Capillary Electrophoresis in Diagnosis and Monitoring of Adenosine Deaminase Deficiency

Abstract

Background:The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attributable to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphorylase (PNP), and S-adenosyl-L-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evaluation of enzyme activities. Methods: Deoxyadenosine metabolites were separated in 30 mmol/L sodium borate-10 mmol/L sodium dodecyl sulfate (pH 9.80) at 25°C on a 60-cm uncoated capillary. For determination of enzyme activities, substrate-product separation and measurements were carried out in 20 mmol/L sodium borate (pH 10.00) at 25°C on a 42-cm uncoated capillary. Results: Deoxynucleotides and deoxyadenosine were readily detectable in erythrocytes and urine, respectively. Both methods were linear in the range 2-500 mol/L (r >0.99). Intra-and interassay CV were <4%. Enzyme activities were linear with respect to sample amounts in the incubation mixture and to incubation time (r >0.99 for both). In erythrocytes from healthy individuals, mean (SD) ADA activity was 5619 (2584) nmol/s per liter of packed cells. In erythrocytes of SCID patients at diagnosis, ADA activity was 56.9 (48.3) nmol/s per liter of packed cells; SAHH activity was also much reduced. PNP activity was similar in patients and controls. Conclusions: CE can be used to test ADA deficiency and enables rapid assessment of ADA expression in hema-

Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency

“…Intervention-free survival (defined as survival without receiving a post-GT SCT or continuous PEG-ADA treatment of $3 months) and infection rates were also assessed. Vector copy number, lymphocyte ADA activity, 21 and red blood cell (RBC) dAXP levels were used to assess engraftment and transgene function. For dAXP analysis, adequate systemic metabolic detoxification was classified as concentrations ,100 nmol/mL dAXP based on observations in patients receiving HLA-matched SCTs.…”

“…For dAXP analysis, adequate systemic metabolic detoxification was classified as concentrations ,100 nmol/mL dAXP based on observations in patients receiving HLA-matched SCTs. [21][22][23][24][25] Immune reconstitution measures included lymphocyte subset counts, 26,27 T-cell receptor excision circle (TREC) analysis, and T cell proliferative capacity. 28 Physical growth was monitored.…”

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

“…21 Adenine ribo and deoxyribonucleotides in plasma samples were measured by high-performance capillary electrophoresis. 21 …”

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

“…by guest www.bloodjournal.org From ADA enzymatic activity Intracellular ADA enzyme activity was analyzed by adenosine to inosine conversion followed by high-performance capillary electrophoresis. 34,35 Red blood cell lysis was performed on BM and spleen samples.…”