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Coupling of an inert polymer to the surface of red cells was examined as a potential means of covering blood group antigens and producing cells that could serve as universal donor cells for transfusion. Effective blockade of red cell antigens was achieved with N-hydroxysuccinimide-activated esters of polyethylene glycol. It was possible to block all antigens tested, but lower concentrations of reactants were required to block peptide-defined antigens than carbohydrate-defined antigens. Red cells remained intact after modification but were significantly damaged. Our results demonstrate the feasibility of antigenic blockade of red cells, but there is a need to reduce damage during coupling reactions to produce viable red cells.

Background: The terminal Gal alpha1,3Galactose (alphaGal) determinant is present on all porcine glycoproteins and glycolipids, but is not expressed by human cells. Consequently human sera contain anti-alphaGal natural antibodies. The human blood group B antigen [Gal alpha1,3(Fuc1,2)Galactose] is differentiated from the alphaGal epitope by the presence of a fucosyl group., Methods: To determine whether the expression of the B antigen has any effect on the level of alphaGal-reactive natural antibodies, equal numbers (n=12) of A, B, AB, and O serum samples were evaluated by ELISA and flow cytometry., Results: A significant reduction in IgG alphaGal reactivity was observed with serum samples from B antigen-expressing donors (B, AB) relative to non-B antigen-expressing donors (A, O)., Conclusions: These results are consistent with the possibility that anti-alphaGal antibodies in non-B antigen-expressing individuals include a subset that is reactive with the structurally related B antigen and that this subset is absent in B and AB individuals.

A highly human leukocyte antigen-sensitized heart transplant candidate underwent immunomodulation with intravenous gamma globulin and cyclophosphamide. His panel reactive antibody screen fell from 64% to 14%. He underwent successful orthotopic heart transplantation with a histoincompatible, T-cell cross-match-negative heart without the development of hyperacute rejection. The combination of intravenous gamma globulin and cyclophosphamide may be a simple and effective means to overcome the human leukocyte antigen-barrier in sensitized heart transplant candidates.

Prostate-specific antigen (PSA) is a kallikrein-like serine protease mainly expressed in the human prostate. It is responsible for the proteolysis of the gel-forming proteins in human semen. Two major extracellular protease inhibitors, alpha-1-antichymotrypsin (ACT) and alpha-2-macroglobulin (AMG) may inactivate PSA escaping from the prostate. The predominant immunodetected form of PSA in serum is complexed to ACT but PSA exists also in a free non-complexed form despite the large excess of inhibitors. The concentrations of PSA in serum are normally less than 4 micrograms/l. but elevated concentrations are found in a majority of patients with prostate cancer (CAP) and the analysis of PSA in serum has become invaluable in the detection and monitoring of patients with CAP. However, it is not an ideal tumor marker in the sense that there are CAP patients with normal PSA concentrations in serum and patients with benign hyperplasia of the prostate (BPH) with elevated PSA concentrations. Analysis of the various PSA forms in serum attracts much interest as there is a higher proportion of PSA in complex with ACT in patients with CAP than in those with BPH. Optimal combinations of monoclonal antibodies have been used to design sensitive noncross-reacting immunoassays for the detection of free PSA, PSA-ACT complexes and the detection of both free PSA and PSA complexes in an equimolar fashion (i.e. total PSA). Several studies have demonstrated that the analysis of the proportions of the free-to-total PSA in serum may increase the diagnostic specificity by 15-20% without significant loss in the sensitivity for detection of CAP.

Background and Objectives: Human monoclonal antibodies have been produced against various Rh antigens. We report here the production of another one against Rh C and the first such antibody against Rh CW., Materials and Methods: Two heterohybridomas secreting IgG human monoclonal antibodies against Rh CW and Rh C (MS-353 and MS-242 respectively) were produced from alloimmunized donors. Their specificity was confirmed by serological testing., Results: MS-353 reacted with all CW-positive phenotypes but not with any CW-negative phenotypes, whereas MS-242 reacted with all CW-positive and C-positive phenotypes. Using 125I-labelled antibodies, the average number of CW sites/red cell was 32,000 (R1WR1W, 15,200 (R1WR1), 19,800 (R1WR2), 15,300 (R1Wr), 26,200 (r'Wr), and the average number of C sites per red cell on equivalent CW/C-positive phenotypes was similar: 22,400 (R1R1), 21,800 (R1WR1), 12,300 (R1R2), 11,700 (R1WR2), 13,400 (R1r), 15,100 (R1Wr), 21,100 (r'r), 18,700 (r'Wr). MS-353 and MS-242 were mutually inhibitory. Both antibodies specifically immunoprecipitated a 33- to 34-kD polypeptide from 125I-surface-labelled cells., Conclusion: MS-353 is suitable for the serological detection of CW-positive cells, and the immunochemical data are entirely consistent with the report that the CW antigen is associated with a point mutation in the RHCE gene [Blood, 1995;86:1196-1201].

The molecular basis of different Kell blood group phenotypes is reviewed. Sequence analysis of the Kell gene (KEL) established that single base substitutions, resulting in amino acid changes, are responsible for the different phenotypes. Most of the amino acid substitutions, with the exception of the one responsible for expression of KEL6 (Jsa), occur at the amino-terminal half of the protein, a domain that has least amino acid homology with a family of zinc endopeptidases, which include neutral endopeptidase 24.11 and endothelin-converting enzyme-1. Some of the genes were expressed in transfected cells and typed with alloantibodies to confirm that the identified mutations are responsible for antigen expression. Clinical applications of Kell blood group genotyping which include prenatal diagnosis to monitor hemolytic disease of the newborn are discussed.

