Journal of Immunological Methods, 154 (1992) 155-161
155
1992 ElsevierSciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06427
Consistency of routine measurements of CD4 ÷, CD8 + peripheral blood lymphocytes J.P. A b o u l k e r a, B. A u t r a n h, K. Beldjord c, F. T o u r a i n e d a n d P. D e b r e b a INSERM SCIO, 16 av. Paul Vaillant Couturier, 04807 l/'dlejui~ France, t, Laboratoire d'lmmunologie Cellulaire et Titndaire, URA CNRS 625, CH Piti~-Sall~tri~re, 75013 Paris, France, c Laboratoire d'lmmunologie, CH Lncnnec, 75015Paris, France, and d Laboratoire d'lmmunologie, CH Cardiologie, 69000 Lyon, France
(Received24 July 1991, revisedreceived16 March 1992, accepted 24 April 1992) In order to evaluate the reliability of CD4 and CD8 T lymphocyte counts in large scale studies, a quality control study was performed in 12 French laboratories. CD4 and CD8 counts, assessed by various haematological and immunological techniques, were compared in order to assess possible differences between the laboratories and the techniques used. Our data showed that (a) the consistency of CD4 measurements was satisfactory since the between-laboratory coefficient of variation for absolute CD4 cell numbers above 2 0 0 / m m 3 was around 15% instead of 5-10% for all laboratories but one; (b) the major sources of variability arose from the use of automatic devices in the two-step measurement procedure: immunophenotyping and haematological counting. These data suggest that muiticentre assays of CD4 and CD8 counts result in some increase in their variability. Nevertheless the results of large multicentric trials can be extrapolated with confidence in the routine c~re of HIV + patients. Together, the results justified the involvement of several experienced laboratories in a clinical trial of HIV-related disease. Key words: Lymphoc~jtenumber; Immunophenotyping; Quality control test; CIM T lymphocyte;CD8 T lymphocyte;Automatic
device
Introduction CD4 and CD8 T lymphocyte blood counts currently serve as routine tests in the follow up of HIV+-infected patients (Schwartz et al., 1985; Lang et al., 1988; Murray et al., 1989; Lang~ et al., 1989). They have been extensively shown to be correlated with the clinical stage of the disease in large multicentric cohort studies (MacDonnel et al., 1990; Volberding et al., 1990) and are now used to determine therapeutic guidelines (Schwartz et al., 1985). In addition, they are increasingly used as a main surrogate marker in Correspondence to: J.P. Aboulker, INSERM SC10, 16 av. Paul Vaillant Couturier, 94807 Villejuif,France.
clinical trials (Collier et al., 1991). In such studies, which are most often multicentric, T lymphocyte subset counts cannot be easily centralised and performed in a single reference laboratory. Thus, it may happen that the samples are tested in as many as several tens of local units. Fluctuations of individual CEM and CD8 counts contrast with the progressive evolution over time of average counts established in groups of hundreds of subjects. The fluctuation may be due to technical errors a n d / o r to individual variations over time of T cell subsets. Errors in absolute CD4 or CD8 T lymphocyte blood counts can occur at two distinct levels: during the enumeration of peripheral blood total lymphocytes by haematological methods, a n d / o r during the determination of
lymphocyte subsets by immunophenotyping. The variability in both total and differential white blood cell counts, depending on the method, has been extensively documented (Goldner et al., 1938; Hughes-Jones et el., 1974; Ruemke et al., 1975; Ruemke, 1977; Carestein Hansen and Stanl, 1984). Nevertheless, in the case of HIV-infected patients, the reliability of methods based on differential countin8 has not received adequate attention. On the other hand, the variations of CD4 or CD8 T lymphocyte percentages by flow cytometry have been assessed experimentally (Hoffman et al., 1980; Morimoto et al., 1985) and are usually known for each parameter in a given laboratory. However, the influence of various factors such as the type of,.~ofluorometer or cell counter, and/or the variation between laboratories which occurs under routine conditions for CIM or CD8 counts in multicentric cohort studies have not been properly analysed and quantified. The aim of the present investigation was to assess the accuracy and variability of CD4 or CD8 cell counts when performed in different laboratories participating in a multicentric trial. The quality control was designed to assess the overall consistency of the CIM and CD8 T lymphocyte counts as they are routinely performed and to analyse the different sources of variability which influence the final result.
Materials and methods Twelve H1V seropositive patients with CIM peripheral lymphocyte counts ranging from 30 to 900/mm s were tested in a single batch. Each blood sample was drawn between 8 and 9 a.m. and was fractionated into duplicate aliquots in order that they could be tested blindly in each laboratory during the subsequent 4 h. A panel of 12 university hospital laboratories located in five French towns (Paris, Lyon, Marseille, Nantes, Nice) participated in the study.
Lymphocyte numeration Total lymphocyte counts were determined by both automatic and microscopic haematoiogical methods.
Automatic measurements using flow cytometry and either morphological or cytochemical criteria were performed in nine of the 12 laboratories. The various items of equipment were as follows: S plus 2, S plus LMG (Conltronics), Ortho ELF 1500 (Ortho Diagnostic systems), HI, H6000 (Teclmicon). In six of these laboratories, differential counts were also determined by microscopy. Two slides were blindly evaluated for each sample, counting 1{~--400 cells per slide. The microscopic method alone was performed in three of the 12 laboratories. One of these used an automatic device for the reading of the slides (Hematrak).
