Improved antibody detection by the use of range expansion and longer filter wavelength in a low ionic strength-protamine sulphate Auto-Analyzer system.

Range expansion, achieved by insertion of a variable resistance between the colorimeter and the recorder together with the use of 550 nm colorimeter filters, has resulted in markedly improved sensitivity for antibody detection, and improved sample identification, in a low ionic strength-protamine sulphate (LISPS) system. Range expansion also permits a lower concentration of red cells to be used, thus economizing on fully typed cells. Glycerol stored frozen cells were found to be only slightly less sensitive than fresh cells in this system.

The effect of heat on the A, B, and H antigenic strength of human red cells.

The reaction of A cells with anti-A, B cells with anti-B, O cells with Ulex europaeus anti-H is reduced by heat (56 degrees C) treatment. This is demonstrated using an automated haemagglutination technique with which minor differences in reactions can be detected.

A structural model for 30 Rh D epitopes based on serological and DNA sequence data from partial D phenotypes.

Both cDNA RHD sequences and reactivity with monoclonal anti-D have been reported in a number of partial D phenotypes, where parts (some epitopes) of the normal D antigen are missing, and anti-D of restricted specificity may be made in response to challenge with normal D positive blood. This paper analyses these reports together and proposes a model for the structure which comprise the epitopes of the Rh D antigen. Some epitopes are proposed to be comprised of continuous peptide sequence within one extracellular loop, whereas others require interactions between two or the extracellular peptide loops.

Human antibody fragments specific for human blood group antigens from a phage display library.

We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

Monoclonal antibodies as blood grouping reagents.

The large volume requirements for high quality ABO and Rh(D) typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents can be prepared, from blends of at least two antibodies, to optimize the intensity of agglutination for slide tests and the potency for the detection of the weaker sub-groups, including Ax and Bw, by tube techniques. New quality control steps have been described for some highly sensitive anti-A/anti-B antibodies to avoid the detection of traces of A on B cells or traces of B on A1 cells, which results from the non-specific activity of A and B transferases. Excellent anti-A,B reagents may also be made by blends of at least two antibodies to optimize both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline Rh(D) typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants, e.g. the epitopes on D category VI cells. However, this can be achieved by blending an IgM anti-D with IgG (polyclonal) anti-D which can detect these types after conversion of negative saline tests to an antiglobulin phase. In addition, high grade Du, D categories and variants can be reliably detected (for typing donors) by selected monoclonal IgM and IgG anti-Ds by use of suitably enhanced tests without the use of an antiglobulin test.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal blood group antibodies.

The large volume requirements for high quality AB0 and RhD typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents are each prepared from blends of two antibodies to optimise the intensity of agglutination for slide tests and the potency for detection of the weaker sub-groups, including Ax and Bw by tube techniques. Excellent anti-A,B reagents must also be made by blends of at least two antibodies to optimise both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline RhD typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants (Cat. VI). However, this can be achieved by blending IgG (polyclonal) anti-D with the IgM anti-D which can detect these types (in donor bloods) after conversion of negative saline tests to an antiglobulin phase. Selected monoclonal anti-complement blended with conventional anti-IgG can provide excellent polyspecific anti-human globulin reagents free of 'false positives' in routine tests. Monoclonal anti-M and -N require careful pH adjustment for optimum reactions. All anti-N reagents must be carefully diluted for reliable detection of heterozyotes, but with freedom of cross reaction with S + MM cells that have the most 'N' that is not a product of the N gene. Specific anti-M monoclonal antibodies can occur and an example is described that does not agglutinate NN cells even as a neat supernatant.

Report of studies on monoclonal anti-IgG antibodies.

Eleven monoclonal anti-IgG antibodies were evaluated by ten laboratories for possible AHG reagent use by the ISBT/ICSH protocol. Three of the anti-IgGs were selected by tests with weak Fya sensitized cells as this represents the best screening test. One of the IgM class anti-IgGs (205) equalled ISBT/ICSH reference reagents (R3P and RIIIM) against weak anti-D, -K and Fya sensitized cells and it reacted with all four subclasses of IgG. Thus, subject to satisfactory stability and extended trials with a wide range of weak antibodies, monoclonal anti-IgGs for AHG reagent use are now a possible alternative to conventional rabbit anti-IgG.

