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A new microcellular cytotoxicity test based on calcein AM release
126
Abstracts
C-7.3 #239
A NEW MICROCELLULAR CYTOTOXICITY TEST BASED ON CALCEIN AM RELEASE. ~Y.aag_XM, Terasaki PI, Rankin GW, Chia D, Zhong HP, and Hardy S, UCLA Tissue Typing Laboratory, Los Angeles, CA. We present a microtest for detecting cell-mediated lysis to replace 51Cr-releas¢, based on the use of the Terasaski lray and a vital dye, calcein AM, which shows less than 15% spontaneous leakage in 4 hrs. at 37°C. The number of target cells needed for this assay is reduced 10 fold when compared with 51Cr-release. This leads to a corresponding 10-fold reduction in the number of effector cells required. Results were read in about 1 minute per tray by using an automated microfluorimeter and were visually confirmed by counterstaining the dead targets with ethidium bromide. NK activity from6 52 normal donors resulted in a mean of 87.33+9.67 lyric units (30% specific lysis/10 ¢ffectors); range 16.43-345.38. As expected, LAK cells, elicited by culturing normal lymphocytes with r-IL2, killed Daudi, K562, and Chang liver targets. Figure 1 shows a semi-log plot of responder cell input per well versus the fraction of negative responding targets for a representative limiting dilution assay. This straight line plot enabled us to determine the CTL-precursor frequency for the indicated HLA typed normal cell pair by # ¢=~,ll/w~l tooo 2000 3000 4000 5000 minimizing chi-square statistical analysis. .° • . , . , . . . . Finally, we obtained a significant positive correlation result from 51Cr-release assays (n=55 E:T combinations; r=0.933; p<.001). ~ 0, p(x*2)=0748 We conclude that this microtest should find R e ~ p o r t d i n g C,e l l - A 2, 2 4 B 4 4 , _ wide application as an alternative to using a # radioactive isotope for studies involving 001 ~t, r n u g l t l n g C e l l - A 2, 2 3 B 7 . 4 2 Figure 1 cell-mediated immunity.
C-7.3 #240
LACK OF CORRELATION BETWEEN CYTOTOXIC T-LYMPHOCYTE PRECURSOR FREQUENCY AND ACUTE GRAFT VERSUS HOST DISEASE 1 2 POST BONE MARROW TRANSPLANT. S Fussell, M Donnellan, M Cooleya, C 1 1 Farrell. NSW Red Cross Blood Transfusion Service and St. Vincent's Hospital2 Sydney, Australia. It has been suggested that the level of patient specific Cytotoxic T-Lymphocyte precursors (CTLp) present in Matched Unrelated Donors (MUD) correlates with the incidence and severity of Graft versus Host Disease (GVHD) post bone marrow transplantation. The CTLp assay uses limiting dilution to determine the number of circulating donor lymphocytes with receptors capable of recognising and responding to antigen present on the patients cells. The CTLp measures the cytotoxic effector cells generated following the proliferative phase of the MLC by the release of SlCr from labelled target ceils. The assay is performed in microtitre trays with responder dilutions ranging from 4 x 104 to 625 cells per well and a constant number (5 x 104) of stimulator cells per well. A minimum of 24 replicate wells are set up at each dilution. The number of negative wells obtained at each dilution is used to calculate the precursor cell frequency. To date, 168 potential matched unrelated bone marrow donors have been tested for 56 recipients of which 20 have proceeded to transplant. 14 of the 20 recipients were evaluable for Acute Graft Versus Host Disease (AGVHD). The patients were divided into 2 groups, those with high donor precursor frequencies (>1:100,000) and those with low donor precursor frequencies (<1:100,000). Clinically significant (grades II-W) AGVHD was observed in recipients of both groups post bone marrow transplantation. Statistical analysis revealed no correlation between high donor precursor frequencies (>1:100,000) and the patient developing clinically significant AGVHD post transplantation (X2 = 1.16). This study casts doubt on the usefulness of the CTLp assay as a predictor of severe acute GVHD post transplantation.