Immlmofluore~ence tests Immunofluorescence (IF) tests were performed using whole blood IF staining and analysis as previously described (Ruemke et al., 1975; Schwartz et al., 1985). In total for the 12 laboratories, three sources of iysing solution were used (Becton Dickinson, Sunnyvale, CA; Coulter, Coultronics; Ortho Diagnostic Systems). The anti-CD4 monoclonal antibodies used were leu3 (Becton Dickinson), T4 (Coulter Coultronics), and OKT4 (Ortho Diagnostic Systems). In one laboratory (lab I), IF tests were also performed with double staining simultest (Becton Dickinson). IF staining was analysed using flow cytometry with the following equipment: Facscan (Becton Dickinson), Facstar (Becton Dickinson), Epics C (Coultronics), Epics Prof'de (Coulter, Couitronics). Ortho Spectrum IIl (Ortho Diagnostic Systems), ATC 2000 (Odam). Statistical methods Duplicate aliquots of each blood sample were tested in each laboratory in order to assess the within-laboratory coefficients of variation. Paired comparisons of the three different sources of MoAbs were performed in three out of the 12 laboratories using duplicate samples. To detect a possible ranking effect of the laboratory testing procedure on the results, the first of the two duplicate aliquots were serially tested first, and the second of them later. To assess overall variability between laboratories, a sample from each patient was tested in each of the 12 laboratories. The 288 aliquots (12 × 2 × 12) were
46.0 9.6
505 441
37.3 8.1
461
6 306 640 336 360 435 646 391 532 690 459 561
39.5 9.2
431
15 287 652 367 377 438 561 378 511 774 415 456
D 30 365 562 342 295 362 581 423 476 832 437 407
50 403 699 381 352 467 578 550 624 887 605 550
21 340 726 245 426 549 619 565 630 822 560 534
F
50.0 9.0
27.2 5.4
598 512 506
33 408 869 420 518 484 760 676 656 1014 672 673
E 24 360 683 286 409 530 726 567 609 809 541 560
H
127.0 24,7
515
33.4 5.9
563
19 86 24 370 341 378 605 921 855 248 192 378 338 381 450 532 420 544 490 928 714 554 509 575 552 350 640 1070 1042 925 680 629 646 464 631 646
G 33 324 846 336 450 527 693 572 615 1053 684 594
27.3 6.3
432
27 304 574 336 325 356 576 480 490 744 459 435
1 56 270 615 294 299 446 594 499 532 748 476 420
30.0 5.4
563
22 336 810 352 480 512 720 621 624 1036 620 627
J
533
30 336 836 391 416 493 684 546 576 945 551 594
41.8 7.6
549
26 343 782 389 464 511 727 570 620 1003 577 549
K 28 380 765 400 441 564 686 540 579 949 558 720
45.0 9.1
497
19 290 737 370 382 458 643 490 552 824 586 557
L 26 282 623 349 421 480 741 540 575 934 490 553
511
3 0 ± 17 347+ 45 742+117 343+ 62 407+ 66 491+ 68 665+ 90 524+ 75 577+ 76 8815:128 571+ 94 553± 04
Mean± betweenlab SD
Significant difference between duplicates in laboratories B. E. and J. In laboratory B there was a difference in total lymphocyte counts. In laboratories E. J there w~s a difference in typing.
Average (col. umn) 601 Within duplicate SD 55.2 CV(%) 9.2
7 360 765 336 390 435 612 460 585 744 493 527
23 384 652 313 312 476 608 389 522 628 496 494
18 401 733 300 464 544 623 516 599 776 619 470
44 320 882 460 561 629 693 572 672 910 805 594
1 2 3 4 5 6 7 8 9 10 11 12
48 432 972 414 464 595 759 600 720 975 646 648
C
B
A
No.
Patient Laboratory
Results of blind duplicate CIM + T lymphocyte determinations (absolute/nun 3) in 12 laboratories
TABLE I
centrally fractionated and labelled by random permutations in order that they could be blindly tested everywhere. Statistical methods included Student's t test (paired comparisons) and two-way analysis of variance (within-laboratory coefficient of variation).
Remits
Blind duplicate absolute CD4 + cell number Table I shows the results of absolute CD4 cell numbers obtained in the 12 laboratories. As can be seen, the reproducibility of the measurement within each laboratory was satisfactory, the coefficients of variation within-lab (standard error of the value divided by its mean) ranging from 5.4% to 9.6%, except for laboratory G. The means of the 12 duplicate CD4 cell determinations within each laboratory ranged from 431 to 6 0 1 / m m 3, suggesting that a substantial systematic difference might be encountered between two laboratories. However the overall between-lab coefficient of variation of individual results ranged from 12% to 18%, except for subject 1 with a very low CD4 cell number (see Table II).
Blind duplicate CD4 and CD8 + percentage Table II shows the mean results obtained for the CD8 percentage of the 12 subjects. Interestingly enough the coefficients of variation of the individual CD8 cell percentage between the 12 laboratories were similar to the values obtained for the CD4 cell percentages. The mean within-lab CD8 count varied from 841 to 1091/mm 3 suggesting a possible 20% systematic difference between two laboratories (data not shown).
