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T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 35, No. 2, pp. 105-108 February, 1961 Copyright © 1961 by The Williams & Wilkins Co. Printed in U.S.A. ELECTRONIC DEMONSTRATION OF BLOOD CELL AGGLUTININS MARK J. HALLORAN, M.D., WILLIAM J. HARRINGTON, M.D., VIRGINIA MINNICH, M.S. AND GRACE K. ARIMURA, B.S. Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri Visual reading of serologic agglutination reactions is subject to the bias of the observer. The availability of electronic instruments capable of distinguishing between particles according to size provided an opportunity for eliminating this subjective factor.4 Antibodies for each of the 3 formed elements of the blood were tested; readily reproducible results which correlated well with predicted values were obtained. The study was designed chiefly as a means of studying platelet agglutinins, however, and this paper includes detailed data only on these. with the following modifications: (1) the silicone used for coating glassware was diluted 1 to 20 with petroleum ether to minimize contamination with droplets of this agent; (2) all distilled water was passed through an ion exchange column primarily to remove any particulate matter, and for the same reason the ethylene diamine tetraacetic acid (disodium) was filtered through Celite Analytical Filter Aidf and Whatman No. 50 paper; (3) 30 rather than 20 min. at 150 g were given to centrifugation of the blood for preparation of the platelet-rich plasma in order to assure complete separation of leukocytes and erythrocytes; (4) All test serums were centifuged at 2000 X g for 20 min., and if lipemic, their clear central portions were carefully pipetted into a clean test tube (a precaution necessary only on samples that had been stored in the cold or frozen, inasmuch as thereafter any neutral fat present tended to separate by flotation). Three-tenths milliliter of a 1:1 dilution of test serum in isotonic saline solution was added to 0.1 ml. of the platelet suspension which contained 300,000 to 400,000 platelets per cu. mm. The mixture was stored for 18 hr. at 5 C , then agitated at 80 r.p.m. on a rotary platform for 5 min. One-tenth milliliter was diluted 1:100 in 10 ml. of isotonic saline solution. METHODS The apparatus used was the Coulter Counter* outfitted with standard equipment for the enumeration of blood cells.1 This instrument records the number and size of particles suspended in an electrolyte solution. As 0.5 ml. of the suspension flows through an aperture, 100 fi in diameter, a change in the resistance between an electrode on each side produces a voltage pulse of a magnitude proportional to the particle size. With appropriate scaler settings all particles admitted, the larger or only the largest may be recognized. Platelet agglutination reactions were performed as previously described,6 but Received, May 16, 1960; revision received, July 5; accepted for publication October 6. Dr. Halloran is Assistant in Medicine, Dr. Harrington is Associate Professor of Medicine, Virginia Minnich is Research Assistant Professor, and Grace Arimura is Research Assistant. Presented at the annual meeting of the Southern Section, American Federation for Clinical Research, New Orleans, La., January 21, 1960. Supported by Grant No. H22 (C14), United States Public Health Service, and by gifts given in memory of Mark Edison, Phillip Hunkel, Bill Burns, and Frederick Jostes. * Model A, Coulter Electronics Company, Chicago, Illinois. Studies on erythrocyte antibodies were performed on a 2 per cent cell suspension. Two-tenths milliliter of appropriate antiserum was added to 0.1 ml. of the suspension prepared either for Rh testing or for the antiglobulin reaction. Standard technics were used. Dilution for reading on the instrument was performed as described for the platelets. Tests for leukocyte agglutinins were conducted according to the method of Brittingham. 