The UK National External Quality Assessment Scheme in Blood Group Serology. ABO and D grouping, antibody screening, direct antiglobulin test and antibody identification 1984-1985.

In seven exercises of blood grouping the overall rates of major error were 0.19% and 0.25% in ABO and D grouping respectively. In ABO grouping this represents an increase in error rate over that observed in 1982-1983 but the increase was due to an unusually high error rate with one particular group A2B cell. An improvement in performance was observed in simple D grouping and was largely due to a lower incidence of false positive grouping of D-negative cells in the antiglobulin test. An improvement in performance observed in D grouping IgG-coated D-negative cells appeared to be due to a better understanding of the problem rather than to any change in serological practice. Error rates in antibody screening were somewhat lower than in 1982-1983 but this may or may not represent an improvement in performance as the test materials were not the same in the two periods. The direct antiglobulin test with IgG-coated cells was reliably performed with polyspecific and with anti-IgG reagents but an excess of false positive results was obtained with anti-C3d. Error rates in antibody identification varied from 0.6% for anti-D to 74% for anti-c + E.

Epitope specificities and quantitative and serologic aspects of monoclonal complement (C3c and C3d) antibodies.

Twenty-two monoclonal antibodies to human C3c and ten to C3d were obtained by hybridization after the immunization of mice with complement-coated human red cells and/or purified human complement components. C3c antibodies were variable in their agglutination reactions with cells coated with C3 by antibody in vitro; more consistent and potent reactions with these cells were observed with anti-C3d, and all anti-C3d reacted with red cells coated with C3 in vivo. Immunoradiometric assays were used to estimate antibody concentration, affinity, and epitope specificity. The antibody content in ascitic fluids varied from less than 0.1 mg per ml to 5.6 mg per ml. The estimated values of antibody affinities for Sepharose-coupled C3 ranged from 2.8 X 10(6) l per M to 5.0 X 10(8) l per M; on average, IgM antibodies had higher affinities than IgG antibodies. Competitive binding assays showed that the monoclonal antibodies recognized at least seven different epitopes, four on the C3c and three on the C3d fragment of C3. When the results of serologic and quantitative assays were compared, no convincing relationship was found between serologic performance and epitope specificity, antibody concentration, or affinity. IgM antibodies generally gave higher agglutination scores than IgG antibodies, and Ig class was the only useful predictor of serologic efficacy.

Anti-granulocyte opsonic activity in sera from patients with systemic lupus erythematosus.

Neutropenia is common in patients with systemic lupus erythematosus (SLE) but mechanisms of cell depletion remain obscure. To investigate the possible autoimmune aetiology of neutropenia in SLE, sera from 31 patients with this disorder were tested for anti-granulocyte activity. Granulocyte-binding immunoglobulins were detected by indirect immunofluorescence, and the ability of patient sera to opsonize granulocytes was determined by measuring the chemiluminescent response of human monocytes to granulocytes sensitized by test sera. Sera from 22 of the 31 patients bound IgG to granulocyte cell membranes and/or to nuclei, but only membrane-binding antibodies opsonized the cells for recognition by monocytes. There was no correlation between neutrophil count and the level of granulocyte-binding IgG as measured by indirect immunofluorescence. In contrast, opsonic activity and neutrophil count were inversely correlated (r = 0.5; P less than 0.05). However, opsonic activity was present in sera from most non-neutropenic patients. In patients with SLE, impaired reticuloendothelial system function may allow sensitized granulocytes to remain in the circulation.

The effect of anticoagulant on platelet associated IgG.

Blood anticoagulated with solid K2EDTA had platelet associated IgG (PAIgG) levels five-fold greater than citrated blood when the platelets were harvested by differential centrifugation but identical PAIgG levels when the platelets were harvested by Percoll density gradient. Furthermore, blood anticoagulated with increasing amounts of solid K2EDTA demonstrated a proportionate increase in both PAIgG and haemolysis whereas the same blood sample anticoagulated with aqueous K2EDTA exhibited neither of these effects. Coulter Counter analysis of platelet poor plasma (PPP) prepared from blood anticoagulated with solid K2EDTA revealed particles with a mean volume greater than normal RBC; such particles were not found in the PPP prepared from citrated blood. While some of these particles could be sedimented through plasma, those remaining were sufficiently buoyant to resist sedimentation unless the plasma was diluted with buffer. Studies with FITC conjugated antisera revealed that the particles could be classified into two types. Type I particles reacted strongly with a monoclonal anti-RBC antibody, weakly with anti-IgG antibody and had the appearance of stroma. Type II particles lacked any apparent biological structure, reacted strongly with anti-IgG antibody but not with monoclonal anti-RBC antibody. Thus, the buoyant IgG-rich particles found in blood anticoagulated with solid K2EDTA appear to contribute to the high PAIgG levels associated with this anticoagulant. Consequently, solid K2EDTA should not be used to anticoagulate blood destined for PAIgG measurement.

