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Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype
Journal of Immunological Methods 228 Ž1999. 13–21 www.elsevier.nlrlocaterjim
Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype V. Deneys ) , V. Thiry, N. Hougardy, A.M. Mazzon, P. Leveugle, M. De Bruyere ` UniÕersite´ Catholique de LouÕain, UniÕersity Hospital, Immunohaematology Laboratory, 1200 Brussels, Belgium Received 2 November 1998; received in revised form 13 May 1999; accepted 24 May 1999
Abstract Background and objectiÕes: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. Material and methods: Blood samples from 15 CLL patients were analyzed freshly and after freezing at y1968C, without separation, and thawing. Results were compared by Student’s paired t-test. Results: The phenotype of fresh CLL cells was as follows: CD19q , CD5 q , faint CD20, CD23 " , weak CD22 and sIg, CD37q , HLA-DRq , FMC7y . After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes’s score was modified in 6 cases out of 15 Ž40%.. Conclusion: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chronic lymphocytic leukaemia; Immunophenotype; Cryopreservation; Flow cytometry; Cluster of differentiation
1. Introduction Cryopreservation of haemopoietic cells, for in vivo use or in vitro research purposes, has greatly increased in popularity in recent years. In order to avoid injury of the cells by direct effect of low temperature, and by the intracellular ice crystal formation occurring at high cooling rates ŽKarow and Webb, 1965. most of the procedures involve freezing at a slow cooling rate in the pres-
AbbreÕiations: CLL: Chronic lymphocytic leukaemia; CD: Cluster of differentiation; MFI: Median Fluorescence Intensity ) Corresponding author. Cliniques Universitaires Saint-LucUCL, Clos Chapelle-aux-Champs, 30 BP 30.52B-1200 Brussels. Tel.: q32-2-764 34 47; fax: q32-2-764 30 21; E-mail: [email protected]
ence of cryoprotective agents, such as glycerol, hydroxyethyl starch or dimethylsulfoxide, and storage in liquid nitrogen ŽRosillo et al., 1995. The infusion of cryopreserved haemopoietic stem cells reconstitutes safely and satisfactorily the function of the recipient’s bone marrow, indicating that appropriate cryopreservation does not induce lethal cellular damage. However, exposure to cryoprotectants such as DMSO andror the freezing procedure itself could provoke some cellular chemical and physical modifications. Flow cytometry is the most widely used method for lymphocyte subset characterisation, but many technical factors, including cryopreservation, may influence the assessment of lymphocyte subpopulations ŽRomeu et al., 1992.. Nevertheless, freezing is a practical approach for cell preservation for quality assessment of new monoclonal antibodies or for cell
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 0 8 9 - 7
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V. Deneys et al.r Journal of Immunological Methods 228 (1999) 13–21
exchange in external quality control programs ŽBowman et al., 1996.. The effect of freezing on B cell marker expression is not well documented. The aim of this work was to check the impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.
2.3. Panel of reagents Table 1 shows the combination of fluorescein isothiocyanate ŽFITC.- and phycoerythrin ŽPE.-conjugated reagents used to determine the expression of each antigen reported here. The optimal dilution of these reagents was determined previously Ždata not shown..
2. Material and methods 2.1. Study population
2.4. Cell staining
EDTA anticoagulant whole blood was collected by venipuncture from 15 patients with morphologically and immunologically classic chronic lymphocytic leukaemia ŽCLL. ŽMatutes’s score of 4 or 5, ŽMatutes and Catovsky, 1994... Blood was analyzed freshly and after freezing and thawing. Blood taken from 15 healthy individuals composed the control group and was treated using the same conditions.
2.4.1. Fresh blood A whole blood lysis technique ŽReichert et al., 1991. without fixation was used. 4 ml erythrocyte lysing solution ŽNH 4 Cl ŽMerck. 8.26 g, KHCO 3 ŽMerck. 1 g, EDTA ŽMerck. 186.12 mg, aqua distilla q.s. one litre. were added to 50 ml whole blood, and the mixture vortexed and incubated for 10 min at room temperature. After washing, the cells were incubated with each monoclonal antibody reagent pair, at room temperature for 30 min. The sample was subsequently centrifuged for 5 min at room temperature at 300 g, the supernatant aspirated, and the pellet resuspended in 4 ml saline solution ŽPBS, Gibco.. After vortexing and a second centrifugation Žsimilar conditions., the pellet was resuspended in 200 ml PBS for analysis. The efficiency of red blood cell lysis was checked by staining with CD45 monoclonal antibody. CD45 is a pan leucocyte marker and is absent from the erythrocyte membrane. A proportion of at least 95% CD45 positive living cells was considered to reflect an efficient red blood cell lysis.
2.2. Cell freezing and thawing After erythrocyte NH 4 Cl lysis and washing, cells were resuspended in Minimal Essential Medium ŽMEM, Gibco. supplemented with 2.5% Hepes ŽGibco. and 10% faetal calf serum ŽBiosys., and cryopreserved in DMSO ŽMerck. at 10% final concentration. Vials were first placed at y808C for 24 h and stored thereafter in liquid nitrogen, until the day of analysis. Cells were thawed rapidly by immersion in a waterbath at 378C, diluted in MEM and processed immediately.
Table 1 Panel of monoclonal antibody combinations CD number ŽFITCrPE.
Clone ŽFITCrPE.
