Screening for Paroxysmal Nocturnal Hemoglobinurea with Cytodiff Analysis

Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositolglycan complementation Class A (PIG-A) gene. PIGA mutations in PNH patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency.In PNH, there is a partial or absolute inability to make GPI-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Clinical features of PNH include intravascular hemolysis, bone marrow failure, and thrombosis, all major causes of morbidity and mortality. Flow cytometry (FCM) plays a key role in the laboratory investigation of PNH, and rapid diagnosis of this condition is highly desirable. A definitive diagnosis of PNH can be established by demonstrating the absence of cell membraneGPI-anchored proteins from granulocytes or red blood cells (RBC) according to ICCS Guidelines for the diagnosis and monitoring of PNH by flow cytometry. It has been also described that the expression of CD16 can be decreased on PNH-affected granulocytes.

Recently new method for extended flow WBC differential was introduced by Beckman Coulter. This method uses flow cytometric analysis with CytoDiff™** reagents which is a 5-color/6-marker reagent that provides a 10-part cytometric differential from whole blood specimens and comprises CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7 (Beckman Coulter).

The protocol allows detection of mature neutrophils, total lymphocytes, total monocytes, eosinophils, basophils, immature granulocytes, B lymphocytes, CD16-negative T/NK lymphocytes, CD16-positive T/NK lymphocytes, CD16 positive and CD16 negative monocytes, and blasts cells with lineage orientation. This method also allows the detection of the abnormal antigen expression on WBC, for example low CD16 expression on neutrophils. In case of abnormal low CD16 expression on segmented neutrophils they will be classified as Immature Granulocytes (Imm Gran). The aim of the study was to evaluate the efficacy of CytoDiff** analysis of peripheral blood for PNH screening detecting low CD16 expression on neutrophils.

Methods: EDTA-anticoagulated blood samples from 53 patients with PHN suspicion were prospectively included in the study. Analysis of the PNH clones was conducted in according with international protocol, using CD235a for RBC gating, CD15-PE/CD45-PC7 for granulocyte gating, CD64-PC5/CD45-PC7 for monocyte gating and GPI-anchored proteins CD59-PE, FLAER-FITC/CD24-PC5 and FLAER-FITC/CD14-PE for RBCs, granulocytes and monocytes accordingly.

For extended flow WBC differential analysis the blood samples were stained with the CytoDiff^** panel, lyzed with Versalyse (Beckman Coulter) and 20 000 leucocytes were analyzed on a FC500 Flow Cytometer (Beckman Coulter) using CytoDiff** CXP software.

Results:Totally 53 patients with PHN suspicion were prospectively included in the study. All these patients were characterized by anemia, thrombocytopenia and/or leucopenia. PNH diagnosis was confirmed in 6 patients and in other 7 patients the final diagnosis was aplastic anemia with PNH clone.

For all 13 patients (4 males, 9 females, with median age of 41.5 years) with confirmed presence of PNH clone CytoDiff** reported increased number of Imm Gran (range 3-45%). Microscopy analysis did not detect the presence of Imm Grans in the slide, so we concluded that falsely reported increased Imm Gran count was due to the decreased expression of CD16 on Neutrophils. Good correlation (r=0.9257) was observed between Imm Gran count and the size of granulocytic PNH clone.

Conclusion: Our data demonstrate that CytoDiff** analysis, which is able to detect a wide spectrum of normal and pathological cells in peripheral blood allows determination of CD16-low expression on neutrophils and thus provide efficient screening for suspicious of PNH in patients with anemic syndrome.

** Not available in the United States and other geographies.