244 FLOW AND IMAGE CYTOMETR, Y, Montpellier, December 1992 )

AN OPTIMIZED METHOD FOR ROUTINE HLA-B27 SCREENING USING FLOW CYTOMETRY

HULSTAERT Frank. HANNET Irene, STRAUSS Kenneth

Becton-Dickinson Irmnunocytometry Systems - Europe, 24 Denderstraat, POB13, 9320 Erembodegem - Aalst, BELGIUM

Flow cytometry and monoclonal antibodies are promising tools for HLA-

antigen detection. Various approaches however have been hampered by the

lack of a ready-to-use system for calibration and sample analysis (for

example, in gating of target population and positioning of decision marker).

The new Becton Dickinson system for HLA-B27 screening combines a

software package and reagents (Calibration beads and anti-HLA-B27 FIT(I/

anti-Leu4 (CD3) PE monoclonal antibodies). This system allows for an

optimized calibration of the FACScan and FACStrak flow cytometers for

HLA-B27 screening.

Sample preparation involves a standard lysed whole blood procedure and

requires only one tube per test. Sample analysis proceeds in batch, and

takes approximately one minute per sample. After acquisition, T-

lymphocytes are automatically gated and analyzed for their expression of

the HLA-B27 - antigen.

The median channel number on the FLI axis is calculated and compared to

a lot-encoded decision marker. When the median is lower than the marker,

the sample is reported as HLA-B27 negative. When the median is higher,

confirmation of HLA-B27 positivity with the microcytotoxicity method is

required as the HLA-B27 monoclonal shows some crossreactivity with

other I-ILA-B types.

FLOW CYTOMETRY AS REFERENCE METHOD TO VALIDATE A NEW CD4 / CD8 LYMPHOCYTE COUNTING ASSAY BASED ON IMMUNO-MAGNETIC SEPARATION (IMS)'.

_BES$ON FAURE Isabelle 2, PONCELET Philippe 2, SAMPOL Jos~.l

1 - Laborato i re d 'h~matologie, 2 7 B d J. Moulin, 13005 Marsei l le

2 - b ioCYtex, Marseil le, France.

T4 (T8) lymphocyte enumeration, often required for monitoring immune status, is routinely obtained using flow cytometry (FCM) associated with blood formula. Both clinical labs which treat less than 5 to 10 analysis a day and Third world countries which cannot afford expensive equipment need alternative simple and cheap tests. The immuno-magnetic separation (IMS) is one answer. Thus, blood cells of interest (i.e. CD4 + or CD8 +) are separated using magnetic beads coated with appropriate antibodies and a simple magnet. After lysis and acridine orange staining these can be visualized and counted under the fluorescent microscope as brightly fluorescent nuclei. This IMS assay directly provides an absolute number of CD4 (CD8) lymphocytes / mm 3 blood.

Flow cytometry has been extensively used i) to design the method (1) ii) to validate the assay. Thus, IMS results w e r e compared to those gained from flow cytometry (direct IF protocol) associated with blood formula; CD45 and CD14 controls were used to optimize lymphocyte gating before CD4 and CD8 analysis. The correlation study made on 100 samples, including lymphopenic and AIDS samples, demonstrated a good fit (r > 0,95) between IMS and FCM.

(1) PONCELET P.I ZANNIER A., GORCE O., MASSOT O. BRUNET C. et SAMPOL J. (1990). Biol. cell, 70, 13A- 14A.

* : DYNABEADS® T4/1"8 Quant (Dynal, Oslo, N and Biosys, Compi~gne, F)

COMPARATIVE ANALYSIS OF SINGLE, SIMULTANEOUS DOUBLE AND TREBLE STAINING APPLIED TO CD3 / CD4 / CD8 CHARACTERIZATION IN FLOW CYTOMETRY. PJM..Philip. F. Baudouin, I. Sudaka and J. Bayle. Laboratoire Central d' Hdmatologie, CHU tie Nice, France.

