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Relative and absolute numbers of human lymphocyte subpopulations
Journal of Immunological Methods, 97 (1987) 251-258 Elsevier
251
JIM 04255
Relative and absolute numbers of human lymphocyte subpopulations A comparison of immunofluorescence microscopy and flow cytometric methodologies with special reference to precision and reference values Ernst Kreuzfelder 1, G u a n x i n Shen 1, Ulrich Rodeck 1, Erika K~3nig 2, Werner Luboldt 3, Heinz-Otto Keinecke 4, Norbert Brockmeyer 5 and Norbert Scheiermann 1 I Institute for Medical Virology and Immunology, 2 The Medical Clinic and Policlinic, Department of Hematology, 3 The Bloodbank and Department for Transfusion, 4 Institute for Medical Computer Science and Biomathematics, and 5 Department of Dermatology University of Essen, Medical School, HufelandstraJ3e 55, D-4300 Essen 1, F.R.G. (Received 12 May 1986, revised received 24 July 1986, accepted 20 November 1986)
Lymphocyte subpopulations were determined in blood samples from blood donors (40 women and 45 men) using immunofluorescence microscopy and flow cytometric methodologies. The study demonstrates the value of both methods for the enumeration of lymphocyte subpopulations. The advantages of employing an automated flow cytometer system are better precision and speed. The automated systems require a large initial technical and financial burden and are therefore probably destined to be reserved for the larger laboratory. There is a need for an adequate lymphocyte standard which shows little variation between aliquots and can be used for interlaboratory comparisons. Key words: Lymphocyte subpopulation; Immunofluorescence microscopy; Flow cytometry; Precision data; Reference range; Monoclonal antibody
Introduction
The introduction of immunofluorescence labelled monoclonal antibodies for the determination of lymphocyte subpopulations rapidly resulted in the incorporation of the automated flow cytome-
Correspondence to: E. Kreuzfelder, Institut ft~r Medizinische Virologie und Immunologie, Universit~itsklinJkum Essen (GHS), HufelandstraBe 55, D-4300 Essen 1, F.R.G. Abbreviations: CV, coefficient of variation; FITC, fluorescein isothiocyanate conjugate; GF, green fluorescence; NK, natural killer; PMT, photomultiplier; r, correlation coefficient.
ter in addition to the conventional immunofluorescence microscope technique for the analysis of human blood samples. Over the past 2 years a number of commercial flow cytometers have appeared that, in comparison to previous models, now permit the routine enumeration and analysis of lymphocyte subpopulations. However, it is difficult to evaluate and compare the measurements obtained by different techniques. For this reason values are here presented from a study of blood donors giving both precision data and reference ranges of the relative and absolute numbers of lymphocyte subpopulations determined by microscopic and flow cytometric methodologies.
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
252
Demographic data and blood sampling The blood samples used for the evaluation of 'precision from day to day' were obtained from laboratory personnel. These included three males (ages 28, 29, 41 years) and one female (age 23 years). The data for 'precision in series' and the 'reference range' values were collected from the analysis of blood samples of blood bank donors (collective reference). The age groups and sex of the collective reference is included in Table I. All blood samples were obtained from the donors by phlebotomy between the hours 8 and 9 a.m.
Methods Enumeration
The isolation of lymphocytes was accomplished using Vetren blood samples (1 ml Vetren 200 plus 19 ml blood) layered over Ficoll-Paque (Pharmacia). After washing, the number of harvested cells was adjusted to 7.5 × 10 6 cells/ml. 20 /~1 of this cell suspension was then incubated for 60 min at 4°C together with 20 #1 of each of the selected monoclonal antibodies. The following monoclonal antibodies were selected for evaluation: O K T l l , OKT3 (total T lymphocyte population), helper/inducer T lymphocyte subset OKT4, suppressor/cytotoxic T lymphocyte subset OKT8. The O K T series of monoclonal antibodies are products of Ortho and
were used in a 1 : 15 dilution. Natural killer (NK) cells were labelled using the monoclonal antibody Leu7 from Becton Dickinson. Following the initial incubation phase with the primary antibody the cells were washed and incubated for 60 min at 4°C with a fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse immunoglobulin (Medac) secondary antibody. The labelled cells were examined using a Zeiss fluorescence microscope with the excitation filter BG 12 (absorption filter 50) and total magnification of 625 × . The percent of labelled lymphocytes, a field of 100 lymphocytes, was registered. The same labelled cell suspension was analysed on the Epics C flow cytometer (Coulter Electronics). For this phase of the study an argon laser (250 mW) 488 nm wavelength was used to excite the FITC monoclonal antibodies. During the optical analysis of the green fluorescence (GF) the following instrument settings were used: photomultiplier (PMT) 1200 V, forward angle light scatter photodiode 20 V, and 90 ° light scatter PMT 500 V. The filters installed in the instrument at the time of analysis were: 514 nm long pass dichroic filter (90 ° light scatter), 560 nm short pass dichroic filter (GF), 525 nm band pass absorption filter (GF). The immunofluorescence data was only accepted from those cells which were deemed to be lymphocytes through the fulfilment of other criteria, i.e., size (forward angle light scatter) and structure (90 ° light scatter). The negative controls (reactivity with anti-mouse immunoglobulin) yielded negligible fluorescence
TABLE I D E T A I L S OF T H E C O L L E C T I V E R E F E R E N C E M A T E R I A L U S E D IN THIS STUDY Analysis of blood donor groups with respect to age (yr) and sex (f, female; m, male) Age group a
Female
Male
n b
.g c
Range
n
2
Range
2 (16-30 yr) 3 (31-45 yr) 4 (46-60 yr)
23 11 6
24 yr 40 yr 53 yr
18-30 yr 33-45 yr 48-58 yr
26 12 7
26 yr 38 yr 49 yr
19-29 yr 34-44 yr 46-53 yr
2-4
40
33 yr
18-58 yr
45
33 yr
i 9 - 5 3 yr
2 - 4 (f + m)
85
33 yr
18-58 yr
a Rodeck et al. (1983) grouping. b n = number. c ~ = average.
253 readings. Different numbers of cells were analysed depending on the particular monoclonal antibody used: OKT3 (2000), O K T l l (5000), O K T 4 (5000), O K T 8 (5000) and Leu7 (10000 cells). The O K T l l antibody labelled cells were analysed in the logarithmic mode. Both the immunofluorescence microscopy and the flow cytometer measurements were performed alternatively by three technicians. The absolute cell numbers were calculated from the leucocyte number (Model S Plus II, Coulter Electronics) and the relative number of lymphocytes derived from a differential blood smear.
