932 Royal Postgraduate Medial Shool, Duane Road, London W12 ONN S M Lewis Western Infirmary, Glasgow R M Rowan Toa Medial Eletronis, Kobe, Japan F Kubota Correspondene to: Dr S M Lewis Aepted for publiation 5 July 199 J Clin Pathol 199;43:932-936 Evaluation of a prototype for a referene platelet ounter S M Lewis, R M Rowan, F Kubota Abstrat A semiautomated single hannel aperture-impedane partile ounter was developed as a prototype for a referene platelet ount for assigning values to referene preparations used in automated blood ell ounts. The instrument is equipped with sheath flow and an aperture orifie of 5 gm in diameter and 6 gm in length to eliminate non-axial flow and minimise oinidene errors. Use of fixed volume and red blood ell:platelet ratios obviate dilution errors. The ounter was assessed in aordane with the International Committee for Standardization in Haematology protool for evaluation of automated blood ell ounters. The ounter provided a high level of linearity and preision, aurate oinidene orretion, ontrolled volume, stability and negligible arryover. The aurate ounting of platelets is assuming inreasing importane in the are of patients, partiularly those with thromboytopenia. Examination of the data published by various national external quality assessment bodies, partiularly NEQAS in the United Kingdom, Etaonorme in Frane, and the College of Amerian Pathologists, indiates a ontinuing trend toward whole blood ounting methods. Modern blood ell ounters are apable of a high degree of preision, but there is often onsiderable variation among instruments. Harmonisation between different ounting systems requires alibration by means of referene preparations with aurately assigned values.' Counters whih an provide a suffiiently high level of auray and reproduibility are needed to assign values to referene preparations for use in alibration. A semiautomated single hannel apertureimpedane ounter with a transduer to detet the passage of ells has been developed by Toa Medial Eletronis Ltd for this purpose, and its red ell ounting apaity has been evaluated.2 This instrument seemed to be an eminently suitable referene ounter when used in onjuntion with a proedure for oinidene orretion.3 A further modifiation, based on the aperture-impedane priniple, was designed to provide an instrument (Sysmex Model SSF) for platelet ounting. This has an orifie diameter: length of 5 x 6 gm with a sheath flow whih eliminates non-axial passage of ells, minimises oinidene error, and prevents reirulation at the orifie. Flow rate is ontrolled by a syringe unit whih injets a fixed volume of sample (11-86,l) into the sheath flow in 14 seonds. A pulse height analyser is linked to the ounter to hek and set moving thresholds. A urve is fitted to the data and partiles whih appear under the fitted urve are distinguished from noise level and miroyti red ells, respetively. The reorded data inlude platelet ount, red ell ount, mean platelet volume (MPV) and platelet size distribution. Data reeived from the ounting system pass to a miroomputer for orretion of oinidene error for both red blood ells and platelets by either logarithmi or linear extrapolation as appropriate. The programme for this takes aount of the problem whih ours when more than one ell type is being ounted by the same detetor; it involves alulating the probabilities of oinidene in red blood ell and platelet ross ounting.4 Comparison of the red blood ell with the red ells ounted on the referene ounter' onfirms that the displaed volume has been onstant. It also provides omparison of the diret platelet ount with the red blood ell: platelet ratio proedure originally proposed as a referene method by ICSH.' The ounter was evaluated in London (Laboratory A) and Glasgow (Laboratory B) in aordane with the ICSH protools.5 Methods Whole blood, in K2-EDTA antioagulant, was used for all measurements. The blood was diluted 1 in 5 in Cell pak (Toa Medial Eletronis Ltd). The diluted samples were ontained in Toa DB- 1 pots whih were shown to prevent surfae adhesion of ells and not to influene their size. Counts were delayed until all visible bubbles due to mixing had disappeared. Exept for the study of drift, the ounts were made within the stability period for the diluted samples. For all dilutions, the pipettes used were offiially tested lass A glassware, lean and free from hipping, as defined by ICSH.' Results EFFECT OF DILUTION As the instrument is a ratio ounter evaluating both red ells and platelets, and a omplex oinidene orretion algorithm is inor- J Clin Pathol: first published as 1.1136/jp.43.11.932 on 1 November 199. Downloaded from http://jp.bmj.om/ on 24 September 218 by guest. Proteted by opyright.
