detection of htert protein by flow cytometry

Leukemia (2000) 14, 2176–2181 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00

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BIO-TECHNICAL METHODS SECTION (BTS) BTSLeukemia

Detection of hTERT protein by flow cytometryASM Ali1, R Chopra1,2, J Robertson1 and NG Testa1

Departments of 1Experimental Haematology and 2Medical Oncology, Paterson Institute for Cancer Research, Manchester, UK

Telomerase is a telomere-specific DNA polymerase consistingof protein and RNA components, which is activated in germlinecells and the majority of cancers and serves to counter theconsequences of telomere shortening. The protein component,hTERT, is believed to be the catalytic subunit of human telo-merase and its expression at the mRNA level correlates wellwith telomerase activity in vitro . Current techniques for assay-ing telomerase activity detect only the mean activity in a sam-ple and are unable to isolate specific cell sub-populations. Thisreport describes the development and validation of a cellular,immunofluorescence-based flow cytometry assay that allowsdetection of intranuclear hTERT while maintaining identifiablecell population characteristics. The assay was shown to beboth sensitive to changes in telomerase expression and wassemi-quantitative. In both cell line differentiation experimentsand in primary cells, a good correlation existed between hTERTexpression measured by flow cytometry and telomeraseactivity detected by the telomeric repeat amplification protocol(TRAP). The method developed offers a quick, simple andreproducible cellular-based assay for hTERT expression. Thisassay will provide a useful, new tool for future investigations,facilitating the analysis of hTERT expression in mixed cellpopulations. Leukemia (2000) 14, 2176–2181.Keywords: flow cytometry; hTERT protein; immunofluorescence;telomerase

Introduction

Eukaryotic chromosomes terminate with specialised structuresknown as telomeres.1 These consist of tandemly repeatedDNA sequences that protect the ends of chromosomes andare important in maintaining their stability.2 Because DNApolymerases cannot completely replicate a linear DNA mol-ecule, telomeric DNA at the end of a chromosome may belost with each cell division.3 Critical shortening of telomeres isassociated with cellular senescence.4 Telomerase is a uniquecellular reverse transcriptase involved in telomere mainte-nance.5,6 Its activity is detectable in the majority of cancers7

as well as germline tissues8 and proliferating somatic cells.9

In cancer, telomerase activation is proposed as one mech-anism by which cells can bypass senescence. Structurally, tel-omerase consists of protein and RNA components. One pro-tein component, hTERT, is the catalytic subunit of humantelomerase and its expression at the mRNA level correlateswell with telomerase activity.10 Current methods in use for

Correspondence: R Chopra, Paterson Institute for Cancer Research,Wilmslow Road, Manchester, M20 4BX, UK; Fax: (0161) 446 3940Received 13 March 2000; accepted 23 August 2000

detection of telomerase activity and hTERT expression areprimarily molecular-based techniques. These are well estab-lished and sensitive methods for the measurement of meantelomerase activity in a whole population of cells. Suchmethods cannot, however, measure telomerase activity in cellsub-populations. A telomerase/hTERT negative sub-populationof cells will therefore not be identified within a large popu-lation of positive cells. Conversely, because the TRAP assayis highly sensitive, the activity in a small highly positive popu-lation mixed with a telomerase negative population may beinterpreted as the entire population exhibiting a medium tolow degree of expression. A further disadvantage of existingmethods is that cell lysis is necessary for assay of telomeraseor hTERT mRNA, and therefore, it is no longer possible tocarry out any further or concurrent analysis on the same setof cells.

In human leukaemias, telomerase upregulation has beenshown to occur during disease progression.11,12 It has still tobe clarified whether telomerase activity in the developmentof cancer follows a pattern of ‘reactivation’ where telomeraseis reactivated in previously telomerase negative cells as aresult of critical telomere shortening; or if it follows a patternof ‘expansion and retention’ where cells with active telomer-ase are there from the outset and undergo positive selectionduring the progression of the disease.13,14 However, becausecurrent methods cannot isolate cell populations it has notbeen possible to investigate this satisfactorily. To facilitatesuch investigations, a method that can measure hTERT in sub-populations is necessary.

