Leukemia (2005) 19, 776783 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.

00
www.nature.com/leu

Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome
L Malcovati1, MG Della Porta1, M Lunghi1, C Pascutto1, L Vanelli1, E Travaglino2, M Mafoli1, P Bernasconi1, M Lazzarino1, R Invernizzi2 and M Cazzola1
1 Division of Hematology, University of Pavia Medical School & IRCCS Policlinico S. Matteo, Pavia, Italy; and 2Department of Internal Medicine, University of Pavia Medical School & IRCCS Policlinico S. Matteo, Pavia, Italy

The purpose of this study was to develop a ow cytometric approach to the evaluation of marrow dysplasia in myelodysplastic syndromes (MDS). We rst studied a cohort of 103 MDS patients as well as 46 pathological and healthy controls. Flow cytometry data were expressed as percentage of positive cells. Analysis of erythroid cells showed higher proportions of immature cells (Po0.001) and decreased levels of CD71 expression on nucleated red cells (P 0.02) in MDS. Analysis of myeloid cells showed lower proportions of CD10 and higher proportions of CD56 granulocytes (Po0.001), and increased ratios of immature to mature cells (P 0.007). Since no single immunophenotype could accurately differentiate MDS from other conditions, we used discriminant analysis for generating erythroid and myeloid classication functions using combinations of immunophenotypic parameters. These functions were prospectively validated in a testing cohort of 69 MDS patients and 46 pathological controls. A diagnosis of MDS was obtained in 60/69 cases (87%). No false-positive results were noticed among controls. Signicant correlations between values of these functions and both degree of morphological dysplasia and the International Prognostic Scoring System were found. These ndings indicate that ow cytometry evaluation of marrow dysplasia is feasible and may be useful in the work-up of individual MDS patients. Leukemia (2005) 19, 776783. doi:10.1038/sj.leu.2403680 Published online 24 March 2005 Keywords: myelodysplastic syndrome; ow cytometry; immunophenotyping; CD34; CD71

formation is poor or absent in most of these patients.4 Thus, overt marrow dysplasia, clonal cytogenetic abnormality, and reduced in vitro progenitor cell growth allow a conclusive diagnosis of MDS.1 This combination is, however, found in only some MDS patients, who tend to be those with more advanced disease.5 In many instances, cytogenetics is not informative and BM cultures are not performed, so that the diagnosis of MDS is based entirely and exclusively on morphological criteria. Flow cytometry immunophenotyping, a reliable method for quantitative and qualitative evaluation of hematopoietic cells, is playing an increasingly crucial role in the diagnosis and management of acute leukemias.6 MDS patients have been found to have abnormal expression of several surface antigens, as indicated by either the intensity of uorescence or the percentage of positive cells.7 Findings of recent studies suggest that ow cytometry immunophenotyping might provide useful information in management of MDS patients.812 The purpose of this study was to develop a ow-cytometric approach to the evaluation of erythroid and myeloid dysplasia in patients with MDS.

Materials and methods

Patients and controls

Introduction
Myelodysplastic syndromes (MDS) are abnormal proliferations of hematopoietic stem cells that retain their capacity to differentiate but do so in an inefcient manner, so that bone marrow (BM) and peripheral blood cells show various morphological abnormalities and the numbers of mature blood cells are variably reduced. 1,2 These disorders are characterized clinically by BM failure, followed by a progressive impairment in the ability of myelodysplastic stem cells to differentiate and an increasing risk of evolution into acute myeloid leukemia (AML). Since dysplasia is the pathologic hallmark and various chromosomal abnormalities may be found in these patients, the current diagnostic approach includes peripheral blood and BM morphology, BM histology, and cytogenetics.3 In vitro cultures of hematopoietic progenitors can provide additional useful information for the diagnosis of MDS, since colony
Correspondence: Dr M Cazzola, Division of Hematology, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy; Fax: 39 0382 502250; E-mail: mario.cazzola@unipv.it This study was supported by a grant to MC from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Research Project entitled Genomic analysis of hematopoietic cells in myelodysplastic syndromes and myeloproliferative disorders), Milan, Italy. Received 2 July 2004; accepted 7 January 2005; Published online 24 March 2005

