Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndrome: A Retrospective Validation From a Single Centre.

Abstract Abstract 4846 Myelodysplastic syndrome (MDS) is heterogeneous clonal hematopoietic stem cell disorder characterized by cytopenia(s) and dysplasia in one or more cell lineage. Though flow cytometry (FCM) is an important diagnostic tool in hematopoietic cell disorders, a prominent immunophenotyping feature in MDS may not be determined. In this study, we retrospectively evaluated flow cytometric features of bone marrow samples diagnosed as MDS with clinical and hematological findings. Patients-Method Between Feb 2004 and March 2009, flow cytometric parameters of 73 patients (M/F: 50/23) with MDS were re-analyzed. Median age was 59 years (17-89 ys). Our general principles are to evaluate quality of bone marrow samples, to determine proportion of the cells and features of their light scatter, and to give percentage of the blast. When detected a finding of dysplasia in the first analysis, the second step includes the determination of the maturation of the cells and the presence of the aberrant antigen expression. Results The samples were interpreted as MDS in % 76.7, MDS-RAEB-1 or RAEB-2 in %16.4, myeloproliferative disorder in %1.4 and non-diagnostic in %6.8 of the cases by flow cytometric examination. We detected variable degrees of hypogranulation in myeloid lineage in %82.2 of the samples by the light scatter features of the cells: 85% of severe and 15% of moderate or mild hypogranulation. The ratio of myeloid and lymphoid was changing from 0.3 to 17.5 (median 2). The decreasing of this ratio (<1) was observed in 19.4% of the samples. We detected altered expression of mature granulocyte. These included decreasing or lack of expression in CD15 45/73 (61.4%), CD13 38/70 (54.3%), CD16 53/67 (79.1%), CD11b 51/71 (71.8%), CD24 44/69 (65.2%), CD10 23/72 (31.9%) and MPO 14/72 (19.4%). Besides, bright expression of CD33 in 53.5% of the samples was observed. CD36 and CD56 in myeloid lineage were co-expressed in about 50 % of the samples. In 80.8 % of the samples dysplasia in erythroid compartment could be evaluated: Expression of CD71 according to glycophorin A (ratio <1) was decreased in 23.7 %. When we made similar analysis in the samples without RAEB-1 and -2 as pathological examination of bone marrow, 13.4 % of the samples could not be evaluated in favor of dysplasia. Of the samples with dysplasia hypogranulation, aberrant antigen expression of myeloid lineage and eryhtroid dysplasia were observed in 92.1%, 34.1% and 31.5%, respectively. In conclusion, FCM events may help to the differantial diagnosis of MDS especially when combining with clinical events. Improving of the analysis by focusing on the blast characteristics may be a standard approach to evaluate for low risk MDS. Disclosures No relevant conflicts of interest to declare.

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Decreased CD10-Positive Mature Granulocytes in Bone Marrow From Patients With Myelodysplastic Syndrome

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1152-dcpmgi ◽

2000 ◽

Vol 124 (8) ◽

pp. 1152-1156

Author(s):

Chung-Che Chang ◽

Ronald P. Cleveland

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndrome ◽

Cell Lineage ◽

Surface Marker ◽

Refractory Anemia ◽

Control Group ◽

Light Scatter ◽

Hematologic Disorders ◽

Lymphoma Involvement

Abstract Objective.—We retrospectively examined the maturation of the granulocytic cell lineage in bone marrow specimens from patients with myelodysplastic syndrome (MDS) by flow cytometry using both light scatter and surface marker characteristics, including CD10 and myeloid lineage–associated antigens. Patients.—The 7 MDS cases we studied included 2 patients with refractory anemia (RA), 3 with RA with ringed sideroblasts, 1 with RA with excess of blasts, and 1 unclassified case. Another 7 patients matched for age and sex who received bone marrow aspirates for lymphoma staging (all negative for lymphoma involvement or any other hematologic abnormalities) were selected as the control group. Results.—The percentage of CD10+ mature granulocytes was significantly lower in patients with MDS than in control patients. Additionally, all patients with MDS had less than 50% CD10+ cells in the granulocytic lineage. In contrast, only 1 of the 7 control patients had less than 50% CD10+ cells (P < .01). Conclusions.—These results suggest that flow cytometry might be a useful adjunct in the assessment of patients with suspected MDS. Further studies to correlate CD10+ mature granulocytes from MDS cases with other benign hematologic disorders are indicated to confirm our evaluation.

