Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen.
Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.
van Dam
P A
PA
Department of Gynecologic Oncology, Saint Bartholomew's Hospital, London, England.
Watson
J V
JV
Lowe
D G
DG
Chard
T
T
Shepherd
J H
JH
D000951:Antigens, Neoplasm / Q000032:analysis*; D015925:Cryopreservation / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde; D005833:Genital Neoplasms, Female / Q000737:chemistry / Q000276:immunology / Q000473:pathology; D006801:Humans; D000432:Methanol; D016253:Microscopy, Immunoelectron; D009369:Neoplasms / Q000737:chemistry / Q000276:immunology / Q000473:pathology*; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods*
D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; D000432:Methanol