This study enrolled patients followed at the Division of Hematology, IRCCS Policlinico San Matteo and University of Pavia Medical School, Pavia, Italy. The procedures followed were in accordance with the Ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 1983. The cytological diagnosis of MDS was initially made according to the FAB criteria,13 all cases were then reclassied according to the WHO classication.3 Patients with RAEB-T were excluded from this study. Morphological analysis of marrow specimens was performed by two or three independent cytologists, who were blinded to the cytometric data. A total of 500 BM nucleated cells per sample were assessed. The evaluation of the erythroid lineage was based on the detection of megaloblastic changes, nuclear lobulation, multinuclearity, and cytoplasmic granules/inclusions. In the myeloid lineage, the following abnormalities were considered: bizarre nuclear shape, hypo- or agranularity, nuclear/cytoplasmic asynchrony, and pseudo-Pelger anomaly. Micromegakaryocytes, small binucleated megakaryocytes, megakaryocytes with small round separated nuclei, and megathrombocytes were considered signs of megakaryocytic dysplasia. The degree of erythroid or myeloid dysplasia was dened according to the following morphological scoring system: 0 (absence of dysplasia, 02% of abnormal cells), 1 (mild

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

dysplasia, 39% of abnormal cells), 2 (moderate dysplasia, 10 29% of abnormal cells), and 3 (severe dysplasia, 30% or more abnormal cells). Cytogenetic analysis was performed at diagnosis, using standard G-banding with trypsin-Giemsa staining,14 and karyotypes were classied using the International System for Cytogenetic Nomenclature Criteria.15 FISH studies were performed as described previously.16,17 The International Prognostic Scoring System (IPSS) was employed to dene prognosis according to Greenberg et al.18 This study involved two cohorts of patients and controls: (a) a learning cohort, whose investigations were aimed at dening discriminant immunophenotypic markers; (b) a testing cohort on which we examined whether our discriminant markers were able to reproduce what we had found in the learning cohort.1921

Flow cytometry studies

K3-EDTA BM aspirate specimens were collected and stained within 3 h using a whole-blood lysis technique (FACS lysing , CA, USA) and a panel of solution, Becton Dickinson, San Jose direct conjugated monoclonal antibodies (MoAb) according to standard procedures described previously. Six-parameter, four-color ow cytometry was performed with a FACSCalibur ow cytometer (Becton Dickinson) equipped with a 15-nW argon laser (excitation at 488 nm). Daily instrument quality control, including uorescence standardization, linearity assessment, and spectral compensation, were performed to ensure identical operation from day to day. At least 50 000 events were acquired for each sample in linear mode for side scatter (SSC) and in log mode for uorescent signals; isotype-matched negative controls were used in all assays. Data were collected and analyzed with CellQuest software (Becton Dickinson). The MoAb panel employed was as follows: uorescein isothiocyanate (FITC)-conjugated anti CD3, CD5, CD7, CD15, CD16, CD34, and CD71; phycoerythrin (PE)-conjugated anti CD4, CD8, CD10, CD13, CD19, CD33, CD34, CD56, and antiglycophorin A (GlyA); allophycocyanine (APC)-conjugated CD14, CD19 and CD45; Tri-Color (TC, PE-Cyanine5)-conjugated anti CD45. All MoAbs were purchased from Becton Dickinson except CD45-TC, which was obtained from Caltag Laboratories (Burlingame, CA, USA). In addition, Laser Dye Styryl (LDS751, Molecular Probes, Leiden, The Netherlands) was used for nuclear cell identication. The CD45 marker was tested in each combination to allow a primary gating of BM cell subsets based on CD45 antigen expression and SSC laser light diffraction as described.22 Orthogonal SSC was compared with that obtained for mature granulocytes in healthy control peripheral blood specimen processed in the same day. Cell subsets recognized by this method were blast cells (CD45lowSSClow), lymphocytes (CD45highSSClow), monocytes (CD45highSSCintermediate), and granulocytes (CD45highSSChigh). CD34, CD3, CD19, CD14 and CD33 populations were back-gated to determine whether the analysis gates were appropriate. Nucleated red cells were gated using a combination of CD71/ GlyA/LDS751/CD45. All MoAbs were analyzed on all cell populations dened in the CD45/SSC dot-plot. Gating strategy to identify erythroid precursors was based on nuclear staining (LDS751) to exclude cellular debris and nonviable cells: erythroblasts were dened in LDS751 gate as GlyA / CD45low/SSClow cells.

