**7.4.** *In vitro* **primaquine-induced hemolysis methods**

3 cc of venous blood anti-coagulated by 2% heparin solution (126 mM NaCl, 14 mM Na2HPO4, 1 mM KH2PO4, 13,2 mM glucose, pH 7.4) was collected from healthy and G6PDdeficient persons. The blood was washed three times with sterile heparin solution at 3000 rpm for 10 min. Erythrocytes were resuspended in PBS after that hematocrit was adjusted to 2%. This is the one of the most important steps for detection of *in vitro* primaquine-nduced hemolysis. Primaquine solution was prepared in 0.1 M Tris buffer (pH 7.4). Primaquine concentration was used between 1 and 4 mM in experiments. Different concentrations of primaquine were added into tubes containing 2% erythrocytes that were prepared before. Tubes were then placed and rotated in a rotator tube for 2 hours at 37°C. The rotation speed was less than 2 rpm. This is another important step for detection of *in vitro* primaquineinduced hemolysis. After 2 hours, the supernatant was collected for the heme concentration, which was then determined spectrophotometrically. Hemoglobin released from erythrocyte induction of primaquine-induced hemolysis and compared with complete lysis (100% hemolysis, control group) obtained by adding 5 mM Tris-HCl (Fig. 1) (Allakhverdiev & Grinberg, 1981).

The principle of this method is based on conversion of Hemoglobin (Hb) to cyanmethemoglobin by the addition of KCN and ferricyanide, whose absorbance is measured spectrophotometrically as cyanmethemoglobin at 540 nm versus a standard solution. Supernatant of hemolyzed red blood was diluted four-fold (v/v) with distilled water. On the other hand, the control group was diluted twenty-fold (v/v) with distilled water. After that, 50 μL KCN (10% w/v) and 50 μL potassium ferricyanide (2% w/v) were directly mixed and the color was measured at 540 nm. The standard curve was constructed using the standard cyanmethemoglobin solutions in different concentrations (Bhaskaram*, et al.*, 2003).

This method demonstrated that the *in vitro* model of primaquine-induced hemolysis can be only maintained by using 2% hematocrit in physiological conditions. Primaquine leads to hemolysis at concentrations between 1 and 4 mM. Other factors that induce primaquine-derived hemolysis are exposure time, incubation temperature, drug

**Figure 1.** *In vitro* Primaquine-Induced hemolysis

concentration and amount of oxygen. Despite the fact that there are several methods in order to diagnose G6PD deficiency, these methods do not determine primaquine sensitivity in patients with G6PD deficiency every time. Therefore, lack of primaquinebased treatment by considering only G6PD deficiency can be very dangerous in terms of health of patients with malaria and the epidemiology of the disease. On the other hand, treatment of primaquine-sensitivive individuals with primaquine can cause death. Hence, in the Centers for Disease Control and Prevention (CDC) report (Hill*, et al.*, 2006), it was highlighted that there was an urgent need to develop new *in vitro* methods for determining hemolysis that indicate primaquine sensitivity before treatment of patients with this drug. By considering primaquine-induced hemolysis in patients with G6PD deficiency, it can be determined whether these patients may be treated with primaquine or not. The advantage of this method is that it can determine primaquineinduced hemolysis before treatment with primaquine and its capacity to determine G6PD deficiency.