Three years ago, a 71-year-old female was admitted because of general lymphadenopathy. She had been on medication for hypothroidism, but had no history of pregnancy or blood transfusion. T-cell lymphoma was found. Since then, she has been treated with intermittent chemotherapy. The patient's LWa antigen was depressed during two relapses of the lymphoma and anti-LWa was detected simultaneously on both occasions. When she entered remission after therapy, her LW phenotype converted from LW(a-) to LW(a+), and this was accompanied by the disappearance of anti-LWa. This suggests that LW(a-) was a transient phenotype. In the first relapse, a total of 8 units of D- LW(a+) packed red cells and 20 units of platelets were transfused during 1 month without any haemolytic reaction. In these relapses, nonhaemolytic anaemia, hyper-gamma-globulinaemia and remarkable bone-marrow infiltration with lymphoplasma cells were recognized. She also possessed antithyroglobulin and antimicrosomal antibodies; however, these autoantibodies did not change with the relapses. These results suggests that the recurrent depressions of LWa may be closely related to the underlying disease.

The methods available for separating mixed populations of red blood cells (RBCs) are not completely satisfactory. The purpose of this study was to evaluate the utility of microbead columns for the separation of mixtures of RBCs of different blood groups. Suspensions of RBCs positive for nine different blood group antigens were mixed with RBCs lacking the antigen so that 1%, 5%, 10%, 15%, or 25% at the antigen-positive RBCs were represented. After agglutination by the appropriate antiserum, the antigen-positive RBCs were separated from the antigen-negative RBCs by using microbead columns. The average recovery of the antigen-negative RBCs in the effluent of the columns of the 135 mixtures of RBCs tested was 83% +/- 5% (mean +/- SD). The absence of contaminating antigen-positive RBCs was established serologically and by flow cytometry. The procedure was effective in removing as little as 1% of antigen-positive cells from a mixture. Microbead columns offer a simple and efficient method for separating mixtures of RBCs for biochemical, clinical, and serologic studies.

Objective: To determine the prevalence of T-cryptantigen activation (TCA) and its predictive value for severity of necrotising enterocolitis (NEC) in babies., Study Design: Prospective descriptive., Study Population: Thirty-four babies with NEC were prospectively screened for TCA at Tygerberg Hospital over a 6-month period. TCA screening was done by testing for red blood cell agglutination by the common peanut lectin, Arachis hypogea. Once TCA was confirmed, only washed red cells were administered to the babies and plasma-containing blood products were avoided. NEC was divided into suspected NEC (stage 1), classic NEC (stage 2) and fulminant NEC (stage 3)., Main Outcome Measures: Prevalence of TCA in babies with various stages of NEC; the association between TCA and bowel necrosis, need for surgery and mortality., Results: TCA was positive in 8 (24%) of the babies in this study. Six babies (18%) had stage 1 NEC, 10 (29%) had stage 2 NEC and 18 (53%) had fulminating or stage 3 NEC. All 18 babies with stage 3 NEC required surgery and TCA was present in 8 (47%) of them. Twelve babies (35%) died, 3 with TCA and 9 with no TCA. Babies with TCA had portal venous gas on abdominal radiographs (P = 0.037) and stage 3 NEC (P = 0.003) more often than babies with no TCA., Conclusions: A strong association was noted between TCA and the fulminant form of NEC with bowel necrosis. TCA is a baby with NEC should alert the surgeon to the possibility of severe disease and the need to avoid plasma-containing blood products. Blood banks are urged to introduce routine screening for TCA in all babies with NEC.

Blood group ABH antigens are associated with platelets as intrinsic determinants and extrinsically adsorbed antigens, and exist both on glycosphingolipids and on glycoproteins (GPs). We now provide direct evidence that the blood group ABH antigens are prominently associated with platelet GPIb and GPIIb. By immunoprecipitation, a murine monoclonal anti-A antibody precipitated surface-biotin-labelled blood group A1 platelet membrane proteins with electrophoretic characteristics identical to those of GPIb/IX and GPIIb/IIIa. By immunoblotting of SDS-PAGE separated blood group A1 platelet proteins the monoclonal anti-A antibody bound to proteins with electrophoretic characteristics identical to those of GPIb and GPIIb. When immunoaffinity purified GPIb/IX and GPIIb/IIIa, derived from blood group O, A1 and A2 platelets, were employed for immunoblotting, GPIb and GPIIb only from A1 platelets bound the monoclonal anti-A antibody. By ELISA, wherein monoclonal antibodies specific for GPIb (APl) and the GPIIb/IIIa complex (AP2) were used to capture and hold antigens from platelet lysate, human anti-A antibodies reacted with these proteins derived from blood group A1 platelets; proteins from blood group A2, O and B platelets showed no reactivity. These results indicate that blood group A antigen is associated with GPIb and GPIIb derived from blood group A1 but not A2 platelets.

It has been reported that the response to alloantigens presented by the direct and indirect pathway may be of differential relevance after human kidney transplantation. Accordingly, we monitored these routes in peripheral blood mononuclear cells (PBMC) of heart transplant patients from before transplantation and up to 2 years thereafter in an attempt to find a correlation with the clinical status of the patients. Both before and after transplantation, comparable proportions of PBMC samples reacted in mixed lymphocyte culture to nondepleted donor spleen cells (direct route), but never to donor cells depleted for antigen-presenting cells (indirect route). In contrast, the latter route could easily be activated by a nominal antigen and persisted after transplantation, although the proportion of PBMC samples responding was significantly suppressed, irrespective of the occurrence of rejection. Consequently, complete removal of antigen-presenting cells from the stimulator population in a mixed lymphocyte culture with PBMC as responder is not a suitable tool for measuring indirect presentation of alloantigens, and therefore not relevant for monitoring the immunological status of heart transplant recipients.