The UK National External Quality Assessment Scheme in Blood Group Serology. Compatibility testing 1983-1984: the influence of variables in test procedures on detection of incomplete antibodies.

Error rates in exercises in compatibility testing varied between 3% and 36% over the six years 1979-1984. Evolution of serological procedures was continuous through this period but without clear evidence of improvement in performance of antibody detection although performance in the UK appears to be comparable with that elsewhere. Relationships have been established between a number of variables of reagents and techniques, and their performance. The influence of some variables is reasonably clear but there appear to be interactions between certain variables in their effects on performance, and interpretation is less straightforward. A test for agglutination of enzyme treated cells together with an antiglobulin test appears to be the most reliable combination for detection of incomplete antibodies in compatibility testing although one stage enzyme techniques are an inevitable compromise between reliability and convenience. Compatibility testing should not be considered in isolation from antibody screening in determining the optimum combination of tests for pretransfusion antibody detection.

Anti-platelet opsonic activity in alloimmune and autoimmune thrombocytopenia.

A chemiluminescence technique (CLT) has been developed which measures the interaction between human monocytes and antibody-coated (opsonized) platelets. This technique has an objective end-point, is simple to perform and is of comparable sensitivity to the platelet suspension immunofluorescence test (PSIFT) when used to detect anti-platelet allo-antibodies. In contrast, only 4/20 sera from patients with clinically diagnosed autoimmune thrombocytopenia were opsonic in the CLT, while 8/20 of these same sera bound IgG to platelets in the PSIFT. Only one serum gave positive results in both tests.

A survey of anti-platelet antibody detection--report of the UK Platelet and Granulocyte Serology Working Group, 1985.

In 1985, serum samples were distributed to 27 laboratories within the UK and Eire with the objective of determining the current state of anti-platelet antibody detection. The sera included five unknown samples for blind assessment and four well-characterized reference reagents. The laboratories involved used a wide range of different methods but the most commonly used techniques were the fluorescent antiglobulin test and the enzyme-linked immunosorbent assay. In the trial, all responding laboratories were able to detect potent anti-platelet alloantibodies but there was a 43% incidence of 'false positive' results with samples which had been designated as inert. There were no apparent differences in performance between assays using fluorescein- or enzyme-labelled antiglobulin reagents or between assays which utilized PFA-treated rather than untreated platelets.

Anti-granulocyte opsonic activity and autoimmune neutropenia.

Sera from patients with unexplained neutropenia have been assayed for anti-granulocyte opsonic activity using a chemiluminescence technique which measures the metabolic response of human monocytes to antibody-coated granulocytes. This rapid and simple technique was more sensitive than indirect immunofluorescence in the detection of anti-granulocyte antibodies. Anti-granulocyte opsonic activity was detected in sera from 17 of 31 patients, suggesting that their neutropenia may have had an autoimmune basis. The opsonic activity of five of the 17 sera was increased when granulocytes were sensitized in the presence of fresh serum. Four of these sera bound IgM and C3b to granulocytes in the immunofluorescence test. Human IgG when added to the monocyte suspension medium inhibited monocyte response to IgG antibody-opsonized granulocytes. This inhibition was less when granulocytes were opsonized with sera containing IgM and complement granulocyte-binding activity. This observation may be relevant to the selection of neutropenic patients for therapeutic use of intravenous immunoglobulin.

Production of human monoclonal IgG and IgM antibodies with anti-D (rhesus) specificity using heterohybridomas.

Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh-negative or DB cells. Concentrations of both antibodies reached between 25 micrograms/ml and 50 micrograms/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody.

The UK National External Quality Assessment Scheme in Blood Group Serology. ABO and D grouping and antibody screening 1982-1983.

