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CD8 lymphocyte subsets in infants and children
Bial. Cell (1991),73 PLATElEf PHENOTYPING BY FLOW CYfOMEfRY.
MAGGIPINTO Gianni. WANTEN Nadine, SONDAG Daniele. Servie d'Immuno-hematologie; CHU B-4000 LIEGE. Phenotyping PLA and Bak platelet systems with polyclonal reagents can be performed easily by flow cytometry even when the direct anti-globulin test of platelets is positive; contrarily to Br and lILA which are much more difficult. Br, because the density of antigenic sites being weak, the antibody reaction is proportionally weak; HI..A, because lIlA antig~ns give aspecific reactions with polyclonal as well as with monoclonal antibodies, most probably due to cross-reactions (i.e. B7, B27, B40). We proved successful in setting up a method for the phenotyping of PLA and Bak by flow cytometry. This technique has been experienced for 7 years and works routinely without any problem. More recently, we approached the Br system by flow cytometry and compared the results with tliose obtained by the ~WP A currently considered as reference method. Correlation was excellent (100%) at least when the direct anti-globulin test of platelets was negative. One of the most attractive trait of flow Cy10metry is the short time (less than 3 h) within the results can be obtained (24 h with the MArPA) and its very low cost price. Although MArPA remains the reference method, flow C)1ometry has a complementary and very promising place in a laboratory of platelet immunology,
CDS LYMPHOCYTE SUBSETS IN lNFANTS AND CHILDREN. DENEYS VeroniqJe'. MAllON Ame-Marie', HULSTAERT Frandl'. VANLANGENDONCK Francis'. DE GOLS Hilde'. DE BRUYERE Marc', , Laboratoire d'!mmunobtmatologte Universite Catholique de Louvain. Clos Cbapelle aux Champs 30.52 1200 Bruxelles, Becton Dickinson Immunocytometry System. Europe,
During HIV infection in adults. increased proportions of peripheral hlood CDR lymphocytes also positive for HLA-DR, CDS7 and CD38 have !leen descri!led, proportionally with the evolution of the disease. In vertically infected infants, similar ohservations have heen documented. The aim of this study was to determine normal ranges for CD8 lymphocyte subsets in infants and children. Study population : 62 children coming to the hospital without any infection. tumor or immunodeficiency and 80 adult blood donors were inclued. Methods: the method was a lysed whole blood one for two color immunophenotyping using a FACScan'" flow cytometer. Simultesl'" reagents and SimuiSET'" software. Lymphocytes were first identifed based both on light scatter characteristics and phenotype (CD45+, CDI4-l using the leucoGATE reagent. The monoclonal antibodies used were two color (FITCfPE) reagenl~ : Leu 2 (CD8) I HLA-DR. Leu 7 (CD57) I Leu2, Leu21 Leu 17 (CD38). ~ expressed as % of total CD8 cells: median (2S-7S~ percentilt: range) ~ ~ ---l:.lli adults N' of CASES 17 26 19 SO IILA·DR+ 14(11-21) 15(11-19) 16(12-21> 22114·29) CD57+ 4(0-5) 8(6-16) 17(13-18) 2406-37) CD38+ 94(92-95) 78(67-83) 70(66-74) 46136-53) Conclusion: Ahout aU CD8 lymphocytes in infants are HLA-DR -. CDS7 - and CD38 +. In children. there is an increasing proportion of HLA-DR + and/or CD57 + and CD38 • CD8 cells. Reference ranges of normal phenolYpe are important to evaluate the CD8 lymphocyte subsets in IIIV infection, The number of cases in this study is still insufficient to establish statistical normal values.,
52a
CD11b AND eD45 ON MONOCYTES (~~el DURING HEMODIALYSIS (HD), DELVILLE Jean-Pierre", TIELE~ANS Christian', HUSSON C~cile", MADHOUN Philippe". lAMBRECHTS Annie'. GOLDMAN Michel",VANHERWEGHEM Jean-louis" "Departments of Immunology and Nephrology. CUB-UlB HOpital Erasme, Brussels. Belgium, Expression of the CD11b adhesion-promoting molecule and the leukocyte common antigen (CD45) was investigated in 5 patients during HD on cuprophane (CU) and polyacrylonitrile (PAN). We studied: 1/ circulating MNC (c-MNCI before (to), and after 15 (t15) and 180 minutes (t180) of HD: 2/ the MNC adhering (a-MNC) to the dialyzer which were collected by using a trypsin-EDTA rinsing technique. A drop in c-MNC occurred with both membranes. maximal at t15 with CU and tl80 with PAN, and more important with CU (88 vs 36% p<.005). The number of MNC adhering to the dialyzer - which wa~ g2eater with CU (1777 • 600 vs 60 • 1 10 /m , p<.025) could by no way explain the monocytopenia. After staining with specific fluorescent menoclonal antibodies CDllb and CD45 were studied by flow cytometry (Table), , to t15 t180 a-MNC CD11b CU PAN CD45 CU PAN
206'76 226.44 152116 147.12
415.138" 270142 NS 201115" 140+14 NS
5851225" 6761101' 221120 222'31 "
6681149' 7681122' 337158' 349'33 '
comparison vs to (Wilcoxon) : "=.05 +=.025 '=,005. The upregulation of CD11b can provide a mechanism for MNC sequestration in the circulation during HD while CD45 may reflect mechanisms regulating the cell activation state. The different chronology between eu and PAN suggest that different mechanism might be implicated.