Standard lymphocytes D a t a for the 'precision from day to day' were obtained from both freshly prepared lymphocyte suspensions and from cryopreserved lymphocytes. The method of cryopreservation was a modification of the method published by Lenhard (1983) based on changes in the freezing rate: down to - 2 0 ° C at l ° C / m i n ; to - 4 0 ° at 2 ° C / m i n ; and to - 150°C at 1 0 ° C / m i n . Using the trypan blue technique the number of viable cells in the cryopreserved cell suspensions proved to be in excess of 80%. Statistics The Wilcoxon test was employed in order to obtain the mean comparison of values for the collective reference whilst taking the sex differences into consideration and to check whether inter-operator error was greater than intra-operator error. The differences between the paired microscope assay and Epics C technique derived values were calculated using the sign-level test and Spearman's correlation test was used to compare groups. Differences between coefficient of variation (CV) values were calculated according to Sachs (1984). 'Precision from day to day' CVs were calculated from non-weighted average values. The standard deviation in this case was derived by calculating corrected average values from individual samples. A significance level of 0.05 (two-tailed test) was used throughout. Statistics were performed using an IBM 4331 calculator V M / S P employing the SAS software package.
Results Only when the laser power was greater than 250 mW and the P M T (GF) was adjusted to greater than 800 V was it possible to analyse OKT3 + and OKT8 + cells. N o significant alterations in relative cell counts could be observed by further increases in either P M T or laser power. Since the instrument settings for the PMT and laser power were within the range tested, they were also used for the other parameters to be analysed. Both the Epics C and the microscopy evaluations of relative cell numbers were comparable (with the exception of O K T l l + cells). The following correlation coefficients ( r ) were calculated between the two methods using samples obtained from 15 blood donors: OKT3, r = 0.63; O K T l l , r = 0 . 3 5 ; OKT4, r = 0 . 6 9 ; OKT8, r = 0 . 7 6 ; and Leu7, r = 0.64. There was no significant alteration in these results after the further evaluation of an additional ten donors. Even after analyzing 2000 cells it was evident that the average relative cell number (Epics C) for O K T l l + lymphocytes was significantly higher than the microscopy evaluations for the same samples. In the case of O K T 4 + lymphocytes the opposite was true.
Precision 'Precision in series' data generated from the flow cytometer analysis of a total of 15 data points utilizing isolated lymphocytes from one donor revealed the following CVs for the respective subsets: OKT3 +, CV = 2.6%; O K T l l + , CV -3.1%; OKT4 +, 2.9%; OKT8 +, C V = 4 . 8 % ; and Leu7 + cells, C V = 2 5 . 8 % . Another experiment done to establish intra-versus inter-operator error revealed the data summarized in Table II. The parameters, OKT3 + cells (flow cytometry) and OKT8 + lymphocytes (microscopy) are significantly influenced by operator. With the exception of OKT3 + cells the CVs varied in both experiments. Table III shows the 'precision from day to day' data. It can readily be seen from these data that the CVs also varied with respect to the donors. There was no apparent change in the antigen determinants that could be attributed to cryopre-
254 TABLE II EVALUATION OF WITHIN-SERIES PRECISION 15-fold T-lymphocytes (OKT) enumeration of a donor blood sample performed by laboratory personnel (Nos. 1-3) using microscopy (M) and flow cytometry (E). no.
M/E
OKT3
y
SD
.~
SD
CV
1
M E
58.6 75.4 d
4.3 1.5
7.3 2.0
39.6 49.6
3.9 3.5
9.8 7.1
11.5 d 20.5
2.2 4.8
19.1 23.4
2
M E
58.8 75.5 d
5.8 1.5
9.9 2.0
41.5 50.9
4.1 2.5
9.9 4.9
16.2 d 20.3
3.7 2.6
22.8 11.3
3
M E
62.1 73.6 d
6.9 2.3
11.1 3.1
41.9 50.2
4.9 3.0
11.7 6.0
18.5 d 20.3
4.0 3.9
21.9 19.2
a
OKT4 SD b
CV c
OKT8 CV
a 2: average (%). SD: standard deviation (%). CV: coefficient of variation. d Value significantly influenced by operator. b
servation. This is evident from the results of evaluations carried out before cryopreservation and after thawing (average _+ standard deviation of microscopy values) on one lot of " s t a n d a r d lymphocytes': O K T 3 +, 57% (55 _+ 10%); O K T 4 +, 46% (36 + 7%); O K T 8 + lymphocytes, 14% (17 _+ 6%). The other data in Table I I I was derived from another blood donor. Close analysis of the CVs shows better precision for those values derived from flow cytometry c o m p a r e d to those obtained by microscopy. A significant difference was evident over the low average value range, i.e., parameters O K T 4 +, O K T 8 +, and Leu7 + cells. Person no. 1 (Table III) was remarkable in that the values obtained by the different methodologies resulted in significant differences in the numbers of cells. The O K T 4 / 8 ration obtained by microscopy was 1.9, significantly higher than the ratio of 1.2 derived by cytometry. This person proved to have alterations in lymphocyte morp h o l o g y due to virus infection. Functional testing in vitro, through mitogen stimulation, of the peripheral lymphocytes of this person were also abnormal.
Reference range Table IV includes b o t h relative and absolute cell numbers from blood donors. Microscopic evaluations of O K T 8 + lympho-
cytes (relative and absolute cell count) and O K T 4 / 8 ratios demonstrated significant sex related differences, whereby the male donors showed higher O K T 8 + lymphocyte values, and therefore lower O K T 4 / 8 ratios. Microscopic evaluations of O K T 4 / 8 ratios demonstrated significant age related differences. The values obtained, both from microscopy and flow cytometry (relative and absolute cell count), for N K cells demonstrated higher results rising with increasing age of the subjects. The comparison of average values obtained by microscopy from a study performed in 1982 in our Institute with those from 1985 showed significant higher numbers of O K T 3 + lymphocytes in the study performed in 1985.
Methodology comparison With the exception of the O K T l l * cells ( r = 0.26) the correlation coefficients were between r = 0.58 ( O K T 4 / 8 ) and r = 0.69 (OKT3 + lymphocytes). Flow cytometry gave significantly higher results than microscopy in the determination of O K T l l +, Leu7 + cells, and O K T 4 / 8 ratios. The O K T 4 + lymphocytes, on the other hand, showed lower values. A comparison between the results of microscopic and flow cytometric determinations on samples from 60 patients with different medical
255 TABLE III DAY TO DAY PRECISION T-lymphocytes (OKT) and natural killer cell (NK) enumeration of blood samples from laboratory personnel (nos. 1-4) and a standard lymphocyte preparation (no. 5), performed by microscopy (M) and flow cytometry (E). no.
