Detection of nuclear and membrane antigens by liquid-based cytology following long-term storage of d1 cells, karpas cells, and peripheral blood mononuclear cells

Abstract

Immunofluorescence is the most frequently utilized technique to analyze protein expression. Fixed immunofluorescent cell suspensions, however, can only be stored for a week. We investigated whether liquid-based cytology could be used to detect antigens in cultured cells after a long storage period. Murine and human cells were fixed in PreservCyt solution, stored for various periods, and then used to perform an automated immunocytochemical analysis. Phosphorylation of the nuclear transcription factor Stat-5 induced by IL-7 was detected up to 4 months after IL-7 stimulation. Simultaneous nuclear positivity for the proliferation index MIB-1 and membrane positivity for the CD30 antigen were evident three months after fixation. Liquid-based cytology thus ensures long-lasting nuclear and membrane antigen immunoreactivity and permits the storage of cells from laborious experiments at room temperature for future analyses.

Figure 1.. Nuclear pStat-5 immunoreactivity in IL-7stimulated murine D1 cells

Murine IL-7 dependent D1 cells were deprived of IL-7 for four hours and cultured with medium alone (A,C,E) or with IL-7 (5Ong/ml) for 15 minutes (B,D,F). Cells were fixed in PreservCyt and immunoreactivity for pStat-5 was tested after 24 hours (A, B), 1 month (C, D) and 4 months (E, F). Antigen retrieval before the staining procedure was performed. Representative of three experimen ts (original magnification x 40).

Figure 2.. Nuclear pStat-5 immunoreactivity in IL-7-stimulated human PBL cells

PBL cells were cultured for 5 days in medium containing IL-7 (50ng/ml). Cells were deprived of IL-7 for 36 hours and cultured with medium alone or with IL-7 (50ng/ml) for 15 minutes. Cells were fixed in PreservCyt and immunoreactivity for pStat-5 was determined 1 month after fixation. A-B: the staining was performed after antigen retrieval, pStat-5 nuclear positivity was absent in unstimulated cells (A) and present in IL-7-stimulated cells (B), but the cell were swollen and the cytoplasms damaged. C-D: omission of antigen retrieval did not modify the results and improved cell morphology, unstimulated cells (C), IL-7stimulated cells (D). Representative of three experiments (original magnification x 40).

Figure 3.. Membrane CD30 and nuclear MIB-1 antigen immunoreactivity in ALCL Karpas cells

A, B: ALCL Karpas cells were fixed in PreservCyt and immunoreactivity for CD30 was determined 1 and 5 months after fixation, antigen retrieval was not performed (original magnification x 40); C: Human tonsil, CD30 positive lymphoid cells (positive control) (original magnification x10); D: Human tonsil, omission of the primary antibody (negative control) (original magnification x10); E: Karpas cells were fixed in PreservCyt and immunoreactivity for MIB-1 was determined 5 months after fixation. Antigen retrieval was not performed (original magnification x 40); F: Human tonsil, MIB-1 positive lymphoid cells in the germinal centre (positive control) (original magnification x 10).

Figure 4.. Double staining for membrane CD30 and nuclear MIB-1 inALCL Karpas cells

Single and double staining for CD30 membrane andMIB-1 nuclear antigens in ALCL Karpas cells. A: CD30 membrane immunoreactivity, B: MIB-1 nuclear immunoreactivity, C: double staining for the isotype matched control MoAbs (anti-CDX2 nuclear transcription factor and anti-CD 8 membrane antigen, CDX and CD8 antigens are not expressed by Karpas cells), D: double stainingfor CD30 and MIB-1 antigens. The experiment was performed three months after fixation of Karpas cells. Antigen retrieval was not performed. Representative of three experiments (original magnification x 40).

Figure 5.. Evaluation of single and double immunoreactivity for CD30 and MIB-1 antigens in ALCL Karpas cells.

The number of CD30 and MIB-1 total, single and double negative, and single and double positive cells was evaluated in two double staining experiments, performed as described in Fig.4. The results are expressed as mean number of cells /HPF + SD (10 HPF/ slide, original magnification x 40). No significant differences were observed (P = NS). The number of cells per field was 136 ± 31.2 in experiment 1, and 127.2 ± 16.6 in experiment2.