2 Leukemic leukocytes were t Johns-Manville Products, New York City, New York. 105 106 H A L L O R A N ET Vol. 85 AL. TABLE 1 ELECTBONIC D E T E C T I O N OF BLOOD C E L L AGGLUTININS* I n s t r u m e n t Settings and Readings Visual Readings 15/1 A. B. C. D. E. 29 control patients 11 patients with I T P 3 patients with I T P 3 control patients 22 patients with I T P 1 patient with drug-induced T P (quinine) Without quinine With quinine + + + 36 66 395 182 650 ± ± ± ± ± 15/2 6 18 151 69 195 95 298 101 195 1122 788 1663 ± ± ± ± ± 15/3 19 66 360 307 403 765 988 3971 2422 5823 ± ± ± ± ± 170 500 130 412f 422J 1105§ 750 911 210H I n s t r u m e n t Settings and Readings Erythrocytes Anti-Rho serum Rh-negative cells Rh-positive cells Antiglobulin serum Autoimmune hemolytic erythrocytes Albumin control Antiglobulin serum Visual Readings 30/1 40/1 50/1 + 109 3362 73 1928 40 1107 + 342 1322 124 708 96 410 disease I n s t r u m e n t Settings a n d Readings Leukocytes Normal serum Isoimmune serum Visual R e a d i n g s + 40/1 50/1 60/1 90 1241 67 415 40 163 * The d a t a are a basis for comparison of visual and electronic readings of blood cell immunologic reactions. With appropriate scaler settings, aggregates of blood cells diluted in t h e suspending medium may be recognized, whereas individual cells of the particular line under study are not counted. The figures given indicate the number of aggregates, in 0.5 ml., with a size greater t h a n t h e minimum predetermined by each combination of settings of threshold and aperture current. In the instrument values on platelets 3 points are notable: (1) the lack of significant difference between results with serums of control patients and most patients with I T P whose serums do not contain visually demonstrable platelet agglutinins; (2) the highly significant difference between either control serums or I T P serums which are negative on visual reading, when compared to the I T P serums graded as positive by the visual method; and (3) the finding of 3 positive I T P serums on instrument readings which were negative by t h e standard method. As noted in the text, t h e significance of the platelet aggregation is not established either by the present method or any other technic currently used, but presumably in I T P most instances reflect antibody-mediated agglutination of platelets. t d for average A & B , p > 0.1. t d for average A & E, p < 0.01. § d for average B & E, p < 0.01. H The instrument setting for these two determinations was 70/1. Feb. 1961 ELECTRONIC DETECTION OF ANTIBODIES used in order to minimize erythrocyte contamination; the final concentration of cells was 20,000 per cu. mm. Here also the final reaction mixture was diluted 1:100. Various scaler settings for threshold and aperture current were observed to be necessary in order to make allowance for a range in size of agglutinates in a given sample, and to exclude recognition of unagglutinated cells. For platelets, 3 aperture currents, 1, 2, and 3, were used with a single threshold setting, 15.J Additional threshold settings of 20 and 25 were generally not of value although an exception to the lack of usefulness of higher thresholds in platelet studies will be noted later. For erythrocytes and leukocytes a single setting for aperture current, 1, was used with threshold settings of 30, 40, and 50 for the former, and 40, 50, and 60 for the latter cells. In all instances conventional microscopic evaluations were performed independently on the undiluted samples and later compared with the results obtained on the instrument. RESULTS Platelet agglutinins. The results on serums from 32 randomly selected medical admissions are compared in Table 1 to those obtained on 36 patients with idiopathic thrombocytopenia (ITP) and 1 patient with thrombocytopenia caused by quinine. Two points are notable: (1) the great spread among the values on ITP serums between the 14 samples graded as negative on visual reading and the 22 samples with positive reactions; (2) the little difference between the negative control group and the negative I T P serums. There were three t Empiric settings. Not necessarily interchangeable with other instruments of the same model. Principles discussed in reference 6; with a population of particles of uniform size a decrease in threshold can be precisely off-set by a parallel increase in aperture current. With the systems used herein this reciprocal relation should also obtain; however, when only one setting at a time is varied a predictable change in count does not take place, although it would be possible to determine with a high degree of precision the distribution in size of agglutinates if systematic variation in a setting is made. 107 "false positives" 3 by both methods in the control samples (1 sample from a woman with systemic lupus erythematosus and 2 serums from patients with advanced hepatic cirrhosis). Of possible significance was the finding that 3 out of 14 ITP serums graded negative by visual readings yielded instrument values within the positive range (15/2 readings of 853, 1007, and 1525), whereas there was no increase in the number of positive reactions among