M/E
OKT3 n
OKTll
a
.g b (%)
SD c (%)
CV d (%)
n
Y (%)
SD (%)
CV (%)
1
M E
8 8
69.5 71.5
9.2 9.9
13.2 13.9
7 7
64.3 82.7
11.2 5.2
17.4 6.2
2
M E
6 6
70.7 71.3
1.0 5.5
1.5 7.8
6 6
64.0 77.2
6.5 7.4
10.2 9.6
3
M E
5 6
54.4 56.3
10.5 10.1
19.2 18.0
5 6
53.0 62.2
8.2 14.8
15.5 23.9
4
M E
5 6
65.6 69.2
5.5 3.2
8.5 4.6
5 6
70.2 77.0
8.6 5.0
12.2 6.5
5
M E
12 20
41.2 39.2
7.6 5.3
18.5 13.4
12 18
44.3 58.4
5.8 5.8
13.1 9.9
1-5
M E
36 46
60.3 61.5
7.2 6.5
11.9 10.6
36 43
59.2 71.5
7.5 7.3
12.7 10.2
OKT4 n
OKT8 .~ (%)
SD (%)
CV (%)
8 8
44.5 38.1
8.3 3.9
18.6 10.2
6 6
49.8 43.8
6.9 1.6
6 6
34.2 31.8
5 6
n
Leu7 2 (%)
SD (%)
CV (%)
8 8
23.9 31.6
6.2 4.7
26.0 14.7
13.8 3.7
6 6
12.5 16.8
4.4 5.6
5.0 2.8
14.5 8.8
6 6
24.0 23.3
48.8 48.3
8.6 7.8
17.6 16.2
5 6
12 20
28.5 26.0
9.7 5.9
34.0 22.8
37 46
41.2 37.6
7.8 5.0
18.9 13.3 e
n
~ (%)
SD (%)
CV (%)
8 8
14.0 19.3
7.1 5.8
50.5 30.3
35.3 33.1
5 6
3.2 6.8
1.8 1.7
55.9 25.2
11.4 5.5
47.5 23.6
5 5
9.0 11.8
6.0 6.6
66.2 56.0
15.8 16.7
5.5 4.3
35.1 25.9
5 5
4.0 5.2
2.6 1.6
66.1 31.6
12 20
10.8 10.8
5.8 4.0
54.4 36.7
16 20
7.6 7.0
3.7 4.0
48.5 57.7
37 46
17.4 19.9
6.5 4.3
37.4 21.6 e
39 44
7.6 10.0
4.4 4.2
57.9 42.0 e
a n = number of days. b -x = average. ¢ SD = standard deviation. d CV = coefficient of variation. e Values significantly different from microscopic value.
histories produced
the following correlation coeffi-
OKT3 + and
cients:
r = 0.72;
fered
O K T 3 +,
OKTll
O K T 4 +, r = 0 . 5 8 ; O K T 8 +, r = r = 0.81; a n d O K T
4/8
+,
r = 0.25;
0 . 6 8 ; L e u 7 + cells,
r a t i o s , r = 0.74. O n l y t h e
OKTll
significantly
rived results.
+ lymphocyte from
the
numbers
dif-
flow cytometry
de-
M
E
OKT3 +
82
83
M
E
70
67
M
E
E
84
85
M
83
84
M
E
84
84
M
E
85
17
16
41
45
-
63
64
63
33
.
median (asymmetric distribution).
5 - 9 5 % = 5 - 9 5 percentile.
d Med=
¢ SD = standard deviation.
a n = n u m b e r o f donors. b .~ = a v e r a g e ( n o r m a l d i s t r i b u t i o n ) .
OKT4+/8 ~
Leu7 +
OKT8 +
OKT4 ÷
OKTll +
83
84
-
Lymphocytes
.
.
Leucocytes
a t/
• b
R e l a t i v e cell no.
M/E
Parameter
(E) d e t e r m i n a t i o n s
-
5-29
2-30
24-59
25-64
-
33-92
44-85
40-85
13-52
.
Y _+ 2 S D c .
-23
-23
31
-33
-64
-69
89
-92
-87
-88
-58
1.1
10.6
1.1-14.0
2
0
5
4
17
25
33
18
38
39
10
Range
2.3
2.6
9
6
76
-
Med a
20
-18
86
1 . 3 - 7.3
1 . 4 - 8.2
3
2
-
-
44
-
-
-
5-95% e
-
-
-
-
841
-
1480
1285
1 299
1279
2016
2
-
2648
-
-
119-1563
312
118-2451
249-2349
2 0 5 - 2 353
700-3332
2 + 2 SD
A b s o l u t e cell no.