MAGGIPINTO Gianni. WANTEN Nadine, SONDAG Daniele. Servie d'Immuno-hematologie; CHU B-4000 LIEGE. Phenotyping PLA and Bak platelet systems with polyclonal reagents can be performed easily by flow cytometry even when the direct anti-globulin test of platelets is positive; contrarily to Br and lILA which are much more difficult. Br, because the density of antigenic sites being weak, the antibody reaction is proportionally weak; HI..A, because lIlA antig~ns give aspecific reactions with polyclonal as well as with monoclonal antibodies, most probably due to cross-reactions (i.e. B7, B27, B40). We proved successful in setting up a method for the phenotyping of PLA and Bak by flow cytometry. This technique has been experienced for 7 years and works routinely without any problem. More recently, we approached the Br system by flow cytometry and compared the results with tliose obtained by the ~WP A currently considered as reference method. Correlation was excellent (100%) at least when the direct anti-globulin test of platelets was negative. One of the most attractive trait of flow Cy10metry is the short time (less than 3 h) within the results can be obtained (24 h with the MArPA) and its very low cost price. Although MArPA remains the reference method, flow C)1ometry has a complementary and very promising place in a laboratory of platelet immunology,
CDS LYMPHOCYTE SUBSETS IN lNFANTS AND CHILDREN. DENEYS VeroniqJe'. MAllON Ame-Marie', HULSTAERT Frandl'. VANLANGENDONCK Francis'. DE GOLS Hilde'. DE BRUYERE Marc', , Laboratoire d'!mmunobtmatologte Universite Catholique de Louvain. Clos Cbapelle aux Champs 30.52 1200 Bruxelles, Becton Dickinson Immunocytometry System. Europe,
During HIV infection in adults. increased proportions of peripheral hlood CDR lymphocytes also positive for HLA-DR, CDS7 and CD38 have !leen descri!led, proportionally with the evolution of the disease. In vertically infected infants, similar ohservations have heen documented. The aim of this study was to determine normal ranges for CD8 lymphocyte subsets in infants and children. Study population : 62 children coming to the hospital without any infection. tumor or immunodeficiency and 80 adult blood donors were inclued. Methods: the method was a lysed whole blood one for two color immunophenotyping using a FACScan'" flow cytometer. Simultesl'" reagents and SimuiSET'" software. Lymphocytes were first identifed based both on light scatter characteristics and phenotype (CD45+, CDI4-l using the leucoGATE reagent. The monoclonal antibodies used were two color (FITCfPE) reagenl~ : Leu 2 (CD8) I HLA-DR. Leu 7 (CD57) I Leu2, Leu21 Leu 17 (CD38). ~ expressed as % of total CD8 cells: median (2S-7S~ percentilt: range) ~ ~ ---l:.lli adults N' of CASES 17 26 19 SO IILA·DR+ 14(11-21) 15(11-19) 16(12-21> 22114·29) CD57+ 4(0-5) 8(6-16) 17(13-18) 2406-37) CD38+ 94(92-95) 78(67-83) 70(66-74) 46136-53) Conclusion: Ahout aU CD8 lymphocytes in infants are HLA-DR -. CDS7 - and CD38 +. In children. there is an increasing proportion of HLA-DR + and/or CD57 + and CD38 • CD8 cells. Reference ranges of normal phenolYpe are important to evaluate the CD8 lymphocyte subsets in IIIV infection, The number of cases in this study is still insufficient to establish statistical normal values.,
52a
CD11b AND eD45 ON MONOCYTES (~~el DURING HEMODIALYSIS (HD), DELVILLE Jean-Pierre", TIELE~ANS Christian', HUSSON C~cile", MADHOUN Philippe". lAMBRECHTS Annie'. GOLDMAN Michel",VANHERWEGHEM Jean-louis" "Departments of Immunology and Nephrology. CUB-UlB HOpital Erasme, Brussels. Belgium, Expression of the CD11b adhesion-promoting molecule and the leukocyte common antigen (CD45) was investigated in 5 patients during HD on cuprophane (CU) and polyacrylonitrile (PAN). We studied: 1/ circulating MNC (c-MNCI before (to), and after 15 (t15) and 180 minutes (t180) of HD: 2/ the MNC adhering (a-MNC) to the dialyzer which were collected by using a trypsin-EDTA rinsing technique. A drop in c-MNC occurred with both membranes. maximal at t15 with CU and tl80 with PAN, and more important with CU (88 vs 36% p<.005). The number of MNC adhering to the dialyzer - which wa~ g2eater with CU (1777 • 600 vs 60 • 1 10 /m , p<.025) could by no way explain the monocytopenia. After staining with specific fluorescent menoclonal antibodies CDllb and CD45 were studied by flow cytometry (Table), , to t15 t180 a-MNC CD11b CU PAN CD45 CU PAN
206'76 226.44 152116 147.12
415.138" 270142 NS 201115" 140+14 NS
5851225" 6761101' 221120 222'31 "
6681149' 7681122' 337158' 349'33 '
comparison vs to (Wilcoxon) : "=.05 +=.025 '=,005. The upregulation of CD11b can provide a mechanism for MNC sequestration in the circulation during HD while CD45 may reflect mechanisms regulating the cell activation state. The different chronology between eu and PAN suggest that different mechanism might be implicated.