control serums when instrument values were compared to visual gradings. Two trials were performed on serum from a patient with quinine-induced thrombocytopenia. As is characteristic, the agglutination when the drug was present was gross and composed of few but very large clumps. Inasmuch as there were so few agglutinates, the instrument count was low, although the amplitude of each pulse was high; however, when the threshold was increased from 15 to 70 the background values were minimized, thereby accomplishing a spread that was not present at the lower threshold between the samples without quinine and those to which it had been added. Red cell antibodies. Listed also in Table 1 are the values obtained on erythrocytes. Although only 2 trials were made, it is evident that agglutination reactions involving these antibodies may also be easily detected. Leukocyte antibodies. Agglutination of white blood cells from a patient with chronic myelocytic leukemia was demonstrated when serum from a patient who had received over 100 transfusions was tested (Table 1). Here also the trial was performed only to test a premise. DISCUSSION The use of instruments to replace visual readings in laboratory procedures is desirable if certain criteria are fulfilled. Accuracy and ease of operation are among the major considerations. Both are achieved by the method described herein. The study was designed, however, not with the objective of replacing present methods, but rather with the intent of adding one more precau- 108 H A L L O R A N ET tion against any pitfalls inherent in technics involving subjective factors. The method should not supplant microscopic readings, inasmuch as extraneous particles or extraordinary size of aggregates which can be simply evaluated microscopically are sources of error in instrument readings. Nor does the method distinguish between nonimmunologic aggregation of cells and agglutination owing to antibodies; in this respect a major shortcoming of agglutination reactions in general remains unresolved. A possible advantage of the technic is suggested by the finding of an increased incidence of positive reactions among I T P serums when tests with the instrument are compared to visual readings. Many more serums must be examined, however, before the platelet agglutination as recognized only by the instrument can be related to clinical data and interpreted. Finally, as a corollary to some of the observations described herein it should be noted that aggregation of platelets in blood samples from patients with thrombocytosis introduces a significant error in the electronic enumeration of erythrocytes. Although platelet clumping is common in most blood samples prepared for counting of erythrocytes, the number of aggregates which approximate or exceed the size of red blood cells is generally insignificant unless the platelet count is extremely high. When the latter obtains "red blood cell counts," several hundred thousand per cubic millimeter above the true value may result. Vol. 85 AL. SUMMARY Agglutinins for blood cells can be readily and reproducibly demonstrated on an electronic instrument now available in many hematology laboratories. Advantages and limitations of its adaptation to serology are discussed. SUMMARIO I N INTERLINGUA Agglutininas pro cellulas de sanguine pote esser demonstrate prestemente e reproducibilemente per medio de un instrumento electronic que es currentemente disponibile in multe laboratorios de hematologia. Es discutite le avantages e le limitationes de su adaptation a objectivos de serologia. REFERENCES 1. B B B C H E B , G., SCHNEIDEEMAN, M., AND WILLIAMS, G. Z.: Evaluation of an electronic blood cell counter. Am. J. Clin. Path., 26: 1439-1449, 1956. 2. B R I T T I N G H A M , T . E. AND C H A P L I N , H., JR.: Febrile transfusion reactions caused by sensit i v i t y to donor leucocytes and platelets. J . A. M . A., 165: 819-825, 1957. 3. DAUSSET, J . : Les thrombo-anticorps. Acta Haemat., 20: 185-194, 1958. 4. HALLOBAN, M . J., H A B E I N G T O N , W. J., M I N N I C H , V., AND ARIMIIBA, G. K . : Elec- tronic demonstration of platelet agglutinins. Clin. Res., 8: 53, 1960. (Abstract.) 5. H A B B I N G T O N , W. J., MINNICH, V., AND ARIMURA, G. K . : The autoimmune thrombocytopenias. In TOCANTINS, L. M . : Progress in Hematology, Vol. 1. New York: Grune and Stratton, 1956, p p . 166-192. 6. M A T T E R N , C. F. T., BRACKETT, F . S., AND OLSON, B . J . : Determination of number and size of particles by electrical gating: blood cells. J. Appl. Physiol., 10: 56-70, 1957.