Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.
BACKGROUND
The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay.


METHODS
Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo).


RESULTS
In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients.


CONCLUSIONS
We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.
Dobo
I
I
Laboratoire d'Hématologie, Centre Hospitalier Universitaire d'Angers, Angers, France.
Mossuz
P
P
Campos
L
L
Girodon
F
F
Allégraud
A
A
Latger-Cannard
V
V
Boiret
N
N
Pineau
D
D
Wunder
E
E
Zandecki
M
M
Praloran
V
V
Hermouet
S
S
D001854:Bone Marrow Cells / Q000473:pathology; D016022:Case-Control Studies; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects; D003094:Collagen; D003114:Colony-Forming Units Assay / Q000379:methods / Q000592:standards*; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology*; D015672:Erythroid Precursor Cells / Q000473:pathology*; D006801:Humans; D008747:Methylcellulose; D011087:Polycythemia Vera / Q000175:diagnosis / Q000473:pathology*; D013920:Thrombocythemia, Essential / Q000175:diagnosis / Q000473:pathology*
D016895:Culture Media, Serum-Free; D016207:Cytokines; D008747:Methylcellulose; D003094:Collagen