Blasts were gated in the CD45 vs SSC dot-plot as CD45low SSClow cells. The immature B-cell precursors were identied in the CD45lowSSClow region on the basis of CD10, CD19, and CD34 co-expression, and excluded from the analysis. Analysis of CD34 cells was performed following the cellgating guidelines recommended by the International Society for Hematotherapy and Graft Engineering (ISHAGE)23 and the subsequent modication of the European Working Group of Clinical Cell Analysis (EWGCCA).24 The CD34 cells were characterized using the combination CD34/CD33/CD45. CD34+ B-cell precursors were excluded basing on CD10 and CD19 staining. Furthermore, myeloid cells were analyzed using various MoAb combinations (CD16/CD33, CD16/CD13, CD16/CD45); CD14 was employed to quantify monocytes and to gate-out these cells from the granulocytes. Immunophenotypic data were expressed as percentage of cells positive for one or more antigens. Multiparametric data analysis of antibody staining patterns was used to analyze different trends in antigen expression in the various populations. As the panel of antibodies was modied during the study and additional antibodies were used in the analysis of later cases, data with all the antibodies were not available for all 103 patients of the learning cohort. Samples from three MDS patients were evaluated in order to test the intralaboratory repeatability and reproducibility of specimen preparation, acquisition, and analysis. Briey, for a single specimen, aliquots were prepared in replicate by four independent investigators, who then analyzed, in a blind manner, each specimen three times.

777

Statistical analysis
An analysis of variance comparing means between multiple groups was performed to test each numerical parameter for differences between MDS patients and pathologic and healthy controls, and among MDS subgroups according to the WHO classication. A logarithmic transformation was performed to normalize the distribution of the variables. Post-hoc comparisons were carried out using Scheffes test. Associations between categorical ordinal variables were tested by Fishers exact test or Pearsons chi-square; associations between ordinal and continuous variables were tested by linear regression models. Discriminant analyses were performed to identify the presence of erythroid and myeloid dysplasia, with the aim of differentiating MDS and pathologic controls, and of classifying, together with ow-cytometric blast percentage, MDS into WHO subgroups: refractory anemia, refractory cytopenia, and refractory anemia with excess blasts. A repeated-measure analysis of variance was carried out on data obtained from three MDS samples evaluated by four blinded independent investigators, who analyzed each sample three times, in order to estimate the between-investigator (s2 bi) and within-investigator (or trial-to-trial) (s2 wi) components of variance. All analyses were performed using Statistica 6.0 software (Statsoft Inc., Tulsa, OK, USA) and Microsofts Excel 2000 (Microsoft Inc., Redmond, Washington, DC, USA).

Results
The learning cohort consisted of 103 MDS patients, 27 patients with hematological disorders sharing clinical features with MDS, and 19 healthy BM donors. MDS were classied as refractory anemia (RA) (21 cases), RA with ringed sideroblasts
Leukemia

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

778
(RARS) (15 cases), refractory cytopenia with multilineage dysplasia (RCMD) (14 cases), RCMD with ringed sideroblasts (RCMD-RS) (six cases), refractory anemia with excess blasts 1 (RAEB-1) (14 cases), RAEB-2 (28 cases), MDS with isolated del(5q) (three cases), MDS unclassied (MDS-U) (two cases). Pathological controls included 13 idiopathic or iatrogenic BM hypoplasia, four infective cytopenia, three megaloblastic anemia, four anemia associated with marrow inltration, and three idiopathic thrombocytopenic purpura.

proportion of CD10 granulocytes (Po0.001) and higher proportions of CD56 granulocytes (Po0.001). The analysis of variance for repeated measures showed that between- and

a 104

103 GLYA PE

Flow cytometry analysis of erythroid cells

The erythroid compartment was analyzed and quantied using the CD71/GlyA/LDS751/CD45 combination. A strong correlation was observed between morphological evaluation of nucleated red blood cells and immunophenotypic assessment (r 0.82, Po0.001), ow cytometry tending to underestimate the value of immature erythroid cells (slope: 0.547, intercept: 5.521). A myeloid to erythroid ratio was calculated through ow cytometry as the ratio between the percentage of CD33 cells and of nucleated red cells, which was signicantly associated with the results obtained through morphological analysis, ow cytometry resulting in an overestimation of the ratio in comparison with morphology (median value 2.96 vs 1.97, respectively) (r 0.72, Po0.001; slope: 1.731, intercept: 0.842). Data obtained from the repeated-measure analysis including specimen preparation, acquisition, and analysis by four blinded investigators showed that the between- and the within-investigator components of variance did not affect signicantly the results (P 0.80 and 0.12, respectively). A signicantly higher proportion of immature red cells was noticed in MDS patients than in controls (Po0.001, Table 1). A signicant correlation was found between the degree of erythroid dysplasia (morphological assessment) and proportion of immature red cells (ow cytometry immunophenotypic evaluation) in MDS patients (r 0.53, Po0.001). A quantitative analysis of the GlyA/CD71 pattern showed a lower percentage of GlyA CD71 nucleated red cells in MDS patients than in healthy controls (P 0.02) (Figure 1), with not signicant between- and within-investigator variability (P 0.84 and 0.98, respectively).