TABLE II MEANS AND BETWEEN-LABORATORY STANDARD DEVIATION OF CD4, CD8 AND TOTAL LYMPHOCYTE COUNTS Patient No. 1 2 3 4 5 6 7 8 9 10 11 !2
CD4 ('70.'.a
CD8 (%)
Totallymphocyte (/mm3)
3.2 + 2.1 20.0+1.8 44.2+4.8 17.1+1.7 27.3+2,9 31.3+3.6 19.6+2.4 21,6+ 2.0 38.8+2.4 35.9+3.3 31.0+3.4 32.5+3.4
70.6+ 8.9 52.6+8.4 37.8+8.3 61.2+6.1 51.6+7.2 42,8+6,2 61.34-7.4 62.3+ 6.9 43.5+4.9 45.9+5.5 44.1+5.0 45.6+7.2
970.9+ 235.9 1737.5+165.9 1675.0+158.1 2005.8:t:281.5 1486.4.4-129.8 1573.7+140.8 3394.6+168.1 2426.6+ 242,7 1484.7+154.2 2448.9+220.9 1833.3+138.5 1701.4+174.5
° Mean of the 2x 12 CD4 determinationsobtained in the 12 laboratories+between-laboratoriesstandard deviation of the CD4 determinationsobtained in the 12 laboratories.
CD4 and CD8 percentage determinations according to the type of cytofluorometer Table III shows the percentages of CD4 cells as determined by the various flow cytometers. The means of the 12 duplicate determinations within each laboratory varied from 23.3% to 29.8% for CD4 and from 44.0% to 61.2% for CD8. Differences between the laboratories could be attributed to neither the monoclonal antibodies used, nor to the whole blood lysing procedure, since comparisons performed in three of the 12 laboratories did not show any significant difference in the results obtained with three different reagents (data not shown). On the other hand, when classified according to the type of cytometer used, the results were more consistent sug-
TABLE !il RESULTS OF CD4 AND CD8 DETERMINATIONS (%) IN THE 12 LABORATORIES ACCORDING TO TYPE OF CYTOFLUOROMETER
Laboratory CD4 (%) mean = CD8 (%) mean =
Becton Dickinson D 1 F 23.8 24.7 25.5 48.9 55.3 54.5
J 26.7 50.1
Ortho SpectrumI11 L B H 26.7 27.3 28.2 49.6 57.0 59.1
Odam F 28.2 48,6
= Expressedas the mean of duplicatevalues of the 12 patients in each laboratory.
Coultronics C E 24.8 27.3 45.7 44.0
Mean O 29.3 61.2
A 29.8 45.9
26.9 51.7
159 TABLE IV COMPARISON OF LEUKOCYTE DIFFERENTIAL COUNT (% OF TOTAL LYMPHOCYTES) USING A MICROSCOPE OR AN AUTOMATIC COUNTER Patient No.
% total I~nnphocytesin the differential count Microscope a
Automatic counters b
1 2 3 4 5 6 7 8 9 10 I1 12
22.9 39.7 45A 23A 37.1 28.9 40.8 35.3 34.2 29.1 40.9 37.6
23.1 39.8 47.3 28.1 36.9 28.2 40.5 37.0 33.4 31.7 41.0 39.5
Average
34.6
35.5
ferential c o u n t s o b t a i n e d u s i n g either a microscope (a pool o f 3400 cells c o u n t e d by a p a n e l o f 10 h a e m a t o l o g i s t s ) o r a n a u t o m a t i c device (a pool o f duplicate results o b t a i n e d in n i n e o f t h e 12 laboratories). O n average, t h e lymphocytosig m e a s u r e d by e i t h e r t h e " n o n - a u t o m a t i c " o r t h e a u t o m a t i c t e c h n i q u e s were very similar. S o m e d i s c r e p a n c i e s were, however, o b s e r v e d b e t w e e n individual differential c o u n t s o b t a i n e d by t h e two t e c h n i q u e s in s o m e patients. T h e s e d i f f e r e n c e s w e r e always attributable to t h e lack o f accuracy or reproducibility o f t h e t e c h n i q u e s establishing t h e p e r c e n t a g e o f total iympbocytes by c o u n t i n g a limited n u m b e r o f cells ( < 400) ( d a t a n o t shown),
Blind duplicate total lymphocyte counts according to the type of cell counter used
a Pooled results of the blind duplicate determinations of differential count using a microscope in nine laboratories: total counting of 3400 cells by 10 haematologists. b Pooled results of the duplicate automatic determinations of lymphocyte percentage using flow cytometry counters in nine laboratories.
gesting that machines and not reagents and/or investigators a r e t h e m a i n s o u r c e o f variability b e t w e e n laboratories.
Comparbon of leukocyte differential counts by microscopy or using automatic devices T a b l e IV c o m p a r e s t h e results o f t h e blind d u p l i c a t e d e t e r m i n a t i o n s o f t h e 12 individual dif-
In o r d e r to d e t e r m i n e t h e influence o f total c o u n t s in t h e overall variability o f a b s o l u t e C I ~ a n d C D 8 lymphocyte n u m b e r s , a n d to d e t e c t possible d i f f e r e n c e s b e t w e e n m e t h o d s we c o m p a r e d t h e d a t a o b t a i n e d o n duplicate s a m p l e s with t h e v a r i o u s types o f h a e m a t o i o g i c a l c o u n t e r . T a b l e V s h o w s t h e results o b t a i n e d in t h e 12 laboratories according to t h e type o f m e t h o d a n d m a c h i n e . A s s e e n , t h e coefficients o f variation for t h e total lymphocyte c o u n t r a n g e d , within e a c h laboratory, f r o m 2.2% to 7.0% for a u t o m a t i c m e t h o d s a n d f r o m 5.6% to 11.3% for m a n u a l m e t h o d s . T h e m e a n o f t h e 12 duplicate total lymphocyte d e t e r m i n a t i o n s in e a c h laboratory r a n g e d f r o m 1731 to 2039 l y m p h o c y t e s / m m 3 s h o w i n g that, i n d e p e n d e n t l y o f i m m u n o - t y p i n g , large systematic d i f f e r e n c e s c a n o c c u r b e t w e e n two laboratories at t h e h a e m a t o l o g i c a l m e a s u r i n g stage. T a b l e V clearly s h o w s that similar equip-
TABLE V RESULTS OF BLIND DUPLICATE TOTAL LYMPHOCYTE DETERMINATIONS (ram3) IN THE 12 LABORATORIES ACCORDING TO TYPE OF EQUIPMENT IN ROU FINE HAEMATOLOGICAL USE S Plus S Plus Ortho Technicon S plus 2 +slide +hematrak ELTI500 HI H6000 Laboratot3, B G Lab. mean a 1731.0 1747.0 Within-duplicate SD 195.9 181.8 CV (%) I 1.3 10.4
I 1766.8 93.3 5.3
S Plus S plus LMG + slide
D L C K H F A J E 1795.0 1857.4 1841.7 1926.0 1987.5 1980.1 2033.3 2033.3 2039.6 105.7 86.7 40.8 108.1 67.7 I11.1 141.4 57.7 59.6 5.9 4.7 2.2 5.6 3.4 5.6 7.0 Z8 2.9
a Expressed as the mean of duplicate lymphocytes counts per mm3 of the 12 patients in each laboratow.
