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.
JOURNAL OF MEDICAL RESEARCH PRODUCTION OF LIOPHILIZED QUALITY CONTROL SAMPLES FOR SEVERAL CLINICAL CHEMISTRY TEST Nguyen Quynh Giao1, Dang Quang Huy1, Pham Thi Huong Trang2, Dang Thi Ngoc Dung3, Trinh Thi Phuong Dung1 Faculty of Medical Technology Quality Control Center for Medical Laboratory 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 control material that can be produced using materials from laboratory Plasma samples, anticoagulated by heparin, 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 parameters 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 I INTRODUCTION Statistical quality control (SQC) - an essential part of medical laboratory quality assurance - is a procedure that requires quality control materials Quality control materials are often commercially bought from various manufacturers at relatively high cost by laboratories there is no Vietnamese laboratory able to selfproduce lyophilized plasma samples for Statistical quality control The aim of this research project was to produce lyophilized quality control materials, following the standard guidelines from the World Health Organization II METHODS Self-production of quality control materials using samples collected by laboratories is an- Study setting other method of obtaining quality control mate- The study was conducted at the rial that is commonly used by many laborato- Department of medical laboratory of Hanoi ries around the world This method helps labo- Medical ratories reduce cost and improve the availabil- collection), ity of quality control material At the moment, Embryology (for lyophilization), and National University Hospital Department of (for sample Histology and Geriatric Hospital (for samples measurement) from March to May of 2016 Corresponding author: Nguyen Quynh Giao, Faculty of Medical Technology Email: quynhgiao83107@gmail.com Received: 15/11/2017 Accepted: 12/11/2018 JMR 116 E3 (7) - 2018 Materials and methods Plasma was stored in blood tubes containing Heparin from the Department of Medical JOURNAL OF MEDICAL RESEARCH Laboratories in Hanoi Medical University Hos- show no significant difference, the sample is pital and National Geriatric Hospital still stable In this study, for each selected Selection criteria for subjects were as fol- point of time within the assessment period with lows: common chemistry parameters including the interval of days, one frozen and one ly- creatinine, total cholesterol and AST that are ophilized samples were selected for testing within normal reportable range These samples were then repeatedly tested Exclusion criteria for this study were as follows: samples that test positive for HIV, HBV, and HCV, haemolysed samples, samples with elevated bilirubin levels, cloudy appearance, or insufficient amount of plasma 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 2.1 Homogeneity testing conduct our study Analytes were chosen By the time the sample lot had just been based on several different measurement meth- produced, at least 10 samples (or 10% of the ods A one-point enzymatic kinetic test was total number of samples, whichever was used to measure plasma creatinine, an end greater) be point colorimetric test was used to measure analysed twice The homogeneity assessment total cholesterol and multi-point enzymatic was done either by determining the between- kinetic tests were used to measure AST levels were selected randomly to sample variability using ANOVA-test and t-test with 95% confidence interval and comparing it Statistical analysis to the assessment criteria (ISO 13528), or by Collected data was analyzed using Micro- using statistical comparison tests (Guide 35), soft Office Excel 2013, SPSS 20 The protocol or by using the combination of statistical for evaluation of stability and homogeneity of criteria and objectives (IUPAC) [2 - 4] (Refer to samples was determined according to ISO the Appendix 1) 13528 (2005), IUPAC Harmonized protocol 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 (2006) and ISO guide 35 The acceptable criteria 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] Research ethics from the sample lot and analysed repeatedly The study was carried out in laboratory throughout the assessment period Within the environment, Heparin samples were collected 95% confidence interval, the collected results from the Medical laboratory department of the are statistically compared with the results Hanoi Medical University hospital The study gathered from the homogeneity assessment did not use for interfere with any private infor- step, which are considered to be the initial mation that might violate human rights and concentration of the sample If the results regulations from the ethical committee JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH III RESULTS Quality of lyophilized plasma-based QC sample Figure 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 Homogeneity testing Table Creatinine concentration results from the homogeneity test of lyophilized and frozen samples Lyophilized No Frozen 2 69.45 70.37 68.77 72.66 70.04 70.78 73.23 72.64 71.12 69.3 71.39 71.64 70.59 69.63 68.56 69.53 71.21 70.87 72.93 72.22 71.31 71.16 72.51 69.23 70.18 70.14 71.62 73.06 71.56 71.44 69.79 70.22 70.89 71.17 66.81 69.65 10 71.47 70.89 72.64 70.87 Sample p > 0.05 p > 0.05 p > 0.05 p > 0.05 p > 0.05 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Table shows the creatinine concentration from the homogeneity testing of frozen and lyophilized sample There was no significant difference in the within-sample (2 replicates for 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) Stability test Table Creatinine concentration of lyophilized and frozen QC samples at different points of time Frozen Time period n X SD Day 20 70.68 0.69 Day 10 68.49 0.7 Day 10 70.1 Day 12 10 Day 16 Day 20 Lyophilized n X SD 20 71 1.83 < 0.05 10 71.82 1.66 > 0.05 1.14 > 0.05 10 72.31 1.83 > 0.05 71.15 0.66 > 0.05 10 72.2 1.71 > 0.05 10 70.91 0.96 > 0.05 10 70 1.92 > 0.05 10 70.37 1.12 > 0.05 