Source
CD45rCD14 CD19rCD5 CD37rHLA-DR CD22rCD19 CD19rCD23 FMC7rCD20 KapparCD19 LambdarCD19
2D1rMOP9 B4rT1 BL14rI2 B-ly8rB4 B4rEVBCS-5 FMC7rB1 KapparB4 LambdarB4
Becton Dickinson CoulterrCoulter ImmunotechrCoulter ImmunoQuality ProductsrCoulter CoulterrBecton Dickinson ImmunotechrCoulter DakorCoulter DakorCoulter
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2.4.2. CryopreserÕed cells After thawing, cell concentration was adjusted between 5 and 10 = 10 3 cells per ml. Each monoclonal antibody pair was added to 100 ml cell suspension, the mixture was incubated for 30 min at room temperature, washed twice Žsee above. and resuspended in 300 ml PBS for analysis. Cellular viability was checked by staining with propidium iodide ŽSigma.. A proportion of at least 70% living cells was considered as acceptable. 2.5. Flow cytometry analysis Data were acquired on a FACScan ŽBecton Dickinson Immunocytometry Systems, San Jose, CA. flow cytometer. The instrument set up was checked weekly using QC windows beads ŽFlow Cytometry Standard, San Juan, PR.. Forward scatter and side scatter measurements were made using linear amplifiers, whereas fluorescence measurements were made with logarithmic amplifiers. Flow cytometric twoparameter dot plots and quadrant statistics were generated by Cellquest software ŽBecton Dickinson Immunocytometry Systems, San Jose, CA.. Analysis was performed after manual gating around the lymphocyte population on a forward scatter versus side scatter dot plot. A minimum of 5000 gated events was acquired for each tube. A gate was then put on the B lymphocyte subpopulation ŽCD19 or CD20 positive. ŽFig. 1a–b.. For some markers, CD22, CD20, surface light chain immunoglobulins, median fluorescence intensity ŽMFI. was considered independently. The MFI values were related to channels on a linear scale of 0–1023. Because fluorescence measurements were made using logarithmic amplifiers and because these data were being displayed on a linear scale, the values must be carefully interpreted and a direct comparison of values would be invalid.
Fig. 1. Representative dot plots of the analysis of kappa positive B cells in a normal peripheral blood sample.
value greater than 0.05 was considered as nonsignificant.
3. Results 2.6. Statistical analysis To state the intra-assay variation associated with these analyses, the same sample was tested ten times, and the standard deviation was calculated. Data are presented as mean " standard deviation. Results before freezing and after freezingrthawing were compared using Student’s paired t-test. A p-
Red blood cell lysis on fresh material was very efficient and at least 95% of the cells in the scatter gate were CD45 positive. The intra-assay variation is shown in Table 2a and b. The phenotype of the CLL population before cryopreservation and after freezingrthawing is shown in Table 3 Ža and b., and the phenotype of the
V. Deneys et al.r Journal of Immunological Methods 228 (1999) 13–21
16 Table 2 Intra-assay variation
a. Percentage of positive cells. Results are presented as mean of positive cells"standard deviation. Marker
percent of positive cells
CD19-PE 6.7"0.8 CD5-PEU 21.7"5.7 CD37-FITC 7.2"1.0 HLA-DR-PE 13.6"2.3 CD22-FITCU 97.1"0.9 CD23-PEU 20.8"10.0 CD20-PE 8.7"1.3 Kappa-FITCU 59.8"3.3 Lambda-FITCU 38.2"4.5 To determine the intra-assay variation, the same sample was tested ten times. b. Median fluorescence intensity. Results are presented as mean of median fluorescence channel"standard deviation. Marker
Median fluorescence intensity
CD22-FITCU CD20-PE Kappa-FITCU Lambda-FITCU
458"13 415"27 609"16 608"23
To determine the intra-assay variation, the same sample was tested ten times.
normal B cell population is shown in Table 4 Ža and b.. Individual results of some markers are illustrated in Fig. 2. CD19: In CLL patients, the percentage of CD19 positive cells was not modified after freezingrthawing Ž77% " 18.8, versus 79.2% " 18.4 before freezing.. There was a slight increase of CD19 positive cells in the control group Ž7.7% " 2.5 before freezing, 12.2% " 6.1 after freezingrthawing., but the number of B cells in these subjects was much lower. CD5 in the B cell population: In CLL patients, the percentage of CD5 positive B cells decreased after freezingrthawing Ž80.7% " 17.9, versus 91.6% " 13.7 before freezing.. CD5 positive normal B cells appeared not to be altered by cryopreservation Ž19.9% " 10.6 before freezing, 26.6% " 12.2 after freezingrthawing.. CD37: The percentage of CD37 positive cells was not modified by cryopreservation in CLL patients Ž74% " 17.1 before freezing, 70.3% " 17.1 after freezingrthawing., nor in normal subjects Ž8% " 2.7 before freezing, 9.5% " 4.2 after freezingrthawing.. HLA-DR: The percentage of HLA-DR positive cells was increased after cryopreservation in CLL
Table 3 CLL phenotype before and after cryopreservation a. Percentage of positive cells. Data are presented as mean of positive cells " standard deviation. Marker
Before freezing
After freezing
p-Value
CD19-PE 77 " 18.8 79.2 " 18.4 n.s. CD5-PEU 91.6 " 13.7 80.7 " 17.9 0.001 CD37-FITC 74 " 17.1 70.3 " 17.1 n.s. HLA-DR-PE 75.8 " 16.1 80.6 " 16.3 0.04 CD22-FITCU 7.8 " 7.9 9.8 " 8.2 n.s. CD23-PEU 52.8 " 31 29.9 " 26.7 n.s. CD20-PE 36.9 " 23.3 30.1 " 23.2 n.s. Kappa-FITCU 86.9 " 6.3 85.0 " 8.3 n.s. Lambda-FITCU 83.7 " 10.4 87.0 " 8.0 n.s. 15 samples were analyzed, except for kappa Ž n s 7. and lambda Ž n s 8.. U These markers were analyzed in the B cell population ŽCD19 gating.. b. Median fluorescence intensity. Data are presented as the mean of the median fluorescence channel" standard deviation. Marker U
CD22-FITC CD20-PE Kappa-FITCU Lambda-FITCU
Before freezing
After freezing
p-Value
219 " 29 318 " 59 335 " 82 367 " 79
239 " 24 318 " 50 345 " 94 347 " 74
0.015 n.s. n.s. n.s.
15 samples were analyzed, except for kappa Ž n s 7. and lambda Ž n s 8.. ŽCD19 gating..