ment gives comparable results, again suggesting that the differences observed between laboratories are mainly attributable to the apparatus used. The results obtained by microscopic methods (labs B, 0 and F) are distributed among the results obtained using automatic equipment with no evidence of systematic clustering.
Discussion Absolute CD4 and CD8 T lymphocyte counts are amongst the most important features in the follow up and therapeutic management of HIV + patients (Schwartz et al., 1985; Lang et al., 1988; Murray et al., 1989; Lange et al., 1989). Although routinely performed on whole blood (Caidwell and Taylor, 1986), the reliability of these tests in large scale studies has remained unestablished. We designed and instituted a quality control survey in a large panel of French university laboratories in order to assess the overall variability of the P.sts under routine conditions, and to analyse the various sources of variability which may influence the final results. We compared the CD4 and CD8 counts performed by multiple haematological and immunological techniques. These suggested that: (a) within-lab reproducibility of the absolute CD4 number was good with coefficients of variation below 10% in all of the laboratories involved in the study, except one; (b) overall variability could be divided into two components: immunophenotyping and total lymphocyte counting; even when automatic equipment was used, the lymphocyte counting step contributed almost half of the total valiability; (c) differential counting using a microscope should be avoided for total lymphocyte enumeration because its variability is generally high and its accuracy not good compared to automatic equipment; (d) variability in measurement was probably mainly attributable to the equipment and not to the reagents or sample processing; (e) in this study, between-laboratory coefficients of variation of CD4 absolute counts were around 15% instead to 5-10% within laboratories. These results may be regarded as satisfactory since they are of the same order of magnitude as the variability in total lymphocyte counts.
Although cohort studies of HIV + patients are now performed in several countries including France, this is, to our knowledge, the first quality control study using fresh samples to be organised on such a large scale. This may be related to the difficulties encountered in setting up such studies. While CD4 and CD8 subset characterization by flow cytometry has been extensively studied and reported (Hoffman et al., 1980; Morimoto et al., 1985; Caldwell and Taylor, 1986), no statistical analyses to assess the reliability of these parameters for therapeutic purposes have previously been performed. From our data, it is clear that all steps of the various techniques for determining accurate cell numbers should be properly evaluated in each laboratory. Under such condi. tions, it is not necessary to duplicate CD4 measurements. In addition, our data suggest that the participation of several experienced laboratories in a clinical trial involving HIV infection was not a major source of variation in the results, providing there is adequate control of lymphocyte counting procedures. It was not necessary to group the samples and perform all of the tests in a central place. Thus, our quality control validated the strategy currently used in cohort studies of HIV seropositive patients, and suggested that results concerning CD4 and CD8 numbers established in large multicentric research programmes can be extrapolated with confidence to the routine monitoring of HIV-infected patients.
References Caldwell, C.N. and Taylor, H.M. (1986) A rapid, no wash technic for immunopheno~pic analysis by flow cytometry. Am. J. Clin. Pathol. 86, 600. Carestein Hansen, A. and Stanl, M. (1984) Lymphocytecounting by cell size distribution analysis of leukocyteo compared with conventional blood film differential counts. Scand..I. Clin. Invest. 44, 211. Collier,A.C., Bozzene, S., Coombs, R.W. et al. (1991) A pilot study of low dose zidovudinein human immunodeficiency virus infection. N. Engl. J. Med. 323, 1015. (3oldner, F.M. and Mann, W.N. (1938) The statistical error of the differentialwhite count. Guy's Hosp. Rep. 88, 54. Hoffman, R.A., Kung, P.C., Hansen, W.P., and (3oldstein, (3. (1980) Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc. Natl. Acad. Sci. USA 77, 4914.
Hughes-Jones, N.C., Norlay, 1., Young, J.M.S. and England, J.M. (1974) Differential white cell counts by frequency distributio~ analysis of cell volumes. 3. C!in. Pathol. 27, 623. Lang, W., Perkins, H., Anderson, R.E., Royce, R., Jewell, N. and Winkelstein, W.J. (1988) Patterns of T lymphocyte changes with human immunodeficiency virus infection: from seroconversion to the development ,~t AIDS. J. Acquired Immune Defic. Syndr. 1, 367. Lange, J.M.A., De Wolf, P. and Goudsm;t, J. (1989) Markers for progression in HIV infection. AIDS 3, 5153. MacDonnel, ICB., Chmiel, J.S., Poggensee, L., Wu, S. and Phair, J.P. (1990) Predicting progression to AIDS: combined usefulness of CD4 lymphocyte counts and p24 antigenemia. Am. J. Med. 89, 706. Morimoto, C., Letvin, N.L., Bayd, A.W., Hagan, M., Brown, H.M., Koruacki, M.M. and Schlossn~:~n, S.F. (1985) The isolation and characterization of the human helper inducer T cell subset. J. lmmunoL 134, 3762.