The association of Na+/H+ antiport with 883 alloantigen was studied in patients with rheumatic disease. Simultaneous measurements were made of red cell Na+/H+ exchange and 883 alloantigen in microlymphocytotoxic test with D8/17 monoclonal antibodies in 20 patients with acute rheumatic fever, 20 patients with inactive rheumatic heart disease, 20 patients with atherosclerotic heart disease (stable anginal syndrome), and 20 healthy subjects. The number of 883(+) B cells and the Na+/H+ antiport activity were increased in rheumatic fever compared to healthy controls: 24.8 +/- 0.4 vs. 11.1 +/- 1.0% cells, 431 +/- 43 vs 121 +/- 12 micromol H+/l cells in min, respectively; p < 0.001; and in patients with rheumatic heart disease compared to patients with atherosclerotic heart disease; 25.7 +/- 1.8 vs. 9.8 +/- 1.1% cells, 482 +/- 73 vs. 124 +/- 12 micromol H+/l cells in min, respectively; p < 0.001.

Although their relative importance and interaction are unclear, donor antigen(Ag)*-specific hyporeactivity and allogeneic microchimerism have been associated with improved long-term graft outcome and a lower incidence of chronic rejection in solid organ transplant recipients. We have postulated that a critical level of donor antigen, for a critical time period, is necessary to develop and maintain donor antigen-specific hyporeactivity; both the level and the time may differ by organ transplanted. In our current study, we tested donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in liver and kidney recipients and compared these values with our previous findings in lung recipients. We tested 25 liver recipients at 12 to 29 months posttransplant: 10 (40%) had developed donor antigen-specific hyporeactivity; 5 (20%), peripheral blood allogeneic microchimerism. For all but 1 of the chimeric and hyporeactive recipients, the level of donor cells was very low (< 1:20,000). Five hyporeactive recipients and all 15 donor antigen-responsive recipients did not have detectable levels of peripheral blood microchimerism. No chronic rejection has developed in any of these recipients to date--however, a lower incidence of acute rejection was observed for those recipients with donor antigen-specific hyporeactivity (30% versus 60% without) or with peripheral blood allogeneic microchimerism (20% versus 55% without) (P = ns). These results differ from our previous findings in 19 lung recipients: at 12 to 18 months posttransplant, 35% of them had developed donor antigen-specific hyporeactivity; 47%, peripheral blood allogeneic microchimerism. All donor antigen-specific hyporeactivity recipients as well as some donor antigen-responsive recipients had peripheral blood allogeneic microchimerism. We expanded our current study to include 26 recipients and a quantitative estimate of the level of allogeneic microchimerism. We observed that the hyporesponsive recipients tended to have higher levels of donor cells in their peripheral blood (> 1:6,000) than did the responsive recipients. We previously reported that 22% of kidney recipients had developed donor antigen-specific hyporeactivity at 12 to 18 months posttransplant. In our current study of 33 kidney recipients, we observed peripheral blood allogeneic microchimerism in 7 (21%) at 12 to 18 months posttransplant. The level of donor cells was very low (approximately 1:75,000), with no correlation between donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism at the time point tested. These studies emphasize the organ-specific nature of the development of donor antigen-specific hyporeactivity and the persistence of peripheral blood allogeneic microchimerism. Donor antigen-specific hyporeactivity correlates with very low levels of donor cells in liver recipients, while a higher critical level of donor cells is important in lung recipients. Additional sequential early posttransplant studies are necessary to further define the possible interrelationship between donor antigen and the development and maintenance of donor antigen-specific hyporeactivity.

An anti-Thomsen-Friedenreich (T) antibody-based enzyme-linked immunosorbent assay (ELISA) with high efficacy in human breast carcinoma detection is described. Immunoreactive T epitopes occur in approximately 90% of all carcinomata; all humans have anti-T antibodies, naturally occurring anti-carcinoma antibodies, induced by their own intestinal flora. Carcinoma patients, but not control subjects, show alterations of serum anti-T hemagglutinin levels. Human anti-T antibodies are predominantly IgM. In the protocol presented here, anti-T IgM antibodies are quantitated by ELISA using Immulon 2 wells coated with human blood group O erythrocyte-derived T antigen as solid phase; in addition, total IgM in each serum is quantitated by ELISA in parallel with the anti-T IgM. Inter-assay coefficient of variation was 2% for both ELISAs. Although anti-T IgM values alone distinguish between carcinoma patients and control subjects, use of the quotient, QMe, which also considers total IgM, increases this distinction. For a given serum, QMe was obtained by the formula: QMe = (100 x (anti-T IgM)2/total IgM). Sera of 242 subjects, 117 breast carcinoma patients, 36 benign breast disease patients and 89 healthy persons were analyzed. QMe identified 88% of the breast carcinoma patients: it all six (100%) in situ, 11/13 (85%) Stage I, 48/58 (83%) Stage II and III and 38/40 (95%) Stage IV patients. Sera from 83% of the 36 benign breast disease patients were negative, i.e. within normal range; five of the six positive sera originated from patients with increased long-term risk of breast carcinoma, while sera from 11 other patients with increased carcinoma risk were negative. Overall, 90% of the 125 non-carcinoma control subjects were negative by both anti-T IgM and QMe. In preliminary studies, the ELISA protocol detected 11/14 (79%) patients with carcinomata other than those of the breast. The identification of all six in situ breast carcinoma patients by QMe points to its usefulness in carcinoma detection, especially early.