We have developed such a method for the detection ofintranuclear hTERT. We have validated the measurements inboth human HL60 cells undergoing dynamic changes in telo-merase during differentiation with dimethyl sulfoxide(DMSO), as well as in primary human haemopoietic cells.

Materials and methods

HL60 cell culture and induced differentiation

HL60 cells, derived from a human promyelocytic leukaemiacell line, were cultured in T75 culture flasks (Falcon, BectonDickinson, Franklin Lakes, NJ, USA) in 25 ml of L-glutamine-free RPMI-1640 medium (GIBCO Life Technologies, Paisley,UK) containing 10% fetal calf serum and 2% L-glutamine andstreptomycin and benzylpenicillin. Flasks were incubated ina 37°C environment with 5% CO2.

Bio-technical methods section (BTS)ASM Ali et al

2177HL60 cells were induced to differentiate with DMSO

(1.25% v/v) using standard methods.15 This allowed validationof the sensitivity of the assay because it has been shown byprevious authors that telomerase activity declines withinduced differentiation.16,17 DMSO differentiation of HL60cells was undertaken for 6 days and was confirmed by mor-phological analysis of cytospin preparations. After this period,‘differentiated’ cells were defined as those which wereapproaching the metamyelocyte stage: cells that had lost theirnucleolus, showed a decreased nuclear:cytoplasmic ratio ofapproximately 1:1 with evidence of nuclear lobulation, lessbasophilia and fewer cytoplasmic granules compared to thepromyelocytes.

Mononuclear and neutrophil cell separation

All fresh samples were collected in vacutainers containingEDTA. The blood was diluted 1:1 with phosphate-bufferedsaline (PBS) and the mononuclear cells (MNC) and neutrophilswere isolated from all samples by density gradient centrifug-ation on Ficoll–Hypaque (Lymphoprep, 1.077 g/ml, NycomedPharma, Oslo, Norway). Briefly, the mononuclear cells werecollected and washed with PBS containing 1% fetal calfserum. The red blood cells within the sample of neutrophilswere removed by the addition of red cell lysis solution (OrthoDiagnostic Systems, Raritan, NJ, USA) for 15 min. The cellswere then centrifuged at 400 g for 5 min and re-suspended inPBS (80% purity).

T cell separation

To isolate T cells, magnetic activated cell sorting was carriedout by with the use of a midi-MACS isolation kit and CD3microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany)according to the manufacturer’s guidelines. Enriched samplesof CD3+ T cells were obtained with 80–85% purity.

TRAP assay

For telomerase activity, cells were stored in CHAPS lysis buffer(1 × 105 cells in 200 ml) at −70°C for analysis by the TRAPassay. The lysis buffer contained 0.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate),

Figure 1 Flow cytometry detection of hTERT expression in HL60 cells. A forward and side scatter density plot of HL60 cells in suspensionshowing the gated region is shown in (a). (b) shows the optimal result obtained with 2% paraformaldehyde fixation and Triton-X100 permeabilis-ation followed by gating. The control antibody is represented by the shaded histogram and hTERT by the open histogram. Mean cell fluorescencewas calculated by subtracting the geometric mean fluorescence of the sample with added control antibody from the geometric mean fluorescenceof the sample with hTERT antibody added.

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10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l MgCl2, 1 mmol/lEGTA, 0.1 mmol/l Benzamidine, 5 mmol/l b-mercapto-ethanol, and 1 ng/ml leupeptine.