102

101

100 100 101 102 CD71FITC 103 104

b 104

103 GLYA PE

102

101

100 0 10

101

102 CD71FITC

103

104

Flow cytometry analysis of myeloid cells

Myeloid dysplasia was rst analyzed by quantifying the expression of each MoAb on granulocytes and monocytes. With respect to controls, MDS patients showed a lower

Figure 1 Flow cytometry analysis of erythroid cells: CD71 expression on GyA /LDS751 /CD45low/SSClow cells in a healthy stem cell donor (a) and in an MDS patient (b). The percentage of GyA+ CD71bright erythroid cells is lower in MDS samples than in normal individuals.

Table 1 Descriptive statistics of the cytometric variables collected in different study groups,a and results of the reproducibility (between2 b investigator variance, s2 bi) and repeatability (within-investigator variance, swi) analysis Cytometric variables Nucleated red cells GlyA+CD71+ nucleated red cells CD10+ granulocytes CD56+ granulocytes CD33/CD16 immature/mature cell ratio CD34+ cells CD34+CD33+/CD34+CD33 ratio
a b

Healthy donors 11.5 78.6 36 0.77 0.9 1.5 1.0 (7.414.3) (6387.9) (2538) (0.61) (0.71.2) (12) (0.61)

Pathologic controls 11 66 36 1 1.1 1.5 1.0 (7.520.0) (3178) (28.943.6) (0.71.2) (0.81.4) (12.2) (0.71.3) 26.5 53.0 12 3 1.9 3.0 2.3

MDS (1337) (38.766) (734) (15) (1.13.2) (1.46.4) (1.53.8)

Pc o0.001 0.02 o0.001 o0.001 0.007 0.003 o0.001

s2 bi 0.62 15.94 20.40 0.21 0.018 0.61 0.006

s2 wi 2.66 0.52 17.87 0.47 0.018 0.08 0.004

Immunophenotypic data are expressed as percentage of cells positive for antigens, and summarized by their median and quartiles. These results were obtained via a repeated-measure analysis of variance on data from three MDS samples evaluated by four blinded independent investigators, who analyzed each sample three times. c All the variables were log-transformed before the analysis of variance to normalize the distribution.
Leukemia

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

779
within-investigator variances were not signicant in both CD10 (P 0.11 and 0.98, respectively) and CD56 (P 0.95 and 0.40, respectively) expression analysis. A signicant correlation was found between the proportion of both CD10 and CD56 granulocytes and the degree of myeloid dysplasia as dened by morphological assessment (r 0.38, Po0.001 and r 0.39, P 0.002, respectively). In order to evaluate myeloid maturation, we analyzed the following patterns: CD33 vs CD16, CD45 vs CD16, and CD13 vs CD16. All these patterns were more frequently abnormal in MDS patients than in controls (P from 0.02 to 0.001). For each pattern, we analyzed and quantied the myeloid subpopulations according to different antigenic expression. As shown in Figure 2, MDS patients showed increased proportions of the more immature immunophenotypic compartments (CD33 CD16, CD45 CD16, CD13 CD16) of myeloid cells and decreased proportions of mature granulocytes (CD33 CD16 , CD45 CD16 , CD13 CD16 ). Between- and within-investigator variances were estimated for the CD33 vs CD16 expression analysis. The repeated-measures analysis of variance showed that both these components of the analytical variance did not signicantly affect the result (P 0.94 and 0.95, respectively). For each of these patterns, we calculated an immature to mature cell ratio. Thus, CD33/CD16, CD45/CD16, and CD13/ CD16 ratios were signicantly higher in MDS patients than in controls (P values from 0.02 to 0.001) (Table 1). Signicant positive correlations were found between these ratios and the degree of morphological marrow myeloid dysplasia (r values ranging from 0.30 to 0.67, P values from 0.002 to o0.001).

