology for D+w(Du) Antigen Evaluation?

We performed a flow-cytometric analysis of the expression of D antigen in D+w samples (previously termed D^u). We also analysed a series of D-positive and D-negative (cde phenotype) samples to obtain positive and negative controls, respectively. The evaluation was carried out by immunofluorescence and the intensity of positivity was expressed as mean channel value (MCV) of fluorescence. Results demonstrated that D+w samples have lower expression (less than 1 log) than D-positive cases (p>0.001, Student t test), while cde samples show the same MCVs as negative controls. Moreover, it was also possible to set a grading of D antigen expression and to analyze cases difficult to assess by agglutination only.     

Rh(D) Alloimmunization in the Absence of Exposure to Rh(D) Antigen

An anti-D was detected in the serum of an Rh(D)-negative primigravida. There is no history of previous pregnancies or blood transfusions. The husband and infant are both Rh(D)-negative.     

Demonstration by Flow Cytometry of the Numbers of Residual White Blood Cells and Platelets in Filtered Red Blood Cell Concentrates and Plasma Preparations

Background and objectives: New-generation polyester filters provide significant depletion of white blood cells (WBC) and platelets (PLT) in filtered red blood cell concentrates (FRCC) and in filtered plasma preparations (FP). The aim of this study was to elaborate a sensitive flow cytometric method for monitoring residual WBC and PLT in FRCC and FP. Materials and methods: We determined the number of WBC in 500 μl FRCC of FP using 50 μl of a combination of monoclonal antibodies (MAB) against CD45 (FITC labeled) and CD19 (PE labeled). After lysis of red blood cells, we mixed a specific number of reference beads with the remaining WBC. The number of residual WBC related to the acquisition volume was defined by the acquired reference beads. Using this method, the detection limit (DL) was 3 WBC/μl. Alternative methods used MAB against CD45 (FITC and PerCP labeled) and CD14 (PE labeled) or lymphocyte subsets such as CD3 (FITC labeled) and CD19, CD4, CD8, CD16 and CD56 (PE labeled) in combination with CD45 (PerCP labeled). The DL values were 10 WBC/μl for the CD45/CD14 staining and 0.1 WBC/μl for the determination of both CD3+ and CD19+ lymphocytes. For residual PLT in FRCC or FP, we used an FITC-conjugated MAB against CD41, with reference beads to determine the acquisition volume. PLT were demonstrated in a green-fluorescence (FL1) single histogram after gating in the forward light scatter × 90° light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/μl. Results: Filtration with Pall WBF-1 filters reduces WBC by 4 log and PLT by 3–4 log, resulting in cell counts which are below the critical limit for causing adverse transfusion reactions. Conclusions: Flow cytometry techniques provide a reproducible and objective tool for counting residual WBC and PLT in blood preparations compared with the Nageotte hemocytometer. Absolute numbers of leukocyte and lymphocyte subpopulations are obtainable.     

The Rh Antigen D: Partial D Antigens and Associated Low Incidence Antigens

The expression of the Rh antigen D varies quantitatively and qualitatively (partial D); published information and 15 years' work studying D variants are discussed in this review. D epitopes correspond to the reaction patterns of monoclonal anti-D with partial D antigens. Partial D antigens can be reported in terms of their D epitopes but the epitope profile of cells with a quantitative variant of D (weak D) is difficult to determine reliably by haemagglutination tests. Nine partial D antigens, categories II-VII, DFR and two not previously reported, are identified by their epitope profiles and by association with low incidence antigens. Monoclonal anti-D recognize 16 D epitopes and more epitopes are anticipated. The specificities of polyclonal anti-D made by people with partial D antigens are considered in terms of possible D epitope specificities: recognized epitope specificities, or combination thereof, were not able to account for all observed reaction patterns of anti-D made by immunized individuals with partial D pheno-types. An attempt is made to understand partial D antigens and their associated low incidence antigens in terms of the molecular genetic information available.