M. B. Van Der Weyden

Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Severe combined immunodeficiency and adenosine deaminase deficiency: failure of enzyme replacement therapy.

A first-born baby boy presented at age 3 months with persistent diarrhoea, failure to thrive, and recurrent bacterial and fungal infections. Severe combined immunodeficiency was demonstrated. A deficiency of adenosine deaminase (ADA) activity was suggested by the presence of extensive skeletal abnormalities, and the ADA activity in erythrocyte and leucocyte lysates was < 0.005 nmol/h per mg protein. Culture of ADA-negative peripheral blood mononuclear cells, together with purified calf ADA, did not alter the absent phytohaemagglutinin response. Treatment with immunoglobulin, pentamidine, and co-trimoxazole was started and a programme of ADA enzyme replacement, with infusions of plasma and frozen irradiated erythrocytes, was begun at age 4 months and achieved blood ADA levels in excess of 30 nmol/h per mg haemoglobin. Although resolution of the interstitial pneumonitis and skeletal abnormalities was observed, there was no evidence of immunological reconstitution. The patient died at age 17 months after a parainfluenza pneumonitis. Features of importance in predicting lack of benefit from enzyme replacement by erythrocyte infusion in ADA-negative severe combined immunodeficiency appear to be early clinical presentation with associated severe skeletal abnormalities, a very low level of residual ADA activity in peripheral blood mononuclear cells, and lack of effect of exogenous ADA on the absent in vitro mitogen response of ADA-negative blood mononuclear cells.

Prenatal diagnosis for adenosine deaminase deficiency.

Amniocentesis was performed in two successive pregnancies of the mother of a child with adenosine deaminase (ADA) deficient severe combined immunodeficiency. Assay of ADA in amniotic fluid fibroblasts showed the pregnancies to be normal and homozygous deficient, respectively. These findings were confirmed by the demonstration of a normal level of erythrocyte ADA in the cord blood of the healthy male born of the first pregnancy and by the demonstration of undetectable ADA activity in cord erythrocytes, spleen, liver, and kidney of the abortus of the second pregnancy. Prenatal diagnosis of ADA deficiency appears to be a reliable procedure.

Chlorpromazine-associated haemolysis in anorexia nervosa.

Chlorpromazine therapy in a patient with anorexia nervosa was associated with haemolytic anaemia. In vitro red cell findings included an increased degree of red cell autohaemolysis and a chlorpromazine-dependent Heinz body formation both corrected with exogenous glucose. Activities of red cell enzymes involved with the glycolytic pathway or glutathione metabolism were normal.