The TRAP assay was performed as described previously byKim et al7 with the use of the TRAPeze kit (Oncor, Gaithers-burg, MD, USA). Briefly this was done as follows: 2 ml of testextract was added to a 48 ml reaction mixture containing 5 mlof 10× TRAP buffer (200 mmol/l Tris-HCl (pH 8.3), 15 mmol/lMgCl2, 630 mmol/l KCl, 0.5% Tween 20, 10 mmol/l EGTA);1 ml of 50× dNTP mix (2.5 mmol/l each of dATP, dTTp, dGTPand dCTP); 2 ml of 32P-labelled TS primer, 1 ml of TRAPprimer mix, 0.4 ml of Taq polymerase and 38.6 ml of PCRgrade water at 30°C for 30 min. The products were amplifiedby 30 cycles at 94°C for 30 s and 48°C for 30 s each and wereseparated by electrophoresis on a 12.5% polyacrylamide gel.A manufacturer-supplied positive control, negative control(lysis buffer) and quantification template (TSR8) were also runon each gel. The gel was dried for 1 h at 80°C and thenexposed on a Molecular Dynamics Storm 860 Phosphorim-ager (Molecular Dynamics, Sunnyville, CA, USA) and quant-ified using Image QuaNT software according to the manufac-turer’s instructions provided with the TRAPeze kit.

Immunofluorescent staining for CD 11b

Staining of the CD11b cell differentiation marker was carriedout according to the manufacturer’s guide lines as follows:cultured cells were suspended in cold wash buffer (PBS sup-plemented with 1% FCS and 1% human AB serum to blocknon-specific Fc-mediated binding) at a concentration of2 × 107/ml. Fifty microliter aliquots of cell suspension (1 × 106

cells) were then placed in 5 ml flow cytometry tubes and tothis 50 ml of diluted antibody was added (either 30455X phy-coerythrin-conjugated mouse anti-human CD11b antibody or33815X Mouse IgG1,k isotype control (both from BD Phar-Mingen, San Diego, CA, USA)). Antibody was left to incubatefor 30 min and the cells were then re-suspended and centri-fuged at 400 g in 1 ml of wash buffer twice. The final suspen-sion was then re-suspended in 200 ml of wash buffer for flowcytometry analysis.


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Figure 2 Detection of changes in hTERT protein expression (open histograms) in HL60cells by the developed assay. (a) shows the gradualreduction of hTERT expression during the first 3 days of DMSO-induced differentiation followed by a rapid decline on day 4 and thereafterremaining low. The control cells, which remained undifferentiated, showed positive hTERT expression throughout the 6 day period as shownin (b).

Results

Optimal staining method

Optimal staining was achieved by the following protocol:5 × 106 HL60 cells were taken from a flask, centrifuged at200 g for 10 min, and re-suspended for washing in 10 ml ofwash buffer (PBS supplemented with 1% fetal calf serum). Thecells were again centrifuged at 200 g for 10 min. The cellswere then fixed in 1 ml of 2% paraformaldehyde and incu-bated on ice for 30 min. The cells were then washed again in10 ml of wash buffer and re-suspended in 3ml of 0.5% Triton-X100. After permeabilisation, cells were washed once morein 10 ml of wash buffer and re-suspended in wash buffer at aconcentration of 2 × 107 per ml. Aliquots of the cell suspen-sion (50 ml) were put into 5 ml flow cytometry tubes and tothese 50 ml of diluted antibody was added (either primary SC-

7214 goat anti-hTERT antibody, primary SC-6370 goat anti-TP1 antibody (TP1 is another protein component oftelomerase) or SC-2028 normal IgG control antibody (all fromSanta Cruz Biotechnology, Santa Cruz, CA, USA)) and left toincubate for 40 min at room temperature. A manufacturer’sblocking peptide was also used as an additional control insome experiments. The suspension was then washed twice byaddition of 1 ml of wash buffer and centrifuging at 800 g andfinally re-suspended in 100 ml of wash buffer. A secondarydiluted SC-2024 donkey anti-goat IgG fluorescein isothiocy-anate (FITC)-conjugated antibody (Santa Cruz Biotechnology)was added and left to incubate at room temperature for 30min. The suspension was then washed twice again (in thesame way as after the addition of primary antibody). The sam-ple was then re-suspended in 500 ml of wash buffer and takenfor analysis on the flow cytometer (FACS Vantage/FACScan,Becton Dickinson, San Jose, CA, USA).