U
These markers were analyzed in the B cell population
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Table 4 Normal B cell phenotype before and after cryopreservation a. Percentage of positive cells. Data are presented as means of positive cells " standard deviation. Marker
Before freezing
After freezing
CD19-PE 7.7 " 2.5 12.2 " 6.1 CD5-PEU 19.9 " 10.6 26.6 " 12.2 CD37-FITC 8.0 " 2.7 9.5 " 4.2 HLA-DR-PE 9.8 " 4.1 15.5 " 10.0 CD22-FITCU 93.7 " 3.2 95.0 " 3.6 CD23-PEU 24.1 " 12.4 19.6 " 9.6 CD20-PE 9.9 " 3.7 9.7 " 4.6 Kappa-FITCU 56.4 " 4.0 55.7 " 5.3 Lambda-FITCU 36.5 " 5.6 38.4 " 3.7 15 samples were analyzed. U These markers were analyzed in the B cell population ŽCD19 gating..
p-Value 0.002 n.s. 0.045 0.02 n.s. n.s. n.s. n.s. n.s.
b. Median fluorescence intensity. Data are presented as the mean of the median fluorescence channel" standard deviation. Marker
Before freezing
After freezing
p-Value
CD22-FITCU CD20-PE Kappa-FITCU Lambda-FITCU
457 " 17 429 " 38 566 " 56 603 " 38
448 " 14 420 " 40 596 " 34 600 " 30
0.03 n.s. 0.044 n.s.
15 samples were analyzed. U These markers were analyzed in the B cell population ŽCD19 gating..
patients Ž80.6 " 16.3, versus 75.8% " 16.1 before freezing. and in normal controls Ž15.5% " 10, versus 9.8% " 4.1 before freezing.. CD22 in the B cell population: The CD22 antigen is weak to negative on B CLL cells, and positive on normal B cells. This was confirmed before cryopreservation and after freezingrthawing without pronounced modification ŽCLL group: 7.8% " 7.9 before freezing, 9.8% " 8.2 after freezingrthawing, MFI: 219 " 29 before freezing, 239 " 24 after freezingrthawing; Control group: 93.7% " 3.2 before freezing, 95% " 3.6 after freezingrthawing, MFI: 457 " 17 before freezing, 448 " 14 after freezingr thawing.. CD23 in the B cell population: CD23 is present on a majority of B CLL cells: this was observed before freezing Ž52.8% " 31., but this proportion decreased to less than 30% of CLL cells after freezingrthawing Ž29.9% " 26.7., without reaching a statistical threshold. However, in 6 out of 15 CLL Žsee below, Tables 5 and 7., CD23 was considered to be positive Ž) 30%. in the B cell population before freezing and negative Ž- 30%. after freezingrthawing. The percentage of CD23 positive B cells in normal subjects was much lower and was not modi-
fied by cryopreservation Ž24.1% " 12.4 before freezing, 19.6% " 9.6 after freezingrthawing.. CD20: CD20 was probably the least affected B cell marker after freezingrthawing in terms of the percentage of positive cells ŽCLL group: 36.9% " 23.3 before freezing, 30.1 " 23.2 after freezingr thawing; Control group: 9.9% " 3.7 before freezing, 9.7% " 4.6 after freezingrthawing. as well as in terms of the fluorescence intensity ŽCLL group: 318 " 59 before freezing, 318 " 50 after freezing; Control group: 429 " 38 before freezing, 420 " 40 after freezingrthawing.. KapparLambda: Lambda chain fluorescence appeared to be more intense than kappa chain fluorescence and therefore the light chains were considered separately. In CLL samples, light chains of immunoglobulins were analyzed only in the monoclonal population; for this reason, the number of samples was reduced Žkappa: n s 7, lambda: n s 8.. Like CD20, kappa and lambda appeared not to be affected by cryopreservation in samples from CLL patients ŽKappa: 86,.% " 6.3 before freezing, 85% " 8.2 after freezingrthawing, MFI: 335 " 82 before freezing, 345 " 94 after freezingrthawing; Lambda 83.7% " 10.4 before freezing, 87% " 8 after freez-
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V. Deneys et al.r Journal of Immunological Methods 228 (1999) 13–21
Fig. 2. Results of some markers before freezing Žleft column. and after freezingrthawing Žright column. on peripheral lymphocytes of healthy subjects Ž^. and in CLL patients Žv ..
ingrthawing, MFI: 367 " 79 before freezing, 347 " 74 after freezingrthawing.. FMC7 in the B cell population: Results obtained for FMC7 are not presented here, because FMC7 was not present on these classical CLL cells either before or after cryopreservation.
The Matutes–Catovsky score ŽMatutes and Catovsky, 1994. was calculated on these fifteen chronic lymphocytic leukaemias. The score was interpreted as follows: CD5 and CD23: 1 point if present on at least 30% B cells, 0 point if absent or present on less than 30% B cells; FMC7:
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Table 5 Matutes–Catovsky score before and after freezing ŽMatutes and Catovsky, 1994. Before freezing
After freezing
CLL
CD5
FMC7
CD23
CD22
krl
Total score
CD5
FMC7
CD23
CD22
krl
Total score
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 0 0 1 1 1 0 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 5 5 4 4 5 5 5 4 5 5 5 5 5 5
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 0 0 1 0 0 0 0 1 1 1 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 5 4 4 4 5 4 4 4 4 5 5 5 4 4
1 point if absent or present on less than 30% B cells, 0 point if present on at least 30% B cells; CD23 and kapparlambda fluorescence intensity: 1 point if negative or weak, 0 point if moderate to strong. Results before freezing showed a score of 5 in twelve samples, and 4 in three samples. After cryopreservation, seven cases had still a score of 5, and eight a score of 4 ŽTable 5.. In six cases out of fifteen, the score was modified, always due to the negativity of CD23.
