Observations on the use of the Coulter model D electronic cell counter in clinical haematology

Transcription

1 J. cln. Path. (1963), 16, 1 Observatons on the use of the Coulter model D electronc cell counter n clncal haematology A. N. BLADES AND H. C. G. FLAVELL From the Dorset County Laboratory, Dorchester, Dorset SYNOPSS The use of the Coulter electronc cell countng machne (model D) n a routne pathologcal laboratory s descrbed, as are also the techncal detals nvolved. The prncple of the Coulter counter, model D, s that a suspenson of partcles passes through an orfce, where the partcles nterfere wth the passage of an electrc current, producng pulses on an electronc counter. The crcut can be adjusted to vary the crtcal volume whch wll just produce a pulse. Alteraton of ths 'threshold' value enables all partcles to be counted, or by rasng the threshold, only partcles of progressvely larger ses. Both red and whte cells may be counted. n countng red cells a correcton s necessary because sometmes more than one red cell passes through the orfce at a tme, reducng the true total; a table provded by the manufacturers gves the correctons. RED CELLS Accordng to Brecher, Schnederman, and Wllams (196), the standard error of the red cell count on ths machne s %. Table shows the values obtaned by examnaton of the blood of 1 normal healthy male members of the laboratory staff, usng the oxyhaemoglobn method, a haematocrt centrfuge, the Coulter red cell counter, and from these calcu- Receved for publcaton 3 August 196 Blood No g. Hb = 1% latng the ndces. The consstency of these results at the expected level confrms that the counter s more accurate than the manual methods of countng. Fgure 1 shows a typcal red cell threshold curve obtaned by progressvely rasng the threshold. The left-hand part of the curve forms a plateau, showng that the threshold s suffcently low to count all cells untl t s rased to a value of about 1-. The centre of ths plateau s the best pont for total red cell counts; at the extreme left there s a certan amount of electrcal nterference and false pulses due to partcles of drt, and at the rght-hand end the numbers are begnnng to fall off due to excluson of the smallest red cells. Machnes vary slghtly n the value of ther threshold settngs. From readngs taken at successve values, the number of cells counted at each value can be plotted. f the value at each threshold s then subtracted from the prevous one, a second curve can be constructed, showng the dstrbuton of red cells accordng to volume. Ths curve, also shown n Fg. 1, s farly symmetrcal wth a peak n the mddle, analogous to the type descrbed by Prce-Jones whch was, however, based on the red cell dameter, whereas ths one s based on the red cell volume. t TABLE HAEMATOLOGCAL DATA OF 1 NORMAL PERSONS USED TO CONSTRUCT FG. Haemoglobn Haematocrt: Coulter Red M.C.H.C. Colour ndex Count lnvestgatons n progress suggest that these values are about to 6% too hgh * * *1 M.C.V J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.

2 Observatons on the use of the Coulter model D electronc cell counter n clncal haematology FG. 1. Example ofa normal threshold curve (contnuous lne = R.B.C. n mllons) and the derved curve of se dstrbuton (broken lne = 1,). FG.. Derved curves of the 1 bloods descrbed n the Table. The shaded area ncludes eght. -J 1 1 FG. 1 S. r.. t ' ' THRESHOLD UNTS es s worth notng that a red cell of,u dameter has about twce the volume of a red cell of 6,u dameter. Fgure shows the derved curves from the 1 blood samples ncluded n Table. We have found that a fgure of 7T on the threshold scale s sutable for countng the total red cells, and that for scannng the se dstrbuton t s convenent to proceed n steps of from to. Ths produces the fgures for calculaton of mean cell volume by the method of Magath and Berkson (196). ABNORMAL RED CELL PCTURES Fgures 3,, and show typcal curves from cases of severe ron defcency anaema. The peak s sharp and shfted to the left,.e., large numbers of small red cells are present and lttle else. Fgures 6, 7, and show curves from cases of macrocytc anaema. n these the curve s flattened and the peak s shfted to the rght, ndcatng that cells of all ses are present, but manly large ones. Fgures 9, 1, 11, and 1 are a seres taken on the days ndcated from a boy of 9 ' 1 1 c 1 - d O. 7. FG. * =. \ * * ' '''' THRESHOLD UNTS wth retculosarcoma. Ten days before the frst one, successful treatment was begun wth cyclophosphamde, and the curves show a retculocyte response wth a peak on the rght-hand sde, whch merges n the second one n the man red cell mass, after whch the curve returns to normal. Two examples of polycythaema nvestgated have both shown curves smlar to those wth ron defcency anaema but at about twce the heght,.e., cells are mcrocytc. MEAN CELL VOLUME 19 By the method of Magath and Berkson (196) the factor necessary to convert the threshold unts nto cubc mcrons came to 3-7. To fnd ths nvolves makng red cell correctons on all the separate values, and does not add sgnfcantly to the clncal nformaton obtaned from the graph. We therefore only correct the total red cell value and draw the graphs on the uncorrected fgures. The machne gves an accurate colour ndex and mean cell volume J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.

