Development and stabilization of glucose 6 phosphate dehydrogenase diagnostic kit control material

SUMMARY Glucose 6 phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway, which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain the integrity of erythrocytes and prevent oxidative stress. G6PD deficiency affects more than 400 million people in many regions of the world. It is seen at a rate of 0.5% throughout our country and 8.2% in the Çukurova region. It is not enough to just tell patients that there is a deficiency, because the severity of the symptoms depends on the amount of enzyme activity. For this reason, quantitative methods are important for clinical follow-up, treatment and precautions to be taken. After completing the validation stages of the G6PD activity measurement kit we produced within our university, it started to be used routinely in our laboratory. In our preliminary studies, it was determined that the liquid enzyme control materials we used suffered loss of activity as a result of temperature changes during transfer between centers. This problem was focused on because the control materials used in routine laboratories to check whether correct results are given during the day and to detect analytical errors are important. In our study, we aimed to develop G6PD control material with long stability by using different methods and chemicals. Liquid whole blood, liquid enzyme and lyophilized control materials were studied. As a result of the experiments we conducted at 45°C and +4°C conditions that we planned for liquid whole blood control material, it was concluded that using liquid whole blood as control material would not provide an advantage in long-term storage and transportation conditions between remote centers. Stability follow-ups showed that ACD and CPDA tubes remained stable for 6-8 weeks under +4°C storage conditions. Tubes containing ACD and CPDA can be used in cases where G6PD enzyme activity cannot be studied immediately and samples need to be transported to an external center. In our experiments and interlaboratory studies for liquid enzyme control material, enzyme control materials dissolved in Glycine and GlycineTris buffer remained stable for the targeted periods. Lyophilized control materials are advantageous in routine use in situations requiring long-term stability, such as long-term storage and transfer to remote centers. In our study, lyophilization of hemolysate samples was studied. Stability for 1 week was achieved in control materials that were lyophilized by creating different dilution rates with CPDA solution and dissolved with CPDA at room temperature. With blood samples taken from individuals with deficient and normal G6PD enzyme activity, different levels of control material can be prepared with different dilutions without preparing separate stocks. Some of the experimental sets of lyophilized control materials prepared in our study were stored at -20°C and 4°C. In the stability study data of the thawed materials after being stored for 2 months, it was seen that storage conditions at -20°C would be beneficial. At the same time, it was observed that stability increased by adding lyoprotectants such as albumin and glycerol in both temperature conditions. In our study, a stable control material was developed for the G6PD enzyme activity measurement kit. In addition, our thesis contributed to the literature on the development of control materials for similar enzyme parameters studied in erythrocytes. Our thesis work on the production of G6PD kit and control material produced at our university will be an important step in reducing foreign dependency in domestic and national production and laboratories and will make a great contribution to our country. Keywords: G6PD, diagnostic kit, control material, autoanalyzer