Intra- and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing.

Background: Sutherland et al. recently published the Practical Guidelines for high-sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing.

Methods: Using this approach, we studied the intra- and interlaboratory variability observed in a multicenter study in which fresh blood samples containing three clinically relevant PNH clone sizes within the granulocytic, monocytic, and red blood cell (RBC) populations were shipped to each participating center.

HLA-G5 in the blood of leukemia patients and healthy individuals.

In this work we focused on analysis of HLA-G5 molecules in the blood of patients with B-CLL leukemia and healthy individuals. Using sandwich ELISA, we found total soluble HLA-G, represented by sHLA-G1 and HLA-G5 in most of B-CLL patients while HLA-G5 alone was present only in few cases in both groups. These results lead us to assume that the majority of soluble HLA-G in blood is generated by proteolytic cleavage and shedding of membrane-bound HLA-G1.

Immunity profile in breast cancer patients.

Objectives: Despite the multifactorial pathogenesis of malignant transformation, it is assumed that deficiency in some immune mechanisms plays a considerable role in its development.

Background: Chronically activated immune cells exert tumour-promoting effects directly by influencing the proliferation and survival of neoplastic cells, as well as by indirect modulation of neoplastic microenvironments in favour of tumour progression.

Patients And Methods: We refer to results of two separate investigations that aim to monitor the immune functions in patients with breast cancer.

Normal maturation sequence of immunoglobulin light and heavy chains in hematogone stages 1, 2 and 3 in acute leukemia after treatment.

The cellular diversity of bone marrow samples was studied by using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on acute leukemia blast cells and regeneration of normal B-cells, hematogones. This approach should enhance the ability to study normal hematopoiesis, and to identify and monitor hematopoietic disorders. The study was performed on a homogeneous group of patients (mainly children), all of them after finishing complete therapy for AL, mostly B-ALL.

Hematogones in acute leukemia during and after therapy.

After each leukemia therapy phase, characteristics of normal regenerating B-cells may be reminiscent of and mistaken for a relapse. We compared the incidence and phenotypic characteristics of hematogone stages in a total of 669 bone marrow aspirates from 107 patients with B-ALL, 97 patients of AML, and 27 patients with T-ALL at diagnosis, during, and after therapy. The three individual physiological maturation phases of B-lymphocytes (hematogone stages 1, 2, and 3) were studied by four-color flow cytometry in the course of bone marrow regeneration in leukemia patients.

Increased myeloid precursors in regenerating bone marrow; implications for detection of minimal residual disease in acute myeloid leukemia.

Presented study is focused on exact immunophenotypic definition of myeloid precursors and their following stages in regenerating bone marrow during treatment of ALL/AML for correct interpretation of the immunophenotype results and proper distinction from minimal residual disease (MRD) by multiparameter flow cytometry. This study includes bone marrow samples from 36 controls, 27 patients with AML, 39 patients with B-ALL undergoing therapy who remained in complete remission after treatment and also 30 B-ALL patients one year after the end of therapy. We observed substantial expansion of immature bone marrow populations in the regenerating bone marrows, which were identified by expression of CD34 and/or CD117 markers by 4-color flow cytometry.

The impact of cell heterogeneity and immunophenotypic changes on monitoring minimal residual disease in acute myeloid leukemia.

Monitoring of minimal residual disease (MRD) becomes increasingly important for the more accurate stratification of the therapy in acute leukemia. The purpose of this study was to characterize in detail the phenotypes of heterogeneous population in various AML subtypes and to identify the leukemia associated aberrant phenotype (LAP) in individual patients with AML for precise investigation of MRD. The impact of heterogeneity of pathological populations, the effectiveness of location AML blasts on CD45/SSC dot plots in AML patients during follow-up and phenotype changes on MRD monitoring were evaluated in the second step.

The value of dot plot patterns and leukemia-associated phenotypes in AML diagnosis by multiparameter flow cytometry.

The immunophenotypic features in patients with acute myeloid leukemia (AML) were investigated at diagnosis using a wide antibody panel including progenitor-associated, myeloid and lymphoid markers in quadruple combinations. Analyzed were bone marrow samples from 37 adult and pediatric patients for exact identification of AML blasts according their localization on CD45/SSC dot plots and aberrant immunophenotypes in various subtype of AML. We found the localization of AML blasts on CD45/SSC dot plots, which in combination with immunophenotype profile of blasts allow discrimination of several AML subtypes (M0-M2, M3, M4/M5 and other types).

The knowledge on the 3rd type hematogones could contribute to more precise detection of small numbers of precursor B-acute lymphoblastic leukemia.

Bone marrow hematogones (benign B-lymphocyte precursors) may cause diagnostic problems due to their morphologic and immunophenotypic similarities with neoplastic lymphoblasts. Hematogone populations in presented study containing 358 bone marrow specimens of 251 individuals always exhibited a continuous and complete maturation spectrum of antigen expression typical for normal evolution of B-lineage precursors; lacking aberrant or asynchronous antigen expression. In contrast lymphoblasts of 19 bone marrows of precursors B-ALL patients showed maturation arrest and exhibited several immunophenotypic aberrancies.

The value of multiparameter flow cytometry of cerebrospinal fluid involved by leukemia/lymphoma cells.

The usefulness of multiparameter flow cytometric (FC) analysis of cerebrospinal fluid (CSF) was evaluated in leukemia/lymphoma patients having central nervous system (CNS) involvement of the disease. In 12 specimens of 8 patients with different types of leukemia/lymphoma (one case of T-ALL, 3 cases of early B-cell ALL, one case of AML, and 3 proven or suspicious NHL cases) the presence of pathological clone in CSF has been confirmed or excluded. The phenotypic patterns of CSF cells were defined according to those of bone marrow (BM)/peripheral blood (PB) at diagnosis or during follow-up of the same patients.