2 717
3612
24
-
-
0-
44-
47-
225-
242-
347
292-
734
811
915
974
1842
2180
2962
2752
286- 2749
308-
550-
4 000-11400
Range
162
119
330
288
-
864
-
-
-
6 050
Med
46
24-
84-
87-
412
367
712
752
393-1595
-
-
-
4 2 0 0 - 9 060
5-95%
R e l a t i v e (%) a n d a b s o l u t e cell n u m b e r s ( c e l l s / ~ l b l o o d ) f o r T - l y m p h o c y t e s ( O K T ) a n d n a t u r a l killer cells ( L e u 7 ) in b l o o d d o n o r s u s i n g m i c r o s c o p i c ( M ) a n d flow c y t o m e t r i c
STATISTIC DATA
T A B L E IV
257 Discussion
The microscopic methodology was used as the basic technique for the evaluation of an automated flow cytometer because many data for lymphocyte subpopulation values in a blood donor population were available from the study of Rodeck et al. (1983) performed in our Institute. Optimal results (maximal values and minimal CVs) were achieved when 2000, 5000, 5000, 5000 and 10 000 cells respectively were accumulated for OKT3 +, O K T l l +, OKT4 ÷, OKT8 ÷ and Leu7 +. The average values for O K T l l ÷ lymphocytes were significantly higher, and the OKT4 + lymphocytes were significantly lower by flow cytometric analysis than by the microscopic technique. However, only after processing numerous samples from different persons is it possible to draw clear conclusions concerning the relation between 'precision in series' and number of accumulated cells, particularly in the case of OKT3 + lymphocyte measurements. Accumulated cell numbers were used throughout the study, since this leads to still further time saving when performing flow cytometry. This aspect is only one of the advantages of a flow cytometer, another being the greater precision than can be expected from microscopy determinations. This is especially true for cells present in low frequency, i.e., less than 40%. The parameters for the total T lymphocyte population (OKT3, 11) were found to have a 'precision from day to day' of 11%, whilst the other parameters demonstrated increasingly larger CVs (OKT4 +, 13%; OKT8 ÷, 22%, and Leu7 + cells 42%). Such values were obtained only by using fresh and cryopreserved lymphocytes and a statistical method which normalized the individual averages when calculating the standard deviations. Significant differences were noted in the 'precision from day to day' data due to the samples originating from different individuals. In an attempt to achieve better 'precision from day to day' it was decided to use cryopreserved lymphocytes. This method, even though comparable to the method of Nicholson et al. (1984) with regard to values found before freezing and after thawing of the samples, still does not guarantee homogeneity of the aliquots. Therefore, a statistical method
was employed which normalized the averages from the different individuals and lymphocyte preparations when calculating the standard deviations. Nevertheless, we were still unable to achieve 'precision from day to day' values comparable to those found by Seelig (1984) within one donor: OKT3 +, 5.8%; OKT4 +, 7.8%, and OKT8 + lymphocytes 11.9%. It is interesting to note that a correlation of average values of relative cell numbers also exists between this and other studies (i.e., OKT4 + lymphocytes (Morimoto et al., 1980; Bach et al., 1981; Landy et al. 1983)), although these investigations were performed using other types of flow cytometer. Moreover, the averages of the relative cell numbers were comparable to the results for OKT4 + lymphocytes obtained by Bongers and Bertrams (1984) using the microscopic technique. Other parameters did show minimal variations compared to other studies. Our average values for OKT8 + and Leu7 + cells are relatively low compared to those from Bach et al. (1981) with 26% and Abo et al. (1982) with 12% N K cells. The values obtained here and the ranges stated by Landy et al. (1983) demonstrate a good correlation. Variations are evident in the absolute cell numbers between our results and those from Burton et al. (1983). The values obtained here appear to be somewhat lower than those from Burton and co-workers. This is especially evident for the OKT8 + subset. These discrepancies could be due to differences in the donor population a n d / o r methods. In addition to smoking Burton et al. (1983) have shown that sex and age have an effect upon the distribution of the analysed cell types. Rodeck et al. (1983) and Bongers and Bertrams (1984) were able to demonstrate by microscopic procedures increases in relative cell numbers of OKT3 + and OKT4 + lymphocytes in the female population. Burton and co-workers (1983) using a flow cytometer also reported an increase in absolute cell numbers in the female population. Neither Landy et al. (1983) nor we could reproduce this effect using flow cytometry. We were, however, able to corroborate the findings of Rodeck et al. (1983) regarding the determinations of O K T 4 / 8 ratios by microscopy. Thus the ratio was lower for the male population than for females. In our
258
study, this is due to a significantly higher relative and absolute cell number of OKT8 positive cells in the male population. Neither Bongers and Bertrams (1984), Burton et al. (1983), Landy et al. (1983) nor we could demonstrate a general age related association as was reported by Rodeck et al. (1983). Perhaps this can be attributed to the fact that both Landy et al. (1983) and we restricted our studies to the 20-50 year-old age group. Changes in the above mentioned parameters can be observed in both children and older donors. We, as well as Abo et al. (1982) and Landy et al. (1983), also observed significantly higher absolute numbers of Leu7 positive cells in the aged. Although there were significant differences in average values of OKT3 + lymphocytes observed between our 1982 donors and the 1985 group, the relative values for other parameters (particularly O K T 4 + lymphocytes) remained constant over a period of 5 years. This suggests a remarkable consistency in the functional binding of the various antibodies as well as a stable expression of the corresponding antigens over this time span. Significant differences were found between microscopy and flow cytometry when using the O K T l l , OKT4, and Leu7 monoclonal antibodies. The O K T 4 / 8 ratio also differed significantly. The flow cytometry values for O K T l l + and Leu7 + positive cells were higher and the results for O K T 4 + lymphocytes were lower compared to' the results obtained using microscopy. The results of the two methods did, however, yield correlation coefficients ( r ) between 0.6 and 0.7. Landy and co-workers (1983) found correlation coefficients between 0.8 and 0.9 when using the OKT3, OKT4, and OKT8 antibodies in their study. A possible explanation for the differences between the two studies may be that we used three technicians in our study. Interestingly, with the exception of OKT3 and O K T l l , when patient populations were examined, no differences attributable to the methods were seen. Here, in agreement with Landy et al. (1983), we noted higher correlation coefficients
in the patient group than in the collective reference material. A possible explanation for this phenomenon is the broader range of values seen in the patients. The differences observed between the two methodologies can be explained in part by higher precision of the flow cytometer. During the study we were also able to observe decreased lymphocyte function in one individual with a lymphotropic viral infection without clinical symptoms. In this case 'the precision from day to day' value obtained by flow cytometry also showed a lower value compared to the value obtained by microscopy.
Acknowledgements We wish to express our appreciation to Mrs. Funk, Mrs. Gemlau, and Mrs. Dinkelbach for their excellent technical assistance.
References Abo, T., Cooper, M.D. and Balch, C.M. (1982) J. Exp. Med. 155, 321. Bach, M.-A., Phan-Dinh-Tuy, F., Bach, J.-F., Wallach, D., Biddison, W.E., Sharrow, S.O., Goldstein, G. and Kung, P.C. (1981) J. Immunol. 127, 980. Bongers, V. and Bertrams, J. (1984) J. Immunol. Methods 67, 243. Burton, R.C., Ferguson, P., Gray, M., Hall. J., Hayes, M. and Smart, Y.C. (1983) Diagn. Immunol. 1,216. Landay, A., Gartland, G.L., Abo, T. and Cooper, M.D. (1983) J. Immunol. Methods 58, 337. Lenhard, V. (1983) Lab. Med. 7, 17. Morimoto, C., Reinherz, E.L. Schlossman, S.F., Schur, P.H., Mills, J.A. and Steinberg, A.D. (1980) J. Clin. Invest. 66, 1171. Nicholson, J.K.A., Jones, B.M., Cross, G.D. and McDougal, J.S. (1984) J. Immunol. Methods 73, 29. Rodeck, U., Kuwert, E. and Keinecke, H.-O. (1983) Dtsch. Med. Wochenschr. 49, 1880. Sachs, L. (1984) Statistische Auswertungsmethoden (Springer Verlag, Berlin) p. 216. Seelig, H.P. (1984) Immun. Infekt. 12, 217.
