The improved method for direct basophil counts described by Cooper and Cruickshank was evaluated. Their findings, indicating a small mriance and negligible chamber error, could be confirmed on a larger series of normal individuals

Botha, M.V.

Laubscher, N.F.

Wassermann, H.P.

English

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8 May 1971 S.A. MEDICAL JOURNAL 491ther the non-functional horse gamma-globulin or the func-tional variety (the lymphocytotoxic antibody) should beaccepted as the actual stimuli for the synthesis of antihorseglobulin in the baboon. The effective separation and useof ALG obtained by specific immuno-absorption proce-dures would be of interest to evaluate in future studies.CONCLUSIONSThe need to produce effective, non-toxic antilymphocyteserum is of particular importance in the treatment of organrejection or auto-immune disease. However, it has beenfound extremely difficult to produce a highly specific andsafe immunosuppressive product. A serious side-effect ofantilymphocyte serum is not only its ability to inducethrombocytopenia and anaemia but also its potential tocross-react with many other tissues besides Iymphocytes.ALS made against intact preparations of lymphoid cellswould therefore appear to contain a complex mixture ofantibodies. In the light of this observation it would not bepossible to conclude that the immunosuppressive activityof ALG resides solely in its ability to induce lymphopenia.ALG prepared against a heterogeneous pool of intactIymphocytes represents a mixture of soluble and insolubleantigens in a particularly crude form. Antibodies to sucha mixture of antigens will inevitably cross-react with a widerange of other tissues as shown by Levey and Medawar.'·4In this condition the prime effect at ALG on lymphocytescannot be anticipated to be specific since the antibodywill also be attached to the surface of other tissues, whichin turn alters their functional activity.Using the haemopoietic cell line as an accessible modelto test the sKie-effects of ALG, in place of inaccessible celllines (spleen, kidney, lung) we were able to draw the follow-ing conclusions from our experiments. When two differentpotencies of cytotoxic ALG were compared, it was shownthat the subcutaneous injection of high-titred antibodiesconsistently caused severe haemolytic anaemia and throm-bocytopenia. This damaging effect on the haemopoieticsystem could be reduced by repeated absorptions of thetoxic ALG with red cells. However, in vivo sensitizationof the red cells could not be eliminated unless repeatedabsorptions achieved absolute removal of the red cell anti-body. Only at this point did the ALG no longer sensitizethe red cells in vivo. The absence of red cell antibodyactivity by repeated absorptions also effectively removedthe presence of lymphocyte antibodies. This is consistentwith the contention that red cells and lymphocytes sharecommon antigen determinants.The in vivo application of ALG produced by massivedosage of lymphocytes and intense immunization pro-cedures often created a condition of lymphocytosis inplace of the anticipated lymphopenia. Complete absorp-tion of the cytotoxic antibody with red cells did not affectthe continu~d observation of lymphocytosis (Fig. 2. baboonT 11). On the other hand, absorption of the cytotoxicantibody with a combination of Iymphocytes sensitizedby antilymphocyte serum (antigen-antibody complexes)consistently abolished the induction of lymphocytosis.However, this in vitro manipulation was not found tocreate a reduction of circulating Iymphocytes in vivo, whichmakes it doubtful whether there was any specific immuno-suppressive effect present in the first place. The presenceof immune complexes in the ALG can of course also playa role in the development of nephrotoxic syndromes bylocalizing in the renal glomeruli.The deliberate use of toxic ALG also produced impor-tant information with respect to the behaviour of thecomplement system. This factor was significantly reducedin value as a result of the in vivo interaction of ALG withvarious tissue components possessing shared antigen con-figuration.Although antilymphocyte serum can be successfullyproduced in some horses by repeated injections with hetero-logous intact Iymphocytes, it is still not certain that thesynthesized antibody will be