4. Discussion B cell chronic lymphocytic leukaemia is the most common adult leukaemia in Western countries. Its morphological criteria are most often quite typical. The immunological diagnosis has been greatly improved by a scoring system based on five markers ŽCD5, CD23, FMC7, CD22, surface light chains of immunoglobulins. as described by Matutes and Catovsky Ž1994.. Flow cytometry is an increasingly useful tool for clinical diagnosis, because it can be used to make quantitative distinctions among individual cells on the basis of cell surface markers. It has therefore become imperative to define the standardization and quality control methods for both the instrument and
immunostaining procedures. Quality control requires standardization of sample preparation procedures, reagents and instrumentation. For this purpose, interlaboratory cell exchange is a useful method to validate the results and interpretation. Moreover, establishing a frozen cell control would permit interlaboratory comparisons of flow cytometric data and provide information that could be used for performance evaluations and competency testing ŽMc Coy et al., 1990.. A few reports have suggested changes in the expression of surface antigens of lymphocytes and leukaemic cells following cryopreservation ŽCampos et al., 1988; Harvath, 1988; Romeu et al., 1992; Martı´ et al., 1993; Islam et al., 1995; Lloyd et al., 1995; Rosillo et al., 1995; Fiebig et al., 1997.. The aim of this work was to evaluate Matutes’s scoring system on frozen B-CLL cells and to compare the results with those obtained using fresh cells. Fifteen morphologically classical B-CLL were analyzed and compared to fifteen normal controls. Our results on fresh samples indicated that the phenotype of these neoplastic cells was as follows: CD19 q , CD5 q , faint CD20, CD23q , weak CD22 and surface light chain of immunoglobulin, CD37 q , HLADR q , FMC7y , as described in the literature ŽFerry and Harris, 1997.. Before the development of directly conjugated monoclonal antibodies, preimmune sera were used in
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flow cytometric studies to estimate the level of ‘‘non specific staining’’ of the specific antibody to its target cell. The widespread use of directly conjugated monoclonal antibodies and multiparameter analysis in clinical flow cytometry has reduced the need for a separate ‘‘negative control’’ ŽKeeney et al., 1998.. The last U.S.–Canadian Consensus recommendation on the immunophenotypic analysis of haematological neoplasia by flow cytometry states that ‘‘a strong consensus that the specific isotypic controls provide no useful additional information beyond unstained cells alone, or negative cells in selected antibody combinations’’ ŽSteltzer et al., 1997.. When separation between negative and positive populations is quite clear, the determination of positive events can be unambiguously resolved. But the problem is particularly difficult when quantifying the expression of antigens that exhibit a continuous staining pattern ranging from negative to positive ŽKeeney et al., 1998.. One approach to quantify populations with non-discrete fluorescence signals is to report shifts in the MFI of the population of interest ŽShapiro, 1995.. This requires a standardised instrument setup and calibration of the instrument’s response to fluorescence signals. Interestingly, after freezingrthawing, the proportion of B cells remained unmodified as assessed by the percentage of CD19 and CD37 positive cells. This confirmed the results already described ŽMartı´ et al., 1993; Rosillo et al., 1995.. Table 6 Percentage of CD5 positive B cells in the 15 CLL samples. B cell population was gated on CD19 positiÕe cells Samples
Before freezing
After freezingrthawing
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
98% 99% 59% 99% 90% 93% 99% 58% 97% 93% 99% 99% 99% 97% 95%
83% 96% 52% 91% 65% 75% 94% 35% 88% 93% 97% 84% 96% 87% 75%
Table 7 Percentage of CD23 positive B cells in the 15 CLL samples. B cell population was gated on CD19 positiÕe cells Samples
Before freezing
After freezingrthawing
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
94% 40% 56% 5% 4% 82% 35% 35% 6% 68% 91% 60% 75% 85% 56%
72% 55% 24% 2% 3% 70% 10% 3% 5% 19% 67% 42% 51% 15% 10%
CD5, a T cell marker, is also expressed on a subset of mature B cells, and particularly on B-CLL cells. Its expression decreased after freezingrthawing. Nevertheless, this decrease was not sufficient to modify the interpretation of positivity; indeed, in no case, did the proportion of CD5 positive B cells fall below 30% ŽTable 6.. HLA-DR positive B-CLL cells increased after cryopreservation, probably reflecting cell activation during manipulation. CD20, a pan B marker, was not modified by cryopreservation. Islam et al. Ž1995. described a decrease of CD20 fluorescence intensity on normal B cells, but the cells were separated on a density gradient before cryopreservation, and the same effect was observed on fresh separated mononuclear cells. This decrease was probably due to the gradient density separation rather than to the cryopreservation itself. CD22 fluorescence intensity was slightly modified after freezingrthawing and no change in the expression of light chain of immunoglobulin was observed. CD23 ŽFc´ IIa. expression is restricted to membrane IgM q and IgD q cells ŽBarclay et al., 1997.. It is often present on B-CLL cells and absent from mantle lymphoma cells. In this study, CD23 was expressed on at least 30% B cells of 12 CLL samples before freezing, but was lost on 6 of them Ž50%!.
V. Deneys et al.r Journal of Immunological Methods 228 (1999) 13–21
after freezingrthawing Žno. 3, 7, 8, 10, 14, 15, Table 7.. This observation with this particular CD23 monoclonal antibody has to be controlled with some other CD23 markers. In 6 samples out of 15 Ž40%., the CLL’s score was different after freezingrthawing. In no instance, did this change modify the immunological diagnosis of CLL, because these samples were ‘‘classical’’ and ‘‘typical’’ CLL. However, in mixed CLL ŽCLLrPL. or in B prolymphocytic leukaemia ŽPLL., for example, when the CLL’s score is often less than 3 on fresh cells Žmainly due to FMC7 positivity, and high expression of CD22 andror light chain of immunoglobulins., the loss of CD23 expression on frozen cells could have an influence on the immunological diagnosis, due to mimicry of the mantle cell lymphoma phenotype ŽCD5 positive, CD23 negative, FMC7 positive, high expression of CD22 and of light chain of immunoglobulins.. The results presented here suggest that cryopreservation modifies the flow cytometric analysis of the immunophenotypic profile of human CLL cells. Therefore, the underestimation of some parameters Žespecially the percentage of CD5 and CD23 positive cells. may occur. This should be bourne in mind when performing retrospective immunophenotypic studies with cryopreserved cells.