3 to : -J_ FG U)a - 6- F. 6.. L FG. l. k. t S 3 3 THRESHOLD UNTS L! ' * -! ; *. ' A. N. Blades and H. C. G. Flavell _ '* lb 1' 3 3S THRESHOLD UNTS 1, U) 1 1 c-6 FG. *\.~~~~~~ THRESHOLD UNTS FG. 3. ron-defcency anaema (scale, etc., as Fg. 1). Hb 6%; P.C.V. 33%; R.B.C. -7 m.; M.C.H.C. %; M.C. V. 77,u3; colour ndex -66. FG.. ron-defcency anaema. Hb. 7%; P.C. V. 9%; R.B.C. 3-6 m.; M.C.H.C. 9%; M.C. V.,3; colour ndex 77. FG.. ron-defcency anaema. Hb %; P.C. V. 3%; R.B.C m.; M.C.H.C. 3%; M.C. V. p3; colour ndex -6. J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.

4 Observatons on the use of the Coulter model D electronc cell counter n clncal haematology 161 -j,@-a* Z-@_._.-e-~~~~~~~~~ % U,* THRESHOLD UNTS FG. 6 n Z -J 3 Z FG u - dl a: FG. v.-, THRESHOLD UNTS.1. l j!!! j!j! ;! ~~~'. ; '. k ~~~~~.j' THRESHOLD UNTS *, - - tj c %O U) - U 3 - F. ~~~~~~~~~~ew.-, 1 S 3 3 THRESHOLD UNTS! ;%! ;%!! ~~~~~~~~!.!-- 1- '% - *, FG. 9 1 S 3 3 THRESHOLD UNTS FG. 1 Macrocytc anaema (folc-acd defcency, drug- FG. 6. nduced). Hb 3%; P.C. V.19%; R.B.C. 13 m.; M.C.H.C. 3 %; M. V.C. 13; colour ndex 1L6. FG. 7. Perncous anaema. Hb 67%; P.C. V. o%; R.B.C. -3 m.; M.C.HC. 33%o; M.C.V. JJ9p3; colour ndex 1-3. FG.. Macrocytc anaema. Hb 6%; P.C. V. ; R.B.C. -6 m.; M.C.H.C. 33%; M.C. V. 1,3; colour ndex 1-3. FG. 9. Case G.B. 1 days after begnnng treatment. Retculocyte response peak on R. Hb 3%; P.C. V. 6%; R.B.C m.; M.C.H.C. 3%; M.C.V. 7L3; colour ndex -7. FG. 1. Case G.B. fve days after precedng fgure. Retculocyte peak mergng nto man body. Hb 6%; P.C.V. %; R.B.C. 3-9 m.; M.C.H.C. 3%; M.C.V..3; colour ndex -9. J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.

