Production of liophilized quality control samples for several clinical chemistry test – Nguyen Quynh Dao

JMR 116 E3 (7) - 2018 1 JOURNAL OF MEDICAL RESEARCH PRODUCTION OF LIOPHILIZED QUALITY CONTROL SAMPLES FOR SEVERAL CLINICAL CHEMISTRY TEST Nguyen Quynh Giao 1 , Dang Quang Huy 1 , Pham Thi Huong Trang 2 , Dang Thi Ngoc Dung 3 , Trinh Thi Phuong Dung 1 1Faculty of Medical Technology 2Quality Control Center for Medical Laboratory 3 Department of Biochemistry An essential part of medical laboratory quality assurance is statistical quality control (SQC) which requires the laboratory to analyse quality control materials. Our research focused on lyophilized quality con- trol material that can be produced using materials from laboratory. Plasma samples, anticoagulated by hepa- rin, that had common clinical chemistry parameters including creatinine, total cholesterol and AST, were collected from the Department of Medical Laboratory at the Hanoi Medical University Hospital. All parame- ters were within the normal reportable range. The samples were lyophilized and tested for homogeneity and stability. Homogeneity and stability testing results for lyophilized and frozen control samples for creatinine, total cholesterol and AST showed no significant difference in 20 days across six assessment time points (p > 0.05). The results also indicated that despite the acceptable stability performance within 20 days. The procedure for production of lyophilized quality control material for several clinical chemistry tests showed initial success. Keywords: Statistical quality control, quality control material, homogeneity, stability Corresponding author: Nguyen Quynh Giao, Faculty of Medical Technology Email: [email protected] Received: 15/11/2017 Accepted: 12/11/2018 I. INTRODUCTION Statistical quality control (SQC) - an essen- tial part of medical laboratory quality assur- ance - is a procedure that requires quality con- trol materials. Quality control materials are often commercially bought from various manu- facturers at relatively high cost by laboratories. Self-production of quality control materials using samples collected by laboratories is an- other method of obtaining quality control mate- rial that is commonly used by many laborato- ries around the world. This method helps labo- ratories reduce cost and improve the availabil- ity of quality control material. At the moment, there is no Vietnamese laboratory able to self- produce lyophilized plasma samples for Statis- tical quality control. The aim of this research project was to produce lyophilized quality con- trol materials, following the standard guide- lines from the World Health Organization. II. METHODS 1. Study setting The study was conducted at the Department of medical laboratory of Hanoi Medical University Hospital (for sample collection), Department of Histology and Embryology (for lyophilization), and National Geriatric Hospital (for samples measurement) from March to May of 2016. 2. Materials and methods Plasma was stored in blood tubes contain- ing Heparin from the Department of Medical 2 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Laboratories in Hanoi Medical University Hos- pital and National Geriatric Hospital. Selection criteria for subjects were as fol- lows: common chemistry parameters including creatinine, total cholesterol and AST that are within normal reportable range. Exclusion criteria for this study were as follows: samples that test positive for HIV, HBV, and HCV, haemolysed samples, sam- ples with elevated bilirubin levels, cloudy ap- pearance, or insufficient amount of plasma. 2.1. Homogeneity testing By the time the sample lot had just been produced, at least 10 samples (or 10% of the total number of samples, whichever was greater) were selected randomly to be analysed twice. The homogeneity assessment was done either by determining the between- sample variability using ANOVA-test and t-test with 95% confidence interval and comparing it to the assessment criteria (ISO 13528), or by using statistical comparison tests (Guide 35), or by using the combination of statistical criteria and objectives (IUPAC) [2 - 4] (Refer to the Appendix 1). 