2179Gating was carried out as shown in Figure 1a. Mean cell

fluorescence was calculated by subtracting the geometricmean fluorescence of the sample with added control antibodyfrom the geometric mean fluorescence of the sample withhTERT antibody added (Figure 1b).

hTERT protein expression in differentiating HL60 cells

The change in hTERT protein expression of the differentiatingHL60 as detected by the developed assay is shown inFigure 2a. It can be seen that hTERT protein expression in thedifferentiating HL60 cells declined steeply on day 4 of cultureand remained low for the final 2 days. The controls that didnot have DMSO added to them showed positive expressionthroughout the 6 day period (Figure 2b). As a further control,the expression of TP1, another protein component of telomer-ase, was also assayed using this method and did not changeduring the 6 day study period (data not shown).

When hTERT protein expression was very low a slight nega-tive shift was seen. This was due to a small degree of positiveactivity exhibited by the control normal IgG antibody used(confirmed by the use of manufacturer’s blocking peptide(Figure 3).

TRAP analysis

The PCR-based TRAP assay confirms the association betweenthe expression of hTERT protein as shown by the flow cytome-try assay and differentiation. It is clear that telomerase activityreduces through the 3 day period and again is virtually lostby day 4 (Figure 4a). The data were also quantified using aPhosphorimager according to the manufacturer’s protocol andthese data can be shown to correlate well with the mean cellfluorescence data from the flow cytometry assay (Figure 4band c). Statistical analysis (Pearson’s product momentcorrelation) comparing the quantified data from the two assaysacross the three experiments showed a significant correlation(1% significance level, r2 = 0.66).

Figure 3 Comparison of control antibody with manufacturer’sblocking peptide. Confirmation that the negative shift seen in situ-ations of very low expression of hTERT is due to a small degree ofpositive activity exhibited by the control antibody (shaded) by use ofthe manufacturer’s blocking antibody (faint line). Note that hTERT(dark line) is positive compared to the blocking peptide.

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Figure 4 Validation of flow cytometry method for hTERT detectionby comparison with conventional TRAP assay of telomerase activityin differentiating HL60 cells. (a) shows a representative gel confirmingloss of detectable telomerase activity by day 4 of differentiation, com-pared with maintenance of telomerase activity in undifferentiated con-trols. Quantitative comparison between flow cytometry method andTRAP assay is shown in (b). (Due to large data range, Phosphorimagerunits are shown on a log scale for ease of comparison. Data shownquantifies the gel shown in (a)) The significant correlation of the twomethods is shown in (c).

Analysis of hTERT expression in a mixed cellpopulation

The assay was applied to an artificially created mixed popu-lation of undifferentiated HL60 cells and DMSO differentiated


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cells (day 6). Analysis showed the presence of two popu-lations: one population showed significant positive expressionof hTERT, while the second population showed no significantexpression (Figure 5a). The nature of the two populations(undifferentiated and differentiated) was further confirmed bysurface staining for the CD11b differentiation marker(Figure 5b) on another sample of cells taken from the samemixture. Concurrent dual staining for CD11b and hTERT wasnot possible because the fixation and permeabilising tech-niques required to access the intranuclear hTERT antigeninterfered with the CD11b antibody’s action.

Relative expression of hTERT in varioushaematological cell types

The assay was applied to primary blood cells obtained from10 healthy individuals and six patients with B-CLL. Using thegeometric mean cell fluorescence, it was possible to see therelative expression of the protein in the different cell types, asshown in Figure 6. A clear difference in levels of expressioncan be seen between the malignant and normal cells. Mono-nuclear cells (MNC) derived from patients with advanced B-

Figure 5 Application of the flow cytometry assay to a mixed cellpopulation shows that the difference in hTERT expression betweendifferentiated and undifferentiated HL60 cells in an artificial mix canbe clearly identified by the assay (a). (b) shows further confirmationof the presence of two populations (one differentiated and oneundifferentiated) via use of the CD11b differentiation marker. Controlantibody is represented by the shaded histogram and hTERT by openhistogram. The twin peaks represent the two sub-populations – theintranuclear nature of the hTERT assay means that control antibodyalso shows twin peaks but with less separation.

Figure 6 Application of the flow cytometry assay to primaryhuman cells showing relative hTERT expression compared with undif-ferentiated HL60 cells. The highest levels are detected in advancedCLL and HL60 cells, with low expression in early CLL and normalMNC and T cells. Neutrophils show no detectable hTERT expression.

