Improved method for diagnosis of polycythemia vera based on flow cytometric analysis of autonomous growth of erythroid precursors in liquid culture

Abstract

"Autonomous" development of erythroid colonies in erythropoietin (EPO)-free semi-solid culture has been used as an in vitro assay for diagnosis of polycythemia vera (PV). These colonies, however, are small and poorly hemoglobinized, rendering the assay in many cases unreliable. We report here on the use of a novel assay; it combines a modified culture procedure that maximizes the growth of EPO-independent erythroid cells, and immunofluorescence flow cytometry for their detection and quantitation. Peripheral blood mononuclear cells are cultured for 2-5 days in the presence of a combination of growth factors. During this phase, early erythroid committed progenitors, burst forming units (BFUe), proliferate and differentiate into colony forming units (CFUe)-like progenitors. In the second phase, the latter cells, in the presence of stem cell factor, hemin, and iron-saturated transferrin, continue to proliferate and mature into hemoglobin (Hb)-containing orthochromatic normoblasts. Neither phases contained EPO. The culture produced large, pure, and synchronized erythroid cell populations. The cells were then dually labeled with fluorescent probes, nuclear DNA with thiazole orange and intracellular hemoglobin (Hb) with phycoerythrin-conjugated monoclonal antibodies against human Hb. Cells positive for both labels were assigned as Hb-containing nucleated precursors. The presence of such cells in EPO-free cultures indicated "autonomous growth." None of the EPO-free cultures derived from normal donors or patients with secondary polycythemia contained such cells. Cultures derived from PV patients contained from 5 to 92% "autonomously grown" cells. These culture and analysis methods should minimize false negative results with PV patients and provide objective and quantitative data.

Erythropoiesis In Vitro—A Research and Therapeutic Tool in Thalassemia

“…Examples include red cell aplasia, where EPC fail to grow in the presence of EPO [50], and polycythemia vera where they can grow in the absence of EPO [51]. The assay of the proliferation/differentiation ability of EPC carried out in semi-solid (endogenous colonies) or liquid cultures [52,53] used to be a standard diagnostic test for polycythemia vera. It has been largely replaced by molecular analysis of genes encoding proteins involved in the EPO signaling pathway, particularly Jak-2 [54].…”

A simplified endogenous erythroid colony assay for the investigation of polycythaemia

“…Cultures are typically set up in serum-containing media in the presence and absence of added erythropoietin, using either McLeod's plasma clot technique (Lacombe, Cassadevall & Varet, 1980;Lemoine et al, 1986) or methylcellulose culture technique (Eridani et al, 1983;Reid, Fidler & Kirk, 1988;Partanen et al, 1989;Weinberg et al, 1989;Shih et al, 1998). Other culture techniques such as the use of serumfree culture systems (Eridani et al, 1987;Correa, Eskinazi & Axelrad, 1994) and liquid culture system followed by flow cytometric analysis (Manor, Rachmilewitz & Fibach, 1997) have been described. Whilst these may further improve the diagnostic yield, they are more difficult to apply in a routine haematology laboratory.…”

“…Other culture techniques such as the use of serum‐free culture systems (Eridani et al. , 1987; Correa, Eskinazi & Axelrad, 1994) and liquid culture system followed by flow cytometric analysis (Manor, Rachmilewitz & Fibach, 1997) have been described. Whilst these may further improve the diagnostic yield, they are more difficult to apply in a routine haematology laboratory.…”

Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment concepts

“…Factor (erythropoietin)-independent and erythropoietin-hyperresponsive growth of BFU-E is indicative of (almost diagnostic for) the presence of PV or a related myeloproliferative neoplasm (MPN). 64 , 65 However, the colony-assay is rather tricky and its success depends on the experience of the team performing the test. Thus, although this assay is of practical value (and was previously used as a criterion for PV), it is nowadays only employed routinely in a few specialized centers.…”