2.2. Stability testing After confirming the homogeneity of the produced samples, the process of stability assessment was done following ISO 13528 guideline. One random sample is selected from the sample lot and analysed repeatedly throughout the assessment period. Within the 95% confidence interval, the collected results are statistically compared with the results gathered from the homogeneity assessment step, which are considered to be the initial concentration of the sample. If the results show no significant difference, the sample is still stable. In this study, for each selected point of time within the assessment period with the interval of 4 days, one frozen and one ly- ophilized samples were selected for testing. These 2 samples were then repeatedly tested 10 times for each parameter. 2.3. Measures and instruments We used the LY3-TTE/DM8 lyophilizer, Panasonic refridgerator and Abbott Architect Ci4100 for automatic chemistry analysis to conduct our study. Analytes were chosen based on several different measurement meth- ods. A one-point enzymatic kinetic test was used to measure plasma creatinine, an end point colorimetric test was used to measure total cholesterol and multi-point enzymatic kinetic tests were used to measure AST levels. 3. Statistical analysis Collected data was analyzed using Micro- soft Office Excel 2013, SPSS 20. The protocol for evaluation of stability and homogeneity of samples was determined according to ISO 13528 (2005), IUPAC Harmonized protocol (2006) and ISO guide 35 . The acceptable cri- teria for homogeneity was p > 0.05 for both ANOVA and t-test. The acceptable criteria for stability was p > 0.05 for t-test [2 - 4]. 4. Research ethics The study was carried out in laboratory environment, Heparin samples were collected from the Medical laboratory department of the Hanoi Medical University hospital. The study did not use for interfere with any private infor- mation that might violate human rights and regulations from the ethical committee. JMR 116 E3 (7) - 2018 3 JOURNAL OF MEDICAL RESEARCH III. RESULTS 1. Quality of lyophilized plasma-based QC sample Figure 1. Serology sample before and after freeze-dried The lyophilized samples was soft and dry before reconstitution. Post-reconstitution samples were light yellow colour and clear, with no precipitate or scum formed. 2. Homogeneity testing Table 1. Creatinine concentration results from the homogeneity test of lyophilized and frozen samples Lyophilized Frozen Sample 1 2 1 2 1 69.45 70.37 68.77 72.66 2 70.04 70.78 73.23 72.64 3 71.12 69.3 71.39 71.64 4 70.59 69.63 68.56 69.53 5 71.21 70.87 p > 0.05 72.93 72.22 p > 0.05 6 71.31 71.16 72.51 69.23 7 70.18 70.14 71.62 73.06 8 71.56 71.44 69.79 70.22 9 70.89 71.17 66.81 69.65 10 71.47 70.89 72.64 70.87 p > 0.05 p > 0.05 p > 0.05 No 4 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Graph 1. Creatinine level of lyophilized and frozen QC samples at different points of time The stability test results gathered from day 4, day 8, day 12, day 16 and day 20 showed no significant difference compared to the initial concentration (p > 0.05). The same results were ob- tained for AST and total Cholesterol. Table 1 shows the creatinine concentration from the homogeneity testing of frozen and lyophi- lized sample. There was no significant difference in the within-sample (2 replicates for 1 sample) and the between-samples (1 replicate for 10 samples) results (p > 0.05). The difference between lyophilized and frozen samples in term of Creatinine, Total Cholesterol and AST concentrations were also insignificant (p > 0.05). 