Evaluation of a prototype for a referene platelet ounter porated, the effet of dilution over antiipated working ranges for both red ells and platelets needs to be assessed. Repliate tests (three at eah dilution) were performed to give results at 1 evenly spaed onentrations, between 1% and 1% volumes by dilutions of platelets in autologous platelet poor plasma (A). The auray of pipetting was heked by estimating the haemoglobin onentration at eah dilution by the haemiglobinyanide method using spetrophotometers alibrated with the ICSH Referene Standard.6 Table 1 Effets of dilution on linearity Experiment Slope Interept oeffiient (R) A Low platelets in platelet poor plasma 734 2-612 -9955 A High platelets in platelet poor plasma -956 --79-9977 BI High platelets low red blood ells -99 25.9999 B2 High red blood ells normal platelets -999 5 123 9999 4.61 't4 4-25- M Us I-.; 3-9 3.55-3.21 (1) (2) (3) -25.5-75 1- Conentrution Figure 1 Coinidene orretion Relation of slope (oinidene ounts) to interept (orreted ounts) on linear sale: (A) RBC fy 446-26X (r - 9977); y = 443-24X (r = - 9996); y 352-17X (r --9942)] (B) PLT [y 272-4X (r -9751); y 262-38X (r - 9936); y 235-47X (r= --9926)] = In a seond experiment (B) two different speimens were used, (1) low red blood ells and high platelets for assessing platelets and (2) high red blood ells and normal platelets for assessing red blood ells. These preparations were made artifiially using platelet rih plasma, red ells, and bovine serum albumin in appropriate proportions. The ranges of onentrations were as follows: Experiment A B (1) B (2) Redbloodells(x 112/1)12-4 -7 Platelets ( x 19/1) -7-8 -3 Results are shown in table 1. The results indiate that there was no signifiant deviation from linearity for either red blood ells or platelets throughout the ranges tested. COINCIDENCE CORRECTION To hek the validity of oinidene orretion red blood ells and platelets were measured on a set of speimens at three further serial dilutions, to provide 3/4, 'A2, 1/4 of the initial sample-that is, 1 in 666; 1 in 1; and 1 in 2, respetively. Eah sample was measured three times. Means were plotted on linear and logarithmi sales and extrapolated (figs 1A and B). Analysis of variane (ANOVA) was arried out on the deviation of the mean from the regression line at eah dilution. F was alulated as the ratio of variane of mean from regression line to variane of repliate measurements. There was no evidene of nonlinearity; extrapolation to zero is idential with the theoretial predition. PRECISION 933 Two different experiments were performed: (A) 1 onseutive ounts were made with ontinuous mixing on three samples of low, mid, and high red blood ell and platelet onentrations. The results were as follows: Conentration Total instrument ounts Low Mid High Red blood ells (mean) 48671 93826 176996 CV -52% -61% -51% Platelets (mean) 3773 673 1159 CV 181% 1-34% -72% (B) Within and between bath preision was measured using a two way analysis of variane as desribed by ICSH.5 Twenty samples (ount range: 2-55-5-78 x 112/l for red blood ells; 64-678 x 19/1 for platelets) were analysed in tripliate in three bathes. The results were as follows: J Clin Pathol: first published as 1.1136/jp.43.11.932 on 1 November 199. Downloaded from http://jp.bmj.om/ on 24 September 218 by guest. Proteted by opyright.
