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Increased sensitivity of the MLC test of CML patients
64
Abstracts
#234 5.3
INCREASED
SENSITIVITY
OF THE MLC
TEST
FOR CML PATIENTS
L Dombr~ A Nikaein, Baylor University Medioal Center, Dallas, Texas MLC test for patients with chronic m y e l o c y t i c leukemia (CML} often fails to reveal accurate results. This is due to the presence of a high number of spontaneously p r o l i f e r a t l n g tumor cells. In this study, we have used two different methods to purify lymphocytes. In 17 patients a n d 117 controls, T cells were p u r i f i e d by nylon wool. In 44 patients and 144 controls, T cells were p u r i f i e d by T Lymphokwlk. In the latter group, patient's m o n o n u o l e a r cells (stimulator cells) were also isolated by m o n o n u o l e a r Lymphokwik. Using either of the methods, patient's responses to donors, controls, and the pool of controls were significantly increased. The number of d i s c r e p a n c i e s for each combination comparing the PBL with isolated cells, by either method, with PBL is shown below. Combinations Patient to Donor Donor to Patient Patient to Controls Controls to Patient
_L_~hokwik_ 10/44 (22%) 11/44 (25%) 13/144 (9%) 23/144 (16%)
_ _ _ ~ y l o n Wool 6/37 (16%) 4/37 (11%) 28/117 (24%) 17/117 (15%)
The above observations indicate that the presence of tumor cells in PBL of CML patients interferes with p r o l i f e r a t i o n of T cells in MLC and p u r i f i c a t i o n of lymphocytes is necessary for the accurate i n t e r p r e t a t i o n of results.
#235 5.4
NON-RADIOACTIVE REVERSE DOT BLOT ASSAY FOR HLA-DR TYPING. BF Dully, P DeTogni, DL Phelan, D Chaplin and T Mohanaknm~r, Barnes Hospital, HIA Laboratory and Washington School of Medicine, Depts. of Surgery and Pathology and Howard Hughes Medical Institute, St. Louis, MO Allele specific oligonucleotide (ASO) hybridization following pol~merase chain reaction (PCR) amplification is the desired method of HLA DR typing in cases of leukopenia and poor antigen expression. "Dot blot" PCR based assays attach the amplified sample DNA to a series of nylon membranes and hybridize each me,~rane with one labelled AS0 probe. Here we describe a non-radioactive "reverse dot blot" assay which hybridizes the PCR amplified sample to RS0 probes, UV-crosslinked to nylon membranes. A single hybridization reaction, performed at one temperature in the absence of tetramethylamuonivm chloride, is required to resolve a broad HLA DR typing. Primers encompassing the DRBI gene were used to amplify DNA prepared from Workshop homozygous B-cell lines. The PCR product was labelled during amplification with biotinylated dATP and hybridized to a panel of poly(T) tailed AS0 probes UV-crosslinked to a nylon membrane. The hybridization was detected by binding of streptavidin-alkaline phosphatase to the biotinylated DNA, followed by a chamiluminescent reaction. This technique identified the DRI-DRwI% alleles with the exception of DR1. ~ and 7 ~ broad specificities. The inability to detect these three alleles may be associated with the high biotinylated dATP content in their amplified sequences which prevents ASO probe hybridization. An HLA-DR t ~ i n g can be performed on 100ul of blood in 6-8 hours. The membranes are re-usable after stripping the hybridized PCR sample. Therefore, hhe reverse dot blot procedure is a safe, rapid and specific technology for HLA-DR typing in to a clinical laboratory.
Abstracts
#234 5.3
INCREASED
SENSITIVITY
OF THE MLC
TEST
FOR CML PATIENTS
L Dombr~ A Nikaein, Baylor University Medioal Center, Dallas, Texas MLC test for patients with chronic m y e l o c y t i c leukemia (CML} often fails to reveal accurate results. This is due to the presence of a high number of spontaneously p r o l i f e r a t l n g tumor cells. In this study, we have used two different methods to purify lymphocytes. In 17 patients a n d 117 controls, T cells were p u r i f i e d by nylon wool. In 44 patients and 144 controls, T cells were p u r i f i e d by T Lymphokwlk. In the latter group, patient's m o n o n u o l e a r cells (stimulator cells) were also isolated by m o n o n u o l e a r Lymphokwik. Using either of the methods, patient's responses to donors, controls, and the pool of controls were significantly increased. The number of d i s c r e p a n c i e s for each combination comparing the PBL with isolated cells, by either method, with PBL is shown below. Combinations Patient to Donor Donor to Patient Patient to Controls Controls to Patient
_L_~hokwik_ 10/44 (22%) 11/44 (25%) 13/144 (9%) 23/144 (16%)
_ _ _ ~ y l o n Wool 6/37 (16%) 4/37 (11%) 28/117 (24%) 17/117 (15%)
The above observations indicate that the presence of tumor cells in PBL of CML patients interferes with p r o l i f e r a t i o n of T cells in MLC and p u r i f i c a t i o n of lymphocytes is necessary for the accurate i n t e r p r e t a t i o n of results.
#235 5.4
NON-RADIOACTIVE REVERSE DOT BLOT ASSAY FOR HLA-DR TYPING. BF Dully, P DeTogni, DL Phelan, D Chaplin and T Mohanaknm~r, Barnes Hospital, HIA Laboratory and Washington School of Medicine, Depts. of Surgery and Pathology and Howard Hughes Medical Institute, St. Louis, MO Allele specific oligonucleotide (ASO) hybridization following pol~merase chain reaction (PCR) amplification is the desired method of HLA DR typing in cases of leukopenia and poor antigen expression. "Dot blot" PCR based assays attach the amplified sample DNA to a series of nylon membranes and hybridize each me,~rane with one labelled AS0 probe. Here we describe a non-radioactive "reverse dot blot" assay which hybridizes the PCR amplified sample to RS0 probes, UV-crosslinked to nylon membranes. A single hybridization reaction, performed at one temperature in the absence of tetramethylamuonivm chloride, is required to resolve a broad HLA DR typing. Primers encompassing the DRBI gene were used to amplify DNA prepared from Workshop homozygous B-cell lines. The PCR product was labelled during amplification with biotinylated dATP and hybridized to a panel of poly(T) tailed AS0 probes UV-crosslinked to a nylon membrane. The hybridization was detected by binding of streptavidin-alkaline phosphatase to the biotinylated DNA, followed by a chamiluminescent reaction. This technique identified the DRI-DRwI% alleles with the exception of DR1. ~ and 7 ~ broad specificities. The inability to detect these three alleles may be associated with the high biotinylated dATP content in their amplified sequences which prevents ASO probe hybridization. An HLA-DR t ~ i n g can be performed on 100ul of blood in 6-8 hours. The membranes are re-usable after stripping the hybridized PCR sample. Therefore, hhe reverse dot blot procedure is a safe, rapid and specific technology for HLA-DR typing in to a clinical laboratory.