3. Stability test Table 2. Creatinine concentration of lyophilized and frozen QC samples at 6 different points of time Time period Frozen Lyophilized n SD P n SD p Day 0 20 70.68 0.69 20 71 1.83 Day 4 10 68.49 0.7 0.05 Day 8 10 70.1 1.14 > 0.05 10 72.31 1.83 > 0.05 Day 12 10 71.15 0.66 > 0.05 10 72.2 1.71 > 0.05 Day 16 10 70.91 0.96 > 0.05 10 70 1.92 > 0.05 Day 20 10 70.37 1.12 > 0.05 10 70.22 2.15 > 0.05 X X JMR 116 E3 (7) - 2018 5 JOURNAL OF MEDICAL RESEARCH Table 3. Cholesterol concentration from homogeneity testing of frozen and lyophilized samples Lyophilized Frozen Sample 1 2 1 2 1 4.89 4.92 4.9 4.93 2 4.91 4.96 4.92 4.91 3 4.89 4.91 4.88 4.89 4 4.89 4.92 4.86 4.92 5 4.84 4.93 p > 0.05 4.91 4.93 p > 0.05 6 4.88 4.9 4.89 4.89 7 4.88 4.88 4.82 4.92 8 4.86 4.9 4.85 4.95 9 4.82 4.86 4.83 4.89 10 4.76 4.9 4.88 4.96 p > 0.05 p>0.05 (t-test) p > 0.05 Measure Table 4. AST concentration from homogeneity testing of frozen and lyophilized samples Lyophilized Freeze Sample Measure 1 2 1 2 1 20.96 21.82 21.55 20.62 2 21.2 21.55 20.85 21.73 3 21.75 21.87 21.45 21.2 4 20.59 20.35 21.02 21.31 5 21.56 21.05 p > 0.05 21.14 20.88 p > 0.05 6 21.54 21.15 21.05 21.57 7 20.31 21.52 21.7 20.91 8 21.7 21.35 20.23 20.92 9 21.36 20.69 20.77 21.41 10 21.3 19.2 21.84 20.65 p > 0.05 p > 0.05 p > 0.05 6 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH 4. Lyophilization performance Table 5 describes the lyophilized performance. The average evaporation was 95% of the total weight, which satisfied the requirement of maximum 5% water retained in the sample after lyophi- lization. Therefore, the lyophilization performance was consistent throughout the lot. Table 5. Lyophilization performance Sample Vial (x) Vial + liquid plasma (y) Liquid plasma (y - x) Vial + Ly- ophilized plasma (z) Lyophilized plasma (z - x) ∆ H2O (y - z) %H2O (y-z)/(y-x) 1 32.37 37.3 4.93 32.81 0.44 4.49 91.08% 2 33.52 38.38 4.86 33.95 0.43 4.43 91.15% 3 32.96 38.04 5.08 33.41 0.45 4.63 91.14% 4 32.28 37.22 4.94 32.73 0.45 4.49 90.89% 5 32.91 37.75 4.84 33.34 0.43 4.41 91.12% 6 31.72 36.7 4.98 32.17 0.45 4.53 90.96% 7 32.74 37.79 5.05 33.2 0.46 4.59 90.89% 8 32.9 37.98 5.08 33.36 0.46 4.62 90.94% 9 21.63 26.65 5.02 22.09 0.46 4.56 90.84% 10 21.7 26.69 4.99 22.14 0.44 4.55 91.18% 11 15.22 19.98 4.76 15.64 0.42 4.34 91.18% 12 14.8 19.83 5.03 15.26 0.46 4.57 90.85% 13 21.73 26.75 5.02 22.18 0.45 4.57 91.04% 14 32.38 37.42 5.04 32.84 0.46 4.58 90.87% 15 32.45 37.47 5.02 32.9 0.45 4.57 91.04% 16 32.54 37.39 4.85 32.97 0.43 4.42 91.13% 17 33.23 38.22 4.99 33.68 0.45 4.54 90.98% 18 31.52 36.51 4.99 31.97 0.45 4.54 90.98% 19 32.1 37.03 4.93 32.54 0.44 4.49 91.08% 20 32.63 37.66 5.03 33.08 0.45 4.58 91.05% JMR 116 E3 (7) - 2018 7 JOURNAL OF MEDICAL RESEARCH IV. DISCUSSION Procedure for production of plasma- based lyophilized QC material for clinical chemistry test The study used pooled plasma sample that was clear, with no precipitate and had light yellow colour, which satisfied the requirement for lyophilisation. It was also important to monitor the performance of the lyophilisation process. The ratio of evaporated water re- flected the lyophilisation performance consis- tency of the samples within one lot; it also helped reassure the homogeneity of the initial pooled plasma. The criterion for the remaining water within product is no more than 5%.The lyophilisation vials were weighted on an elec- trical balance (0.0005g accuracy) before and after the freeze-dried process. The results showed that the average evaporated water was 95% in weight. Therefore, the samples within this lot were consistent in lyophilization performance. The final product was soft, dry, and satisfied requirements for analysis post- reconstitution [1]. This result demonstrated that the procedure taken was appropriate and the plasma sample lot was completely lyophi- lized and was ready for the homogeneity and stability test. Homogeneity In this study, there was no significant differ- ence in the Creatinine concentration within- sample (2 repeats for 1 sample) and