Development and Prospective Validation of a Standardized Flow Cytometric Assay of Peripheral Blood Neutrophil Myeloperoxidase Expression for Ruling Out Myelodysplastic Syndromes.
Development and Prospective Validation of a Standardized Flow Cytometric Assay of Peripheral Blood Neutrophil Myeloperoxidase Expression for Ruling Out Myelodysplastic Syndromes.
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal bone marrow
 neoplasms that predominate in the elderly, with a median age at diagnosis of 70 years. The
 diagnosis of MDS relies on peripheral blood cytopenia and morphologic dysplasia for one or
 more hematopoietic cell lineage. Cytopenia is evidenced with hemogram while dysplasia
 requires bone marrow aspirate, which is an invasive procedure .

 Considering the low prevalence of disease among subjects referred for suspected MDS, many
 patients are exposed to unnecessary bone marrow aspiration-related discomfort and harms.
 Therefore, an objective assay based on a peripheral blood sample that accurately
 discriminates MDS from other cytopenia etiologies is highly desirable.

 We have previously developed and refined a flow cytometric analysis protocol for quantifying
 neutrophil MPO expression in peripheral blood at three university-affiliated hospitals (i.e.,
 Clermont-Ferrand, Saint-Etienne, and Grenoble) (Raskovalova et al, Hematologica 2019). We
 found that the robust coefficient of variation (RCV, computed as the robust standard
 deviation divided by the median) within an individual subject was the best parameter in
 discriminating patients with versus without MDS.

 Although promising, flow cytometric analysis of neutrophil MPO expression in peripheral blood
 is technically complex, time consuming, and not standardized. Hence, its performance requires
 specific expertise and the results show substantial variability. A single ready-to-use tube
 with lyophilized antibodies would have the potential to standardize the measurement of
 neutrophil MPO expression in peripheral blood across laboratories, with results available
 within 30-60 min in routine practice.

 In this study, the investigators hypothesize that a standardized and semi-automatic flow
 cytometric assay of neutrophil MPO expression in peripheral blood could accurately rule out
 MDS and obviate the need for bone marrow aspiration and biopsy, with sensitivity and negative
 predictive value estimates approaching 100%.

 In this observational diagnostic accuracy study, burden will be null for recruited patients.
 No specific intervention is assigned to participants. All diagnostic testing, procedures, and
 medication ordering are performed at the discretion of attending physicians. A test result
 will have no impact on patient management. .Compliance with current guidelines disseminated
 by the French Haute Autorité de Santé (HAS) will be advocated for the diagnostic work-up of
 patients with suspected MDS. No follow-up visits are planned in this cross-sectional study.
The primary objective of MPO-MDS-develop study is to estimate the discriminative accuracy
 (i.e., area under the ROC curve along with 95% CI) of a standardized and semi-automatic flow
 cytometric assay of neutrophil MPO expression in peripheral blood for the diagnosis of MDS in
 adult patients.

 The secondary objectives are to assess:

 1. intra-laboratory reproducibility (intra- and inter-assay precision) for intra-individual
 RCV measurement of neutrophil MPO expression in peripheral blood;

 2. specimen stability for intra-individual RCV measurement of neutrophil MPO expression in
 peripheral blood at different time points in comparison with H0 (stored at 4°C and at
 room temperature);

 3. inter-laboratory reproducibility for intra-individual RCV measurement of neutrophil MPO
 expression in peripheral blood;

 4. the negative predictive value for intra-individual RCV measurement of neutrophil MPO
 expression in peripheral blood (with a prespecified threshold of 30.0%) in ruling out
 MDS in consecutive adult patients.

 The MPO-MDS-Develop project is a multicenter, cross-sectional, diagnostic accuracy study of
 an index test by comparison with a reference standard, in unselected consecutive patients.

 Screening: All consecutive patients referred to the immuno-hematology lab at the study sites
 for suspicion of MDS will be screened for eligibility. A lab physician will review inclusion
 and exclusion criteria, using computerized medical and laboratory records.

 Recruitment: Participants will be included in the study once all the screening activities
 have been conducted and only if the patient meets all inclusion and none exclusion criteria.
 The consent for flow cytometry analysis of peripheral blood sample and data collection
 through chart review will be sought under a regime of "non-opposition" (opt-out): after
 appropriate written information is delivered, cross-sectional data will be collected except
 in case of opposition from the patient. All patients included in the study will be assigned a
 unique patient identification number. This number will be used to identify the patient
 throughout the study.

 Index test: Flow cytometry analysis of neutrophil myeloperoxidase expression in peripheral
 blood will be performed using a single ready-to-use tube with lyophilized antibodies
 developed in partnership with Becton Dickinson France SAS and blinded to the reference
 standard.

 Reference standard: The diagnosis of MDS will be established according to the World Health
 Organization (WHO) classification, based on clinical data, peripheral blood cytopenia,
 cytomorphology of peripheral blood and bone marrow aspirate, and cytogenetic analysis. The
 criteria for MDS diagnosis are 1) the presence of ≥10% dysplastic cells in any hematopoietic
 lineage, 2) the exclusion of acute myeloid leukemia (defined by the presence of ≥20%
 peripheral blood or bone marrow blasts), and 3) the exclusion of reactive etiologies of
 dysplasia. Cytopenia is defined by hemoglobin concentration <10 g/dL, platelet count
 <100x109/L, and/or absolute neutrophil count <1.8 x109/L. Yet a diagnosis of MDS could be
 made with milder levels of cytopenia. Idiopathic cytopenia of uncertain significance (ICUS)
 is defined by unexplained mild persistent cytopenia for at least 6 months and the failure to
 establish the diagnosis to MDS according to published guidelines. Consistent with WHO
 classification, MDS subcategorization will rely on the degree of dysplasia (unilineage versus
 multilineage), blast percentages, presence of ring sideroblasts, and cytogenetic analysis
 (del(5q)). The criteria for CMML diagnosis are 1) the presence of persistent peripheral blood
 monocytosis ≥1 x109/L, and 2) monocyte accounting for more than 10% of the white blood cell
 differential count. Evaluation of bone marrow cytomorphology will be performed prospectively
 by experienced hematopathologists who are blinded to the index test results.

 Patients with confirmed suspicion of MDS: Participants for whom the diagnosis of MDS (or
 CMML) is confirmed by the reference standard will be categorized as patients with confirmed
 suspicion of MDS.

 Patients with unconfirmed suspicion of MDS: Participants for whom the diagnosis of MDS (or
 CMML) is ruled out by the reference standard will be categorized as patients with unconfirmed
 suspicion of MDS. This latter subgroup will include patients with ICUS, as defined in
 accordance with published guidelines.

 Follow-up: No follow-up visit is planned in this cross-sectional diagnostic accuracy study.
Inclusion Criteria:

 -  Age at enrollment ≥18 years

 -  Clinical suspicion of MDS

 -  Indication for bone marrow examination

 -  ≥1 peripheral blood cytopenia defined by hemoglobin concentration <12 g/dL for female
 and <13g/dL for male patients, platelet count <150 x109/L, absolute neutrophil count
 <1.8 x109/L

 -  Inpatient and outpatient care patients

 Exclusion Criteria:

 -  Refusal to participate

 -  History of or active documented MDS

 -  Enrollment in intensive or critical care unit

 -  Incarcerated or individuals protected by French regulation (Article L1121.5 and
 following, Code de la Santé Publique)

 -  Not affiliated with social security system

 -  Previous enrollment in the study.