Acknowledgements The authors wish to thank Salus Sanguinis Foundation for the financial support of this work, and Mrs. Annie Robert for her statistical help.
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Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype V. Deneys ) , V. Thiry, N. Hougardy, A.M. Mazzon, P. Leveugle, M. De Bruyere ` UniÕersite´ Catholique de LouÕain, UniÕersity Hospital, Immunohaematology Laboratory, 1200 Brussels, Belgium Received 2 November 1998; received in revised form 13 May 1999; accepted 24 May 1999
Abstract Background and objectiÕes: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. Material and methods: Blood samples from 15 CLL patients were analyzed freshly and after freezing at y1968C, without separation, and thawing. Results were compared by Student’s paired t-test. Results: The phenotype of fresh CLL cells was as follows: CD19q , CD5 q , faint CD20, CD23 " , weak CD22 and sIg, CD37q , HLA-DRq , FMC7y . After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes’s score was modified in 6 cases out of 15 Ž40%.. Conclusion: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chronic lymphocytic leukaemia; Immunophenotype; Cryopreservation; Flow cytometry; Cluster of differentiation
1. Introduction Cryopreservation of haemopoietic cells, for in vivo use or in vitro research purposes, has greatly increased in popularity in recent years. In order to avoid injury of the cells by direct effect of low temperature, and by the intracellular ice crystal formation occurring at high cooling rates ŽKarow and Webb, 1965. most of the procedures involve freezing at a slow cooling rate in the pres-
AbbreÕiations: CLL: Chronic lymphocytic leukaemia; CD: Cluster of differentiation; MFI: Median Fluorescence Intensity ) Corresponding author. Cliniques Universitaires Saint-LucUCL, Clos Chapelle-aux-Champs, 30 BP 30.52B-1200 Brussels. Tel.: q32-2-764 34 47; fax: q32-2-764 30 21; E-mail: [email protected]
ence of cryoprotective agents, such as glycerol, hydroxyethyl starch or dimethylsulfoxide, and storage in liquid nitrogen ŽRosillo et al., 1995. The infusion of cryopreserved haemopoietic stem cells reconstitutes safely and satisfactorily the function of the recipient’s bone marrow, indicating that appropriate cryopreservation does not induce lethal cellular damage. However, exposure to cryoprotectants such as DMSO andror the freezing procedure itself could provoke some cellular chemical and physical modifications. Flow cytometry is the most widely used method for lymphocyte subset characterisation, but many technical factors, including cryopreservation, may influence the assessment of lymphocyte subpopulations ŽRomeu et al., 1992.. Nevertheless, freezing is a practical approach for cell preservation for quality assessment of new monoclonal antibodies or for cell
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 0 8 9 - 7
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exchange in external quality control programs ŽBowman et al., 1996.. The effect of freezing on B cell marker expression is not well documented. The aim of this work was to check the impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.
2.3. Panel of reagents Table 1 shows the combination of fluorescein isothiocyanate ŽFITC.- and phycoerythrin ŽPE.-conjugated reagents used to determine the expression of each antigen reported here. The optimal dilution of these reagents was determined previously Ždata not shown..
2. Material and methods 2.1. Study population
2.4. Cell staining
EDTA anticoagulant whole blood was collected by venipuncture from 15 patients with morphologically and immunologically classic chronic lymphocytic leukaemia ŽCLL. ŽMatutes’s score of 4 or 5, ŽMatutes and Catovsky, 1994... Blood was analyzed freshly and after freezing and thawing. Blood taken from 15 healthy individuals composed the control group and was treated using the same conditions.
2.4.1. Fresh blood A whole blood lysis technique ŽReichert et al., 1991. without fixation was used. 4 ml erythrocyte lysing solution ŽNH 4 Cl ŽMerck. 8.26 g, KHCO 3 ŽMerck. 1 g, EDTA ŽMerck. 186.12 mg, aqua distilla q.s. one litre. were added to 50 ml whole blood, and the mixture vortexed and incubated for 10 min at room temperature. After washing, the cells were incubated with each monoclonal antibody reagent pair, at room temperature for 30 min. The sample was subsequently centrifuged for 5 min at room temperature at 300 g, the supernatant aspirated, and the pellet resuspended in 4 ml saline solution ŽPBS, Gibco.. After vortexing and a second centrifugation Žsimilar conditions., the pellet was resuspended in 200 ml PBS for analysis. The efficiency of red blood cell lysis was checked by staining with CD45 monoclonal antibody. CD45 is a pan leucocyte marker and is absent from the erythrocyte membrane. A proportion of at least 95% CD45 positive living cells was considered to reflect an efficient red blood cell lysis.
2.2. Cell freezing and thawing After erythrocyte NH 4 Cl lysis and washing, cells were resuspended in Minimal Essential Medium ŽMEM, Gibco. supplemented with 2.5% Hepes ŽGibco. and 10% faetal calf serum ŽBiosys., and cryopreserved in DMSO ŽMerck. at 10% final concentration. Vials were first placed at y808C for 24 h and stored thereafter in liquid nitrogen, until the day of analysis. Cells were thawed rapidly by immersion in a waterbath at 378C, diluted in MEM and processed immediately.
Table 1 Panel of monoclonal antibody combinations CD number ŽFITCrPE.
Clone ŽFITCrPE.