251
JIM 04255
Relative and absolute numbers of human lymphocyte subpopulations A comparison of immunofluorescence microscopy and flow cytometric methodologies with special reference to precision and reference values Ernst Kreuzfelder 1, G u a n x i n Shen 1, Ulrich Rodeck 1, Erika K~3nig 2, Werner Luboldt 3, Heinz-Otto Keinecke 4, Norbert Brockmeyer 5 and Norbert Scheiermann 1 I Institute for Medical Virology and Immunology, 2 The Medical Clinic and Policlinic, Department of Hematology, 3 The Bloodbank and Department for Transfusion, 4 Institute for Medical Computer Science and Biomathematics, and 5 Department of Dermatology University of Essen, Medical School, HufelandstraJ3e 55, D-4300 Essen 1, F.R.G. (Received 12 May 1986, revised received 24 July 1986, accepted 20 November 1986)
Lymphocyte subpopulations were determined in blood samples from blood donors (40 women and 45 men) using immunofluorescence microscopy and flow cytometric methodologies. The study demonstrates the value of both methods for the enumeration of lymphocyte subpopulations. The advantages of employing an automated flow cytometer system are better precision and speed. The automated systems require a large initial technical and financial burden and are therefore probably destined to be reserved for the larger laboratory. There is a need for an adequate lymphocyte standard which shows little variation between aliquots and can be used for interlaboratory comparisons. Key words: Lymphocyte subpopulation; Immunofluorescence microscopy; Flow cytometry; Precision data; Reference range; Monoclonal antibody
Introduction
The introduction of immunofluorescence labelled monoclonal antibodies for the determination of lymphocyte subpopulations rapidly resulted in the incorporation of the automated flow cytome-
Correspondence to: E. Kreuzfelder, Institut ft~r Medizinische Virologie und Immunologie, Universit~itsklinJkum Essen (GHS), HufelandstraBe 55, D-4300 Essen 1, F.R.G. Abbreviations: CV, coefficient of variation; FITC, fluorescein isothiocyanate conjugate; GF, green fluorescence; NK, natural killer; PMT, photomultiplier; r, correlation coefficient.
ter in addition to the conventional immunofluorescence microscope technique for the analysis of human blood samples. Over the past 2 years a number of commercial flow cytometers have appeared that, in comparison to previous models, now permit the routine enumeration and analysis of lymphocyte subpopulations. However, it is difficult to evaluate and compare the measurements obtained by different techniques. For this reason values are here presented from a study of blood donors giving both precision data and reference ranges of the relative and absolute numbers of lymphocyte subpopulations determined by microscopic and flow cytometric methodologies.
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
252
Demographic data and blood sampling The blood samples used for the evaluation of 'precision from day to day' were obtained from laboratory personnel. These included three males (ages 28, 29, 41 years) and one female (age 23 years). The data for 'precision in series' and the 'reference range' values were collected from the analysis of blood samples of blood bank donors (collective reference). The age groups and sex of the collective reference is included in Table I. All blood samples were obtained from the donors by phlebotomy between the hours 8 and 9 a.m.
Methods Enumeration
The isolation of lymphocytes was accomplished using Vetren blood samples (1 ml Vetren 200 plus 19 ml blood) layered over Ficoll-Paque (Pharmacia). After washing, the number of harvested cells was adjusted to 7.5 × 10 6 cells/ml. 20 /~1 of this cell suspension was then incubated for 60 min at 4°C together with 20 #1 of each of the selected monoclonal antibodies. The following monoclonal antibodies were selected for evaluation: O K T l l , OKT3 (total T lymphocyte population), helper/inducer T lymphocyte subset OKT4, suppressor/cytotoxic T lymphocyte subset OKT8. The O K T series of monoclonal antibodies are products of Ortho and
were used in a 1 : 15 dilution. Natural killer (NK) cells were labelled using the monoclonal antibody Leu7 from Becton Dickinson. Following the initial incubation phase with the primary antibody the cells were washed and incubated for 60 min at 4°C with a fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse immunoglobulin (Medac) secondary antibody. The labelled cells were examined using a Zeiss fluorescence microscope with the excitation filter BG 12 (absorption filter 50) and total magnification of 625 × . The percent of labelled lymphocytes, a field of 100 lymphocytes, was registered. The same labelled cell suspension was analysed on the Epics C flow cytometer (Coulter Electronics). For this phase of the study an argon laser (250 mW) 488 nm wavelength was used to excite the FITC monoclonal antibodies. During the optical analysis of the green fluorescence (GF) the following instrument settings were used: photomultiplier (PMT) 1200 V, forward angle light scatter photodiode 20 V, and 90 ° light scatter PMT 500 V. The filters installed in the instrument at the time of analysis were: 514 nm long pass dichroic filter (90 ° light scatter), 560 nm short pass dichroic filter (GF), 525 nm band pass absorption filter (GF). The immunofluorescence data was only accepted from those cells which were deemed to be lymphocytes through the fulfilment of other criteria, i.e., size (forward angle light scatter) and structure (90 ° light scatter). The negative controls (reactivity with anti-mouse immunoglobulin) yielded negligible fluorescence
TABLE I D E T A I L S OF T H E C O L L E C T I V E R E F E R E N C E M A T E R I A L U S E D IN THIS STUDY Analysis of blood donor groups with respect to age (yr) and sex (f, female; m, male) Age group a
Female
Male
n b
.g c
Range
n
2
Range
2 (16-30 yr) 3 (31-45 yr) 4 (46-60 yr)
23 11 6
24 yr 40 yr 53 yr
18-30 yr 33-45 yr 48-58 yr
26 12 7
26 yr 38 yr 49 yr
19-29 yr 34-44 yr 46-53 yr
2-4
40
33 yr
18-58 yr
45
33 yr
i 9 - 5 3 yr
2 - 4 (f + m)
85
33 yr
18-58 yr
a Rodeck et al. (1983) grouping. b n = number. c ~ = average.
253 readings. Different numbers of cells were analysed depending on the particular monoclonal antibody used: OKT3 (2000), O K T l l (5000), O K T 4 (5000), O K T 8 (5000) and Leu7 (10000 cells). The O K T l l antibody labelled cells were analysed in the logarithmic mode. Both the immunofluorescence microscopy and the flow cytometer measurements were performed alternatively by three technicians. The absolute cell numbers were calculated from the leucocyte number (Model S Plus II, Coulter Electronics) and the relative number of lymphocytes derived from a differential blood smear.