CLL (Binet stages B and C) showed higher hTERT expressionthan MNC from patients with early B-CLL (Binet stage A). Theundifferentiated HL60 cell line showed a level of expressionintermediate between early and late CLL. In addition, it couldbe seen that normal MNC and T cells also exhibited a lowlevel of hTERT expression with only neutrophils showing nodetectable hTERT expression.

Discussion

We describe an immunofluorescence flow cytometry-basedassay for measurement and quantification of the hTERTcomponent of telomerase. This method will allow sub-popu-lations of cells to be separated according to hTERT expressionprior to further manipulation, which is not possible usingcurrent techniques of telomerase detection.

The sensitivity of the assay and its ability to detect changesin hTERT expression was established using HL60 cells that hadbeen induced to differentiate with DMSO. Differentiation withDMSO has previously been shown to result in decreasedhTERT mRNA expression using RT PCR.18,19 No data on rela-tive protein expression have been published previously. It canbe clearly seen that the level of hTERT protein expressiondecreases after the first 3 days of differentiation, very sharplyon day 4 and little further on days 5 and 6 (Figure 2a). Thisobservation correlates well with the TRAP assay which alsoshows an almost complete loss of telomerase activity on day4 (Figure 4a). The correlation between the TRAP assay andhTERT protein expression would suggest that the hTERT anti-body used is detecting functional isoforms of hTERT.

The TP1 component of telomerase, however, showed nosignificant change in expression during differentiation, addingfurther weight to the proposition that hTERT protein is thecatalytic rate-limiting sub-unit of telomerase10,18 and that itsexpression at both the mRNA and protein level can be usedas a diagnostic indicator of telomerase activity. Further vali-dation of sensitivity was obtained by carrying out an analysisof a mixed cell population of differentiated and undifferen-tiated cells where it could be seen that the assay was able tovisualise and clearly identify the difference in hTERTexpression between the two populations. Surface staining of


2181the CD11b differentiation marker gave further confirmation ofthe differentiation status of the two populations.

The analysis of hTERT protein expression by flow cytometryshowed the general applicability of the assay and also enableda relative quantification of telomerase activity in differentcomponents of blood. The lack of hTERT expression in neutro-phils correlates well with previous findings which have gener-ally shown that telomerase activity is not present in neutro-phils.20,21 The low expression of hTERT protein in normalmononuclear cells and T cells is also consistent with previousfindings of telomerase activity in normal blood leukocytes andmay explain the continuous age-related telomere shorteningin T cells despite telomerase expression.21 The importance ofdeveloping such a sensitive assay is further underlined by thefact that hTERT mRNA expression is not readily detected byNorthern blotting.18

The analysis of mononuclear cells (MNC) derived frompatients suffering from early and advanced stage B cellchronic lymphocytic leukaemia (B-CLL) shows an increase inhTERT activity according to the stage of the disease. This find-ing is in agreement with previous studies of CLL, which havesuggested that, in NZB mice, telomerase activity correlateswith disease stage22 and in humans it is associated with bothdisease progression and probability of survival.12

To conclude, we have developed a quick, safe and sensitiveassay for hTERT protein expression. hTERT expression as mea-sured by this assay correlates well with telomerase activity andthe assay allows a degree of relative quantification. The anti-bodies used in this study were polyclonal, but as monoclonalantibodies to hTERT become commercially available, itshould be possible to increase the sensitivity and reliability ofthe assay even further. It is hoped that the multi-parameternature of flow cytometry and its ability to identify cellular sub-populations will enable a fuller understanding of the mech-anisms of activation of telomerase and hTERT during diseaseprogression and allow concurrent analysis of other cellularproteins which may be involved in its regulation.

Acknowledgements

ASM Ali is supported by the University of Manchester and theJHG Holt Scholarship and Special Exhibition in Medicine. RChopra is supported by the Christie Hospital LeukaemiaEndowment fund and Chugai Pharmaceuticals. J Robertson issupported by the Leukaemia Research Fund. NG Testa issupported by the Cancer Research Campaign.

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detection of htert protein by flow cytometry

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