Source
CD45rCD14 CD19rCD5 CD37rHLA-DR CD22rCD19 CD19rCD23 FMC7rCD20 KapparCD19 LambdarCD19
2D1rMOP9 B4rT1 BL14rI2 B-ly8rB4 B4rEVBCS-5 FMC7rB1 KapparB4 LambdarB4
Becton Dickinson CoulterrCoulter ImmunotechrCoulter ImmunoQuality ProductsrCoulter CoulterrBecton Dickinson ImmunotechrCoulter DakorCoulter DakorCoulter
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2.4.2. CryopreserÕed cells After thawing, cell concentration was adjusted between 5 and 10 = 10 3 cells per ml. Each monoclonal antibody pair was added to 100 ml cell suspension, the mixture was incubated for 30 min at room temperature, washed twice Žsee above. and resuspended in 300 ml PBS for analysis. Cellular viability was checked by staining with propidium iodide ŽSigma.. A proportion of at least 70% living cells was considered as acceptable. 2.5. Flow cytometry analysis Data were acquired on a FACScan ŽBecton Dickinson Immunocytometry Systems, San Jose, CA. flow cytometer. The instrument set up was checked weekly using QC windows beads ŽFlow Cytometry Standard, San Juan, PR.. Forward scatter and side scatter measurements were made using linear amplifiers, whereas fluorescence measurements were made with logarithmic amplifiers. Flow cytometric twoparameter dot plots and quadrant statistics were generated by Cellquest software ŽBecton Dickinson Immunocytometry Systems, San Jose, CA.. Analysis was performed after manual gating around the lymphocyte population on a forward scatter versus side scatter dot plot. A minimum of 5000 gated events was acquired for each tube. A gate was then put on the B lymphocyte subpopulation ŽCD19 or CD20 positive. ŽFig. 1a–b.. For some markers, CD22, CD20, surface light chain immunoglobulins, median fluorescence intensity ŽMFI. was considered independently. The MFI values were related to channels on a linear scale of 0–1023. Because fluorescence measurements were made using logarithmic amplifiers and because these data were being displayed on a linear scale, the values must be carefully interpreted and a direct comparison of values would be invalid.
Fig. 1. Representative dot plots of the analysis of kappa positive B cells in a normal peripheral blood sample.
value greater than 0.05 was considered as nonsignificant.
3. Results 2.6. Statistical analysis To state the intra-assay variation associated with these analyses, the same sample was tested ten times, and the standard deviation was calculated. Data are presented as mean " standard deviation. Results before freezing and after freezingrthawing were compared using Student’s paired t-test. A p-
Red blood cell lysis on fresh material was very efficient and at least 95% of the cells in the scatter gate were CD45 positive. The intra-assay variation is shown in Table 2a and b. The phenotype of the CLL population before cryopreservation and after freezingrthawing is shown in Table 3 Ža and b., and the phenotype of the
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16 Table 2 Intra-assay variation
a. Percentage of positive cells. Results are presented as mean of positive cells"standard deviation. Marker
percent of positive cells
CD19-PE 6.7"0.8 CD5-PEU 21.7"5.7 CD37-FITC 7.2"1.0 HLA-DR-PE 13.6"2.3 CD22-FITCU 97.1"0.9 CD23-PEU 20.8"10.0 CD20-PE 8.7"1.3 Kappa-FITCU 59.8"3.3 Lambda-FITCU 38.2"4.5 To determine the intra-assay variation, the same sample was tested ten times. b. Median fluorescence intensity. Results are presented as mean of median fluorescence channel"standard deviation. Marker
Median fluorescence intensity
CD22-FITCU CD20-PE Kappa-FITCU Lambda-FITCU
458"13 415"27 609"16 608"23
To determine the intra-assay variation, the same sample was tested ten times.
normal B cell population is shown in Table 4 Ža and b.. Individual results of some markers are illustrated in Fig. 2. CD19: In CLL patients, the percentage of CD19 positive cells was not modified after freezingrthawing Ž77% " 18.8, versus 79.2% " 18.4 before freezing.. There was a slight increase of CD19 positive cells in the control group Ž7.7% " 2.5 before freezing, 12.2% " 6.1 after freezingrthawing., but the number of B cells in these subjects was much lower. CD5 in the B cell population: In CLL patients, the percentage of CD5 positive B cells decreased after freezingrthawing Ž80.7% " 17.9, versus 91.6% " 13.7 before freezing.. CD5 positive normal B cells appeared not to be altered by cryopreservation Ž19.9% " 10.6 before freezing, 26.6% " 12.2 after freezingrthawing.. CD37: The percentage of CD37 positive cells was not modified by cryopreservation in CLL patients Ž74% " 17.1 before freezing, 70.3% " 17.1 after freezingrthawing., nor in normal subjects Ž8% " 2.7 before freezing, 9.5% " 4.2 after freezingrthawing.. HLA-DR: The percentage of HLA-DR positive cells was increased after cryopreservation in CLL
Table 3 CLL phenotype before and after cryopreservation a. Percentage of positive cells. Data are presented as mean of positive cells " standard deviation. Marker
Before freezing
After freezing
p-Value
CD19-PE 77 " 18.8 79.2 " 18.4 n.s. CD5-PEU 91.6 " 13.7 80.7 " 17.9 0.001 CD37-FITC 74 " 17.1 70.3 " 17.1 n.s. HLA-DR-PE 75.8 " 16.1 80.6 " 16.3 0.04 CD22-FITCU 7.8 " 7.9 9.8 " 8.2 n.s. CD23-PEU 52.8 " 31 29.9 " 26.7 n.s. CD20-PE 36.9 " 23.3 30.1 " 23.2 n.s. Kappa-FITCU 86.9 " 6.3 85.0 " 8.3 n.s. Lambda-FITCU 83.7 " 10.4 87.0 " 8.0 n.s. 15 samples were analyzed, except for kappa Ž n s 7. and lambda Ž n s 8.. U These markers were analyzed in the B cell population ŽCD19 gating.. b. Median fluorescence intensity. Data are presented as the mean of the median fluorescence channel" standard deviation. Marker U
CD22-FITC CD20-PE Kappa-FITCU Lambda-FITCU
Before freezing
After freezing
p-Value
219 " 29 318 " 59 335 " 82 367 " 79
239 " 24 318 " 50 345 " 94 347 " 74
0.015 n.s. n.s. n.s.
15 samples were analyzed, except for kappa Ž n s 7. and lambda Ž n s 8.. ŽCD19 gating..