Flow cytometry analysis of CD34 cells

The percentages of CD34 cells and CD34 CD33 cells were signicantly higher in MDS patients than in controls (P 0.006 and Po0.001, respectively). Linear regression analysis showed a close relationship between the degree of erythroid and myeloid dysplasia (morphological assessment) and the proportion of CD34 cells (r 0.31, P 0.001 and r 0.48, Po0.001, respectively). A ratio between the CD34 CD33 and CD34 CD33 cells was calculated (Figure 3), signicantly higher in MDS patients than in controls (Po0.001, Table 1), and positively correlated with both erythroid and myeloid dysplasia (r 0.27, P 0.006 and r 0.40, Po0.001, respectively). Repeatedmeasures analysis of variance showed that neither the between-investigator nor the within-investigator components of variance signicantly affected the results of CD34 (P 0.27 and 0.30, respectively) and CD34/CD33 analyses (P 0.94 and 0.95, respectively).

a 104

10 CD33 PE

102

101

100 10 0 10 1 10 2 CD16 FITC 10 3 10 4

Flow cytometry analysis of blast cells

Blast cells were identied as CD45lowSSClow cells on the CD45/ SSC dot-plot and conrmed with a back-gating technique. Immature B-cell precursors were identied in the CD45low SSClow region on the basis of CD10, CD19, and CD34 coexpression, and excluded from the analysis. The percentage of CD45lowSSClow blast cells was 2% in our controls and between 0.19 and 20% (median 4%) in MDS patients (Po0.001). A strong positive linear correlation between the proportion of BM blast cells estimated by morphology and the proportion of blast cells gated on the dot-plot CD45/SSC was noticed (r 0.85, Po0.001; slope: 0.929, intercept: 0.625). A positive linear correlation was also found between the proportion of blast cells estimated by cytometry and the proportion of both CD34 (r 0.86, Po0.001; slope: 0.766, intercept: 0.302) and CD34 CD33 BM cells (r 0.84, Po0.001; slope: 0.599, intercept: 0.048). Repeated-measure analysis of variance showed that between-investigator and within-investigator variances did not signicantly affect the results of such an analysis (P 0.90 and 0.67, respectively).

b 104

103 CD33 PE

102

101

100 10
0

10 1

10 2 CD16 FITC

10 3

10 4

Discriminant analysis: erythroid dysplasia

Discriminant analysis was performed with the aim of distinguishing MDS patients from controls based on ow cytometry estimates that were previously found to be related to erythroid dysplasia. A classication function was obtained as a linear
Leukemia

Figure 2 Flow cytometry analysis of myeloid cells: analysis of CD33 vs CD16 pattern. Increased proportions of myeloid cells in the more immature immunophenotypic compartments (CD33 CD16) and reduced proportions of mature granulocytes (CD33 CD16 ) are visible in MDS patients (b) compared to normal controls (a).

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

780
Table 2 Monoclonal antibody combinations used for the analysis of erythroid and myeloid marrow dysplasia Hematopoietic compartment Erythroid lineage Myeloid lineage CD34+ cells Antibody combination CD71/GLYA/LDS751/CD45 CD16/CD33/CD45 CD10/CD45 CD56/CD45 CD34/CD33/CD45

Table 3

Erythroid and myeloid classication functionsa Erythroid function coefcients 3.3161 7.0227 12.4856 0.2125 F F F 19.0721 Myeloid function coefcients 9.1799 4.4653 F F 4.9654 3.0147 10.8109 17.4053

Cytometric variables CD34+ cells CD34+CD33+/ CD34+CD33 ratio Nucleated red cells GlyA+CD71+ nucleated red cells CD10+ granulocytes CD56+ granulocytes CD16/CD33 immature to mature cell ratio Constant
a

The following classication functions were evaluated. Erythroid function: YE 3.3161 log(CD34+ cells)+7.0227 log(CD34+CD33+/ CD34+CD33ratio)+12.4856 log(nucleated red cells)+0.2125 log(GlyA+CD71+nucleated red cells)19.0721. Myeloid function: YM 9.1799 log(CD34+ cells)+4.4653 log(CD34+CD33+/CD34+CD33 ratio)+4.9654 log(CD10+ granulocytes)+3.0147 log(CD56+granulocytes)+10.8109 log(CD16/CD33 immature to mature cell ratio)17.4053.

Figure 3 Flow cytometry analysis of CD34 cell subsets: determination of CD34 CD33 to CD34 CD33 cell ratio. The ratio was signicantly higher in MDS patients (b) than in normal controls (a).

combination of the following immunophenotypic variables (logtransformed): percentage of BM CD34 cells, CD34 CD33 /CD34 CD33 cell ratio, nucleated red cell percentage, and GyA/CD71 expression. Antibody combinations used for the analysis of parameters of interest are specied in Table 2. In order to classify a subject, the function must be evaluated using the patients values of the selected parameters: the patient will be classied as having MDS if the obtained value is greater than zero or as not having MDS if the value is lower than zero. Table 3 shows the erythroid classication function. Within the MDS group, only patients with denitive morphological evidence of erythroid dysplasia were included in the discriminant analysis. As mentioned previously, the antibody panel evolved during the study, and therefore not all patients were valuable for the discriminant analysis. Consequently, the discriminant function was tailored on a total of 68 subjects, 32 MDS patients and 36 pathologic controls. Of the 32 MDS patients, 30 (93.7%) were correctly classied.
Leukemia

