Domingo, E. (Esther)

Merino, J. (Juana)

Moreno, C. (Cristina)

Panizo, C. (Carlos)

Paramo, J.A. (José Antonio)

Sanchez-Ibarrola, A. (Alfonso)

Dadun, University of Navarra

Enhanced sensitivity of flow cytometry for routine assessment of minimal residual diseaseIn a recent paper by Béné and Kaeda,1 technicalapproaches for minimal residual disease (MRD) assess-ment are extensively reviewed. PCR-based studies haveproved to be 1-log more sensitive than flow cytometry(FC). For this reason, they are increasingly being preferredfor MRD analysis, especially at the end of therapy or posthematopoietic stem cell transplantation.1,2 It would bevaluable to develop MRD flow cytometry assays with thislevel of sensitivity that could be applied routinely. In thepresent work, we analyzed MRD+ samples with a level ofinfiltration below the limit of detection of routine FC,which is accepted as 10-4 and comes from the standardacquisition of 2-5×105 leukocytes.3-11 At least 10-fold moreleukocytes must be acquired to increase sensitivity by 1-log; this large number of leukocytes can be acquired easi-ly in digital cytometers by acquiring several individualtubes stained with the same combination of monoclonalantibodies, and putting them in a single file. Because thetime of acquisition for each individual tube is notincreased, no problems of cellular aggregation arise. Using this approach, we acquired 6 million leukocytesfrom each sample using a FACSCanto flow cytometer(Becton-Dickinson). The stability of fluorescent parame-ters between tubes was verified (Online SupplementaryFigure S1). The study was performed with the event rateused routinely in our laboratory, i.e. 2,000 events/sec. Toexplore the possibility of decreasing acquisition time, weanalyzed the influence of increasing the event rate on thepercentage of electronic aborts (due mainly to coincidenceevents), and compared MRD measurements in samplesacquired at different event rates (Online SupplementaryTable S1). We found that the acquisition event rate couldbe increased reliably to at least 4,000 events/sec. Usingthis event rate, the time of acquisition of 6 million leuko-cytes is suitable for routine measurements. We assessed that this approach enhanced sensitivity by1-log by comparing MRD analysis by FC with real-timequantitative PCR for BCR-ABL1p190 transcripts in serialdilutions of a Philadelphia chromosome-positive B-II acutelymphoblastic leukemia sample (Table 1A). We thenselected multiple myeloma (MM) and B-cell chronic lym-phocytic leukemia (BCLL) MRD+ samples with about0.01% infiltration and diluted them 10-fold with normalleukocytes. As shown in Table 1B, MRD was detectedaccurately in all diluted samples (10-5 infiltration).Observed values were as expected ±10% in all cases.This increase in sensitivity did not compromise thespecificity of the technique. Ten million leukocytes fromthe bone marrow of patients (n = 3) without hematologicneoplasias and from the peripheral blood of healthydonors (n = 3) were acquired and blindly tested for thepresence of events with a myelomatous or B-CLL pheno-type, respectively. The results were unambiguously nega-tive (Online Supplementary Figure S2). We also acquired 10million normal leukocytes and searched for events withphenotypic characteristics of other hematologic malignan-cies, such as follicular lymphoma and hairy cell leukemia,with negative results.In addition to the total number of leukocytes analyzed,the sensitivity of FC depends on the number of neoplasticevents that must be detected in order to obtain precisemeasurements.Based on the theory that rare events follow a Poissondistribution, it is accepted that 50-100 events are neededto reliably measure the frequency of a population.3,7,8,11,12However, to our knowledge, no studies have really meas-ured the influence of the number of detected tumoral cellson MRD measurement precision. Accordingly, we