Report of studies on monoclonal antibodies (MABS) to complement components.

Twenty-five monoclonal antibodies (MABS) to complement components were evaluated according to the ISBT/ICSH protocol by twelve laboratories. Seven detected some form of C3c, but one of them, 174, did not react with EiC3b, although it was positive with 'EC3b' (Fruitstone). 174 may detect some form of enzyme sensitive C3b antigen, but C3a was not evaluated (present on 'EC3b' Fruitstone). Twelve of the antibodies were anti-C3d, one was an anti-C3g and five were anti-C4c.

The A1 (B) phenomenon: a monoclonal anti-B (BS-85) demonstrates low levels of B determinants on A1 red cells.

A monoclonal anti-B (BS 85) that reacts strongly with red cells from weak B variants (B3, Bint and Bv) has demonstrated the presence of a trace of B on A1 red cells. The agglutination of group A1 red cells by an anti-B antibody is called the A1 (B) phenomenon and is the converse of the B(A) phenomenon seen with certain monoclonal anti-A antibodies. Fragile A1 (B) agglutination is best seen by spin-tube techniques and A1 red cells negative in saline tests are agglutinated by albumin and protease enzyme-enhanced tests, but no reactions are seen with A2 red cells. The A1 (B) reaction is specifically inhibited by B substance, and D-galactose and the galactose-containing sugars melibiose and lactose. Red cells from B variants showed differential inhibition patterns with various sugars. A1 transferase levels were normal even in the strongest A1 (B) reactive blood samples, although the plasma H transferase levels and H status of these red cells were elevated. This is in contrast to the B(A) phenomenon which is associated with elevated levels of B transferase. It is suggested that A1(B) overlapping specificity can occur because of a combination of higher H activity (and thus more H sites) together with normal levels of A transferase activity as they are 20% higher than normal levels of B transferase. The production of anti-B reagents free of the A1 (B) phenomenon with BS-85 is achieved by suitable dilution using quality control tests with protease-treated A1 red cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Subtypes of i demonstrated by the use of atypical Ii cell types and inhibition studies.

Haemagglutination, absorption and elution studies with several anti-i sera and atypical Ii red cell types and Oh cells were carried out and three subtypes of specificity were demonstrated. The fourth i subtype was demonstrated by an inhibition method using human serum and amniotic fluid as a source of i substance.

Association of polymyalgia rheumatica and giant-cell arteritis with HLA-B8.

Histocompatibility antigens were determined in 30 patients with temporal arteritis, 27 patients with polymyalgia rheumatica, and 216 normal blood donors. HLA-B8 was significantly more common in patients with polymyalgia rheumatica (59%) and temporal arteritis (50%) than in the controls (27%). The findings of HLA-A10 in 26% of the patients with polymyalgia rheumatica compared with only 10% of the controls may be associated with the suggested immunological pathogenesis of the condition.

Monoclonal antibodies to Rh D--development and uses.

Monoclonal anti-D has proved impossible to make in rodent systems. Human monoclonal anti-D has been produced using EBV transformed peripheral B cells, coupled with fusions to myeloma cell lines. More recently molecular biology techniques have been used to produce monoclonal anti-D. The range of monoclonal anti-D produced is considered. The selection of monoclonal anti-D for use as blood grouping reagents for typing donors and recipients is reviewed--all types of D positive should be typed as positive when donors are considered. However, DVI patients should be typed as D negative. Considerations for the development of monoclonal anti-D for prophylactic use are reviewed.

Monoclonal anti-D specificity and Rh D structure: criteria for selection of monoclonal anti-D reagents for routine typing of patients and donors.