Murray, H.W., Godbalt, J.H., Gwrita, IC and Roberts, R.B. (1989) Progression to AIDS in patients with lymphadenopathy or AIDS related complex: reappraisal of risk and predictive factors. Am. J. Med. 86, 533. Ruemke, C.L. (1977) The statiscally expected variability in differential leukocyte counting. In: J.A. Koepke (Ed.), Differential Leukocytes Counting, p. 39. Ruemke, C.L., Besemer, P.D. and Kirik, DJ. (1975) Normal values al~d least significance differences for diffential leukocytes counts. J. Chron. Dis. 28, 661. Schwartz, K., Visscher, B., Detels, R., Taylor, J., Nishanian, P. and Pahey, J.L. (1985) Immunologicalchanges in lymphadenopathy virus positive and negative symptomless male homosexuals: two years of observation. Lancet ii, 831. Volberding, P.A., Lagakos, S.W., Koch, M.A. et al. (1990) Zidovudine in asymptomatic human immunodeficiency virus infection. N. EngL J. Med. 322, 941.
155
1992 ElsevierSciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06427
Consistency of routine measurements of CD4 ÷, CD8 + peripheral blood lymphocytes J.P. A b o u l k e r a, B. A u t r a n h, K. Beldjord c, F. T o u r a i n e d a n d P. D e b r e b a INSERM SCIO, 16 av. Paul Vaillant Couturier, 04807 l/'dlejui~ France, t, Laboratoire d'lmmunologie Cellulaire et Titndaire, URA CNRS 625, CH Piti~-Sall~tri~re, 75013 Paris, France, c Laboratoire d'lmmunologie, CH Lncnnec, 75015Paris, France, and d Laboratoire d'lmmunologie, CH Cardiologie, 69000 Lyon, France
(Received24 July 1991, revisedreceived16 March 1992, accepted 24 April 1992) In order to evaluate the reliability of CD4 and CD8 T lymphocyte counts in large scale studies, a quality control study was performed in 12 French laboratories. CD4 and CD8 counts, assessed by various haematological and immunological techniques, were compared in order to assess possible differences between the laboratories and the techniques used. Our data showed that (a) the consistency of CD4 measurements was satisfactory since the between-laboratory coefficient of variation for absolute CD4 cell numbers above 2 0 0 / m m 3 was around 15% instead of 5-10% for all laboratories but one; (b) the major sources of variability arose from the use of automatic devices in the two-step measurement procedure: immunophenotyping and haematological counting. These data suggest that muiticentre assays of CD4 and CD8 counts result in some increase in their variability. Nevertheless the results of large multicentric trials can be extrapolated with confidence in the routine c~re of HIV + patients. Together, the results justified the involvement of several experienced laboratories in a clinical trial of HIV-related disease. Key words: Lymphoc~jtenumber; Immunophenotyping; Quality control test; CIM T lymphocyte;CD8 T lymphocyte;Automatic
device
Introduction CD4 and CD8 T lymphocyte blood counts currently serve as routine tests in the follow up of HIV+-infected patients (Schwartz et al., 1985; Lang et al., 1988; Murray et al., 1989; Lang~ et al., 1989). They have been extensively shown to be correlated with the clinical stage of the disease in large multicentric cohort studies (MacDonnel et al., 1990; Volberding et al., 1990) and are now used to determine therapeutic guidelines (Schwartz et al., 1985). In addition, they are increasingly used as a main surrogate marker in Correspondence to: J.P. Aboulker, INSERM SC10, 16 av. Paul Vaillant Couturier, 94807 Villejuif,France.
clinical trials (Collier et al., 1991). In such studies, which are most often multicentric, T lymphocyte subset counts cannot be easily centralised and performed in a single reference laboratory. Thus, it may happen that the samples are tested in as many as several tens of local units. Fluctuations of individual CEM and CD8 counts contrast with the progressive evolution over time of average counts established in groups of hundreds of subjects. The fluctuation may be due to technical errors a n d / o r to individual variations over time of T cell subsets. Errors in absolute CD4 or CD8 T lymphocyte blood counts can occur at two distinct levels: during the enumeration of peripheral blood total lymphocytes by haematological methods, a n d / o r during the determination of
lymphocyte subsets by immunophenotyping. The variability in both total and differential white blood cell counts, depending on the method, has been extensively documented (Goldner et al., 1938; Hughes-Jones et el., 1974; Ruemke et al., 1975; Ruemke, 1977; Carestein Hansen and Stanl, 1984). Nevertheless, in the case of HIV-infected patients, the reliability of methods based on differential countin8 has not received adequate attention. On the other hand, the variations of CD4 or CD8 T lymphocyte percentages by flow cytometry have been assessed experimentally (Hoffman et al., 1980; Morimoto et al., 1985) and are usually known for each parameter in a given laboratory. However, the influence of various factors such as the type of,.~ofluorometer or cell counter, and/or the variation between laboratories which occurs under routine conditions for CIM or CD8 counts in multicentric cohort studies have not been properly analysed and quantified. The aim of the present investigation was to assess the accuracy and variability of CD4 or CD8 cell counts when performed in different laboratories participating in a multicentric trial. The quality control was designed to assess the overall consistency of the CIM and CD8 T lymphocyte counts as they are routinely performed and to analyse the different sources of variability which influence the final result.