U
These markers were analyzed in the B cell population
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Table 4 Normal B cell phenotype before and after cryopreservation a. Percentage of positive cells. Data are presented as means of positive cells " standard deviation. Marker
Before freezing
After freezing
CD19-PE 7.7 " 2.5 12.2 " 6.1 CD5-PEU 19.9 " 10.6 26.6 " 12.2 CD37-FITC 8.0 " 2.7 9.5 " 4.2 HLA-DR-PE 9.8 " 4.1 15.5 " 10.0 CD22-FITCU 93.7 " 3.2 95.0 " 3.6 CD23-PEU 24.1 " 12.4 19.6 " 9.6 CD20-PE 9.9 " 3.7 9.7 " 4.6 Kappa-FITCU 56.4 " 4.0 55.7 " 5.3 Lambda-FITCU 36.5 " 5.6 38.4 " 3.7 15 samples were analyzed. U These markers were analyzed in the B cell population ŽCD19 gating..
p-Value 0.002 n.s. 0.045 0.02 n.s. n.s. n.s. n.s. n.s.
b. Median fluorescence intensity. Data are presented as the mean of the median fluorescence channel" standard deviation. Marker
Before freezing
After freezing
p-Value
CD22-FITCU CD20-PE Kappa-FITCU Lambda-FITCU
457 " 17 429 " 38 566 " 56 603 " 38
448 " 14 420 " 40 596 " 34 600 " 30
0.03 n.s. 0.044 n.s.
15 samples were analyzed. U These markers were analyzed in the B cell population ŽCD19 gating..
patients Ž80.6 " 16.3, versus 75.8% " 16.1 before freezing. and in normal controls Ž15.5% " 10, versus 9.8% " 4.1 before freezing.. CD22 in the B cell population: The CD22 antigen is weak to negative on B CLL cells, and positive on normal B cells. This was confirmed before cryopreservation and after freezingrthawing without pronounced modification ŽCLL group: 7.8% " 7.9 before freezing, 9.8% " 8.2 after freezingrthawing, MFI: 219 " 29 before freezing, 239 " 24 after freezingrthawing; Control group: 93.7% " 3.2 before freezing, 95% " 3.6 after freezingrthawing, MFI: 457 " 17 before freezing, 448 " 14 after freezingr thawing.. CD23 in the B cell population: CD23 is present on a majority of B CLL cells: this was observed before freezing Ž52.8% " 31., but this proportion decreased to less than 30% of CLL cells after freezingrthawing Ž29.9% " 26.7., without reaching a statistical threshold. However, in 6 out of 15 CLL Žsee below, Tables 5 and 7., CD23 was considered to be positive Ž) 30%. in the B cell population before freezing and negative Ž- 30%. after freezingrthawing. The percentage of CD23 positive B cells in normal subjects was much lower and was not modi-
fied by cryopreservation Ž24.1% " 12.4 before freezing, 19.6% " 9.6 after freezingrthawing.. CD20: CD20 was probably the least affected B cell marker after freezingrthawing in terms of the percentage of positive cells ŽCLL group: 36.9% " 23.3 before freezing, 30.1 " 23.2 after freezingr thawing; Control group: 9.9% " 3.7 before freezing, 9.7% " 4.6 after freezingrthawing. as well as in terms of the fluorescence intensity ŽCLL group: 318 " 59 before freezing, 318 " 50 after freezing; Control group: 429 " 38 before freezing, 420 " 40 after freezingrthawing.. KapparLambda: Lambda chain fluorescence appeared to be more intense than kappa chain fluorescence and therefore the light chains were considered separately. In CLL samples, light chains of immunoglobulins were analyzed only in the monoclonal population; for this reason, the number of samples was reduced Žkappa: n s 7, lambda: n s 8.. Like CD20, kappa and lambda appeared not to be affected by cryopreservation in samples from CLL patients ŽKappa: 86,.% " 6.3 before freezing, 85% " 8.2 after freezingrthawing, MFI: 335 " 82 before freezing, 345 " 94 after freezingrthawing; Lambda 83.7% " 10.4 before freezing, 87% " 8 after freez-
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Fig. 2. Results of some markers before freezing Žleft column. and after freezingrthawing Žright column. on peripheral lymphocytes of healthy subjects Ž^. and in CLL patients Žv ..
ingrthawing, MFI: 367 " 79 before freezing, 347 " 74 after freezingrthawing.. FMC7 in the B cell population: Results obtained for FMC7 are not presented here, because FMC7 was not present on these classical CLL cells either before or after cryopreservation.
The Matutes–Catovsky score ŽMatutes and Catovsky, 1994. was calculated on these fifteen chronic lymphocytic leukaemias. The score was interpreted as follows: CD5 and CD23: 1 point if present on at least 30% B cells, 0 point if absent or present on less than 30% B cells; FMC7:
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Table 5 Matutes–Catovsky score before and after freezing ŽMatutes and Catovsky, 1994. Before freezing
After freezing
CLL
CD5
FMC7
CD23
CD22
krl
Total score
CD5
FMC7
CD23
CD22
krl
Total score
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 0 0 1 1 1 0 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 5 5 4 4 5 5 5 4 5 5 5 5 5 5
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 0 0 1 0 0 0 0 1 1 1 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 5 4 4 4 5 4 4 4 4 5 5 5 4 4
1 point if absent or present on less than 30% B cells, 0 point if present on at least 30% B cells; CD23 and kapparlambda fluorescence intensity: 1 point if negative or weak, 0 point if moderate to strong. Results before freezing showed a score of 5 in twelve samples, and 4 in three samples. After cryopreservation, seven cases had still a score of 5, and eight a score of 4 ŽTable 5.. In six cases out of fifteen, the score was modified, always due to the negativity of CD23.