934 Figure 2 Instrument stability, using monosised latex preparations: RM165 and RM166 refer to the ertified referene materials (CRM) of the European Communities Bureau of Referene. Figure 3 Speimen stability: (A) platelet ounts at room temperature; (B) platelet ounts at 4 C; (C) red blood ells at room temperature; (D) red blood ells at 4 C. CARRY-OVER In both laboratories arry-over was assessed using the Broughton method,7 as reommended by ICSH.5 The results were as follows: Red blood ells Platelets Laboratory A Laboratory B -1% -42% 1-3% -1% DRIFT A mixture of latex partiles of 2- gm (surrogate platelets) and 4-8 gm (surrogate red ells) diameter (BCR Certified Referene Material No CRM 165 and CRM 166, respetively; Community Bureau of Referene of the Commission of the European Communities, Brussels) was used over an eight hour period to assess instrument stability. The material was measured five times in eah hour. The overall reproduibility of the data from this experiment is a measure of drift or its absene. Results are displayed graphially in fig 2. This suggested a slight drift, espeially with Certified Referene Materials 165, a one-way analysis of variane gave a CV of 1 34% for red ells and 1-27% for platelets with no measurement outside this range; thus no signifiant instrument drift had ourred. U'n 34-32 75, &-- 9:3 1:3 RM166 (4.8pm for RBC) RM165 (2-Opm for PLT) 11:3 12:3 13:3 14:3 15:3 16:3 17:3 Time 61 = x 45-3 3- IU IL 42-4- 3 38- -,36~ - ISOI 75-1 Lewis, Rowan, Kubota SPECIMEN STABILITY An experiment was designed to assess the stability of red blood ells and platelets in speimens with inreasing time after olletion and any effet of time lapse on ounts obtained. Seven speimens were seleted from the routine workload inluding examples of low and high platelet ount. Aliquots from eah were stored at room temperature and at 4 C and measured hourly during the first six hours and subsequently at 24, 48, and 72 hours following venesetion (fig 3). Results of platelet ounts are displayed in table 2. COMPARABILITY Platelet ounts obtained from the Model SSF were ompared with those obtained by differential entrifugation89 by single hannel aperture-impedane ounters and with those obtained by a standardised visual mirosopy method' with phase ontrast (in Laboratory A) or ordinary light mirosopy (in Laboratory B). Red ell ounts derived from the SSF were ompared with those from a semiautomated single hannel aperture-impedane instrument using thresholding speifiations defined by ICSH (1988).' Studies were performed in both laboratories using similar protools with the following results: Number of Sample number observers Referene for visual red ell LowMid High ounting ounter LabA 1 2 1 1 Toa2 LabB 13 1 14 3 Coulter ZF Comparisons were assessed when results were stratified into those falling into the low, the middle, or the high parts of the range.,-.u 5. OA o 4' u 3. J 1.J 1 2 3 4 5 6 7 6 -,; 45- : 3-8 X 5 -J o. I... * 1 2 3 4 5 6 7 (hours) 2 4, 6 Iō 5 'C 4-8 6-I U&3- M2- l' IB (D _. 1 2 3 4 5 6 7 _~~~- = 1 2 3 4. 5 6 7 (hours) J Clin Pathol: first published as 1.1136/jp.43.11.932 on 1 November 199. Downloaded from http://jp.bmj.om/ on 24 September 218 by guest. Proteted by opyright.
Evaluation of a prototype for a referene platelet ounter 935 Table 2 Stability ofplatelet ounts on storage Room temperature 4 C Hours 1 2 3 4 5 6 7 1 2 3 4 5 6 7 247 262 33 339 145 583 35 251 266 327 339 15 577 36 1 255 266 328 348 151 583 4 26 256 32 333 15 61 42 2 251 257 34 342 146 561 47 249 264 232 344 16 589 35 3 26 279 337 355 16 593 42 25 268 255 338 15 573 41 4 253 266 349 346 15 561 35 246 272 22 342 155 615 33 5 255 268 332 355 151 577 44 252 256 195 347 16 599 45 6 26 261 333 346 153 569 4 255 268 191 328 148 61 35 24 257 272 333 358 152 568 43 249 255 179 347 158 587 38 48 262 273 337 356 155 634 42 245 254 158 344 149 59 35 72 264 277 338 389 24 614 78* 26 28 172 348 16 69 43 CV% 2-2 2-65 1-79 3-96 19 46 27-4 2-6 3-2 26-8 1.9 3-3 2-2 23-4 Eah vertial olumn indiates ount obtained at, 1, 2, 3, 4, 5, 6, 24, 48 and 72 hour after venesetion. Speimen 3 at 4 C shows fall in ount by two hours; this was due to agglutination not adequately dispersed by mixing. *Exluding this aberrant result, CV was 9-66%. graphs of platelets ounts by SSF and the visual referene method are shown in fig 4. of visual and entrifugation methods are shown in fig 5. Regression analysis has been done separately 7 6 5 O 4 u- 3 2 1 1 2 36 46 56 66 7 Mean platelet ounts ( x 19/l) Low Mid High Overall Lab A Centrifuge 99 28 555 34 (n = 4) Visual 88 291 576 312 SSF 93 293 586 316 Lab B Centrifuge 1 275 524 38 (n = 37) Visual 93 263 544 31 SSF 85 268 519 299 with the visual and entrifugation methods with the following results: (a) With visual ounting method: Slope Y-inter df t test LabA *992 1-1 2-14 39-1 199 (NS) Lab B *9936-95 3-25 36 2-623 (p = -5) (b) with entrifugation method: LabA 9973 1-8 -11-21 39-4-42 (p = -1) LabB 9933 1-2 -15-97 36 2-233 (p = 5) (36 df) = 2-28 at p = 5, 2-719 at p =*1 (39 df) = 2-23 atp = 5, 2-78 atp = 1 'CY-x 4 4' : ~~~Visual (x tog/o l) AV 4w lo DW MV' Visual (xlo /1) Figure 4 Comparison ofplatelet ounts by SSF and Figure 5 Comparison ofplatelet ounts by visual and visual referene method in Lab A: (A) [r = 992; entrifugation referene methods in Lab A: (A) [r = interept 2 1378; slope + 1 78] and Lab B: 999; interept = 13 4844; slope = + 9311] and (B) [r -9936; interept 3 2512; slope = Lab B: (B) [r = -9868; interept = 22-6858; slope = +.9537j. +921]. E J Clin Pathol: first published as 1.1136/jp.43.11.932 on 1 November 199. Downloaded from http://jp.bmj.om/ on 24 September 218 by guest. Proteted by opyright.