deter-mined the coefficient of variation (CV) of the percentageof MRD in samples from patients with MM, B-CLL and T-cell lymphoproliferative disorders (T-CLPD) obtainedhaematologica | 2010; 95(4) 691Letters to the EditorTable 1. Accuracy of minimal residual disease assessment in sam-ples with infiltration <10-4. Sample Sample diluted 10-foldNumber of acquired leukocytes 0.5 6(millions)MM 1 MRD (%) 0.0127 0.00127# Cluster events 94 54MM 2 MRD (%) 0.017 0.0017# Cluster events 87 100MM 3 MRD (%) 0.012 0.00118# Cluster events 82 87MM 4 MRD (%) 0.02 0.0018# Cluster events 107 99B-CLL 1 MRD (%) 0.015 0.0014# Cluster events 78 114B-CLL 2 MRD (%) 0.024 0.0022# Cluster events 124 181B-CLL 3 MRD (%) 0.025 0.0024# Cluster events 139 151B-CLL 4 MRD (%) 0.022 0.0024# Cluster events 122 157Sample Sample seriallydiluted 10-fold% MRD by FC 1.5 0.16 0.016 0.001% MRD by RQ-PCR 4.9 0.33 0.025 0.001(A) Minimal residual disease (MRD) levels measured by flow cytometry (FC) orBCRABL1p190 transcripts quantitation by real-time quantitative PCR (RQ-PCR) in seri-al dilutions of a Philadelphia chromosome-positive B-II acute lymphoblasticleukemia bone marrow sample. MRD results by FC are expressed as percentage oftotal leukocytes. MRD results by RQ-PCR are expressed as BCR-ABL1p190 copy numberper 100 molecules of GUS. (B) MRD levels measured by FC in samples with 10-5 infil-tration: MRD+ samples with about 0.01% infiltration from 4 patients with multiplemyeloma (MM) and from 4 with B-cell chronic lymphocytic leukemia (B-CLL) wereassessed. The percentage of MRD for each sample (“Sample”) represents the meanof 5 independent measurements. In each measurement, 0.5 million leukocytes wereacquired. These MRD+ samples were diluted 10-fold with normal leukocytes in orderto obtain tumoral infiltrations in the range of 10-5 (“Sample diluted 10-fold”). The per-centage of MRD in diluted samples was measured by acquiring 6 million leukocytes.The number of malignant events detected in each measurement is shown.BA% MRD by RQ-PCR% MRD by FC0.0001 0.001 0.01 0.1 1 101010.10.010.0010.0001©Ferrta Storti Foundationwhen detecting increasing numbers of events in the malig-nant cluster. As shown in Table 2, the CVs of the B-CLLand T-CLPD samples were very close to those predictedby the Poisson distribution. Fifty to 60 events wererequired to obtain a CV less than 15%. Strikingly, in MMsamples, the CVs were around 10% regardless of the sizeof the cluster, even when as few as 20 malignant eventswere detected (possibly because it is easier to identifymalignant cells from MM than from other hematologicmalignancies, since myelomatous plasma cells usuallyoccupy a space in which background events are scarce).In summary, acquiring 6 million leukocytes is feasiblewith a digital cytometer on a routine basis. Because detec-tion of 50-60 malignant cells is required to get a CV lessthan 15%, a sensitivity of 1×10-5 is achieved. Being able toroutinely apply MRD FC assays with high sensitivitywould be very valuable, especially in cases where molec-ular techniques cannot be used.Esther Domingo,1 Cristina Moreno,1 Alfonso Sánchez-Ibarrola,1 Carlos Panizo,2 José Antonio Páramo2and Juana Merino11Department of Immunology, Clínica Universidad de Navarra,University of Navarra; 2Department of Haematology, ClínicaUniversidad de Navarra, University of Navarra, Spain.Key words: minimal residual disease, flow cytometry, sensitivity,specificity, precision, coefficient of variation.Correspondence: Juana Merino, Department of Immunology,Clínica Universidad de Navarra, University of Navarra, Avda. PioXII 36, 31008 Pamplona, Spain. E-mail: jmerino@unav.es. Citation: Domingo E, Moreno C, Sánchez-Ibarrola A, Panizo C,Páramo JA, and Merino J. Enhanced sensitivity of flow cytometry forroutine assessment of minimal residual disease. Haematologica.2010; 95:691-692. doi:10.3324/haematol.2009.018911References1. Béné