Endogenous erythroid and megakaryocytic colony formation in serum-free, cytokine-free collagen gels

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disor ders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV, 76 patients) or essential thrombocythemia (ET, 27 patients) were grown in collagen-based, serum-free, cytokine-free s emisolid medium. Colony analysis at day 8 or 10 showed that this collagen a ssay is specific, as endogenous growth of erythroid colonies was never obse rved in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (C ML) patients. Endogenous erythroid colony formation was observed in 53.3% o f patients suspected of PV, with only 15.4% of positive cultures for patien ts with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for p atients with greater than or equal to 2 minor or 1 major PV criterion. Simi larly, endogenous growth of erythroid colonies was found in 44.4% of patien ts suspected of ET, with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with greater than or equal to 2 ET criter ia. In addition, we found that in collagen gels, tests of erythropoietin (E PO) hypersensitivity in the presence of 0.03. or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be: used to detect PV or ET, as these tests were positive for, respectivel y, 21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retro spective analysis suggests that collagen assays are more sensitive than met hylcellulose assays to assess endogenous growth of erythroid colonies. In s ummary, serum-free collagen-based colony assays are simple and reliable ass ays of endogenous growth of erythroid colonies in myeloproliferative diseas es. They also appear to be more sensitive than methylcellulose-based assays .