Background: Although hemagglutination techniques have proved worthwhile since many years in immunohematology, they also have several disadvantages. They are manual and subjective visual methods, which make it difficult to quantitate red cell antibodies or surface antigens. Flow cytometric analysis overcomes these limitations because of its ability to analyze individual populations of cells by sensitive, reproducible, and objective methods., Materials and Methods: Washed red cells from regular blood donors and patients were analyzed natively and after treatment with enzymes (sialidase, protease) or pneumococcal polysaccharides, using monoclonal Rh antibodies, human 7s-immunoglobulin, and FITC-labeled anti-human IgG or FITC anti-T lectin. The fluorescence intensity of single red cells was determined in the Ortho Cytoron Absolute flow cytometer., Results: We determined the optimal test conditions and normal values by investigation of 50 blood donors. The fluorescence intensity of untreated red cells proved to be constant and therefore was used to adjust the instrument. Furthermore, the data of experimental adsorptions of red cells with pneumococcal antigens, sialidase (Vibrio cholerae) and protease (papain) as well as data from patients suffering from chronic HBV infection and autoimmune hemolytic anemia and acute pancreatitis are presented. A special software program was developed for statistical analysis and graphical presentation of the raw data. The computer program permits to analyze results from different experiments or from different dates and depicts them comparatively in overlay histograms, which may be useful in a serial study of changes of the antibody concentration or antigen expression., Conclusion: The flow cytometric analysis of red cells proves to be a simple, rapid, reproducible, and objective method for antigen and antibody quantitation. Furthermore, this technique may be a useful new tool for the investigation of acute, infection-associated hemolytic anemia.

Transfusion medicine has become a multi-disciplinary field with many recent technical developments and the flow cytometer has had a significant impact in transfusion medicine, especially at the level of platelet immunobiology. Many routine tests in platelet immunology are now performed by flow cytometry laboratories, including assessment of platelet-associated allo- and auto-antibodies and complement components. Platelet analysis by flow cytometry has been applied to detection of platelet antigens, platelet surface-bound proteins, platelet activation, measurement of reticulated platelets, intracellular calcium studies, and the measurement of platelet microparticles in vivo and in vitro. This review will focus on the use of the flow cytometer in these applications in investigations of platelet immunology.

Flow cytometric analysis of blood leukocytes is currently used for both routine clinical measurements as well as for cutting edge research applications. This technology has enabled rapid and accurate determination of leukocyte antigens and quantitative analysis of leukocyte subsets, tests of leukocyte function, determination of the presence of antineutrophil and antilymphocyte antibodies in plasma and on cells, measurement of CD34+ hematpoietic stem cells in peripheral blood and bone marrow samples, measurement of apoptosis, and detection of virus-infected leukocytes. This review will focus on the use of the flow cytometer for investigations of blood leukocytes in transfusion medicine.

A series of methods and assay systems was designed, using both mouse monoclonal antibodies and purified human polyclonal antibodies, by which alterations in the antigenic properties, and potentially therefore in the immunogenic properties, of FVIII concentrates could be identified. Those methods could be applied to the pre-clinical evaluation of FVIII concentrates. It has become evident that very subtle alterations in FVIII can have dramatic effects on its antigenicity and immunogenicity and it is suggested that an analysis of FVIII preparations, using reagents such as mouse monoclonal antibodies, should be carried out as a pre-clinical evaluation. It is also thought that any haemophiliac patient is at risk of developing anti-FVIII antibodies, even though he had been considered as tolerant to the infusion of FVIII. The diversity of FVIII concentrates now available on the market has multiplied the chances of encountering a product that would give an immune response in some patients and this further stresses the need for a careful check of FVIII concentrates.

The various blood group related carbohydrate structures which are in clinical use as circulating tumour makers are reviewed. Their location on carbohydrate chains and their structural characteristics are shown, and their clinical performance in various malignant diseases is reviewed. The available data on their sensitivity, specificity and predictive value are shown; and carcinomas of the pancreas, ventricle, colon-rectum and ovary are identified as diseases in which these markers can be of good benefit for follow-up. Future research should be devoted to studies of the function of these structures, and to studies of their gene-transcription.

Previous studies suggest that serum levels of the mucin-associated sialyl Lewis(a) (Le(a)) antigen (NeuAC alpha 2-3 Gal beta 1-3 [Fuc alpha 1-4]GlcNAc beta 1...) correlate with the pulmonary status of cystic fibrosis (CF) patients who have Le(a) or Lewis(b) blood types and can form the antigen. However, there is little information on serum sialyl Le(a) antigen levels in CF patients or normal children younger than 9 y. We measured serum antigen levels using the MAb 19-9 in normal term neonates, CF infants and young children, and infants and children who had bronchopulmonary dysplasia or asthma. The mean serum sialyl Le(a) antigen level of the CF patients was 46.7 U/mL, significantly above (p < 0.01) the mean levels of the three other groups. The mean serum sialyl Le(a) levels of the three non-CF groups were not significantly different from one another or from published normal values. We conclude that serum sialyl Le(a) antigen levels are elevated early in CF, but are normal in asthma and bronchopulmonary dysplasia patients.

The past year has seen further advances in our understanding of the molecular biology of the most abundant erythrocyte proteins associated with blood group antigens (band 3, the glycophorins, and the Rh antigen-related proteins). There have also been several important developments in the structural and functional identification of some of the less abundant antigens. These developments include the association of the Colton antigens with the erythrocyte water channel, aquaporin; the cloning of the Duffy antigen and its identification as a chemokine receptor; the cloning of the Kx antigen, which is associated with McLeod syndrome; and the cloning of the LW, CD47, and Xga antigens.