Standard lymphocytes D a t a for the 'precision from day to day' were obtained from both freshly prepared lymphocyte suspensions and from cryopreserved lymphocytes. The method of cryopreservation was a modification of the method published by Lenhard (1983) based on changes in the freezing rate: down to - 2 0 ° C at l ° C / m i n ; to - 4 0 ° at 2 ° C / m i n ; and to - 150°C at 1 0 ° C / m i n . Using the trypan blue technique the number of viable cells in the cryopreserved cell suspensions proved to be in excess of 80%. Statistics The Wilcoxon test was employed in order to obtain the mean comparison of values for the collective reference whilst taking the sex differences into consideration and to check whether inter-operator error was greater than intra-operator error. The differences between the paired microscope assay and Epics C technique derived values were calculated using the sign-level test and Spearman's correlation test was used to compare groups. Differences between coefficient of variation (CV) values were calculated according to Sachs (1984). 'Precision from day to day' CVs were calculated from non-weighted average values. The standard deviation in this case was derived by calculating corrected average values from individual samples. A significance level of 0.05 (two-tailed test) was used throughout. Statistics were performed using an IBM 4331 calculator V M / S P employing the SAS software package.
Results Only when the laser power was greater than 250 mW and the P M T (GF) was adjusted to greater than 800 V was it possible to analyse OKT3 + and OKT8 + cells. N o significant alterations in relative cell counts could be observed by further increases in either P M T or laser power. Since the instrument settings for the PMT and laser power were within the range tested, they were also used for the other parameters to be analysed. Both the Epics C and the microscopy evaluations of relative cell numbers were comparable (with the exception of O K T l l + cells). The following correlation coefficients ( r ) were calculated between the two methods using samples obtained from 15 blood donors: OKT3, r = 0.63; O K T l l , r = 0 . 3 5 ; OKT4, r = 0 . 6 9 ; OKT8, r = 0 . 7 6 ; and Leu7, r = 0.64. There was no significant alteration in these results after the further evaluation of an additional ten donors. Even after analyzing 2000 cells it was evident that the average relative cell number (Epics C) for O K T l l + lymphocytes was significantly higher than the microscopy evaluations for the same samples. In the case of O K T 4 + lymphocytes the opposite was true.
Precision 'Precision in series' data generated from the flow cytometer analysis of a total of 15 data points utilizing isolated lymphocytes from one donor revealed the following CVs for the respective subsets: OKT3 +, CV = 2.6%; O K T l l + , CV -3.1%; OKT4 +, 2.9%; OKT8 +, C V = 4 . 8 % ; and Leu7 + cells, C V = 2 5 . 8 % . Another experiment done to establish intra-versus inter-operator error revealed the data summarized in Table II. The parameters, OKT3 + cells (flow cytometry) and OKT8 + lymphocytes (microscopy) are significantly influenced by operator. With the exception of OKT3 + cells the CVs varied in both experiments. Table III shows the 'precision from day to day' data. It can readily be seen from these data that the CVs also varied with respect to the donors. There was no apparent change in the antigen determinants that could be attributed to cryopre-
254 TABLE II EVALUATION OF WITHIN-SERIES PRECISION 15-fold T-lymphocytes (OKT) enumeration of a donor blood sample performed by laboratory personnel (Nos. 1-3) using microscopy (M) and flow cytometry (E). no.
M/E
OKT3
y
SD
.~
SD
CV
1
M E
58.6 75.4 d
4.3 1.5
7.3 2.0
39.6 49.6
3.9 3.5
9.8 7.1
11.5 d 20.5
2.2 4.8
19.1 23.4
2
M E
58.8 75.5 d
5.8 1.5
9.9 2.0
41.5 50.9
4.1 2.5
9.9 4.9
16.2 d 20.3
3.7 2.6
22.8 11.3
3
M E
62.1 73.6 d
6.9 2.3
11.1 3.1
41.9 50.2
4.9 3.0
11.7 6.0
18.5 d 20.3
4.0 3.9
21.9 19.2
a
OKT4 SD b
CV c
OKT8 CV
a 2: average (%). SD: standard deviation (%). CV: coefficient of variation. d Value significantly influenced by operator. b
servation. This is evident from the results of evaluations carried out before cryopreservation and after thawing (average _+ standard deviation of microscopy values) on one lot of " s t a n d a r d lymphocytes': O K T 3 +, 57% (55 _+ 10%); O K T 4 +, 46% (36 + 7%); O K T 8 + lymphocytes, 14% (17 _+ 6%). The other data in Table I I I was derived from another blood donor. Close analysis of the CVs shows better precision for those values derived from flow cytometry c o m p a r e d to those obtained by microscopy. A significant difference was evident over the low average value range, i.e., parameters O K T 4 +, O K T 8 +, and Leu7 + cells. Person no. 1 (Table III) was remarkable in that the values obtained by the different methodologies resulted in significant differences in the numbers of cells. The O K T 4 / 8 ration obtained by microscopy was 1.9, significantly higher than the ratio of 1.2 derived by cytometry. This person proved to have alterations in lymphocyte morp h o l o g y due to virus infection. Functional testing in vitro, through mitogen stimulation, of the peripheral lymphocytes of this person were also abnormal.
Reference range Table IV includes b o t h relative and absolute cell numbers from blood donors. Microscopic evaluations of O K T 8 + lympho-
cytes (relative and absolute cell count) and O K T 4 / 8 ratios demonstrated significant sex related differences, whereby the male donors showed higher O K T 8 + lymphocyte values, and therefore lower O K T 4 / 8 ratios. Microscopic evaluations of O K T 4 / 8 ratios demonstrated significant age related differences. The values obtained, both from microscopy and flow cytometry (relative and absolute cell count), for N K cells demonstrated higher results rising with increasing age of the subjects. The comparison of average values obtained by microscopy from a study performed in 1982 in our Institute with those from 1985 showed significant higher numbers of O K T 3 + lymphocytes in the study performed in 1985.
Methodology comparison With the exception of the O K T l l * cells ( r = 0.26) the correlation coefficients were between r = 0.58 ( O K T 4 / 8 ) and r = 0.69 (OKT3 + lymphocytes). Flow cytometry gave significantly higher results than microscopy in the determination of O K T l l +, Leu7 + cells, and O K T 4 / 8 ratios. The O K T 4 + lymphocytes, on the other hand, showed lower values. A comparison between the results of microscopic and flow cytometric determinations on samples from 60 patients with different medical
255 TABLE III DAY TO DAY PRECISION T-lymphocytes (OKT) and natural killer cell (NK) enumeration of blood samples from laboratory personnel (nos. 1-4) and a standard lymphocyte preparation (no. 5), performed by microscopy (M) and flow cytometry (E). no.
