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.
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.
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.
Titration studies with IgG anti-A antibodies revealed similar activity at both 4 and 37°C. The saline activity of IgG anti-A antibodies generally increased as the papain titre increased with both cord and adult A, and A, cells. Sera with papain titres >256 had saline titres in the range 32-256 at 37°C.Estimations of the IgG anti-A content of 'immune' anti-A sera made by titration following partial neutralization revealed a decreasing quantitation of the results with increasing saline IgG anti-A titre, as shown by comparison to the 2-ME method.
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