A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders.
[3H]thymidine uptake by NFS-60 cells in microcultures was found to increase in a linear fashion with the increasing doses of purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). Such increases were found neither with rhG-CSF samples pretreated with rabbit anti-rhG-CSF serum nor with other human colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (hGM-CSF) or macrophage colony-stimulating factor (hM-CSF). Based on these findings, sera from normal persons and patients with severe infections or various hematological disorders were tested after dialysis using this system in order to determine whether G-CSF levels in sera can be estimated or not. In ten normal persons, five patients with acute myelogenous leukemia (AML M1, M2, and M3), five with myelodysplastic syndrome, and four with chronic myelogenous leukemia, no increases in [3H]thymidine uptake were found within the dose range of 0.4 microliters to 50 microliters. In contrast, linear dose responses parallel to a G-CSF standard curve were observed in one patient with a severe bacterial infection, four with aplastic anemia, two with acute myelomonocytic leukemia (AMMoL) (M4), and two with idiopathic neutropenia tested. From the standard curve, the probable levels of G-CSF were calculated as follows: approximately 200 pg/ml with infection, 130-220 pg/ml with aplastic anemia, 150 and 200 pg/ml with AMMoL, and 1120 and 1200 pg/ml with idiopathic neutropenia. The activities of sera were reduced by the anti-rhG-CSF serum pretreatment in the same way as documented in the case of rhG-CSF. Furthermore, the level in a patient with a severe infection became undetectable soon after elimination of the infection and blood neutrophil counts had returned to normal. These findings indicate that the microbioassay system will be useful for measuring circulating G-CSF levels which would fluctuate in accord with requirements for stimulating neutrophil production or with abnormal production of hG-CSF.
Shirafuji
N
N
Department of Hematology-Oncology, University of Tokyo, Japan.
Asano
S
S
Matsuda
S
S
Watari
K
K
Takaku
F
F
Nagata
S
S
D000328:Adult; D000818:Animals; D001424:Bacterial Infections / Q000097:blood* / Q000378:metabolism; D001790:Blood Physiological Phenomena; D001855:Bone Marrow Diseases / Q000097:blood* / Q000378:metabolism; D002460:Cell Line; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000097:blood* / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000097:blood; D013936:Thymidine / Q000378:metabolism
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D013936:Thymidine