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Direct Compatibility Testing by N J Clin Pathol: first published as 10.1136/jcp.11.4.311 on 1 July 1958. Downloaded from J. clin. Path. (1958), 11, 311. DIRECT COMPATIBILITY TESTING BY N. A. F. YOUNG From St. Margaret's Hospital, Epping (RECEIVED FOR PUBLICATION NOVEMBER 29, 1957) Despite the rapid development of knowledge it The Indirect Coombs Test (A.H.G.) still remains fundamental that mistakes within the Minimum Incubation Period for Reliable Results.- ABO and Rhesus systems are responsible for vir- Twenty-five sera were tested in triplicate, all tests tually all fatal mismatched transfusions. It therefore being standardized so that 4 vol. of serum was cannot be too highly stressed that the careful used to sensitize 1 vol. 50% suspension of washed preliminary grouping of the bloods of all patients packed Group 0 Rjr cells. Incubation periods of for transfusion is of prime importance, irrespective 10, 30, and 120 minutes were used. The test sera of whether there is a previous record of their blood ranged in potency from extremely weak ones, giving group or not. This should always be regarded as only a trace albumin agglutination, to strong sera copyright. an essential part of any cross-maiching routine, with albumin titres up to 1/128. and in urgent cases Stratton's (1955) " sandwich Readings were made by the usual tile method and technique" is highly recommended for rapid Rh the reactions timed with a stopwatch. typing. If no agglutination had occurred at the end of The " emergency crossmatch " presents a problem, seven minutes a negative result was recorded. and the vital question which needs answering is: All tests gave positive results although some of http://jcp.bmj.com/ What can safely be accepted as the minimum the " 10-minute incubation " ones were rather weak. incubation period which will not impair the effi- The usual reaction in these cases was a fine granu- ciency of the particular compatibility tests being larity first appearing between two and four minutes, used ? gradually becoming clear cut, but still tending to The concept of gradual red cell sensitization retain a homogeneous pink background at the end progressively increasing with time is fallacious. of seven minutes. Antigen-antibody union takes place quite rapidly, The reactions of the " 30-minute incubation tests'" and, after a relatively short time, which varies were all strong and clear cut. They were as good on September 28, 2021 by guest. Protected slightly with different methods, further prolonged as those obtained by longer incubation and in a incubation does not increase the sensitivity or surprising number of instances they were somewhat render the detection of very weak antibodies better defined than the corresponding two-hour significantly more likely. This is shown by the agglutinations. investigations which are detailed later, and the There seems little doubt that the optimal incuba- results obtained by Lewis and Chown (1957) using tion time for complete sensitization is much shorter a 10-minute albumin agglutination technique than is generally appreciated. Using the indirect substantiate this view. Coombs for direct compatibility testing, it is con- The ideal cross-matching technique should provide sidered that an incubation time of 30 minutes is a simple, easily performed procedure which is not adequate. In an emergency, a 10- or 15-minute only highly sensitive but which at the same time Coombs test is an extremely valuable procedure will yield reliable results when used routinely. which is capable of detecting very weak sensitization. Certain practical points have been investigated, and on the basis of the findings obtained a simple Effect of Variable Serum-cell Ratios on Sensitivity direct matching procedure, which is not time- of Coombs Reaction.-The Coombs reactions consuming but still combines the advantages of obtained after sensitizing red cells for a standard both trypsin and Coombs testing, is recommended. incubation period of 30 minutes using serum : cell J Clin Pathol: first published as 10.1136/jcp.11.4.311 on 1 July 1958. Downloaded from 312 Ar. A. F. YO UNG ratios varying from 2 :1 to 10: 1 have shown no test was weakly positive. In sera 2 and 3 the significant differences. It is therefore apparent that, tile method was negative at 10 minutes whereas the although theoretically it might be expected that the tube showed positive agglutination after this period addition of a greater volume of serum would increase of incubation. the amount of antibody available for each red cell, Further experience in a large number of con- in practice the effect is minimal. The probable trolled tests has led to the conclusion that for routine explanation of this is the fact that, although the usage the tube technique gives more consistent total amount of antibody is thereby increased, the resuilts and is distinctly more sensitive than the tile actual concentration of antibody molecules per unit method. There does not appear to be any tendency volume of serum remains precisely the same. to false agglutination and in all cases the negative A Modified Tube Technique.