In seven surveys of blood grouping the overall rates of major error were 0.12% and 0.37% for uncomplicated ABO and D grouping respectively. Of 17 errors of ABO grouping, 13 were errors of transposition or interpretation and four were apparently technical. Of 52 errors of D grouping, 20 appeared to be errors of transposition or interpretation and 32 were apparently technical. Of the 32 technical errors of D grouping, 31 were D-negative grouped as Du (29) or D-positive (2) and most of these errors were due to misgrouping in the antiglobulin test. Causes of error in D grouping by antiglobulin test include anti-Bg and other contaminating immune antibodies, residual unabsorbed anti-A and the inherently high rate of false positive results obtained in the antiglobulin test. In view of the lack of benefit of Du testing to blood recipients or to pregnant women and of the possible adverse consequences of misgrouping D-negative patients as Du or D-positive, it is recommended that Du testing be abandoned in these groups of patients. The surveys of antibody screening demonstrated lack of standardisation and error rates similar to those previously reported in the UK for compatibility testing.

Proficiency testing in immunohaematology in Ontario, Canada, and in the United Kingdom: a comparative study.

A comparative study of proficiency testing models in immunohaematology has been carried out between the United Kingdom National External Quality Assessment Scheme and the Laboratory Proficiency Testing Program of the Ontario Medical Association, using material supplied by both programmes to laboratories in the United Kingdom and Ontario. The results suggest that the general standard of performance in immunohaematology practice is similar in the two jurisdictions and that, where clear differences are seen, these reflect differences in technique or in educational emphasis.

The UK national external quality assessment scheme in blood group serology. Compatibility testing 1981-1982: performance and practice.

The design of exercises of compatibility testing was modified in 1981 in order better to accommodate participants' serological practices. Ten reference laboratories were also enrolled in order to determine the 'correct' results for each exercise. In 1981-1982 3.5 to 36% of participants missed incompatibilities in exercises in which undiluted antibodies were issued and this did not represent an improvement over performance obtained in 1979-1980. Surveys were undertaken of antiglobulin test procedures and revealed that serological practices continue to change, old techniques are being modified, new techniques are being employed but standardization shows little overall improvement. Surveys of quality control procedures and of cross-match procedures for agglutination in albumin and for agglutination of enzyme treated cells show equal lack of standardization.

The detection of anti-granulocyte antibodies by chemiluminescence.

Ten sera containing polyspecific anti-HLA antibodies, a rabbit antihuman leucocyte antiserum and a monoclonal anti-granulocyte antibody were used in the development of a semi-quantitative technique for the detection of anti-granulocyte antibodies. The assay measures the metabolic response of human monocytes to antibody coated (opsonized) granulocytes. Immune complexes were opsonic only in the presence of fresh human serum. Monocyte response was strongly inhibited by normal human serum and purified free immunoglobulin. The assay is simple, rapid and sensitive, and all antibodies investigated were readily detected.

Quantitative quality control of antiglobulin reagents.

Double antibody radioimmunoassays have been developed for the quantification of anti-IgG, anti-C3, anti-C3c, anti-C3d and anti-C4 antibodies and for the determination of their binding constants. Assays were undertaken on 53 polyspecific antiglobulin reagents obtained from a variety of commercial and public sources. Concentrations of anti-IgG varied from 1.2 to 12.8 micrograms/ml in commercial products and from 0.4 to 6.0 micrograms/ml in public products. Concentrations of anti-C3 and anti-C3c varied from 0.1 to 1.0 micrograms/ml in most commercial products but in public products concentrations varied by more than 100-fold from 0.02 to 6.5 micrograms/ml. Concentrations of anti-C3d varied from 0.05 to 0.7 micrograms/ml in most commercial products and from less than 0.01 to 1.3 micrograms/ml in public products. Concentrations of anti-C4 varied from less than 0.01 to 0.18 micrograms/ml in commercial products and from less than 0.01 to 0.08 micrograms/ml in public products. Mean binding constants for commercial products were: anti-IgG 6.6 x 10(9) l/mol, anti-C3 4.6 x 10(9) l/mol, anti-C3c 5.3 x 10(9) l/mol, anti-C3d 0.4 x 10(9) l/mol and anti-C4 4.9 x 10(9) l/mol. Relationships were found between results obtained in quantitative assays of specific antibodies and independently performed serological assessments of potency. Anti-IgG was present in suboptimal concentrations for agglutination in several public products and anti-C3 and anti-C3c were in suboptimal concentrations for agglutination in many public and commercial products.

The UK national external quality assessment scheme in blood group serology. Compatibility testing 1979-1980: trends and performance in relation to practice in antiglobulin testing.