M/E
OKT3 n
OKTll
a
.g b (%)
SD c (%)
CV d (%)
n
Y (%)
SD (%)
CV (%)
1
M E
8 8
69.5 71.5
9.2 9.9
13.2 13.9
7 7
64.3 82.7
11.2 5.2
17.4 6.2
2
M E
6 6
70.7 71.3
1.0 5.5
1.5 7.8
6 6
64.0 77.2
6.5 7.4
10.2 9.6
3
M E
5 6
54.4 56.3
10.5 10.1
19.2 18.0
5 6
53.0 62.2
8.2 14.8
15.5 23.9
4
M E
5 6
65.6 69.2
5.5 3.2
8.5 4.6
5 6
70.2 77.0
8.6 5.0
12.2 6.5
5
M E
12 20
41.2 39.2
7.6 5.3
18.5 13.4
12 18
44.3 58.4
5.8 5.8
13.1 9.9
1-5
M E
36 46
60.3 61.5
7.2 6.5
11.9 10.6
36 43
59.2 71.5
7.5 7.3
12.7 10.2
OKT4 n
OKT8 .~ (%)
SD (%)
CV (%)
8 8
44.5 38.1
8.3 3.9
18.6 10.2
6 6
49.8 43.8
6.9 1.6
6 6
34.2 31.8
5 6
n
Leu7 2 (%)
SD (%)
CV (%)
8 8
23.9 31.6
6.2 4.7
26.0 14.7
13.8 3.7
6 6
12.5 16.8
4.4 5.6
5.0 2.8
14.5 8.8
6 6
24.0 23.3
48.8 48.3
8.6 7.8
17.6 16.2
5 6
12 20
28.5 26.0
9.7 5.9
34.0 22.8
37 46
41.2 37.6
7.8 5.0
18.9 13.3 e
n
~ (%)
SD (%)
CV (%)
8 8
14.0 19.3
7.1 5.8
50.5 30.3
35.3 33.1
5 6
3.2 6.8
1.8 1.7
55.9 25.2
11.4 5.5
47.5 23.6
5 5
9.0 11.8
6.0 6.6
66.2 56.0
15.8 16.7
5.5 4.3
35.1 25.9
5 5
4.0 5.2
2.6 1.6
66.1 31.6
12 20
10.8 10.8
5.8 4.0
54.4 36.7
16 20
7.6 7.0
3.7 4.0
48.5 57.7
37 46
17.4 19.9
6.5 4.3
37.4 21.6 e
39 44
7.6 10.0
4.4 4.2
57.9 42.0 e
a n = number of days. b -x = average. ¢ SD = standard deviation. d CV = coefficient of variation. e Values significantly different from microscopic value.
histories produced
the following correlation coeffi-
OKT3 + and
cients:
r = 0.72;
fered
O K T 3 +,
OKTll
O K T 4 +, r = 0 . 5 8 ; O K T 8 +, r = r = 0.81; a n d O K T
4/8
+,
r = 0.25;
0 . 6 8 ; L e u 7 + cells,
r a t i o s , r = 0.74. O n l y t h e
OKTll
significantly
rived results.
+ lymphocyte from
the
numbers
dif-
flow cytometry
de-
M
E
OKT3 +
82
83
M
E
70
67
M
E
E
84
85
M
83
84
M
E
84
84
M
E
85
17
16
41
45
-
63
64
63
33
.
median (asymmetric distribution).
5 - 9 5 % = 5 - 9 5 percentile.
d Med=
¢ SD = standard deviation.
a n = n u m b e r o f donors. b .~ = a v e r a g e ( n o r m a l d i s t r i b u t i o n ) .
OKT4+/8 ~
Leu7 +
OKT8 +
OKT4 ÷
OKTll +
83
84
-
Lymphocytes
.
.
Leucocytes
a t/
• b
R e l a t i v e cell no.
M/E
Parameter
(E) d e t e r m i n a t i o n s
-
5-29
2-30
24-59
25-64
-
33-92
44-85
40-85
13-52
.
Y _+ 2 S D c .
-23
-23
31
-33
-64
-69
89
-92
-87
-88
-58
1.1
10.6
1.1-14.0
2
0
5
4
17
25
33
18
38
39
10
Range
2.3
2.6
9
6
76
-
Med a
20
-18
86
1 . 3 - 7.3
1 . 4 - 8.2
3
2
-
-
44
-
-
-
5-95% e
-
-
-
-
841
-
1480
1285
1 299
1279
2016
2
-
2648
-
-
119-1563
312
118-2451
249-2349
2 0 5 - 2 353
700-3332
2 + 2 SD
A b s o l u t e cell no.