Here, we analyzed the immunological phenotypes (CD3, CD4, CD8) from 20 patients investigated in a in flow cytometry clinical laboratory by using Single (S), Simultaneous Double (SDS) and Treble (STS) immunofluores- cent Staining. The lymphocytes had been isolated by lysed whole blood using "Q-Prep" (Coultronics, Hialeah, FL, USA) on the Forward / Side Scatter parameters of the flow cytometer (Coulter-Epics Profile II). Optical filters separate by wavelenghth the light from the laser stream intersection. Only fluorescence from Fluorescein-lso-Thio-Cyanate (FITC) is transmitted by the 525 nm band pass filter (PIN 3814036), Phyco-Erythrin (PE) being transmitted by the 575 nm band pass filter (P/N 3814066) and the new Energy Coupled Dyes (ECD) from Coultronics (Hialeah, FL, USA) by the 635 nm band pass filter (P/N 3814069). The objective of this study was to establish whether FITC, PE or ECD conjugated monoclonal antibodies used single (CD4; CDS; CD3), both paired in SDS (CD3 vs CD8; CD4 vs CD8) or associated together in a STS (giving other paired CD3 vs CD4; CD3 vs CDS; CD4 vs CDS) give identical or different percentages of fluorescence according to the staining test used for flow cytometry cell counting. Concur- rently, the mean of fluorescent in all configurations tested (S, SDS and STS) for each patient, was analyzed using the same monoclonal antibodies di- rectly conjugated. Each immunological phenotype was realized in this study with the same selected tests as well as the same selected color compensa- tions. Our results showed that percentage of positive cells are identical, whatever the flurochrome and the method used: FITC-CD3 = 74.2 +13.4 (S); FITC-CD3 = 73.6 + 14 (SDS) and ECD-CD3 = 73.6 + 13.3 (STS); FITC-CD4 = 9.1+7.5 (S); PE-CD4 = 10.2+8.5 (SDS); PE-CD4 = 10.6+8.4 (STS); FITC-CD8 = 60.4+14.8 (S); PE-CD8 = 60. I+15 (SDS); FITC-CD8 = 59.7+14.7 (STS).Comparison of positive cell percentages for each CD tested in single, in simultaneous both paired determined within double and treble staining, give correlations higher than 0.98 for CD8, 0.94 for CD4 and 0.97 for CD3. Conversely, no correlations were observed between the fluorescence means of single, double or treble immunofluorescent staining. This observation excludes today the use of any simultaneaous double or tre- ble staining in flow cytometry for an antigenic quantification. Conversely, selected tests defined for simultaneous double or treble staining, may be used to enumerate all positive cells showing antigenic coexpressions since no dispersion were observed when compared to the single ones. Thus, SDS and STS immunofluoreseent staining may be used to follow the immunolo- gical phenotype of leukocytic cell subpopulations in any clinical disorders.

QUANTITATIVE CELL-SURFACE IMMUNO- PHENOTYPE : A NEW DIMENSION FOR

IMMUNO-CYTOMETRY. APPLICATIONS AND TECHNOLOGY TRANSFER VIA QIFIKI'I -rM

(1,3)PONCELE T Philippe, (2)LAVABRE-BERTRAND Thierry, (1)(3 EORG E Fran~oise,

(1) Lab. Immuno-H~rnatologie - Facu/t6 de Pharrnacia , 13005 Marsei/la (2) Sen,. Maladies du Sang, CHR Lapeyronia, 34100 Montpellier

(3) bioCYtax, 140 ch. Arm~e d'Afrique, 13010 Marsellle

Although immunofluorescence flow cytometry is widely used to define and count cell subsets using monoclonal antibodies, little attention has been paid to quantitative aspects of antibody binding. "How to put the -merry into immunofluorescence flow cytometry ?" (1). This requires new tools to standardize IF measurements and to report results in terms of numbers of antibody binding sites per cell, moving from relative arbitrary units to absolute physical values. In addition to standards adapted to direct IF (F.C.S.C., USA), QIFIKIT TM (bioCYtex, Marseille, F) appears as a practical answer for indirect IF protocols using un-conjugated MAbs. The Quantitative IF Indirect (QIFI) assay (2) has been already applied in 3 International Leucocyte Typing workshops as a clustering tool (3, 4). It has also permitted the definition of "quantitative immuno-phenotype" for leukemic cells (3, 5) leading to new data with diagnostic, physiopathology and prognostic values (6). Normal density values for the main human leucocyte antigens are being recorded (7) as a basis before studying pathological states. The concept of "quantitative immuno-phenotype" can provide information directly related to the functional activity of cells, to the effect of inducers or culture conditions and potentially influence immunologic manipulat ions such as immuno-physical separations, bone marrow purging and in vivo MAb therapy (8). 1) SHAPIRO H.M. (1991), Cytometry Supp. 3, 70. 2) PONCELET P. et el. (1985), J. Immunol. Math., 85, 65. 3) Same (1986), In : Leucocyte Typing II, p. 329. 4) Same (1987), in : Leucocyte Typing III, p. 341. 5) DUPERRAY C. at al. (1989), Blood, 73. 566. 6) LAVABRE-BERTRAND et al. (1990), Blood, 76, Supp. 1, 1427. 7) Same (1991), Cytometry Supp. 3, 418 A. 8) LAURENT G. et el. (1986), Blood, 67, 1680.