Surveys of antiglobulin test procedures and reagents were undertaken in 1979-1980 as part of a national external quality assessment scheme in compatibility testing. An extraordinary lack of standardization was revealed. In addition, practices underwent considerable changes over this period. The use of tube and of low ionic strength solution (LISS) techniques increased whilst the use of tile and of albumin-antiglobulin techniques declined. Performance in compatibility test exercises was significantly better with tube techniques than with tile techniques in 9/22 incompatibilities. Performance with LISS techniques was occasionally significantly better than with normal ionic strength techniques. Performance with albumin-antiglobulin techniques was occasionally significantly worse than in the absence of albumin. To varying extents significant relationships were found between performance and cell concentration, serum/cell concentration ratio, antiglobulin reagent and method of reading the results.

The UK National External Quality Assessment Scheme in Blood Group Serology, Compatibility testing 1979-1980.

Seven proficiency tests in compatibility testing were issued in 1979 to 1980. In each exercise participants were required to test five sera, most of which contained alloantibodies, against three samples of red cells by three designated techniques, namely agglutination in saline at room temperature, agglutination in albumin at 37 degrees C and the antiglobulin test at 37 degrees C. Comparability was achieved by scoring of results. Incompatibilities were missed by 3.5 to 22% of participants in five exercises in which undiluted antibodies were issued. There was no significant improvement in performance over two years. Antibodies which were issued diluted, but detectable by antiglobulin test, were missed with high frequency.

A double antibody radioassay for the detection of platelet associated IgG.

A double antibody radioimmunoassay has been developed for the detection of platelet associated IgG (PAIgG). The assay employs conventional radioassay technology and, as such, readily may be adopted by laboratories familiar with radioassay techniques. The assay is sensitive to 0.6 ng IgG and can be undertaken on 10(6)-10(7) platelets, or fewer in the case of thrombocytopenia with raised PAIgG. The assay can be completed within 2 working days. Normal PAIgG levels in EDTA blood were 0.8 to 5.7 micrograms IgG/10(9) platelets. However, normal levels depended upon the anticoagulant used for blood collection and were on average five-fold higher for blood taken into EDTA than for blood taken into ACD; normal range 0.1-1.2 micrograms/IgG 10(9) platelets. PAIgG was assayed on blood taken simultaneously into ACD and EDTA from 15 patients with chronic idiopathic thrombocytopenia (ITP). PAIgG was raised in 14 out of 15 EDTA samples, but in only 11 of 15 ACD samples. EDTA was therefore regarded as the anticoagulant of choice. A total of 20 of 23 (87%) patients with chronic ITP had PAIgG values above the normal range. The PAIgG in chronic ITP was weakly related to platelet count, weakly and inversely related to platelet volume, but was independent of serum IgG concentration.

Radioassays of blood group M, N and T (Thomsen-Friedenreich) antigens.

Radioassays employing the double-antibody or Farr techniques were developed for the M, N and T antigens. Blood group glycoproteins were isolated by butanol extraction of red cell stroma and iodinated by the chloramine-T technique. The final purity of glycoprotein was over 75% as judged by radioimmunoassay (RIA). T activation of glycoprotein was obtained with neuraminidase. A specific RIA was obtained for the M antigen and was sensitive to approximately 10 ng of glycoprotein or glycopeptide. In the RIA system rabbit anti-M displayed a higher affinity for M glycoprotein than for M glycopeptide. A RIA that was entirely specific for the N antigen, could not be obtained. A radioassay, obtained for the T antigen with peanut agglutinin in the Farr technique, was sensitive to approximately 100 ng of T antigen and was readily inhibitable by monosaccharides. A RIA, obtained for the T antigen with rabbit anti-T, was entirely specific and sensitive to approximately 1 ng of T activated glycoprotein or glycopeptide but was not inhibitable by monosaccharides.

Automated antibody detection in a low ionic strength (glycine) medium.

Modifications were introduced into the low ionic strength system of Lalezari. Glycine was added in place of acidified glucose and the sensitisation and aggregation phases were segregated. In the automated glycine system a stable baseline is readily obtained as strong cell aggregation, with risk of cell adhesion to coil walls, is restricted to the last few turns of the aggregation phase. The automated glycine system is sensitive to a concentration of 0.0015 microgram/ml of anti-D diluted in saline and to 0.006 microgram/ml of anti-D in serum. Antibodies of a broad range of specificity are detectable.