K12 is a high-prevalence antigen, with no known antithetical partner, that is associated with the Kell blood group system. We report studies on the 5th propositus (MS) with the K:-12 phenotype and the second case to show that the K12 is inherited. The anti-K12 in his serum did not destroy antigen-positive incompatible red cells transfused on at least 3 occasions over 6 years. Red cell membranes from MS possessed the Kell protein that was indistinguishable from control membranes. K12 antigen was shown by immunoprecipitation to be on a protein with an apparent molecular mass of 93,000 and by monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay to be on the Kell protein in proximity to K/k and Jsa/Jsb antigens. These data remove the K:-12 phenotype from its current Kell-related (or para Kell) status and elevates the K12 antigen to a bona fide member of the Kell blood group system.

Production of murine monoclonal antibodies to the low prevalence MNS antigen Henshaw (He; MNS6) has enabled more detailed study of this antigen. Using these directly hemagglutinating anti-He, red blood cells (RBCs) from 1695 people of African origin were screened in the USA and England. The prevalence of He+ samples among these donors was 2.1%. In Natal, blood samples from 1218 black donors were screened with rabbit anti-He. The prevalence of He+ donors in this population was 7.0%. Immunoblotting confirmed that the He antigen is carried on an erythrocyte membrane component with a molecular mass that is indistinguishable from glycophorin B. Hemagglutination and immunoblotting demonstrated that ten of 56 He+ samples tested more extensively had a reduced expression of the He antigen. The majority of He+ RBCs were S+; those He+ RBC samples that were S-s+ more frequently had a weakened expression of He.

A 47-year-old native American (TOU) was admitted to hospital for hip surgery. His serum agglutinated all red blood cells (RBCs) tested except Ko and DTT-treated RBCs and was weakly reactive with RBCs known to have a weak expression of Kell antigens, namely Kmod, McLeod, Kp(a+b-) (KEL:3,-4) and K:-13 (KEL:-13) phenotypes. RBCs from three siblings, a son and a daughter were incompatible with TOU's antibody. TOU's RBCs had the common Kell phenotype: K-k+Kp(a-b+c-)Ku+Js(a-b+)Ul(a-)K:11,-17K:14,-24K:12,13,18,19, 22,-23(KEL:-1,2,-3,4,5,-6,7,-10,11,12,13,14,-17,18,19,-21,22,-23,-24). Since TOU's RBCs were not agglutinated by an unidentified Kell-related antibody (IAN), tests were performed to show that TOU and IAN were mutually compatible. IAN is a Latino female hospitalised for a hysterectomy. The TOU antigen was shown to be located on the Kell glycoprotein by a monoclonal antibody immobilisation of erythrocyte antigen (MAIEA) assay. The unique pattern of reactivity obtained with TOU and IAN antibodies using this assay indicated the TOU epitope to be in an area remote from other Kell antigens, namely K, k, Kpa, Kpb, Kpc, Ku, Jsa, Jsb, U1a, K11, K12, K13, K14, Wka, K18, K19, K22 and K24 (KEL1, KEL2, KEL3, KEL4, KEL5, KEL6, KEL7, KEL11, KEL12, KEL13, KEL14, KEL17, KEL18, KEL4, KEL5, KEL6, KEL7, KEL11, KEL12, KEL13, KEL14, KEL17, KEL18, KEL19, KEL21, KEL22 and KEL24) but close to the low-incidence antigen K23 (KEL23).(ABSTRACT TRUNCATED AT 250 WORDS)

Ten red cell samples lacking the high incidence Gya antigen were found to have the previously undescribed Do(a-b-) phenotype. Fifteen Hy- red cell samples were Do(a-b+) with weak expression of Dob and 6 Jo(a-) red cell samples were Do(a+) with weak expression of Doa. Five of the 6 Jo(a-) samples had extremely weak expression of Dob. The sixth Jo(a-) was Do(b-). Immune precipitates were prepared from radio-iodinated antigen-positive red cells with anti-Gya, -Hy, -Doa and Dob. Immunoblotting of these immune precipitates with affinity-purified anti-Gya and anti-Dob under non-reducing conditions revealed similar broadly migrating bands of M(r) 48,700-59,750, suggesting that the Doa and Dob antigens are carried on the same glycoprotein as Gya and Hy. The phenotypically associated high incidence Joa antigen has previously been shown to reside on the Gya/Hy glycoprotein. Enzyme-treated and chemically modified red cells tested with anti-Doa, -Dob, -Gya, -Hy and -Joa gave the same pattern of reactivity. We propose that Gya, Hy and Joa become part of the Dombrock blood group system and that, henceforth, the Gya/Hy-active glycoprotein be renamed the Dombrock-active glycoprotein. The Gy(a)-Hy- Jo(a-) phenotype constitutes the 'null' phenotype within the Dombrock system.

Blood group antigens have provided tools for investigation of the red cell surface and been very useful as genetic markers in family, population and forensic studies. Precise definition of phenotypes is very important. Application of MAIEA (monoclonal antibody-specific immobilisation of erythrocyte antigen), a recently reported technique, to identify antigens and to assign red cell antigens to a particular membrane component is described: location of Knops system antigens on CR1 is confirmed and provisional assignment of Cromer system antigens to the different short consensus regions of decay accelerating factor (DAF) is described. Variability of red cell antigen expression is considered. The possibility is discussed that factors other than alterations in Rh genes may be responsible for some Rh variant phenotypes. Some C variants, two of which are associated with low incidence antigens, are described. The relationship of Xga with the quantitative polymorphism of 12E7 antigen is reconsidered in light of some recent immunochemical studies.

