243

OPTIMIZATION OF THE CONDITIONS FOR SIMULTANEOUS ANALYSIS OF MYC NUCLEAR ONCOPROTEIN EXPRESSION AND CELLULAR DNA CONTENT BY FLOW CYTOMETRY. P E.L/_SSON Isabe.lle(1), LIZARD G~rard(2), MUTIN Mirei l le(2), CHARDONNET Yvette(1). (1)/NSERM U346, /~av R; (2)centre de cytofluorom~trie -/NSERM U80, Pav P, H&pita/ Edouard Herriot 69437 L YON codex 03.

In order to analyse myc nuclear oncoprolein in epithelial tumors, we first determined optimal conditions to detect and quantify simultaneously myc protein expression and total cellular DNA content in epithelial cell lines derived from cervical carcinomas (HeLa, CaSki, SiHa). The cells were used either unfixed, or fixed in acetone-methanol (1:1, 5 min. at 4°C) or 0.5% paraformaldehyde (15 to 30 min. at 4°C). To allow the monoclonal antibody specific to c-myc oncoprotein to enter nuclei, cells were treated either with 0.3% saponine (15 min. at 4"C) or with 0.1% triton X100 (3 min. at 4°C). Myc protein-antibody complexes were revealed with a FITC- conjugate. Cells were further digested with RNase and incubated in propidium iodide for evaluation of cellular DNA content. Fluorescent stainings were quanlilied by dual parameter analysis with flow cytometry on a FACSTAR Plus tlow cylomeler. A faint.signal of myc protein was observed in unfixed cells whereas it was detected both in paraformaldehyde and acetone-methanol fixed cells. Fixation with paraformaldehyde allowed the analysis of single cells and was prefered to acetone-methanol fixation that frequently induced aggregates. Myc protein was detected both in saponine and triton-treated cells, the fluorescence intensity beeing higher after 30 min. than after 15 min. el fixation. Transmission electron microscopy showed that nuclear ullraslructure was altered by saponine and was quite well preserved with triton. Thus, optimal condit ions to detect myc protein were fixation with paraformaldehyde and treatment with triton; in such conditions, the analysis of cell cycle was possible. Myc protein expression was higher in G2-M than in G0-Gt cells. No significant variation in the expression of myc protein was seen between cells in exponential growth phase and subconfluent cells.

USE OF FLOW CYTOMETRY TO DETECT INTERMEDIATE FILAMENTS FROM CENTRAL

NERVOUS SYSTEM

DUFAY Nathalie °, PACALET Merielle', TOURAINE Franq:oise " and BELIN Marie-Fran~:oise °

°INSERM CJF 90-10 and *lmmunobiology Laboratory, Neurological Hospital, BP Lyon Montchat 69394, L YON

CEDE)( 03, FRANCE.

Vimentin, neurofilaments and Glial Fibrilliary Acidic protein (GFAP) are intermediate filament proteins of the cell cytoskeleton. They are distributed in a specific manner in central nervous system (CNS) cells. Thus vimenlin is a specilic marker for immature cells whereas neurofilaments and GFAP are selectively localized in cells that have differentiated as neurons and glia respectively.

The specific expression of these molecules makes them useful markers in the differential diagnosis of CNS tumors. To date, analysis is mainly performed by immunohistochemistry on tissue sections. We have developed an alternative method in which cell suspensions are immunostained and analysed by flow cytometry (Profile, COULTER). We illustrated our method with results obtained using CNS tumor cell lines (TE-671, Dev, C6,1RM32). Various methods of fixation and/or permeabilization (paraformaldehyde, ethanol, methanol, triton X-100 and saponin) were compared. Double-staining with propidium iodide was also performed and allowed the analysis of intermediale filament expression during the cell cycle.

The best results were obtained using saponin permeabilization without fixation which is rapid, avoid cell agregation, and does not denature cell surface antigens.

In conclusion, such a method could be successfully applied to study various cytoplasmic antigens and help wilh CNS turnout diagnosis.

OPTIMIZED CD4, CD8 AND CD3 ABSOLUTE COUNTS USING THE FACSCount TM INSTRUMENT

STRAUSS Kenneth, HULSTAERT Frarlk, HANNET Irene

Becton-Dickinson Immunocytontetry 2~vstems - Europe, 24 Denderstraat, POB I 3, 9320 Erembodegem - Aalst, BELGIUM

The FACSCount TM is a compact, dedicated system recently announced by

Becton-Dickinson for obtaining absolute CD4, CD8 and CD3 cell counts. It is a

stand-alone system with no requirements for ancillary, technologic.'il or laboratory

facilities. The instrument platform is mobile and is designed for field use. No

previous lab experience is required of the operator, and approximately 3 hours of

training are needed before beginning instrument operation. Minimal handling of

potentially biohazardous samples is required and the unit-dose reagent

configuration is designed to facilitate field transport of samples. Methods

controls, cross calibration and system error-flagging are key components of the

system, as are propriatory reagents and reference standards. The system uses a

no lyse, no wash, no centrifuge technology. In muhicenter field trials with

prototype units the correlation with current flow-based technology has been

excellent, even at low absolute CD4 T cell counts found in some HlV-infected

patients. The FACSCount TM is expected to reduce the cost of CD4 counting by

three-to four-fold over the cost of current CD4 counting technology.

A LARGE MULTICENTER EVALUATION OF TIlE BECTON DICKINSON HLA-B27 SCREENING SYSTEM.

HUI.STAERT Frank, HANNET Irene, STRAUSS Kenneth

Becton-Dickinson Immunocytometry Systems - Europe, 24 Denderstraat, POB I3, 9320 Erembodegem - Aalst, BELGIUM

A new flow cytometric HLA-B27 screening system from Becton Dickinson

was compared, in three large reference laboratories, to the classic

microcytotoxicity assay for I-ILA- typing. An identical panel of 14 antisera (2x

negative control, Ix B5, 5x B7, lx B14, Ix B22, 5x B27, Ix B37, lx positive

control fall from Fresenius]) was used at the three sites. Standardized criteria

were used for the interpretation of nticrocytotoxicity readings.

The flow cytometric system used in all sites consisted of a FACScan TM flow

cytometer and the I-[LA-B27 screening system (including software and

reagents). The same decision threshold for HLA-B27 positivity was used at all

sites. 1573 samples were analysed using both methods. Acceptable

microcytotoxicity results were obtained for 1435 of these samples. In 4 cases

(0.25 %) the gating failed on the flow cytometer. Of the 1431 remaining

samples, 260 (18.2 %) were HLA-B27 positive by classic microcytotoxicity.

All of these samples also screened positive with the flow cytometric assay.

Observed sensitivity of the HLA-B27 screening system was thus 100 %. 30

HLA-B27 negative samples, most of which were I-[LA-B7 positive, tested

falsely positives using the flow cytometry screening method. The resuhin~

specificity of the screening method was thus 95% or ~eater in all sites.