Flow Cytometric Analysis of Peripheral Blood Neutrophil Myeloperoxidase Expression for Ruling Out Myelodysplastic Syndromes: Protocol for a Diagnostic Accuracy Study

The primary objective of the MPO-MDS-Valid study is to estimate the discriminative accuracy (i.e., area under the ROC curve point estimate along with 95% confidence interval [CI]) of the intra-individual RCV for neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of MDS in consecutive patients.

The secondary objectives are:

1. To estimate the negative predictive value of the intra-individual RCV for neutrophil myeloperoxidase expression in peripheral blood (with a prespecified threshold of 30.0%) for ruling out MDS in consecutive patients.
2. To estimate the prevalence of alternate diagnoses established by bone marrow cytomorphology among true negative patients (i.e., patients with intra-individual RCV value lower than 30.0% and for whom MDS was ruled out by the reference method).

Study design: The MPO-MDS-Valid study project is a multicenter diagnostic accuracy study of an index test by comparison with a reference standard in unselected consecutive patients. An institutional review board (Comité de Protection des Personnes Nord Ouest III) reviewed and approved the study protocol and the information form, prior to study initiation. No specific intervention is assigned to participants. All diagnostic testing, procedures, and medication ordering are performed at the discretion of attending physicians. The index test result will have no impact on patient management.

Screening: All consecutive patients referred for suspicion of MDS to the immuno-hematology lab at the study sites 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, 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 cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood will be blinded to the reference standard. Myeloperoxidase expression in the peripheral blood neutrophil population within an individual subject will be expressed as RCV. The intra-individual RCV is calculated as the robust standard deviation divided by the median. The robust standard deviation is a function of the deviation of individual data points to the median of the study population. Intra-individual RCV is expressed as percentage and reflects the variability in myeloperoxidase expression in the peripheral blood neutrophil population within an individual subject.

Reference standard: At each study site, cytomorphological evaluation of bone marrow aspirate will be performed by experienced hematopathologists blinded to the index test.

Morphologic assessment may be complemented by bone marrow flow cytometric score, karyotype, and molecular profiling, where relevant. The reference criteria for MDS are 1) the presence of ≥10% dysplastic cells in any hematopoietic lineage on bone marrow aspirate, and 2) the exclusion of acute myeloid leukemia (defined by the presence of ≥20% bone marrow blasts). Consistent with WHO classification, MDS subcategorization will be based on the degree of dysplasia (unilineage versus multilineage), blast percentages, presence of ring sideroblasts, and cytogenetic analysis (del(5q)).

The reference criteria for chronic myelomonocytic leukemia (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. Idiopathic cytopenia of uncertain significance (ICUS) will be defined by unexplained mild persistent cytopenia for 4 to 6 months and the failure to establish the diagnosis of MDS according to reference criteria.

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.