Signicant positive correlations were observed between the numerical value of the erythroid classication function and both the degree of the erythroid marrow dysplasia assessed by morphology (r 0.61, Po0.001), and the IPSS (r 0.56, P 0.001). Out of 36 control subjects, 34 (94.4%) were correctly classied. The two false positives were patients with megaloblastic anemia. In all, 20 MDS patients had isolated macrocytosis or normomacrocytic anemia with not denitive evidence of erythroid dysplasia at the time of diagnosis. In these cases, the diagnosis of MDS was based upon the presence of cytogenetic abnormalities (12 patients) and/or defective in vitro colony formation. In all these cases, the diagnosis of MDS was conrmed by follow-up investigations. These patients were not included in the discriminant analysis: using the erythroid discriminant function, a diagnosis of erythroid dysplasia was formulated in 12 out of 20 of these patients with nondiagnostic erythroid morphology.

Discriminant analysis: myeloid dysplasia

A discriminant function based on percentage of BM CD34 cells, myeloid/lymphoid ratio, CD10 and CD56 expression on myeloid cells, and a CD33/CD16 ratio of immature to mature myeloid cells was estimated in order to distinguish MDS patients with myeloid dysplasia from MDS patients without myeloid involvement or controls. Antibody combinations used for the analysis of parameters of interest are shown in Table 2. All variables were log-transformed to normalize their

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

781
distributions. The myeloid classication function obtained (Table 3) was then integrated with the immunophenotypic blast count, based on the dot-plot CD45/SSC and FSC/SSC, and conrmed with a back-gating technique, to discriminate among refractory cytopenia, RAEB-1, and RAEB-2. A total of 31 MDS patients and 23 controls were included in this analysis. Only two subjects were incorrectly classied using the myeloid discriminant function (6.5%). No false positives were detected among control subjects. Strong positive relationships were found between the value of the myeloid discriminant function and both the degree of morphological myeloid dysplasia (r 0.79, Po0.001), and the IPSS (r 0.67, Po0.001). MDS-U and a mild myeloid dysplasia had a negative myeloid function. Seven MDS patients had an incorrect WHO subgroup assignment. No false-positive cases were noticed among 46 pathologic controls (Figure 4). In this cohort, a denite morphologic diagnosis of MDS, based upon marrow aspirate concurrently used for cytometry, was obtained in 44 out of 69 MDS patients (64%). Among patients with a conclusive morphology, a ow-cytometric diagnosis of MDS was formulated in 41 of 44 cases (93%), while in patients with an indeterminate morphology ow-cytometric analysis allowed a diagnosis in 19 of 25 cases (76%).

Discussion

Prospective validation of the discriminant functions

A prospective study for validating the obtained classication functions was then carried out on a series of 115 consecutive, unselected patients referred to our Institution for mono- or multilineage cytopenia. The denitive diagnosis was of MDS in 69 cases (20 RA, 10 RARS, 10 RCMD, four RCMD-RS, eight RAEB1, 14 RAEB-2, three MDS unclassied). The remaining 46 patients had idiopathic BM aplasia (four cases), iatrogenic BM hypoplasia (14 cases), anemia associated with marrow inltration (15 cases), transient pancytopenia (one case), megaloblastic anemia (ve cases), iron deciency anemia (three cases), and anemia of chronic inammation or associated with liver disease (four cases). A correct diagnosis was obtained in 60 out of 69 MDS cases (87%). Six patients with refractory anemia (two with ringed sideroblasts) had a negative erythroid function. Two patients showed a very low value of the erythroid function (Figure 4), one affected with a MDS with marrow brosis, the other one with a hypoplastic MDS. One patient with RCMD with a highly hypocellular BM aspirate had a negative result in both the erythroid and myeloid functions. Two patients with In this study, we used a simple form of ow cytometry immunophenotyping that provides percentages of positive cells. As previously found by other investigators,8,9,11 no single immunophenotypic parameter proved able to discriminate accurately between MDS and other pathological conditions. Therefore, we performed discriminant analyses using several immunophenotypic parameters: this allowed us to dene classication functions that proved useful in distinguishing MDS from other conditions both in the learning and the testing cohorts. In addition, these functions provided a reliable evaluation of erythroid and myeloid dysplasia in MDS patients. A major problem in immunophenotypic analysis of MDS is evaluation of the erythroid dysplasia because of limited availability of antibodies specic for this lineage. In our study, we used a lyse-no wash technique with the aim of simplifying the approach and of facilitating its inclusion in the work-up of MDS patients, though this technique could potentially affect nucleated red cell estimate. A regression analysis of cytometric vs morphological analysis of nucleated red cell showed that an underestimation of the immature erythroid cells is obtained by