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CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors

Sao Paulo Medical Journal ◽

10.1590/s1516-31802009000100004 ◽

2009 ◽

Vol 127 (1) ◽

pp. 12-18 ◽

Cited By ~ 8

Author(s):

Jerusa Martins Carvalho ◽

Marlon Knabben de Souza ◽

Valéria Buccheri ◽

Cláudia Viviane Rubens ◽

José Kerbauy ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Antigen Expression ◽

Cross Sectional Study ◽

Cross Sectional ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Before And After ◽

Marrow Cells

CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.

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Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples

Analytical Cellular Pathology ◽

10.1155/1998/341340 ◽

1998 ◽

Vol 16 (3) ◽

pp. 151-159 ◽

Cited By ~ 51

Author(s):

Luis Escribano ◽

Alberto Orfao ◽

Jesús Villarrubia ◽

Beatriz Díaz-Agustín ◽

Carlos Cerveró ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Mast Cells ◽

Antigen Expression ◽

Hematologic Malignancies ◽

Healthy Controls ◽

Flow Cytometric ◽

Hla Dr ◽

Flow Cytometric Study

The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p= 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases – CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogenous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.

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Hhe results of different antibodies application for CD1a expression evaluation in pediatric t-lineage all

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2018-17-4-23-26 ◽

2019 ◽

Vol 17 (4) ◽

pp. 23-26 ◽

Cited By ~ 1

Author(s):

A. S. Aksenova ◽

O. I. Illarionova ◽

D. V. Litvinov ◽

S. A. Kashpor ◽

A. M. Popov

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Lymphoblastic Leukemia ◽

Antigen Expression ◽

Expression Data ◽

Multicolor Flow Cytometry ◽

Tumor Cell Population ◽

Multicenter Trials ◽

Flow Cytometric ◽

Expression Evaluation

CD1a antigen expression is an important prognostic factor in T-lineage acute lymphoblastic leukemia (T-ALL), thus standardized approach for this antigen expression detection is crucial for multicenter trials. The use of different antibodies in laboratories could lead to wrong decisions for patients management. The aim of the present study was to analyze the results of flow cytometric bone marrow investigation in children with T-ALL using different CD1a-directed antibodies. The bone marrow samples from 31 children (8 girls and 23 boys) with T-ALL aged from 1 to 16 years (median age 7) were studied by multicolor flow cytometry including two different antibodies against CD1a (BL6 и SK9). There were no significant differences in the immunophenotyping results. However, the CD1a-positivity of tumor cell population was visible in the dot plot better when BL6 antibody was used. In addition, two patients with discordant CD1a expression data were founded. Therefore, we antibody BL6 was recommended for routine T-ALL immunophenotyping.

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The Diagnostic Role of Flow Cytometry in Idiopathic Cytopenia of Unknown Significance (ICUS): A Single-Center Analysis of 79 Patients

Blood ◽

10.1182/blood-2018-99-114517 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3105-3105

Author(s):

Konstantinos Dimopoulos ◽

Olga Kristina Hansen ◽

Jakob Werner Hansen ◽

Lene Sjö ◽

Leonie Saft ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Board Of Directors ◽