MC, Kaeda JS. How and why minimal residual disease stud-ies are necessary in leukemia: a review from WP10 and WP12 ofthe European LeukaemiaNet. Haematologica 2009; 94(8):1135-50.2. Campana D. Minimal Residual Disease in Acute LymphoblasticLeukemia. Semin Hematol 2009;46(1):100-6.3. Craig FE, Foon KA. Flow cytometric immunophenotyping forhematologic neoplasms. Blood 2008;111(8):3941-67.4. Paiva B, Vidriales M, Cerveró J, Mateo G, Pérez JJ, Montalbán MA,et al. Multiparameter flow cytometric remission is the most rele-vant prognostic factor for multiple myeloma patients who undergoautologous stem cell transplantation. Blood 2008;112(10):4017-23.5. Krampera M, Perbellini O, Vincenzi C, Zampieri F, Pasini A, ScupoliMT, et al. Methodological approach to minimal residual diseasedetection by flow cytometry in adult B-lineage acute lymphoblas-tic leukemia. Haematologica 2006;91(8):1109-12.6. De Tute RM, Jack AS, Child JA, Morgan GJ, Owen RG, RawstronAC. A single-tube sixcolour flow cytometry screening assay for thedetection of minimal residual disease in myeloma. Leukemia2007;21(9):2046-9.7. Rawstron AC, Villamor N, Ritgen M, Böttcher S, Ghia P, ZehnderJL, et al. International standardized approach for flow cytometricresidual disease monitoring in chronic lymphocytic leukaemia.Leukemia 2007;21(5):956-64.8. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA,Bumbea H, et al. Report of the European Myeloma Network onmultiparametric flow cytometry in multiple myeloma and relateddisorders. Haematologica 2008;93(3):431-8.9. Borowitz MJ, Devidas M, Hunger SP, Bowman WP, Carroll AJ,Carroll WL, et al. Clinical significance of minimal residual diseasein childhood acute lymphoblastic leukemia and its relationship toother prognostic factors: a Children's Oncology Group study.Blood 2008;111(12):5477-85.10. Dworzak MN, Gaipa G, Ratei R, Veltroni M, Schumich A, MagliaO, et al. Standardization of Flow Cytometric Minimal ResidualDisease Evaluation in Acute Lymphoblastic Leukemia: MulticentricAssessment is Feasible. Cytometry B Clin Cytom 2008;74(6):331-40.11. Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L, et al.Establishment and validation of a standard protocol for the detec-tion of minimal residual disease in B lineage childhood acute lym-phoblastic leukemia by flow cytometry in a multi-center setting.Haematologica 2009;94(6):870-4.12. Clinical Laboratory Standards Institute. Enumeration ofImmunologically Defined Cell Populations by Flow Cytometry;Approved guideline-2nd edition document H42-A2. 2007.CLSI/NCCLS, Wayne, Pennsylvania.692 haematologica | 2010; 95(4)Letters to the EditorTable 2. Precision of minimal residual disease assessment accord-ing to the number of events in the malignant cluster.# Cluster MRD CVevents (%) (%)MMCase 1 19 0.0127 943 0.013 1094 0.0127 10Case 2 30 0.018 1044 0.017 1087 0.017 9Case 3 25 0.012 1038 0.011 1082 0.012 10Case 4 39 0.021 854 0.02 10107 0.02 10Case 5 22 0.0027 942 0.003 1091 0.003 8B-CLLCase 1 26 0.016 2036 0.014 1978 0.015 11Case 2 37 0.023 1860 0.023 14124 0.024 7Case 3 37 0.022 1355 0.023 9139 0.025 10Case 4 36 0.021 962 0.023 12122 0.022 10Case 5 31 0.049 355 0.051 7130 0.05 9T-CLPDCase 1 30 0.04 1250 0.038 12272 0.04 4Case 2 25 0.025 2047 0.024 1379 0.026 11Case 3 48 0.031 1286 0.032 8186 0.032 8Case 4 57 0.031 12106 0.031 8193 0.03 2Minimal residual disease (MRD) was measured in samples from 5 patients withmultiple myeloma (MM), 5 patients with B-cell chronic lymphocytic leukemia (B-CLL), and 4 patients with T-cell lymphoproliferative disorders (T-CLPD) by detectingthree different levels of malignant cells (approximately 20-30, 40-60, and 80-200).Results are presented as the mean of 5 independent trials. The CV was calculated as(standard-deviation/mean) x 100.©Ferrata Storti Foundation

Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease

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