Figure 4 Scatterplot of the myeloid vs erythroid classication function values for each patient of the testing cohort, according to diagnosis. Values above zero in a classication function (erythroid or myeloid) indicate the presence of dysplasia in the corresponding lineage.
Leukemia

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

782
ow cytometry, resulting in a higher myeloid to erythroid ratio with respect to morphological analysis. However, a strong linear correlation between morphological and immunophenotypic evaluation of nucleated red cells was found, and owcytometric analysis was demonstrated to be repeatable and reproducible, without signicant differences among investigators who processed and repeatedly analyzed samples in a blind manner. Dys-synchronous expression of CD71 vs glycophorin A on red cell precursors, a nding previously reported by others,8 was the only consistent erythroid abnormality in our study. By contrast, various immunophenotypic abnormalities of myeloid cells have been previously recognized through the use of different MoAbs.8,11,12 In this study, MDS patients showed increased proportions of myeloid cells in the more immature immunophenotypic compartments and decreased proportions of mature granulocytes. Thus, we dened a ratio of immature to mature cells, which was markedly increased in MDS patients and signicantly correlated with the degree of morphological myeloid dysplasia. As far as CD34 cells are concerned, as previously reported by Del Can izo et al,11 we found a signicantly higher percentage of CD34 hematopoietic progenitor cells. The ratio between the CD34 CD33 and CD34 CD33 cells was signicantly higher in the MDS group than in the controls. The immunophenotypic analysis of hematopoietic compartments allowed us to estimate discriminant functions to differentiate between patients with MDS and those with other pathologies, including disorders similar to MDS, such as megaloblastic anemia and BM hypoplasia. A crucial point in immunophenotype in MDS is blast count. In our study, we observed a strong correlation between the proportion of blast cells estimated by morphology and the proportion of cells into the blast gating. A signicant correlation between the proportion of CD34-positive cells analyzed by a single-platform standardized method and the percentage of BM blasts evaluated by cytometry was also noticed. Finally, in our experience blast count was not signicantly affected by between- and withininvestigator variability. Therefore, we decided to integrate erythroid and myeloid discriminant functions with blast count obtained on the dot-plot CD45/SSC in order to classify MDS into WHO subgroups. Our immunophenotypic approach allowed us to correctly classify more than 90% of MDS with denitive morphological marrow dysplasia in the learning cohort and was validated in the prospective analysis of a testing cohort of patients. Moreover, ow-cytometric analysis has been demonstrated to be sensitive also in a high proportion of cases with a nondiagnostic morphology. This seems to suggest that variables related to sample quality, such as peripheral blood contamination or hypocellularity, are less signicant in immunophenotypic analysis than in morphological evaluation. Finally, the quantitative evaluation provided by the discriminant functions reected the severity of marrow dysplasia. In conclusion, an accurate ow cytometry evaluation of marrow dysplasia was achieved in this study with a panel of eight MoAbs, including MoAb against CD45, CD10, CD16, CD33, CD34, CD56, CD71, and GlyA-PE. This immunophenotypic analysis showed intralaboratory repeatability and reproducibility, and can be performed at a reasonable cost. Both these features make this approach potentially useful in the work-up of patients with MDS. The classication functions developed by us can be easily inserted in a Microsofts Excel sheet, so that their use by clinicians is extremely simple. Although interlaboratory variability has been demonstrated to be a problematical issue in ow-cytometric immunophenotyping, such an approach might
Leukemia

be a potentially useful tool for multicenter cytometric studies aimed at dening prognostic factors and predictors of response in MDSs.25,26