Myelodysplastic Syndromes ◽

Diagnostic Value ◽

Low Grade ◽

Severe Dysplasia ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Introduction and aims The term ICUS is used to describe cases with persistent cytopenia (more than 6 months) without evidence of dysplasia in the bone marrow (BM) smear, and normal cytogenetics. Flow cytometry is currently a standard tool for the diagnosis of myelodysplastic syndromes (1). Our goal was to evaluate the efficacy of flow cytometry in detecting bone marrow dysplasia in the absence of abnormal morphology. Materials and methods A total of 79 patients with ICUS and 12 patients with MDS were analyzed with a standardized flow cytometry panel (2). Data were analyzed using two different algorithms; the Ogata algorithm (3) and Wells algorithm (4). For each of the algorithms, samples were classified as suggestive for dysplasia when scoring equal to or higher than 2. Furthermore, all ICUS patients were screened for mutations using targeted sequencing of 20 genes (DNMT31, TET2, EZH2, SRSF2, CBL1, SF3B1, UAF1, GATA2, IDH1, IDH2, CEBPa1, ZRSR2, TP53, KRAS, NRAS, ETV6, RUNX1, JAK2 and ASXL1) and 57/71 samples were additionally evaluated for signs of dysplasia by two independent hematopathologists. Results The diagnostic value of flow cytometry in MDS was confirmed by our data; the Wells algorithm was slightly superior to Ogata algorithm in predicting MDS (10/12 patients (83.3%) vs. 7/12 patients (58.3%) respectively, fig. 1A) and was therefore the main algorithm used for the further analysis of the ICUS patients. Flow cytometry was suggestive of dysplasia in 34/79 (43%) patients with ICUS. Interestingly, in patients with at least one detected mutation, flow cytometry was positive in 23/42 (54.8%) of the cases, while it was positive in 8/9 (88.9%) patients with more than two mutations (fig. 1C). There was no higher frequency of abnormal flow cytometry in patients with higher risk mutations. Additionally, for the 57 patients evaluated for dysplastic changes, flow cytometric abnormalities were more frequent (p= 0.05) in the 22 patients with moderate/severe dysplasia (15/22 patients or 68.2%, fig. 1D). After a median follow-up of 20 months (range: 3 - 90), a total of eight patients progressed to either MDS, CMML or AML. Interestingly, these eight patients had flow cytometric abnormalities suggestive of dysplasia before developing hematological malignancy. In conclusion, flow cytometry can identify a subgroup of ICUs patients with a higher mutational burden, dysplastic changes and a higher risk for progression to MDS or a more aggressive myeloid disease and has a place in the diagnostic evaluation of patients with idiopathic cytopenia. References: Porwit A, Van De Loosdrecht AA, Bettelheim P, Eidenschink Brodersen L, Burbury K, Cremers E, et al. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes - Proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Leukemia. 2014;28(9):1793-8. van Dongen JJM, Lhermitte L, Böttcher S, Almeida J, van der Velden VHJ, Flores-Montero J, et al. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia [Internet]. 2012 Sep [cited 2014 Nov 28];26(9):1908-75. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3437410&tool=pmcentrez&rendertype=abstract Porta MGD, Picone C, Pascutto C, Malcovati L, Tamura H, Handa H, et al. Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: Results of a European LeukemiaNET study. Haematologica. 2012;97(8):1209-17. Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, et al. Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood. 2003;102(1):394-403. Figure 1. Figure 1. Disclosures Hansen: Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees. Grønbæk:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

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Correcting Flow Cytometric Blast Counts for Blood Contamination in Bone Marrow Aspirates.

Blood ◽

10.1182/blood.v110.11.4613.4613 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4613-4613

Author(s):