The Rh blood group system is the next most important to the ABO system in terms of its clinical significance in blood transfusion. It is vital to the safe, efficient practice of transfusion medicine that Rh D phenotyping tests are selected, executed and interpreted correctly. However, the Rh D blood group antigen has been shown to be subject to many phenotypic variations, and different reagents and typing techniques vary in their ability to detect these variants. The range of D-positive phenotypes are reviewed in terms of their reactivity with monoclonal antibody reagents and their clinical significance. In view of the available evidence, it is suggested that patient typing can be safely achieved by the duplicate use of one high-avidity or two very similar IgM monoclonal anti-D reagents that detect most variants except category DVI in simple tube or microplate saline tests. Antiglobulin testing for weak D should not be carried out on patient samples. Donor typing can be safely achieved by the use of the same monoclonal, used in parallel with a polyclonal anti-D reagent that detects DVI on sensitive automated equipment.

The computer crossmatch: a safe alternative to the serological crossmatch.

The crossmatch has evolved from including a wide range of techniques through a test purely to eliminate ABO incompatibility (immediate spin) to computer crossmatching in which no serological testing is carried out and validation ensures the correct ABO/RhD type blood is issued. The crossmatch was always considered to be the most important feature of the compatibility test and in particular the antiglobulin phase; however, there are potential risks associated with serological and computer crossmatching including technical and procedural errors. The use of immediate spin and computer crossmatch change the emphasis for safety of the compatibility test from the crossmatch to the antibody screen. UK guidelines have now been published describing the features necessary for the introduction of computer crossmatching. Computer crossmatching is used by many institutions in various countries. It is considered safe practice and brings benefits to the laboratory and the patient. Compatibility testing is only one element of the blood transfusion procedure; the others are equally as important and include correct patient identification at the time of collection of the blood sample and at the administration of the blood transfusion.

A simple method for detecting complement-fixation by autologous platelets in autoimmune thrombocytopenic purpura.

A new modification of the microtitre complement fixation test, (CFT), is described for the detection of platelet-bound antibodies (PBA). The test was positive in 12 out of 16 patients, (75%), with active autoimmune thrombocytopenic purpura (AITP). It was negative in four patients who were in remission of AITP when tested, in 10 patients with non-immune thrombocytopenia and in 51 normal blood donors. This is a semi-quantitative method in which suspensions of the patients' own platelets consume complement and therefore prevent the lysis of sensitised sheep red cells (SRBC). Sera from some of these cases were also tested for serum anti-platelet antibody (SPA) and immune complexes. The possible mechanisms and the relevance of positive results are discussed.

HLA-B27 and frozen shoulder.

Histocompatibility antigens were determined in 38 patients with frozen shoulder and 216 normal blood-donors. HLA-B27 was significantly more common in patients with frozen shoulder (42%) than in the controls (10%). The distribution of the other 19 histocompatibility antigens was similar in the patient and control groups. This result may be associated with the suggested immunological pathogenesis of the condition.

Further evidence for the presence of A antigen on group B erythrocytes through the use of specific exoglycosidases.

Certain antibodies have shown the ability to detect small amounts of A antigenic structures on certain group B cells. These rare cells that reverse group as B, are designated here as B(A) cells. Among the anti-A antibodies capable of detecting these cells are MHO4 (an IgM murine monoclonal antibody) and polyclonal anti-A derived from blood group O donors. The latter (anti-A, B) have been adsorbed exhaustively with normal B cells, to deplete the serum of antibodies to group B antigen. The cell specificity detected by these antibodies can be removed only by an alpha-N-acetylgalactosaminidase (A-zyme) but not by an alpha-galactosidase (B-zyme). Inhibition studies show that these reactions can be inhibited by A secretor saliva and cannot be inhibited by B secretor saliva. Moreover, papain treatment of normal group B cells not previously agglutinable with these antibodies, now causes these cells to become reactive, and this specificity, too, is removed only by A-zyme. These results suggest that low levels of blood group A antigen are being recognized by these antibodies and that these structures can exist not only on B(A) cells but on all group B erythrocytes.

A new monoclonal anti-A. Culture supernatants with the performance of hyperimmune human reagents.

By proper selection for good growth and high avidity, we have prepared a new anti-A monoclonal antibody producing cell line that gives culture supernatants as potent as US-licensed commercial hyperimmune human reagents and which meet USA FDA standards without the need for concentration. The production and use of this reagent is cost effective and makes it a candidate to replace conventional anti-A typing reagents.