specifically reactive for lym-phoid tissue because it is known to cross-react with manyother tissues. This limitation affects our knowledge of itsin vivo activity when used as an immunosuppressive re-agent.Investigations are now in progress to isolate and assessthe immunological properties of substances obtained fromwithin the lymphocyte.We wish to th1nk Drs J. A. M. White. M. A. Rogers andB. C. Wess~ls for the'r vlluabie co-operation in the collectionof baboon thor~cic duct Iymphocytes. This work was sUP1J0rtedin Dlrt bv the South African Medical Research Council (Grant9/3/12-537).REFERENCESI. Brody. J. I. ami l3eizer, L. H. (1964): J. Lab. Clin. Med .. 63, 819.2. Rosenfield. R. E.. Rubinstein, P .. Lalezari. P.. Dausset. J. and VanRood, J. J. (1967): Vox Sang. (Basel), 13, 461.3. Levey, R. H. and Medawar, P. B. (1966): Ann. N.Y. Acad. Sci .. 129,164.4. Idem (1966): Proc. Nat. Acad. Sci. (Wash.), 56, 1130.5. Eijsvoogel, V. P., Du Bois, M. J. G. J., Schellekens, P. Th. A. andVan Loghem, J. J. (1969): Transplantation Proceedings, I, 408.6. Wessels, B. C. (1971): J. S. Afr. Vet. Med. Assoc. (in the press).7. Mittal, K. K., Mickey, M. R., Singal, D. P. and Terasaki, P. I.(1968): Transplantation, 6, 913.8. Grobbelaar, B. G., Brain, P. and Rogers, A. J. (1970): Lancet. I, 1120.9. Malek, P., Kole, J., Haskova, V. and Zastava, V. (1969): Transplanta-tion, 8, 333.10. Dacie, J. V. and Lewis, S. M. (1963): Practical Hematology. NewYork: Gmne & Stratton.11. Alien, P. Z., Berger, B. M. and Helms, C. M. (1968): J. ImmunoJ.,101, 1023.12. Austen, K. F. and Russell, P. S. (1968): Ann. N.Y. Acad. Sei., 129,657.13. Bloeh-Shtaeher, N., Hirsehhorn, K. and Uhr, J. W. (1968): Clin.Exp. ImmunoJ., 3, 889.EVALUATION OF AN IMPROVED METHOD FOR DIRECT BASOPHIL COUNTS*H. P. WASSERMANN, N. F. LAUBSCHER AND M. V. BOTHA, Pigment Metabolism Research UniT of The SouthAfrican Medical Research Council and the University of Slellenbosch, and the National Research Institutefor Mathemalical Sciences, PretoriaSUMMARYThe improved method for direct basophil counts de-scribed by COJoper and Cruickshank was evaluated. Their'Date recei"Od: 14 December 1970.-====:::::::::::::::::=::;=:.::=======~=---~~findings, indicating a small mriance and negligible cham-ber error, could be confirmed on a larger series of normalindividuals.492 S.-A. MEDIESE TYDSKRIF 8 Mei 1971There is little doubt that one of the contributing factorsto the scantiness of knowledge on basophil leucocytefunction is the lack of a satisfactory direct countingmethod to enumerate these cells in capillary and venousblood.The indirect method, making use of differential countsto calculate the 'absolute' number of basophils as a per-centage of the total leucocyte counts, is satisfactory onlywhen at least 2000 cells are classified in the differentialcount."Since the first description' of a method for direct baso'phil counts in 1953, several modifications'·4 of the methodpoints to its inadequacies. Although all methods employtoluidine blue for metachromatic staining of basophils,our own experience and that of others, found the twomain problems to be the diffuse staining due to watersolubility of the polysaccharides in basophil granules andthe clotting and aggregation of blood platelets, frequentlyassociated with degranulation of basophils:These problems were overcome by Cooper and Cruick-shank' by adding cetylpyridinium chloride and aluminiumsulphate to the toluidine blue. The former lyses erythro-cytes and complexes with mucopolysaccharides, renderingthem relatively insoluble in water. The latter accentuatesthe metachromatic staining by acting as a mordant. Theuse of EDTA in saline as anticoagulant prevents aggrega-tion and platelet clumping.METHODTo assess the improved method of Cooper and Cruick-shank' we employed the same experimental set-up asused by them to assess errors associated with pi pettingand counting. An attempt to reduce pipetting errors wasmade by using a screw-attachment attached to the pipettesfor measuring out the diluents. This was compared withpipetting done by mouth through a rubber tube in a sepa-rate experiment on a single blood sample.Blood was obtained from a fingerprick in 37 individuals.The first drop was wiped away and 0·02 ml of the secondand subsequent drops were taken with 3 pipettes. Thiswas added to 0·08 ml 