Michael R. Loken ◽

Sung-Chao Chu ◽

Wayne K. Fritschle ◽

Dian-Kun Li ◽

Denise A. Wells

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Flow Cytometric Analysis ◽

Myeloid Cells ◽

Light Scatter ◽

Blood Contamination ◽

Simple Method ◽

Flow Cytometric ◽

Biopsy Specimens ◽

Blast Count

Abstract An accurate blast count is pivotal in the diagnosis, classification and prognosis of patients with myelodysplasia. Blast counts in all previous classification schemes are based on morphologic assessment of marrow aspirates with a poor correlation to blast counts determined by flow cytometry. A significant problem in blast enumeration by flow cytometry is the variable hemodilution of the marrow during collection for flow cytometric analysis. Blast counts can vary depending on which aspirate tube is used for flow analysis, e.g., 2.4%, 1st 5ml tube; 0.62%, 2nd 5ml tube; 0.58% 3rd 5ml tube. Morphologists circumvent this problem by selecting a region for assessment close to a spicule with minimal blood dilution. Cell surface antigens can be used to distinguish mature cells found in blood as distinct from immature cells identified in marrow. CD16 intensity on neutrophils reaches a maximum at the band/segmented stage of development with a low coefficient of variation, thereby becoming a marker for mature myeloid forms. A simple method to distinguish immature from mature myeloid cells was developed to assess extent of blood contamination in marrow aspirates using a combination of CD16, CD13, and CD45. The average mature neutrophil content of a marrow was determined from phenotypically normal bone marrow biopsy specimens, assumed to have minimal blood contamination. The proportion of dimCD16 cells gated on the myeloid cells based on CD45 and right angle light scatter in 31 biopsy specimens was 82% (range 69–93, SD=6.2) (Figure 1). A value of 80% (rather than 82%) was used for the subsequent calculations to correct for the excess mature neutrophils found in an aspirate as compared to the biopsies (Corrected Blasts = [80 / % dim CD16 myeloid] x determined blast count). To test this hypothesis bone marrow aspirates were diluted with blood at different ratios to mimic blood marrow hemodilution. Blasts (defined as CD45 dim, low right angle light scatter, HLA-DR positive, CD11b negative) were determined for the various dilutions, then corrected based solely on the proportion of dim CD16 myeloid cells (Figure 2). A marrow from an MDS case was also diluted (1:5 v/v) with blood for comparison. The original marrow contained 80% dim CD16 myeloid cells with a blast count of 9.2%. After dilution, only 12% dim CD16 cells were detected with 1.1% blasts, however upon correction (6.67), the blast count was 7.3%, close to the original determination. This approach may provide for more standardization and consistency in the determination of blast counts in MDS marrow specimens using flow cytometric analysis. Figure 1, CD16 of marrow myeloid cells. Figure 1,. CD16 of marrow myeloid cells. Figure 2, Uncorrected/Corrected Blast Count Figure 2,. Uncorrected/Corrected Blast Count

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FLOW CYTOMETRY IN MYELODYSPLASTIC SYNDROME. STEP FORWARD.

Liječnički vjesnik ◽

10.26800/lv-141-7-8-27 ◽

2019 ◽

Vol 141 (7-8) ◽

pp. 214-219

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Flow Cytometric Analysis ◽

Lymphoid Tissues ◽

Hematopoietic Stem ◽

Diagnosis And Prognosis ◽

Flow Cytometric ◽

Increased Risk ◽

Made In

Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal diseases originating from altered (malignant) hematopoietic stem cells. Clinically, it manifests as cytopenia in peripheral blood, dysplastic changes in one or more cell lines in bone marrow and increased risk of evolution to acute myeloid leukemia (AML). Acquired chromosomal aberrations are detected in 40-50% of MDS patients. According to WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Lion 2008), MDS diagnosis is based on the morphological interpretation of found dysplastic changes, the number of blasts, the presence of ring sideroblasts in bone marrow and the established cytogenetic abnormalities. The progress made in recent years in immunophenotyping of hematopoietic progenitor cells and mature cells of dysplastic bone marrow gives to multiparameter flow cytometric analysis (MFC), although initially included as an optional technique, an opportunity to become a standard part of the integrated MDS diagnosis and prognosis.

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Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia

Blood ◽

10.1182/blood.v112.11.4107.4107 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4107-4107

Author(s):

Hwee Yong Lim ◽

Marjorie Farley ◽

Carl Wittwer ◽

Charles Parker

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Surface ◽

Peripheral Blood ◽

Bone Marrow Failure ◽

Flow Cytometric Analysis ◽

Small Populations ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Bone Marrow Failure Syndromes

Abstract PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham’s test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had >5% GPI-AP− RBCs, while 18 cases (33%) had >10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had >10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham’s test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with >10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham’s test and the sucrose lysis test as screening assays for PNH.

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