After decades of studying the human blood groups by serological and, more recently, biochemical techniques, analysis of blood group genes at the molecular level has confirmed that a variety of different genetical events have given rise to the vast complexity of blood group systems. In order to illustrate this 4 blood group systems have been selected: ABO and H, involving carbohydrate determinants, and MNS and Rh, involving predominantly protein antigens. The molecular basis of the A1, A2, B, and O groups, and of the rare H-deficiency phenotypes will be described. The Sta antigen of the MNS system will be discussed in order to illustrate the variety of different genetic mechanisms that can give rise to a single rare antigen. Finally, recent work on the molecular basis of the polymorphic Rh antigens, D, C, c, E, and e, and on some rare Rh phenotypes, Rhnull, D--, and r's, will be explained briefly in order to emphasize the complexity of blood group genetics.

Investigation of a mild case of hemolytic disease of the newborn has led to recognition of a 'new' low-incidence red cell antigen, WARR (ISBT No. 700.55). Data gleaned from two kindreds, both with Native American heritage, exclude WARR from the MNS, Lutheran, Lewis, Duffy, Kidd, Xg, Chido/Rodgers, Kx and Gerbich blood group systems. Serologic or genetic evidence suggests it is not part of the Kell or Yt systems.

The biochemical relationship between the red cell antigens Xga and the MIC2 gene product, CD99--previously designated the 12E7 antigen--has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xga-immunoprecipitates with anti-Xga has shown that Xga antigen is carried on a broad band of apparent molecular weight (Mr)) 24,500-29,500, which consists of a dark stained component at M(r)24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-1 and NaM123 which also recognise CD99, distinguished two bands of M(r) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(r) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xga protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xga antigen and CD99 are associated in the membrane.

Using segregation and population analysis of serological data prepared in our laboratory, we have tentatively defined five OLA antigenic specificities: CO1.1, CO1.2, CO2.1 and CO2.2, whose genetic control would fit a model of two class I OLA loci (CO1.1 and CO1.2 first locus, CO2.1 and CO2.2 second locus, and CO3.1, that could represent a subtype of CO2.2. Alloantisera were obtained from Spanish Merino sheep by intrafamilial cross-immunization and from serum of primiparous and multiparous ewes. Screening of alloantisera for antilymphocyte reactivity was carried out by a two-step dye exclusion microcytotoxicity test against 216 animals belonging to different local breeds: Spanish Merino, Merino Landschaft, Manchega and Churra. Lymphocyte antigen specificities were characterized by cluster and correlation analysis, as well as family studies. Comparison of CO alloantisera with reference reagents from Scotland (CA reagents) showed an excellent correlation between CO1.1 and CA2, and CO2.1 and CA5. The reagents described can be used in various breeds, a characteristic important for their future applications.

Blood group antigens (BGAs) are chemical moieties on the red blood cell (RBC) membrane. Some BGAs (e.g., A, B, H, Lewis, P, I) are widely distributed throughout the body and may not be primarily erythroid antigens. Statistical correlations with ABO blood groups and disease have been made for years and have been highly controversial. It is not known if BGAs have a biological function. There are increasing reports of BGAs [e.g., Le(x) (an isomer of Le(a)), Le(y) (an isomer of Le(b)), T, Tn, "A-like"] appearing as "new" antigens on malignant tissue. Their presence and membrane density appears to correlate with the metastatic potential of the tumor. This often parallels loss of normal BGAs (e.g., ABH) from the tissue. Some of these antigens have been shown to influence the humoral and cellular response and have been used in assays to determine preclinical cancer, and in tumor immunotherapy. Interactions of some parasites and bacteria with human cells have been shown to depend on the presence of certain BGAs. P. vivax malarial parasites only enter human RBCs when the Fy6 Duffy blood group protein is present on the RBCs. Certain E. coli will only attach to the epithelial cells of the urinary tract if P or Dr BGAs are present in the epithelial cells. The P antigen is also the RBC receptor for Parvovirus B19. Leb has recently been found to be the receptor for H. pylori in the gastric tissue. The high frequency BGA, AnWj, is the RBC receptor for H. influenzae. BGAs have been shown to be associated closely with some important complement proteins. Ch/Rg BGAs have been found not to be true BGAs but are RBC-bound C4 (C4d). Knops/McCoy/York BGAs have been located on the C3b/C4b receptor (CR1). The high frequency BGAs of the Cromer (Cr) system are located on decay accelerating factor (DAF or CD55). Cartwright (Yt) BGAs are located on RBC acetylcholinesterase molecules. DAF and acetylcholinesterase are on phosphatidylinositol-glycan (PIG) linked proteins. When the PIG anchor is missing from RBCs, as in paroxysmal nocturnal hemoglobinuria, the affected RBCs lack all Cr, Yt, JMH, Hy/Gy, Do and Emm BGAs. The most important ligand for P, E and L selectins is sialyl-Le(x). This interaction is the tethering stage that start the leukocytes' journey from the circulation into the tissue. It appears that malignant cells may move through tissue in a similar way and may explain the close association of Le(x) with metastasis. Thus, there are increasing data suggesting a biological role for BGAs unrelated to the RBC.