Materials and methods Twelve H1V seropositive patients with CIM peripheral lymphocyte counts ranging from 30 to 900/mm s were tested in a single batch. Each blood sample was drawn between 8 and 9 a.m. and was fractionated into duplicate aliquots in order that they could be tested blindly in each laboratory during the subsequent 4 h. A panel of 12 university hospital laboratories located in five French towns (Paris, Lyon, Marseille, Nantes, Nice) participated in the study.
Lymphocyte numeration Total lymphocyte counts were determined by both automatic and microscopic haematoiogical methods.
Automatic measurements using flow cytometry and either morphological or cytochemical criteria were performed in nine of the 12 laboratories. The various items of equipment were as follows: S plus 2, S plus LMG (Conltronics), Ortho ELF 1500 (Ortho Diagnostic systems), HI, H6000 (Teclmicon). In six of these laboratories, differential counts were also determined by microscopy. Two slides were blindly evaluated for each sample, counting 1{~--400 cells per slide. The microscopic method alone was performed in three of the 12 laboratories. One of these used an automatic device for the reading of the slides (Hematrak).
Immlmofluore~ence tests Immunofluorescence (IF) tests were performed using whole blood IF staining and analysis as previously described (Ruemke et al., 1975; Schwartz et al., 1985). In total for the 12 laboratories, three sources of iysing solution were used (Becton Dickinson, Sunnyvale, CA; Coulter, Coultronics; Ortho Diagnostic Systems). The anti-CD4 monoclonal antibodies used were leu3 (Becton Dickinson), T4 (Coulter Coultronics), and OKT4 (Ortho Diagnostic Systems). In one laboratory (lab I), IF tests were also performed with double staining simultest (Becton Dickinson). IF staining was analysed using flow cytometry with the following equipment: Facscan (Becton Dickinson), Facstar (Becton Dickinson), Epics C (Coultronics), Epics Prof'de (Coulter, Couitronics). Ortho Spectrum IIl (Ortho Diagnostic Systems), ATC 2000 (Odam). Statistical methods Duplicate aliquots of each blood sample were tested in each laboratory in order to assess the within-laboratory coefficients of variation. Paired comparisons of the three different sources of MoAbs were performed in three out of the 12 laboratories using duplicate samples. To detect a possible ranking effect of the laboratory testing procedure on the results, the first of the two duplicate aliquots were serially tested first, and the second of them later. To assess overall variability between laboratories, a sample from each patient was tested in each of the 12 laboratories. The 288 aliquots (12 × 2 × 12) were
46.0 9.6
505 441
37.3 8.1
461
6 306 640 336 360 435 646 391 532 690 459 561
39.5 9.2
431
15 287 652 367 377 438 561 378 511 774 415 456
D 30 365 562 342 295 362 581 423 476 832 437 407
50 403 699 381 352 467 578 550 624 887 605 550
21 340 726 245 426 549 619 565 630 822 560 534
F
50.0 9.0
27.2 5.4
598 512 506
33 408 869 420 518 484 760 676 656 1014 672 673
E 24 360 683 286 409 530 726 567 609 809 541 560
H
127.0 24,7
515
33.4 5.9
563
19 86 24 370 341 378 605 921 855 248 192 378 338 381 450 532 420 544 490 928 714 554 509 575 552 350 640 1070 1042 925 680 629 646 464 631 646
G 33 324 846 336 450 527 693 572 615 1053 684 594
27.3 6.3
432
27 304 574 336 325 356 576 480 490 744 459 435
1 56 270 615 294 299 446 594 499 532 748 476 420
30.0 5.4
563
22 336 810 352 480 512 720 621 624 1036 620 627
J
533
30 336 836 391 416 493 684 546 576 945 551 594
41.8 7.6
549
26 343 782 389 464 511 727 570 620 1003 577 549
K 28 380 765 400 441 564 686 540 579 949 558 720
45.0 9.1
497
19 290 737 370 382 458 643 490 552 824 586 557
L 26 282 623 349 421 480 741 540 575 934 490 553
511
3 0 ± 17 347+ 45 742+117 343+ 62 407+ 66 491+ 68 665+ 90 524+ 75 577+ 76 8815:128 571+ 94 553± 04
Mean± betweenlab SD
Significant difference between duplicates in laboratories B. E. and J. In laboratory B there was a difference in total lymphocyte counts. In laboratories E. J there w~s a difference in typing.
Average (col. umn) 601 Within duplicate SD 55.2 CV(%) 9.2
7 360 765 336 390 435 612 460 585 744 493 527
23 384 652 313 312 476 608 389 522 628 496 494
18 401 733 300 464 544 623 516 599 776 619 470
44 320 882 460 561 629 693 572 672 910 805 594
1 2 3 4 5 6 7 8 9 10 11 12
48 432 972 414 464 595 759 600 720 975 646 648
C
B
A
No.
Patient Laboratory
Results of blind duplicate CIM + T lymphocyte determinations (absolute/nun 3) in 12 laboratories
TABLE I
centrally fractionated and labelled by random permutations in order that they could be blindly tested everywhere. Statistical methods included Student's t test (paired comparisons) and two-way analysis of variance (within-laboratory coefficient of variation).