-With the develop- controls have shown no trace of agglutination. ment of a simplified trypsin-saline and trypsin- Stability of Stored Diluted A.H.G. Serum.--The Coombs compatibility routine in mind, the results instability of A.H.G. serum and its inability to of the Coombs test performed in tubes were com- withstand repeated thawing and refreezing are well- pared with those obtained by the more generally recognized drawbacks which can cause anomalous adopted tile method. For this comparative trial a results. Mollisoni (1951) states: "Attention must series of very weak albumin agglutinating anti-Rh be drawn to the fact that anti-human globulin sera sera was used. A parallel series of tests was made diluted in saline lose their potency very rapidly by the tile method and by a modified tube technique even when stored at -- 20' C. The diluted reagent described here. should be prepared freshly each day...." This Method. Unit volumes of 100' washed test cell has not been the author's experience nor that of suspensions were sensitized with 4 vol. of serum. Tests his colleague Dr. F. Marsh (1957), to whom he is were set up in triplicate for incubation periods of 10, indebted for the results of a two-year trial using 30, and 120 minutes respectively. After incubation they stored diluted antiglobulin sera. Diluted serum copyright. were washed three times in saline, the final supernatant stored at 20 C., placed in a water-bath for a couple being completely removed so as to leave a dry button of of minutes and used for testing, then immediatelv red cells. Three drops of suitably diluted A.H.G. replaced in the refrigerator, will withstand thawing reagent were added to each tube. The tubes were vigorously shaken to mix the cells, allowed to stand at for at least seven times without significant loss of bench temperature for two minutes, and then centrifuged its ability to agglutinate weakly sensitized red cells. at 1,500 r.p.m. for two minutes. Readings were made Such diluted reagent has been kept and used regu- http://jcp.bmj.com/ by gently resuspending the cells and microscoping if larly once daily for periods up to 10 to 14 days necessary. without deterioration. Reagent made up and used at weekly intervals remained stable and still strongly Results.-In most instances the results by tile agglutinated sensitized cells when finally tested for and tube techniques were similar, but in some cases the seventh time after being stored for eight weeks. the tube test gave clearer results and was distinctly Initially storage results were not constant or more sensitive for the detection of minimal sen- reliable, and it was noted that odd A.H.G. sera sitization. The results obtained in four antenatal would deteriorate much more rapidly and unexpec- on September 28, 2021 by guest. Protected patients whose sera contained very weak antibodies tedly, as Mollison states. Experience showed that are shown in Table I, and it will be noted that in these failures were attributable to two causes. In one case the tile test was negative whereas the tube the first place, the extreme importance of avoiding TABLE I any trace of globulin contamination in the saline RESULTS IN TILE AND TUBE TESTS ON FOUR ANTENATAL used as the diluent had not been sufficiently appre- SERA ciated. Secondly, it was found that if the A.H.G. serum from which the diluted reagent was made Serum Incubation Time N. Method had itself began to deteriorate, then the diluted 10 Min. 30 Min. 120 Min. reagent would be quite unstable. Provided meticu- Tile Uniform fine - lous care is taken in the preparation of the diluted granularitv Tube -7 reagent from a fully potent A.H.G. serum which 2 Tile Neg. Neg. Neg. has not started to deteriorate, then this reagent Tube Weak Weak Weak can be stored at -- 200 C. for long periods. It will 3 Tile Neg. Very weak Weak --- granularity withstand thawing up to at least seven times; nor Tube Weak - does there appear to be any loss of specificity and 4 Tile Neg. Doubtful Weak Tube false agglutinations have not been encountered. The great advantage and convenience of having a J Clin Pathol: first published as 10.1136/jcp.11.4.311 on 1 July 1958. Downloaded from DIRECT COMPATIBILITY TESTING 313 reliable reagent immediately available for use, albumin (the diluent effect of serum excluded). It particularly for night emergencies, can be appre- would seem therefore that, if albumin is used for ciated. compatibility testing, a 10-minute centrifugation Storage of Sensitized Cells Suitable for Coombs test is to be preferred, since the results of tests on a Control.-Washed, packed, sensitized red cells may wide selection of sera have clearly indicated that be stored at 60 C.
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