Abstracts
C-7.3 #239
A NEW MICROCELLULAR CYTOTOXICITY TEST BASED ON CALCEIN AM RELEASE. ~Y.aag_XM, Terasaki PI, Rankin GW, Chia D, Zhong HP, and Hardy S, UCLA Tissue Typing Laboratory, Los Angeles, CA. We present a microtest for detecting cell-mediated lysis to replace 51Cr-releas¢, based on the use of the Terasaski lray and a vital dye, calcein AM, which shows less than 15% spontaneous leakage in 4 hrs. at 37°C. The number of target cells needed for this assay is reduced 10 fold when compared with 51Cr-release. This leads to a corresponding 10-fold reduction in the number of effector cells required. Results were read in about 1 minute per tray by using an automated microfluorimeter and were visually confirmed by counterstaining the dead targets with ethidium bromide. NK activity from6 52 normal donors resulted in a mean of 87.33+9.67 lyric units (30% specific lysis/10 ¢ffectors); range 16.43-345.38. As expected, LAK cells, elicited by culturing normal lymphocytes with r-IL2, killed Daudi, K562, and Chang liver targets. Figure 1 shows a semi-log plot of responder cell input per well versus the fraction of negative responding targets for a representative limiting dilution assay. This straight line plot enabled us to determine the CTL-precursor frequency for the indicated HLA typed normal cell pair by # ¢=~,ll/w~l tooo 2000 3000 4000 5000 minimizing chi-square statistical analysis. .° • . , . , . . . . Finally, we obtained a significant positive correlation result from 51Cr-release assays (n=55 E:T combinations; r=0.933; p<.001). ~ 0, p(x*2)=0748 We conclude that this microtest should find R e ~ p o r t d i n g C,e l l - A 2, 2 4 B 4 4 , _ wide application as an alternative to using a # radioactive isotope for studies involving 001 ~t, r n u g l t l n g C e l l - A 2, 2 3 B 7 . 4 2 Figure 1 cell-mediated immunity.
C-7.3 #240
LACK OF CORRELATION BETWEEN CYTOTOXIC T-LYMPHOCYTE PRECURSOR FREQUENCY AND ACUTE GRAFT VERSUS HOST DISEASE 1 2 POST BONE MARROW TRANSPLANT. S Fussell, M Donnellan, M Cooleya, C 1 1 Farrell. NSW Red Cross Blood Transfusion Service and St. Vincent's Hospital2 Sydney, Australia. It has been suggested that the level of patient specific Cytotoxic T-Lymphocyte precursors (CTLp) present in Matched Unrelated Donors (MUD) correlates with the incidence and severity of Graft versus Host Disease (GVHD) post bone marrow transplantation. The CTLp assay uses limiting dilution to determine the number of circulating donor lymphocytes with receptors capable of recognising and responding to antigen present on the patients cells. The CTLp measures the cytotoxic effector cells generated following the proliferative phase of the MLC by the release of SlCr from labelled target ceils. The assay is performed in microtitre trays with responder dilutions ranging from 4 x 104 to 625 cells per well and a constant number (5 x 104) of stimulator cells per well. A minimum of 24 replicate wells are set up at each dilution. The number of negative wells obtained at each dilution is used to calculate the precursor cell frequency. To date, 168 potential matched unrelated bone marrow donors have been tested for 56 recipients of which 20 have proceeded to transplant. 14 of the 20 recipients were evaluable for Acute Graft Versus Host Disease (AGVHD). The patients were divided into 2 groups, those with high donor precursor frequencies (>1:100,000) and those with low donor precursor frequencies (<1:100,000). Clinically significant (grades II-W) AGVHD was observed in recipients of both groups post bone marrow transplantation. Statistical analysis revealed no correlation between high donor precursor frequencies (>1:100,000) and the patient developing clinically significant AGVHD post transplantation (X2 = 1.16). This study casts doubt on the usefulness of the CTLp assay as a predictor of severe acute GVHD post transplantation.