To avoid the well-known shortcomings of phenotyping granulocytes for the NA antigens using NA-specific human sera, a DNA-based method to determine the NA genotype was developed. Genomic DNA was isolated from blood cells or serum, amplified by polymerase chain reaction (PCR), immobilized on nylon membrane and genotyped using digoxigenin-labeled, sequence-specific oligonucleotides (SSO). The genotyping results of whole blood samples from 54 and of serum from 20 individuals correlated perfectly with our phenotyping using the antigen capture assay MAIGA. In three cases with the phenotype "NA-null" no hybridization of the NA-specific oligonucleotides occurred. These data show that SSO is a reliable method for NA genotyping especially if only small volumes of blood or even only serum probes are available.

The neutrophil-specific antigen NC1 is defined by an antibody in the serum of a mother who gave birth to a child with alloimmune neonatal neutropenia. NC1 has been reported to be associated with the neutrophil-specific antigen NA2, but the precise relation of NC1 and NA2 remained unclear. Therefore, we investigated the serum using the antigen capture assay MAIGA and the granulocyte (GIFT) and lymphocyte (LIFT) immunofluorescence tests. In GIFT, no NA association was observed. In LIFT, serum antibodies bound preferably to lymphocytes with the HLA antigens HLA-B7 and cross-reacting antigens. In MAIGA, an antibody specific for the NA2 variant of the granulocyte Fc gamma-receptor III was observed. The NA2 specificity was confirmed by testing granulocytes from 40 further different donors. This indicates that the NC1 and NA2 antigens are identical. A positive GIFT result but a negative one in LIFT using cells of an NA2-negative typed individual suggest the presence of an additional, non-NA2-specific granulocyte antibody.

Pooled platelet concentrates (PC) prepared by the platelet-rich plasma (PRP) method were filtered with three different filters and stored for 8 days at room temperature. The effect of filtration on leukocyte contamination, platelet concentration, and the in vitro function, morphology, metabolism and activation of platelets were studied. Eight pools of 20 PRP-PC were used, each pool was split into 4 equal volumes; 3 were filtered over a PL50HF, a PL-10A and a Bio P10 filter, the 4 served as a control. After filtration, leukocyte counts exceeded 3 x 10(5) in none of the pooled PC. Platelet loss induced by filtration was about 17%. During storage, no differences in pH, PCO2, and lactate and glucose concentration were found between the filtered and the unfiltered units, nor were any differences observed between filtered and unfiltered pooled PC in aggregation upon stimulation with collagen and/or ADP, adhesion capacity to collagen in flowing blood, nucleotide content of the platelets and nucleobase concentration in the plasma, expression of activation-dependent antigens, or platelet morphology as observed by light microscopy and by the swirling effect. Selective removal of beta-thromboglobulin (22%) by the PL50HF filter was observed. Pooled PC prepared by the PRP-method can be filtered and stored for 8 days without detrimental effect on platelet function, metabolism or activation.

Neonatal alloimmune thrombocytopenia (NAIT) was first described in the 1950s. The disease results from maternal antiplatelet antibodies against surface antigens on fetal platelets due to feto-maternal platelet incompatibility. Although NAIT is transient, during its active phase thrombocytopenia can cause intracranial hemorrhage with grave neurological sequelae or death. We characterized the alloimmune antibodies in 29 mothers and found anti-HPA-1a (in 11 cases), anti-HPA1b (1), anti-HPA-5a (1) and anti-HPA-5b (5). In 11 women no specific antibodies were identified despite characteristic disease in the newborns. 3 neonates in the anti-HPA-1a, and 1 in the anti-HPA-1b groups were born with intracranial hemorrhage and are severely disabled. We described a case of successful prenatal treatment by a combination of steroids, high-dose immunoglobulins and intraumbilical platelet transfusion in a mother known to have anti-HPA-1, whose previous child was severely affected. It is important to examine antibodies in any woman whose neonate has thrombocytopenia of unknown etiology.

Despite numerous attempts to increase the neutrophil count of infants with alloimmune neonatal neutropenia, no therapy has been consistently effective. We describe two infants with alloimmune neutropenia who had a rapid and prolonged increase in neutrophil number after treatment with granulocyte colony-stimulating factor (G-CSF). Patient 1 had antibody directed against the neutrophil antigen NA2. He received three daily doses of G-CSF, and within 2 days his neutrophil count increased from 0.350 x 10(9) to 3.584 x 10(9)/L (350 to 3584/mm3). Despite cessation of treatment the neutrophil count remained in the normal range. Patient 2 had antibody to the neutrophil antigen NA1, and received six daily doses of G-CSF. Within 4 days his neutrophil count increased from 0.477 x 10(9) to 4.320 x 10(9)/L (477 to 4320/mm3) and remained in the normal range for 11 days after the last dose of G-CSF. We recommend that treatment with G-CSF be considered for selected infants with alloimmune neutropenia.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

A child with hyporegenerative anaemia was chronically transfused with group 0 Rh-negative blood. As typing with anti-D showed a mixed-field pattern, the patient's red blood cells (RBC) were identified as D positive. Due to the transfused ccddee RBC, it was impossible to determine by simple agglutination whether c was present on the patient's RBC. To resolve this question, two-colour indirect immunofluorescence was performed using D as a marker of the patient's RBC. Likewise, Duffy and Kidd antigens could be determined on the patient's RBC despite multiple transfusions.