Remits
Blind duplicate absolute CD4 + cell number Table I shows the results of absolute CD4 cell numbers obtained in the 12 laboratories. As can be seen, the reproducibility of the measurement within each laboratory was satisfactory, the coefficients of variation within-lab (standard error of the value divided by its mean) ranging from 5.4% to 9.6%, except for laboratory G. The means of the 12 duplicate CD4 cell determinations within each laboratory ranged from 431 to 6 0 1 / m m 3, suggesting that a substantial systematic difference might be encountered between two laboratories. However the overall between-lab coefficient of variation of individual results ranged from 12% to 18%, except for subject 1 with a very low CD4 cell number (see Table II).
Blind duplicate CD4 and CD8 + percentage Table II shows the mean results obtained for the CD8 percentage of the 12 subjects. Interestingly enough the coefficients of variation of the individual CD8 cell percentage between the 12 laboratories were similar to the values obtained for the CD4 cell percentages. The mean within-lab CD8 count varied from 841 to 1091/mm 3 suggesting a possible 20% systematic difference between two laboratories (data not shown).
TABLE II MEANS AND BETWEEN-LABORATORY STANDARD DEVIATION OF CD4, CD8 AND TOTAL LYMPHOCYTE COUNTS Patient No. 1 2 3 4 5 6 7 8 9 10 11 !2
CD4 ('70.'.a
CD8 (%)
Totallymphocyte (/mm3)
3.2 + 2.1 20.0+1.8 44.2+4.8 17.1+1.7 27.3+2,9 31.3+3.6 19.6+2.4 21,6+ 2.0 38.8+2.4 35.9+3.3 31.0+3.4 32.5+3.4
70.6+ 8.9 52.6+8.4 37.8+8.3 61.2+6.1 51.6+7.2 42,8+6,2 61.34-7.4 62.3+ 6.9 43.5+4.9 45.9+5.5 44.1+5.0 45.6+7.2
970.9+ 235.9 1737.5+165.9 1675.0+158.1 2005.8:t:281.5 1486.4.4-129.8 1573.7+140.8 3394.6+168.1 2426.6+ 242,7 1484.7+154.2 2448.9+220.9 1833.3+138.5 1701.4+174.5
° Mean of the 2x 12 CD4 determinationsobtained in the 12 laboratories+between-laboratoriesstandard deviation of the CD4 determinationsobtained in the 12 laboratories.
CD4 and CD8 percentage determinations according to the type of cytofluorometer Table III shows the percentages of CD4 cells as determined by the various flow cytometers. The means of the 12 duplicate determinations within each laboratory varied from 23.3% to 29.8% for CD4 and from 44.0% to 61.2% for CD8. Differences between the laboratories could be attributed to neither the monoclonal antibodies used, nor to the whole blood lysing procedure, since comparisons performed in three of the 12 laboratories did not show any significant difference in the results obtained with three different reagents (data not shown). On the other hand, when classified according to the type of cytometer used, the results were more consistent sug-
TABLE !il RESULTS OF CD4 AND CD8 DETERMINATIONS (%) IN THE 12 LABORATORIES ACCORDING TO TYPE OF CYTOFLUOROMETER
Laboratory CD4 (%) mean = CD8 (%) mean =
Becton Dickinson D 1 F 23.8 24.7 25.5 48.9 55.3 54.5
J 26.7 50.1
Ortho SpectrumI11 L B H 26.7 27.3 28.2 49.6 57.0 59.1
Odam F 28.2 48,6
= Expressedas the mean of duplicatevalues of the 12 patients in each laboratory.
Coultronics C E 24.8 27.3 45.7 44.0
Mean O 29.3 61.2
A 29.8 45.9
26.9 51.7
159 TABLE IV COMPARISON OF LEUKOCYTE DIFFERENTIAL COUNT (% OF TOTAL LYMPHOCYTES) USING A MICROSCOPE OR AN AUTOMATIC COUNTER Patient No.
% total I~nnphocytesin the differential count Microscope a
Automatic counters b
1 2 3 4 5 6 7 8 9 10 I1 12
22.9 39.7 45A 23A 37.1 28.9 40.8 35.3 34.2 29.1 40.9 37.6
23.1 39.8 47.3 28.1 36.9 28.2 40.5 37.0 33.4 31.7 41.0 39.5
Average
34.6
35.5
ferential c o u n t s o b t a i n e d u s i n g either a microscope (a pool o f 3400 cells c o u n t e d by a p a n e l o f 10 h a e m a t o l o g i s t s ) o r a n a u t o m a t i c device (a pool o f duplicate results o b t a i n e d in n i n e o f t h e 12 laboratories). O n average, t h e lymphocytosig m e a s u r e d by e i t h e r t h e " n o n - a u t o m a t i c " o r t h e a u t o m a t i c t e c h n i q u e s were very similar. S o m e d i s c r e p a n c i e s were, however, o b s e r v e d b e t w e e n individual differential c o u n t s o b t a i n e d by t h e two t e c h n i q u e s in s o m e patients. T h e s e d i f f e r e n c e s w e r e always attributable to t h e lack o f accuracy or reproducibility o f t h e t e c h n i q u e s establishing t h e p e r c e n t a g e o f total iympbocytes by c o u n t i n g a limited n u m b e r o f cells ( < 400) ( d a t a n o t shown),
Blind duplicate total lymphocyte counts according to the type of cell counter used
a Pooled results of the blind duplicate determinations of differential count using a microscope in nine laboratories: total counting of 3400 cells by 10 haematologists. b Pooled results of the duplicate automatic determinations of lymphocyte percentage using flow cytometry counters in nine laboratories.
gesting that machines and not reagents and/or investigators a r e t h e m a i n s o u r c e o f variability b e t w e e n laboratories.