References
1 Jacobs A. Myelodysplastic syndromes: pathogenesis, functional abnormalities, and clinical implications. J Clin Pathol 1985; 38: 12011217. 2 Alessandrino EP, Amadori S, Cazzola M, Locatelli F, Mecucci C, Morra E et al. Myelodysplastic syndromes: recent advances. Haematologica 2001; 86: 11241157. 3 Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classication of the myeloid neoplasms. Blood 2002; 100: 22922302. 4 Flores-Figueroa E, Gutierrez-Espindola G, Guerrero-Rivera S, Pizzuto-Chavez J, Mayani H. Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation. Leuk Res 1999; 23: 385394. 5 Cazzola M, Ponchio L, Rosti V, Farina G, Pedrotti C, Bergamaschi G. Diagnostic approach to the myelodysplastic syndromes. Leukemia 1992; 6 (Suppl 4): 1922. 6 Bene MC, Bernier M, Castoldi G, Faure GC, Knapp W, Ludwig WD et al. Impact of immunophenotyping on management of acute leukemias. Haematologica 1999; 84: 10241034. 7 Elghetany MT. Surface marker abnormalities in myelodysplastic syndromes. Haematologica 1998; 83: 11041115. 8 Stetler-Stevenson M, Arthur DC, Jabbour N, Xie XY, Molldrem J, Barrett AJ et al. Diagnostic utility of ow cytometric immunophenotyping in myelodysplastic syndrome. Blood 2001; 98: 979987. 9 Maynadie M, Picard F, Husson B, Chatelain B, Cornet Y, Le Roux G et al. Immunophenotypic clustering of myelodysplastic syndromes. Blood 2002; 100: 23492356. 10 Ogata K, Nakamura K, Yokose N, Tamura H, Tachibana M, Taniguchi O et al. Clinical signicance of phenotypic features of blasts in patients with myelodysplastic syndrome. Blood 2002; 100: 38873896. 11 Del Canizo MC, Fernandez E, Lopez A, Vidriales B, Villaron E, Arroyo J et al. Immunophenotypic analysis of myelodysplastic syndromes. Haematologica 2003; 88: 402407. 12 Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM et al. Myeloid and monocytic dyspoiesis as determined by ow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood 2003; 102: 394403. 13 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposals for the classication of the myelodysplastic syndromes. Br J Haematol 1982; 51: 189199. 14 Bernasconi P, Astori C, Cavigliano PM, Boni M, Malcovati L, Calatroni S et al. Cytogenetic and FISH analyses in ve patients with hypoplastic bone marrow. Leukemia 2000; 14: 13221323. 15 Anonymous. An International System for Human Cytogenetic Nomenclature (1985) ISCN 1985. Report of the Standing Committee on Human Cytogenetic Nomenclature. Birth Defects Orig Artic Ser 1985; 21: 1117. 16 Bernasconi P, Cavigliano PM, Boni M, Calatroni S, Klersy C, Giardini I et al. Is FISH a relevant prognostic tool in myelodysplastic syndromes with a normal chromosome pattern on conventional cytogenetics? A study on 57 patients. Leukemia 2003; 17: 21072112. 17 Bernasconi P, Boni M, Cavigliano PM, Calatroni S, Brusamolino E, Passamonti F et al. Acute myeloid leukemia (AML) having evolved from essential thrombocythemia (ET): distinctive chromosome abnormalities in patients treated with pipobroman or hydroxyurea. Leukemia 2002; 16: 20782083. 18 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 20792088. 19 Fukushima PI, Nguyen PK, OGrady P, Stetler-Stevenson M. Flow cytometric analysis of kappa and lambda light chain expression in evaluation of specimens for B-cell neoplasia. Cytometry 1996; 26: 243252.

Flow cytometry immunophenotyping in myelodysplastic syndromes L Malcovati et al

783
20 Landay AL, Muirhead KA. Procedural guidelines for performing immunophenotyping by ow cytometry. Clin Immunol Immunopathol 1989; 52: 4860. 21 Stelzer GT, Marti G, Hurley A, McCoy Jr P, Lovett EJ, Schwartz A. U.S.Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by ow cytometry: standardization and validation of laboratory procedures. Cytometry 1997; 30: 214230. 22 Lacombe F, Durrieu F, Briais A, Dumain P, Belloc F, Bascans E et al. Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 1997; 11: 18781886. 23 Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform ow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 1998; 34: 6170. 24 Brando B, Barnett D, Janossy G, Mandy F, Autran B, Rothe G et al. Cytouorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. Cytometry 2000; 42: 327346. 25 Alessandrino EP, Amadori S, Barosi G, Cazzola M, Grossi A, Liberato LN et al. Evidence- and consensus-based practice guidelines for the therapy of primary myelodysplastic syndromes. A statement from the Italian Society of Hematology. Haematologica 2002; 87: 12861306. 26 Schiller GJ. Myelodysplasiatherapeutic response to novel therapy and the need for new diagnostic groups. Leukemia 2003; 17: 11831185.

Leukemia