2 717
3612
24
-
-
0-
44-
47-
225-
242-
347
292-
734
811
915
974
1842
2180
2962
2752
286- 2749
308-
550-
4 000-11400
Range
162
119
330
288
-
864
-
-
-
6 050
Med
46
24-
84-
87-
412
367
712
752
393-1595
-
-
-
4 2 0 0 - 9 060
5-95%
R e l a t i v e (%) a n d a b s o l u t e cell n u m b e r s ( c e l l s / ~ l b l o o d ) f o r T - l y m p h o c y t e s ( O K T ) a n d n a t u r a l killer cells ( L e u 7 ) in b l o o d d o n o r s u s i n g m i c r o s c o p i c ( M ) a n d flow c y t o m e t r i c
STATISTIC DATA
T A B L E IV
257 Discussion
The microscopic methodology was used as the basic technique for the evaluation of an automated flow cytometer because many data for lymphocyte subpopulation values in a blood donor population were available from the study of Rodeck et al. (1983) performed in our Institute. Optimal results (maximal values and minimal CVs) were achieved when 2000, 5000, 5000, 5000 and 10 000 cells respectively were accumulated for OKT3 +, O K T l l +, OKT4 ÷, OKT8 ÷ and Leu7 +. The average values for O K T l l ÷ lymphocytes were significantly higher, and the OKT4 + lymphocytes were significantly lower by flow cytometric analysis than by the microscopic technique. However, only after processing numerous samples from different persons is it possible to draw clear conclusions concerning the relation between 'precision in series' and number of accumulated cells, particularly in the case of OKT3 + lymphocyte measurements. Accumulated cell numbers were used throughout the study, since this leads to still further time saving when performing flow cytometry. This aspect is only one of the advantages of a flow cytometer, another being the greater precision than can be expected from microscopy determinations. This is especially true for cells present in low frequency, i.e., less than 40%. The parameters for the total T lymphocyte population (OKT3, 11) were found to have a 'precision from day to day' of 11%, whilst the other parameters demonstrated increasingly larger CVs (OKT4 +, 13%; OKT8 ÷, 22%, and Leu7 + cells 42%). Such values were obtained only by using fresh and cryopreserved lymphocytes and a statistical method which normalized the individual averages when calculating the standard deviations. Significant differences were noted in the 'precision from day to day' data due to the samples originating from different individuals. In an attempt to achieve better 'precision from day to day' it was decided to use cryopreserved lymphocytes. This method, even though comparable to the method of Nicholson et al. (1984) with regard to values found before freezing and after thawing of the samples, still does not guarantee homogeneity of the aliquots. Therefore, a statistical method
was employed which normalized the averages from the different individuals and lymphocyte preparations when calculating the standard deviations. Nevertheless, we were still unable to achieve 'precision from day to day' values comparable to those found by Seelig (1984) within one donor: OKT3 +, 5.8%; OKT4 +, 7.8%, and OKT8 + lymphocytes 11.9%. It is interesting to note that a correlation of average values of relative cell numbers also exists between this and other studies (i.e., OKT4 + lymphocytes (Morimoto et al., 1980; Bach et al., 1981; Landy et al. 1983)), although these investigations were performed using other types of flow cytometer. Moreover, the averages of the relative cell numbers were comparable to the results for OKT4 + lymphocytes obtained by Bongers and Bertrams (1984) using the microscopic technique. Other parameters did show minimal variations compared to other studies. Our average values for OKT8 + and Leu7 + cells are relatively low compared to those from Bach et al. (1981) with 26% and Abo et al. (1982) with 12% N K cells. The values obtained here and the ranges stated by Landy et al. (1983) demonstrate a good correlation. Variations are evident in the absolute cell numbers between our results and those from Burton et al. (1983). The values obtained here appear to be somewhat lower than those from Burton and co-workers. This is especially evident for the OKT8 + subset. These discrepancies could be due to differences in the donor population a n d / o r methods. In addition to smoking Burton et al. (1983) have shown that sex and age have an effect upon the distribution of the analysed cell types. Rodeck et al. (1983) and Bongers and Bertrams (1984) were able to demonstrate by microscopic procedures increases in relative cell numbers of OKT3 + and OKT4 + lymphocytes in the female population. Burton and co-workers (1983) using a flow cytometer also reported an increase in absolute cell numbers in the female population. Neither Landy et al. (1983) nor we could reproduce this effect using flow cytometry. We were, however, able to corroborate the findings of Rodeck et al. (1983) regarding the determinations of O K T 4 / 8 ratios by microscopy. Thus the ratio was lower for the male population than for females. In our
258
study, this is due to a significantly higher relative and absolute cell number of OKT8 positive cells in the male population. Neither Bongers and Bertrams (1984), Burton et al. (1983), Landy et al. (1983) nor we could demonstrate a general age related association as was reported by Rodeck et al. (1983). Perhaps this can be attributed to the fact that both Landy et al. (1983) and we restricted our studies to the 20-50 year-old age group. Changes in the above mentioned parameters can be observed in both children and older donors. We, as well as Abo et al. (1982) and Landy et al. (1983), also observed significantly higher absolute numbers of Leu7 positive cells in the aged. Although there were significant differences in average values of OKT3 + lymphocytes observed between our 1982 donors and the 1985 group, the relative values for other parameters (particularly O K T 4 + lymphocytes) remained constant over a period of 5 years. This suggests a remarkable consistency in the functional binding of the various antibodies as well as a stable expression of the corresponding antigens over this time span. Significant differences were found between microscopy and flow cytometry when using the O K T l l , OKT4, and Leu7 monoclonal antibodies. The O K T 4 / 8 ratio also differed significantly. The flow cytometry values for O K T l l + and Leu7 + positive cells were higher and the results for O K T 4 + lymphocytes were lower compared to' the results obtained using microscopy. The results of the two methods did, however, yield correlation coefficients ( r ) between 0.6 and 0.7. Landy and co-workers (1983) found correlation coefficients between 0.8 and 0.9 when using the OKT3, OKT4, and OKT8 antibodies in their study. A possible explanation for the differences between the two studies may be that we used three technicians in our study. Interestingly, with the exception of OKT3 and O K T l l , when patient populations were examined, no differences attributable to the methods were seen. Here, in agreement with Landy et al. (1983), we noted higher correlation coefficients
in the patient group than in the collective reference material. A possible explanation for this phenomenon is the broader range of values seen in the patients. The differences observed between the two methodologies can be explained in part by higher precision of the flow cytometer. During the study we were also able to observe decreased lymphocyte function in one individual with a lymphotropic viral infection without clinical symptoms. In this case 'the precision from day to day' value obtained by flow cytometry also showed a lower value compared to the value obtained by microscopy.
Acknowledgements We wish to express our appreciation to Mrs. Funk, Mrs. Gemlau, and Mrs. Dinkelbach for their excellent technical assistance.
References Abo, T., Cooper, M.D. and Balch, C.M. (1982) J. Exp. Med. 155, 321. Bach, M.-A., Phan-Dinh-Tuy, F., Bach, J.-F., Wallach, D., Biddison, W.E., Sharrow, S.O., Goldstein, G. and Kung, P.C. (1981) J. Immunol. 127, 980. Bongers, V. and Bertrams, J. (1984) J. Immunol. Methods 67, 243. Burton, R.C., Ferguson, P., Gray, M., Hall. J., Hayes, M. and Smart, Y.C. (1983) Diagn. Immunol. 1,216. Landay, A., Gartland, G.L., Abo, T. and Cooper, M.D. (1983) J. Immunol. Methods 58, 337. Lenhard, V. (1983) Lab. Med. 7, 17. Morimoto, C., Reinherz, E.L. Schlossman, S.F., Schur, P.H., Mills, J.A. and Steinberg, A.D. (1980) J. Clin. Invest. 66, 1171. Nicholson, J.K.A., Jones, B.M., Cross, G.D. and McDougal, J.S. (1984) J. Immunol. Methods 73, 29. Rodeck, U., Kuwert, E. and Keinecke, H.-O. (1983) Dtsch. Med. Wochenschr. 49, 1880. Sachs, L. (1984) Statistische Auswertungsmethoden (Springer Verlag, Berlin) p. 216. Seelig, H.P. (1984) Immun. Infekt. 12, 217.