Biotin-Labeled RBC Survival in Thalassemia and Impact of Treatment.

Abstract

Determination of red blood cell (RBC) survival in the peripheral blood, to evaluate the efficacy of pharmacological agents that augment fetal hemoglobin (HbF) and total hemoglobin (Hb) in patients with beta-thalassemia, has not been implemented. We report the use of biotin labeling, a nonisotopic, safe and effective method to determine the erythrocyte lifespan, proven to be equivalent to 51Cr.We studied RBC survival in 9 Hemoglobin E-β-thalassemia (ET) patients, 4 thalassemia intermedia (TI) patients and compared RBC survival to normal control. Repeat measures were performed in 3 ET patients while on hydroxyurea (HU) treatment at 15–19 mg/Kg/day. RBC from 30 ml blood were ex-vivo biotinylated under sterile conditions using EZ-Link Sulfo-NHS-Biotin (Pierce), and re-infused to the patient as described before (Franco et al,1998). At set time points, the number of biotinylated cells was determined in small blood samples by flowcytometry using fluorescently labeled strepavidin. All patients tolerated the procedure well without an indication of re-infusion induced infection or anti-biotin antibody related hemolysis. The presence of biotinylated cells in the circulation was followed until only 0.05-0.08% biotinylated cells could be detected. The data were mathematically fitted to 100–100*[1-(1/T)*t]exp(-kt), where t is the time point, T is the extinction time, and k the exponential rate of RBC removal. The control data showed a linear removal rate (k=0), and a T of 100 days (R=0.95), consistent with a normal red cell life span. In the 9 ET and 4 TI patients, a faster disappearance of biotinylated cells was noted, and the number of surviving (biotinylated) cells in the population followed an exponential pattern, consistent with random removal (k=0.033, R= 0.97). Mean survival was 43±11 and 36±15 days, respectively. The 3 patients who received 10–12 months of HU treatment had a mean 0.8 gr/dL increase in their Hb and 20% increase in HbF. However, no difference was found in the RBC survival curves of these patients (k=0.034, R= 0.97), as compared to the non treated patients. One patient who was treated with HU and Erythropoietin (500u/Kg x3/wk) and had a 1.6 gr/dL increase in hemoglobin and RBC survival analysis showed the biotinylated cells to follow a similar survival curve. While these results cannot exclude a longer survival in selected cells, specifically those containing higher HbF, the overall effect of HU treatment has not proven to prolong the lifespan of labeled thalassemia erythrocytes.These results indicate that biotin label provides RBC survival characteristics in thalassemia patients. The method has advantages over 51Cr, including lack of radioactivity, avoiding labeling elution, low expense and recovery of labeled cells after prolonged time in the circulation. Moreover, the method is a valuable tool to measure effect of various treatments on the survival characteristics of the thalassemia erythrocytes. Preliminary findings indicate that despite the effect of HU on an increase in HbF and a mild to moderate increase in total Hb, the overall thalassemic erythrocyte lifespan has not increased, suggesting other mechanisms for these results including erythroid expansion, improved effective erythropoiesis and cell selection.