between- sample (1 run for 10 samples) (p > 0.05). The similar results were obtained for Cholesterol and AST. Therefore, it was concluded that the lyophilized samples were completely homoge- nous and could be tested for stability. Stability Creatinine concentration changes through out the assessment period compared to the initial value showed that despite the fact that both types of preservation indicated accept- able stability at least 20 days. Based on the result, lyophilized samples were proven to have high stability for Creatinine. This report is similar to the results obtained by Rixin Jamtsho and his research team [5]. Total cholesterol results showed similar results regarding the stability of both lyophi- lized and frozen samples within the period of 20 days, with very low variable across different points of time (variable of 0 - 0.2%). This pa- rameter, however, indicated that the lyophi- lized samples were more stable. According to J. Maurukas, the concentra- tion of Cholesterol in lyophilized serum were stable up to 5 weeks under -20 0 C condition. The finding from this study is also in line with Maurukas’s results [6]. The results of changing AST activity in comparison to the initial value demonstrated that although both frozen and lyophilized sam- ples were stable within the period of 20 days. The lyophilized showed less variation com- pared to the initial value . According to P.D.Divya and K.K. Jayavardhannan, activity AST in goat serum stable up to 11 11 days under 4 0 C and 14 days under -20 0 C condition [7]. The research only covered a short period of time and had not considered different stor- ing conditions for both freeze and lyophilized samples. Further research with larger number of samples and scales are needed to provide more accurate decision. 8 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH V. CONCLUSION The procedure for production of lyophilized quality control material for several clinical chemistry tests showed initial success. Further research on longer period for stability test is needed to improve upon and confirm the cur- rent data. REFERENCES 1. Ngo Thin Duy, Trinh Binh, Pham Duong Tuan, Do Phan Trung (2007). Research on development of fresh plasma lyophilization for clinical treatment using LY3- TTE/DM8 lyophilizer. Journal of Medical Research, 49(3). 2. ISO 15328 (2015). Statistical methods for use in proficiency testing by interlaboratory comparison. 3. Thompson M., Stephen LR Ellison., Wood R (2006). The international harmonized protocol for the proficiency testing of analytical chemistry laboratories (IUPAC Technical Report). Pure and Applied Chemistry, 78(1), 145 - 196. 4. ISO Guide 35 (2006). Reference materi- als -General and statistical principles for certi- fication. 5. Jamtsho R (2013). Stability of Lyophi- lized Human Serum for Use as Quality Control Material in Bhutan. Indian J Clin Biochem, 28 (4), 418 - 421. 6. Maurukas J (1978). Process for prepar- ing biological compositions for use as refer- ence controls in diagnostic analyses 7. Divya PD., Jayavardhanan KK (2010). Effect of Temperature and storage time on Hepatobiliary enzyme activities in Goat serum. Veterinary World, 3(6), 277 - 279. JMR 116 E3 (7) - 2018 9 JOURNAL OF MEDICAL RESEARCH Plasma pool 1 Store at -20 0 C Defreeze, mix, filter 20 eppendorf tubes V = 0.5 mL Prepare frozen sample, store at -20 0 C Randomly select 10 samples, run 2 repeats per sample Perform homogeneity test Take 1 sample every 4 days, run 10 repeats Perform stability test Compare Compare Selected plasma Plasma pool 2 Store at -20 0 C Re-analyze biochemis- try parameters Defreeze, mix, filter Plasma pool 3 Divide the plasma pool 20 glass vials V = 5 mL Prepare lyophilized sam- ples, store at -20 0 C Randomly select 10 sam- ples, reconstitute, run 2 repeats per sample Perform homogeneity test Take 1 sample every 4 days, run 10 repeats Perform stability test Appendix 1. Research process