0'1 o/~ EDTA previously deliveredinto Wassermann tubes. To the anticoagulant in the Was-sermann tubes was added 0'1 ml of the cetylpyridiniumchloride-toluidine blue-aluminium sulphate solution, andthe tube gently shaken. Two Fuchs-Rosenthal countingchambers were filled from each of the three samples ob-tained from each individual with a Pasteur pipette and thestained basophils counted. For each individual we there-fore had 6 separate counts.STATISTICAL ANALYSISA three-factor analysis of variance was carried out on thecounts. The counting chambers (C) and patients (P) aretwo factors occuring together throughout, while the threepipette samples (S) are nested within patients. The threepipette samples were considered as random sample froman infinite population of possible blood samples from eachpatient. The patients were considered as a random samplefrom an infinite population of possible patients. Thecounting chambers were two specific chambers from whichconclusions were to be drawn.If YIJK represents the number of basophils per cubicmillimetre blood from the k-th sample of patient i, ascounted in chamber j, then i=l ... 37. j=I,2 andk= 1,23 in this set of data. A suitable model for analysisof variance would then be:'YIJl-\:= u+a?+aJ c+ aIKs+arJPc + aIJKc,s+ erJK,where the' a's are main effects and interactions of therandom factors while a C is the only fixed effect. The erJKare the error components which are assumed as inde-pendent and normally distributed. The analysis of variancefor this model is shown in Table 1.TABLE I. ANALYSIS OF VARIANCESource of Sum of Degrees of Mean Slim of\'orituioll sllllare~ freedom squares F-ceSfSBetween patients 81481'56 36 2263-38 34·7Between countingchambers 100·81 JOO·81 1'77Between samples(within patients) 4825'50 7':' 65·21P X C interaction 2045'13 36 56·81 1·09cxS interaction(within patients) 3 871-44 74 52·32The residual variance is thus 52·32. The mean; x, ofany future patient, calculated from 6 observations in 2counting chambers, would thus have a standard errorof (52'32/6) =2,95. A confidence interval for the truemean of a future patient would then be x±2·95 t, wheret refers to student's [ with 74 degrees of freedom.The components of the total variance that can beattributed to samples and counting chambers are in therelation of 47·9: 1. The mean counts for the two count-ing chambers were 33·4 and 34·7 and the mean for thetotal sample of patients was 34·1.In the experiment where 10 counts were performedover 6 counting nets on dilutions made by the screw-attachment, and the same procedure carried out on dilu-tions made by mouth-pipetting, no significant advantageis apparent in either procedure. The means and standarddeviations were 2·7 ±1'49 and 2·5 ±1'45 cells per countingnet respectively.DISCUSSIONThis evaluation of Cooper and Cruickshank's improvedmethod for direct counting of basophils confirms theirfindings" The small variance found by them could beconfirmed on this larger series of patients and makes themethod very suitable to detect minor changes in basophilcounts. There appears to be no method at present avail-able to further reduce the major potential source of error.namely that inherent to pipette-dilutions. Like the originalauthors, we also believe that the improved staining tech-nique is responsible for the reduction of total error.As -suggested by Cruickshank and Haye,' the tendency'to translate basophil function in terms of mast cellbehaviour during the whole of this century' can, with thisimproved method, now be changed to have 'a new lookat basophils in their own right'.REFERENCES1. ~io060i J. E. and James. G. W. (1953): Proc. Soc. Exp. BioI. (N.Y.),2. B';seila: A. W. A. (1959): Acta med. scand., 163, 525.3. ~orsman. H. (1957): Acta derm.-venereol. (Srockh.), 37, 121.':'. Kovacs. G. S. (1961): Folia haemat. (Lpz.), 5, 166.5. Coope.r, J. R. ~nd Cruickshank, C. N. D. (1966): J. Clin. Path.. 19, 402.6. Scheffe, H. (19)9): The Anal}'sis of Variance, pp. 178 - 188 and 27.:. - 289.I ew York: John W,ley & Sons.7. ~r4.ickshank. c. N. D. and Haye, K. R. (196): J. Invest. Derm., SI.-

Evaluation of an improved method for direct Basophil counts

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Evaluation of an improved method for direct Basophil counts

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