IgG anti-Jka/Jkb antibodies are unlikely to fix complement.

Antibodies in the Kidd blood group system show a great deal of serological variability, are notoriously elusive and hence evoke difficulties in detection. However, they have been regularly reported as causing severe immediate or delayed haemolytic transfusion reactions and this clinical potential has been largely attributed to their complement binding ability. In initial investigations on 43 anti-Jka/Jkb sera with a range of titres of IgG antibody only a few seemed to fix complement, though following repeated tests on 20 of these sera a further five were shown to bind complement, making a total of 12 (27.9%) showing evidence of complement binding. Twenty-three sera were unavailable for re-testing. Subsequent tests revealed that only those sera which showed direct agglutination or were positive with an anti-IgM reagent in an indirect antiglobulin test (IAT) fixed complement. Evaluations on the IgG fractions of six selected potent anti-Jka sera failed to reveal any complement-fixing ability although all the original sera bound complement avidly and contained variable amounts of IgM antibody, some at very low subagglutinating levels. These findings challenge past perceptions and give cause for reflection on the changing methodologies and strategies which could unduly compromise the detection of these potentially clinically damaging antibodies.

An explanation and the clinical significance of the failure of microcolumn tests to detect weak ABO and other antibodies.

Shear forces are proposed to explain the failure of antiglobulin and 'neutral' (no antiglobulin) microcolumn tests at 37 degrees C to detect weak ABO incompatibilities and other weak antibodies, clearly detectable by spin-tube methods. These shear forces can be minimized in a microcolumn test using a biphasic centrifugation phase. Although this biphasic test is not suitable for routine use, it may be of use as an investigational method for reference laboratories. This failure of microcolumn test to detect weak ABO incompatibilities is of little clinical significance as the antibodies are dubiously active at 37 degrees C.

BCSH-NIBSC anti-D reference reagent for antiglobulin tests: the in-house assessment of red cell washing centrifuges and of operator variability in the detection of weak, macroscopic agglutination. British Committee for Standards in Haematology. National Institute for Biological Standards and Control.

A batch of an anti-D preparation, reference 91/608, has been prepared for the preparation of red cells weakly sensitized with IgG that can reveal inhibition of the antiglobulin test by one volume of human serum, diluted 1:1000. The preparation provides an objective assessment of red cell washer efficacy and the confidential, in-house assessment of operator variability in detecting weak but definite macroscopic agglutination by blind, replicate tests. Red cell washer efficacy and poor operator reading procedures causing disruption of weak agglutination are two major causes of false-negative antiglobulin tests; neither are adequately detected by the common quality-control procedure of adding strongly IgG-sensitized red cells ('Coombs control cells') to apparently negative antiglobulin tests. However, weakly IgG-sensitized red cells do offer a valuable control function that can detect some degree of cell washer inefficiency and reading errors although such cells are not a substitute for the more sensitive replicate testing. Test protocols are provided to assess the efficacy of cell washing machines and operator skills in the detection of weak but definite macroscopic agglutination.

Replicate tests for the detection and correction of errors in anti-human globulin (AHG) tests: optimum conditions and quality control.

Replicate blind AHG tests with weak IgG anti-D sensitised red cells revealed that 32% of workers caused 5 per cent or more false negative errors by using excessive agitation in reading techniques. The common quality control procedure of adding strongly sensitised cells to all negative AHG tests cannot reveal this type of error. Furthermore, strongly sensitised control cells may create an illusion of safety because AHG giving a false negative test with weak antibody in a serum sample may still show a reassuringly strong positive in the control test. "In-house" assessment of all staff and automatic cell-washers by blind replicate tests is recommended as an effective way of improving AHG test performance, thus reducing many of the errors involving false negative AHG tests seen year after year in External Proficiency Trials.

Use of monoclonal anti-C3 antibodies to characterise the fragments of C3 that are found on erythrocytes.

Using monoclonal antibodies to C3 it has been shown that the red blood cells of patients with cold haemagglutinin disease carry on their cells C3d,g (alpha-2D-globulin) rather than C3d. C3d,g seems to be the final product of in vivo C3 activation in fluid phase and on red cells. The cleavage of C3dg to C3d and C3g does not appear to occur in vivo either in the fluid phase or on red cell bound C3bi. In vitro C3-coated red cells prepared by antibody or low ionic strength techniques produce cells with C3d and C3bi as the predominant C3 fragment, whereas the Fruitstone technique in which coating occurs by the alternative pathway has principally C3b. The activity of C3 cleaving enzymes in whole serum is strongly influenced by the ionic conditions of the serum.

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.