936 Lewis, Rowan, Kubota of visual and entrifugation methods were as follows: Slope Y-inter df t-test Lab A 999-93 13-48 39 1-87 LabB *9868-92 22-68 36 337 Disussion Automated platelet ounting is now generally performed on whole blood, the platelets being identified by a size disrimination proedure. It is therefore desirable to have a referene method whih is also apable of ounting platelets on whole blood speimens to assign values to referene preparations for use in alibration. The instrument desribed in this study is intended for this purpose. The ommon auses of inauray are dilution error, oinidene ounts, ounting loss of small platelets and inadequate disrimination. To minimise systemati errors there is a sheath flow, 5 gm (diameter) x 6 gm (length) orifie, extrapolation orretion, and, to avoid dilution errors, red blood ells and platelets are ounted proportionately in a fixed volume. The instrument was evaluated in aordane with the ICSH protool for the evaluation of automated blood ell ounters.' The results indiate that this instrument is apable of a high level of linearity and preision, aurate oinidene orretion, ontrolled volume, stability and negligible arry-over. Comparison with established methods was satisfatory, but minor differenes are likely to be a refletion of variability by the traditional tehniques rather than due to any inauray by the SSF. Thus the SSF seems to be eminently suitable as a andidate referene instrument for platelet ounting. 1 ICSH. The assignment of values to fresh blood used for alibrating automated blood ell ounters. Clin Lab Haematol 1988;1:23-12. 2 Lewis SM, England JM, Rowan RM, Kubota F. Standardisation for haemoytometry. Lab Pratie 1989;38:68-9. 3 Lewis SM, England JM, Kubota F. Coinidene orretion in red blood ell ounting. Phys Med Biol 1989;34: 1239-46. 4 Groner W, Epstein E. Counting and sizing of blood ells using light sattering. In: van Assendelft OW, England JM, eds. Advanes in haematologial methods: The blood ount. Boa Raton, Florida: CRC Press, 1982:74-84. 5 ICSH. Protool for evaluation of automated blood ell ounters. Clin Lab Haematol 1984;6:69-84. 6 ICSH. Reommendations for referene method for haemoglobinometry in human blood and speifiations for international haemoglobinyanide referene preparation (3rd edition). Clin Lab Haematol 1987;9:73-9. 7 Broughton PMG, Gowenlok AN, MCormak JJ, Neil DW. A revised sheme for the evaluation of automated instruments for use in linial hemistry. Ann Clin Biohem 1974;11:27-18. 8 Cousins S, Lewis SM. A rapid and aurate differential entrifugation method for platelet ounts. J Clin Pathol 1982;35: 114-16. 9 Lewis SM, Skelly JV, Cousins S. Automated platelet ounting-a re-evaluation of the sedimentation methods. Clin Lab Haematol 1981;3:215-22. 1 Lewis SM, Wardle J, Cousins S, Skelly JV. Platelet ounting-development as a referene method and a referene preparation. Clin Lab Haematol 1979;1:227-37. J Clin Pathol: first published as 1.1136/jp.43.11.932 on 1 November 199. Downloaded from http://jp.bmj.om/ on 24 September 218 by guest. Proteted by opyright.