Comparbon of leukocyte differential counts by microscopy or using automatic devices T a b l e IV c o m p a r e s t h e results o f t h e blind d u p l i c a t e d e t e r m i n a t i o n s o f t h e 12 individual dif-
In o r d e r to d e t e r m i n e t h e influence o f total c o u n t s in t h e overall variability o f a b s o l u t e C I ~ a n d C D 8 lymphocyte n u m b e r s , a n d to d e t e c t possible d i f f e r e n c e s b e t w e e n m e t h o d s we c o m p a r e d t h e d a t a o b t a i n e d o n duplicate s a m p l e s with t h e v a r i o u s types o f h a e m a t o i o g i c a l c o u n t e r . T a b l e V s h o w s t h e results o b t a i n e d in t h e 12 laboratories according to t h e type o f m e t h o d a n d m a c h i n e . A s s e e n , t h e coefficients o f variation for t h e total lymphocyte c o u n t r a n g e d , within e a c h laboratory, f r o m 2.2% to 7.0% for a u t o m a t i c m e t h o d s a n d f r o m 5.6% to 11.3% for m a n u a l m e t h o d s . T h e m e a n o f t h e 12 duplicate total lymphocyte d e t e r m i n a t i o n s in e a c h laboratory r a n g e d f r o m 1731 to 2039 l y m p h o c y t e s / m m 3 s h o w i n g that, i n d e p e n d e n t l y o f i m m u n o - t y p i n g , large systematic d i f f e r e n c e s c a n o c c u r b e t w e e n two laboratories at t h e h a e m a t o l o g i c a l m e a s u r i n g stage. T a b l e V clearly s h o w s that similar equip-
TABLE V RESULTS OF BLIND DUPLICATE TOTAL LYMPHOCYTE DETERMINATIONS (ram3) IN THE 12 LABORATORIES ACCORDING TO TYPE OF EQUIPMENT IN ROU FINE HAEMATOLOGICAL USE S Plus S Plus Ortho Technicon S plus 2 +slide +hematrak ELTI500 HI H6000 Laboratot3, B G Lab. mean a 1731.0 1747.0 Within-duplicate SD 195.9 181.8 CV (%) I 1.3 10.4
I 1766.8 93.3 5.3
S Plus S plus LMG + slide
D L C K H F A J E 1795.0 1857.4 1841.7 1926.0 1987.5 1980.1 2033.3 2033.3 2039.6 105.7 86.7 40.8 108.1 67.7 I11.1 141.4 57.7 59.6 5.9 4.7 2.2 5.6 3.4 5.6 7.0 Z8 2.9
a Expressed as the mean of duplicate lymphocytes counts per mm3 of the 12 patients in each laboratow.
ment gives comparable results, again suggesting that the differences observed between laboratories are mainly attributable to the apparatus used. The results obtained by microscopic methods (labs B, 0 and F) are distributed among the results obtained using automatic equipment with no evidence of systematic clustering.
Discussion Absolute CD4 and CD8 T lymphocyte counts are amongst the most important features in the follow up and therapeutic management of HIV + patients (Schwartz et al., 1985; Lang et al., 1988; Murray et al., 1989; Lange et al., 1989). Although routinely performed on whole blood (Caidwell and Taylor, 1986), the reliability of these tests in large scale studies has remained unestablished. We designed and instituted a quality control survey in a large panel of French university laboratories in order to assess the overall variability of the P.sts under routine conditions, and to analyse the various sources of variability which may influence the final results. We compared the CD4 and CD8 counts performed by multiple haematological and immunological techniques. These suggested that: (a) within-lab reproducibility of the absolute CD4 number was good with coefficients of variation below 10% in all of the laboratories involved in the study, except one; (b) overall variability could be divided into two components: immunophenotyping and total lymphocyte counting; even when automatic equipment was used, the lymphocyte counting step contributed almost half of the total valiability; (c) differential counting using a microscope should be avoided for total lymphocyte enumeration because its variability is generally high and its accuracy not good compared to automatic equipment; (d) variability in measurement was probably mainly attributable to the equipment and not to the reagents or sample processing; (e) in this study, between-laboratory coefficients of variation of CD4 absolute counts were around 15% instead to 5-10% within laboratories. These results may be regarded as satisfactory since they are of the same order of magnitude as the variability in total lymphocyte counts.
Although cohort studies of HIV + patients are now performed in several countries including France, this is, to our knowledge, the first quality control study using fresh samples to be organised on such a large scale. This may be related to the difficulties encountered in setting up such studies. While CD4 and CD8 subset characterization by flow cytometry has been extensively studied and reported (Hoffman et al., 1980; Morimoto et al., 1985; Caldwell and Taylor, 1986), no statistical analyses to assess the reliability of these parameters for therapeutic purposes have previously been performed. From our data, it is clear that all steps of the various techniques for determining accurate cell numbers should be properly evaluated in each laboratory. Under such condi. tions, it is not necessary to duplicate CD4 measurements. In addition, our data suggest that the participation of several experienced laboratories in a clinical trial involving HIV infection was not a major source of variation in the results, providing there is adequate control of lymphocyte counting procedures. It was not necessary to group the samples and perform all of the tests in a central place. Thus, our quality control validated the strategy currently used in cohort studies of HIV seropositive patients, and suggested that results concerning CD4 and CD8 numbers established in large multicentric research programmes can be extrapolated with confidence to the routine monitoring of HIV-infected patients.
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