4. Discussion B cell chronic lymphocytic leukaemia is the most common adult leukaemia in Western countries. Its morphological criteria are most often quite typical. The immunological diagnosis has been greatly improved by a scoring system based on five markers ŽCD5, CD23, FMC7, CD22, surface light chains of immunoglobulins. as described by Matutes and Catovsky Ž1994.. Flow cytometry is an increasingly useful tool for clinical diagnosis, because it can be used to make quantitative distinctions among individual cells on the basis of cell surface markers. It has therefore become imperative to define the standardization and quality control methods for both the instrument and
immunostaining procedures. Quality control requires standardization of sample preparation procedures, reagents and instrumentation. For this purpose, interlaboratory cell exchange is a useful method to validate the results and interpretation. Moreover, establishing a frozen cell control would permit interlaboratory comparisons of flow cytometric data and provide information that could be used for performance evaluations and competency testing ŽMc Coy et al., 1990.. A few reports have suggested changes in the expression of surface antigens of lymphocytes and leukaemic cells following cryopreservation ŽCampos et al., 1988; Harvath, 1988; Romeu et al., 1992; Martı´ et al., 1993; Islam et al., 1995; Lloyd et al., 1995; Rosillo et al., 1995; Fiebig et al., 1997.. The aim of this work was to evaluate Matutes’s scoring system on frozen B-CLL cells and to compare the results with those obtained using fresh cells. Fifteen morphologically classical B-CLL were analyzed and compared to fifteen normal controls. Our results on fresh samples indicated that the phenotype of these neoplastic cells was as follows: CD19 q , CD5 q , faint CD20, CD23q , weak CD22 and surface light chain of immunoglobulin, CD37 q , HLADR q , FMC7y , as described in the literature ŽFerry and Harris, 1997.. Before the development of directly conjugated monoclonal antibodies, preimmune sera were used in
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flow cytometric studies to estimate the level of ‘‘non specific staining’’ of the specific antibody to its target cell. The widespread use of directly conjugated monoclonal antibodies and multiparameter analysis in clinical flow cytometry has reduced the need for a separate ‘‘negative control’’ ŽKeeney et al., 1998.. The last U.S.–Canadian Consensus recommendation on the immunophenotypic analysis of haematological neoplasia by flow cytometry states that ‘‘a strong consensus that the specific isotypic controls provide no useful additional information beyond unstained cells alone, or negative cells in selected antibody combinations’’ ŽSteltzer et al., 1997.. When separation between negative and positive populations is quite clear, the determination of positive events can be unambiguously resolved. But the problem is particularly difficult when quantifying the expression of antigens that exhibit a continuous staining pattern ranging from negative to positive ŽKeeney et al., 1998.. One approach to quantify populations with non-discrete fluorescence signals is to report shifts in the MFI of the population of interest ŽShapiro, 1995.. This requires a standardised instrument setup and calibration of the instrument’s response to fluorescence signals. Interestingly, after freezingrthawing, the proportion of B cells remained unmodified as assessed by the percentage of CD19 and CD37 positive cells. This confirmed the results already described ŽMartı´ et al., 1993; Rosillo et al., 1995.. Table 6 Percentage of CD5 positive B cells in the 15 CLL samples. B cell population was gated on CD19 positiÕe cells Samples
Before freezing
After freezingrthawing
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
98% 99% 59% 99% 90% 93% 99% 58% 97% 93% 99% 99% 99% 97% 95%
83% 96% 52% 91% 65% 75% 94% 35% 88% 93% 97% 84% 96% 87% 75%
Table 7 Percentage of CD23 positive B cells in the 15 CLL samples. B cell population was gated on CD19 positiÕe cells Samples
Before freezing
After freezingrthawing
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
94% 40% 56% 5% 4% 82% 35% 35% 6% 68% 91% 60% 75% 85% 56%
72% 55% 24% 2% 3% 70% 10% 3% 5% 19% 67% 42% 51% 15% 10%
CD5, a T cell marker, is also expressed on a subset of mature B cells, and particularly on B-CLL cells. Its expression decreased after freezingrthawing. Nevertheless, this decrease was not sufficient to modify the interpretation of positivity; indeed, in no case, did the proportion of CD5 positive B cells fall below 30% ŽTable 6.. HLA-DR positive B-CLL cells increased after cryopreservation, probably reflecting cell activation during manipulation. CD20, a pan B marker, was not modified by cryopreservation. Islam et al. Ž1995. described a decrease of CD20 fluorescence intensity on normal B cells, but the cells were separated on a density gradient before cryopreservation, and the same effect was observed on fresh separated mononuclear cells. This decrease was probably due to the gradient density separation rather than to the cryopreservation itself. CD22 fluorescence intensity was slightly modified after freezingrthawing and no change in the expression of light chain of immunoglobulin was observed. CD23 ŽFc´ IIa. expression is restricted to membrane IgM q and IgD q cells ŽBarclay et al., 1997.. It is often present on B-CLL cells and absent from mantle lymphoma cells. In this study, CD23 was expressed on at least 30% B cells of 12 CLL samples before freezing, but was lost on 6 of them Ž50%!.
V. Deneys et al.r Journal of Immunological Methods 228 (1999) 13–21
after freezingrthawing Žno. 3, 7, 8, 10, 14, 15, Table 7.. This observation with this particular CD23 monoclonal antibody has to be controlled with some other CD23 markers. In 6 samples out of 15 Ž40%., the CLL’s score was different after freezingrthawing. In no instance, did this change modify the immunological diagnosis of CLL, because these samples were ‘‘classical’’ and ‘‘typical’’ CLL. However, in mixed CLL ŽCLLrPL. or in B prolymphocytic leukaemia ŽPLL., for example, when the CLL’s score is often less than 3 on fresh cells Žmainly due to FMC7 positivity, and high expression of CD22 andror light chain of immunoglobulins., the loss of CD23 expression on frozen cells could have an influence on the immunological diagnosis, due to mimicry of the mantle cell lymphoma phenotype ŽCD5 positive, CD23 negative, FMC7 positive, high expression of CD22 and of light chain of immunoglobulins.. The results presented here suggest that cryopreservation modifies the flow cytometric analysis of the immunophenotypic profile of human CLL cells. Therefore, the underestimation of some parameters Žespecially the percentage of CD5 and CD23 positive cells. may occur. This should be bourne in mind when performing retrospective immunophenotypic studies with cryopreserved cells.
Acknowledgements The authors wish to thank Salus Sanguinis Foundation for the financial support of this work, and Mrs. Annie Robert for her statistical help.
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