5 16 A. N. Blades and H. C. G. Flavell 1 COUNTNG OF WHTE CELLS Whte cell counts are reproducble on ths machne wth a standard error of 7- % (Rchar and Breakell, 199) and duplcate counts can be run off at the rate of one a mnute. Aganst ths must be set the add- n/ \ tonal work nvolved n preparng dlutng fluds, and addtonal care n ppettng, whch s now the, 6 greatest error. Z * TECHNCAL DETALS U - \ ANTCOAGULANTS As a routne we have used venous blood taken nto plastc bottles contanng Ant-clot', a heparn-lke substance. Blood treated wth Wntrobe's ; > *s antcoagulant very occasonally causes the orfce to become blocked due to oxalate crystals. We have found \*+that ". the total number of red cells remans constant n! \ *>. whole blood treated wth ant-clot and kept at C. for * -.~ hours. * DLUTON OF BLOOD FOR COUNTNG The dlutons are S b 3 UN T made n two steps. The frst s 1/ made by ppettng THRESHOLD UNTS cu. mcro-ltres of blood nto 1 ml. of 9% albumn- FG. 11. Case G.B. four days after Fg. 1. Retculocytes salne. From ths a second dluton s made of 1/, by absorbed nto man mass. Hb 6%; P.C. V. 3%; R.B.C. takng - ml. nto a further ml. of albumn-salne. The 3 1 m.; M.C.H.C. 9%; M.C. V. 9,u3; colour ndex -9. frst dluton s then lysed wth Saponn and used for the whte cell count, the second dluton for the red cell count. When wpng the tp of the ppette, small peces of lens tssue are most satsfactory. Red cells reman stable n a dluton of 1/ n pure 9% salne or n albumn-salne for hours, but n a O dluton of, n pure -9 % salne red cells swell and lyse (see Fg. 13). A phosphate buffer soluton contanng sequestrene (see Fg. 1) was nferor to 9 % salne contanng 1 part n 3, of albumn (see Fg. 1). The graph of R.B.C.s n salne shows clearly the tendency r t. * of red cells to swell and lyse. - ;Weconclude that the total red cell count s reproducble, ; after dluton n albumn-salne for seven hours, although / \ four hours s a good workng optmum; when the red cell f X se dstrbuton s to be scanned, as short a tme as 36 - possble should elapse. _ ; PREPARATON AND DSPENSNG OF DLUTNG FLUDS Stock ; s. % soluton A. R. sodum chlorde s bottled n amounts U of ltres wth a plastc gvng-set used as an ext. Glass- n _ \s dstlled water s used to dlute t to a concentraton of.9%. Ths s ttrated n bulk, fltered through a sntered glass flter fller (Matburn unt no. F wth P flter), bottled n ml. amounts n Brtsh Standard blood! '. transfuson bottles, and autoclaved at lb. for mnutes. The closures must be tghtly screwed before autoclavng or the concentraton of salt wll rse, and t s _*s % safer to do ths overnght to allow t to cool slowly. One part n 3, of bovne albumn s then added, and the bottle suspended upsde down above an automatc THRESHOLD UNTS FG. 1. Case G.B. 1 days after Fg. 11. Hb %; R.B.C. 7 m.; return to normal. ppette (1 or ml.) connectng t by a plastc gvng-set. For countng, we dspense 1 ml. amounts daly nto 'Clnton Laboratores, Calforna. J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.

6 . U) - 3. co- Observatons on the use of the Coulter model D electronc cell counter n clncal haematology THRESHOLD UNTS FG FG. 1 ) THRESHOLD UNTS U) - J THRESHOLDS UNTS FG. 1 FG. 13. Effect ofpure 9% salne on R.B.C.s n dluton of 1 n,, showng threshold curves at ntervals oftwo hours (contnuous lne), three hours (short dashes), seven hours (dots), and hours (long dashes). There s a progressve ncrease n large cells,.e., swellng, untl at hours haemolyss s evdenced by a reducton n total numbers. FG. 1. Effect ofphosphate buffered salne wth sequestrene on R.B.C.s, as n Fg. 13. Stable up to three hours. Legend as for Fg. 13. FG. 1. Effect of albumned salne on R.B.C.s. Stable up to seven hours, total stable up to hours. glass flat-bottomed tubes of about 3 ml. capacty wth plastc stoppers. Albumn-salne produced by ths method gves a blank count at a threshold level of about 3 R.B.C.s per ml. Commercally prepared salne usually gves counts of under per ml. Regular checks should be made of the blank count as ths gves an ndcaton of whether the apparatus s becomng contamnated. CLEANNG OF GLASSWARE After use the glass pots are rnsed n tap water, soaked n -3 % Haemosol for 3 mnutes, rnsed n dstlled water, and dred upsde down n an oven. The plastc stoppers are smlarly cleaned, but dred at 37 C. Ppettes are rnsed n Haemosol, dstlled water and ether-meths, and dred. WHTE CELL COUNTNG Whte cells are counted n the 1/ dluton after the addton of Saponn. SAPONN Ths s a % soluton of whte Saponn (Hopkn and Wllams) of whch 1 drop s added to the frst dluton, after the preparaton of the second dluton, to lyse the red cells. Ths takes four mnutes, and the whte cells should then be counted, as after a further nterval they also begn to dsappear. We are ndebted to Messrs. Coulter for nformaton and techncal data, to Mrs. R. Harrs for clercal assstance and to the Wessex Regonal Board for fnancal assstance and encouragement. REFERENCES 163 Brecher, G., Schnederman, M., and Wllams, G. Z. (196). Amer. J. cln. Path., 6, 139. Magath, T. B., and Berkson, J. (196). bd., 3, 3. Rchar, W. J., and Breakell, E. S. (199). bd., 31, 3. J Cln Pathol: frst publshed as /jcp on 1 March Downloaded from on 9 June 1 by guest. Protected by copyrght.