Earlier studies showed that Moluccella laevis lectin, which has anti-Tn specificity, reacts more strongly with native or desialylated blood group N glycophorin A than with the respective glycophorins of blood group M. We now present results indicating that Vicia villosa B4 anti-Tn lectin, which does not show detectable reaction with untreated glycophorins or erythrocytes, reacts better with desialylated blood group N antigen than with asialo M antigen. This was demonstrated by three assays: (1) agglutination of asialoerythrocytes; (2) binding of biotinylated lectin to asialoerythrocytes immobilized on ELISA plates; and (3) inhibition of lectin binding to asialo-agalactoglycophorin with asialoglycophorins M and N. These results supply further support for the conclusion that glycophorin of blood group N has more GalNAc residues unsubstituted with Gal (Tn receptors) than glycophorin of blood group M.

Blood group antigen-related oligosaccharides have been implicated in growth regulation, cell mobility control and adhesion; we are therefore interested in the localization of receptors for these oligosaccharides in tumour cells. Labelled neoglycoconjugates that carry synthetic sugar structures are suitable tools to determine: whether such binding sites are present in human lung cancer; whether structural alterations of the glycoligand part will affect extent of binding; and whether cell type-associated alterations can be detected. Sections from 121 cases of lung cancer, representing small cell and non-small cell lung carcinoma, mesothelioma and metastases from extrapulmonary primary carcinomas were used to study the binding of nine synthetic AH- and Le-related oligosaccharides. Probes with fucose-alpha 1-3/4-N-acetylglucosamine-beta 1-R, an A-like disaccharide and 3'-sulfated galactose as ligand appear to bind less well to small cell than to non-small cell lung cancer cases, whereas Lec-disaccharide distinguishes mesothelioma from metastatic carcinoma. The latter ligand, A-like disaccharide and H (type III)-like trisaccharide exhibit evident cell type-associated differences in extent of binding. Thus, tailor-made neoglycoconjugates constitute a promising class of histopathological tools that warrants further study.

Plasma antithrombin III activity and concentration were measured simultaneously on COBAS MIRA-S by colorimetry using a synthetic substrate and by immunoturbidimetry, respectively, in normal subjects (n = 30), in patients with liver disease (n = 37), and in those with thrombosis (n = 76). The activity/concentration ratio of plasma antithrombin III, in which concentration was represented as the ratio to the mean concentration for normal, was evaluated in each group. Results showed that the ratio was significantly lower in patients with liver diseases (0.76 +/- 0.18, mean +/- SD, p < 0.01) and in thrombotic patients (0.89 +/- 0.18, p < 0.01) than that in normal (1.07 +/- 0.06), demonstrating the predominance of antithrombin III molecules with low activity in the former groups. This simple and rapid (< 10 min) method is applicable for detecting such the constitutional change in plasma antithrombin III.

An RNA-based method has been developed to genotype donors for the granulocyte-specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fc-gamma-receptor III-1 sequence-specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR-RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.

This report presents the results of two National Workshop exercises which were designed to evaluate interlaboratory and interassay variation in sensitivity of techniques used for the detection of platelet-reactive alloantibodies. Most workshop participants used the platelet immunofluorescence test. Sensitivity of this assay was improved when fluorescence was measured by flow cytometer rather than by microscope. The MAIPA assay was found to be highly sensitive but required considerable technical expertise, and the choice of antiglycoprotein IIb/IIIa (CD61/41) monoclonal antibody was found to have a significant effect on its ability to detect anti-HPA-1a.

A highly sensitive and specific sandwich enzyme immunoassay for human liver-specific antigen (LSA) was developed and its forensic application using LSA as a marker for the determination of liver injury were examined. The LSA was purified from human liver by immunoaffinity chromatography. Polystyrene ball coated with affinity-purified rabbit anti-human LSA IgG was incubated with the human LSA and then with affinity-purified anti-human LSA Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl) propionic acid as a hydrogen donor. The detection limit for human LSA was 0.52 pg per assay. The serum LSA levels determined by this assay in healthy male and female adults were 1.5-1.6 ng/ml and 0.7-1.0 ng/ml, respectively. The recovery of LSA added to 5 microliters and 10 microliters serum samples was estimated to be 53.1-55.5% and 70.6-74.8%, respectively, and no difference in recovery between serum from males and females was observed. LSA antigenic activity in bloodstains containing LSA was detectable after storage for 30 days at room temperature. High levels of LSA were proved to exist in forensic samples taken from stabbed livers, and it was clearly possible to differentiate between samples from stabbed livers and those originating from other stabbed organs. These findings demonstrate that determination of LSA from forensic samples is useful for detecting liver injuries.

We have developed a method for the quantitative analysis of Lewis antigens on human red blood cells (RBC) using immunofluorescence labeling and flow cytometry. Initially, Lewis a and Lewis b (Le(a) and Le(b)) antigens were labeled with monoclonal anti-Le(a) or anti-Le(b) antibodies followed by labeling with the fluorescein isothiocyanate (FITC)-conjugated second antibody. This method was not sensitive enough to identify the Lewis antigens on RBC, although the FITC method is very commonly used for antigens on white blood cells. Next, we selected the enhanced labeling technique using the avidin-biotin procedure. Biotinylated anti-mouse IgM was used for the second label and the reaction with R-phycoerythrin (RPE)-conjugated streptavidin followed to produce the fluorescence. The method was found to be effective for our objectives. From the results analyzed by the enhanced labeling technique, differences were not found in either the levels of the antigen-positive percentage and the peak mean channel of Le(a) antigens on RBC in the groups of blood type O and A (in ABO system). On the other hand, both the levels of Le(b) antigens on RBC were higher in the groups of blood type O than in those of blood type A. We found both Le(a) and Le(b) antigens on RBC from a few blood type O subjects. We conclude that enhanced labeling and flow cytometry constitute a useful technique for the determination of Lewis antigens on RBC and that this method enables the precise quantification of such antigens.