10 70.22 2.15 > 0.05 P p Graph 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 obtained for AST and total Cholesterol JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Table Cholesterol concentration from homogeneity testing of frozen and lyophilized samples Lyophilized Measure Frozen 2 4.89 4.92 4.9 4.93 4.91 4.96 4.92 4.91 4.89 4.91 4.88 4.89 4.89 4.92 4.86 4.92 4.84 4.93 4.91 4.93 4.88 4.9 4.89 4.89 4.88 4.88 4.82 4.92 4.86 4.9 4.85 4.95 4.82 4.86 4.83 4.89 10 4.76 4.9 4.88 4.96 Sample p > 0.05 p > 0.05 p > 0.05 p>0.05 (t-test) p > 0.05 Table AST concentration from homogeneity testing of frozen and lyophilized samples Lyophilized Measure Freeze 2 20.96 21.82 21.55 20.62 21.2 21.55 20.85 21.73 21.75 21.87 21.45 21.2 20.59 20.35 21.02 21.31 21.56 21.05 21.14 20.88 21.54 21.15 21.05 21.57 20.31 21.52 21.7 20.91 21.7 21.35 20.23 20.92 21.36 20.69 20.77 21.41 10 21.3 19.2 21.84 20.65 Sample p > 0.05 p > 0.05 p > 0.05 p > 0.05 p > 0.05 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Lyophilization performance Table Lyophilization performance Sample Vial (x) Vial + liquid plasma (y) Liquid plasma (y - x) Vial + Lyophilized plasma (z) Lyophilized plasma (z - x) ∆ H2O (y - z) %H2O (y-z)/(y-x) 32.37 37.3 4.93 32.81 0.44 4.49 91.08% 33.52 38.38 4.86 33.95 0.43 4.43 91.15% 32.96 38.04 5.08 33.41 0.45 4.63 91.14% 32.28 37.22 4.94 32.73 0.45 4.49 90.89% 32.91 37.75 4.84 33.34 0.43 4.41 91.12% 31.72 36.7 4.98 32.17 0.45 4.53 90.96% 32.74 37.79 5.05 33.2 0.46 4.59 90.89% 32.9 37.98 5.08 33.36 0.46 4.62 90.94% 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% Table 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 lyophilization Therefore, the lyophilization performance was consistent throughout the lot JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH IV DISCUSSION Procedure for production of plasmabased lyophilized QC material for clinical chemistry test Creatinine concentration changes through out the assessment period compared to the initial value showed that despite the fact that both types of preservation indicated acceptable stability at least 20 days The study used pooled plasma sample that Based on the result, lyophilized samples 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- were proven to have high stability for Creatinine This report is similar to the results obtained by Rixin Jamtsho and his research team [5] flected the lyophilisation performance consis- Total cholesterol results showed similar tency of the samples within one lot; it also results regarding the stability of both lyophi- helped reassure the homogeneity of the initial lized and frozen samples within the period of pooled plasma The criterion for the remaining 20 days, with very low variable across different water within product is no more than 5%.The points of time (variable of - 0.2%) This pa- lyophilisation vials were weighted on an elec- rameter, however, indicated that the lyophi- trical balance (0.0005g accuracy) before and lized samples were more stable after the freeze-dried process The results According to J Maurukas, the concentra- showed that the average evaporated water tion of Cholesterol in lyophilized serum were was 95% in weight Therefore, the samples stable up to weeks under -200C condition within this lot were consistent in lyophilization The finding from this study is also in line with performance The final product was soft, dry, Maurukas’s results [6] and satisfied requirements for analysis postreconstitution [1] This result demonstrated that the procedure taken was appropriate and the plasma sample lot was completely lyophilized and was ready for the homogeneity and stability test The results of changing AST activity in comparison to the initial value demonstrated that although both frozen and lyophilized samples were stable within the period of 20 days The lyophilized showed less variation compared to the initial value According to Homogeneity P.D.Divya and K.K Jayavardhannan, activity In this study, there was no significant differ- AST in goat serum stable up to 11 11 days ence in the Creatinine concentration within- under 40C and 14 days under -200C condition sample (2 repeats for sample) and between- [7] sample (1 run for 10 samples) (p > 0.05) The The research only covered a short period similar results were obtained for Cholesterol of time and had not considered different stor- and AST Therefore, it was concluded that the ing conditions for both freeze and lyophilized lyophilized samples were completely homoge- samples Further research with larger number nous and could be tested for stability of samples and scales are needed to provide Stability JMR 116 E3 (7) - 2018 more accurate decision JOURNAL OF MEDICAL RESEARCH Wood R (2006) The international harmonized V CONCLUSION protocol for the proficiency testing of analytical 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 current data chemistry laboratories (IUPAC Technical Report) Pure and Applied Chemistry, 78(1), 145 - 196 ISO Guide 35 (2006) Reference materials -General and statistical principles for certification Jamtsho R (2013) Stability of Lyophi- REFERENCES lized Human Serum for Use as Quality Control Ngo Thin Duy, Trinh Binh, Pham Duong Tuan, Do Phan Trung (2007) Research on development of fresh plasma lyophilization for clinical treatment using LY3TTE/DM8 lyophilizer Journal of Medical Research, 49(3) Material in Bhutan Indian J Clin Biochem, 28 (4), 418 - 421 Maurukas J (1978) Process for preparing biological compositions for use as reference controls in diagnostic analyses ISO 15328 (2015) Statistical methods Divya PD., Jayavardhanan KK (2010) for use in proficiency testing by interlaboratory Effect of Temperature and storage time on comparison Hepatobiliary enzyme activities in Goat serum Thompson M., Stephen LR Ellison., Veterinary World, 3(6), 277 - 279 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Appendix Research process Selected plasma Plasma pool Store at -200C Defreeze, mix, filter Re-analyze biochemistry parameters Plasma pool Store at -200C Defreeze, mix, filter Plasma pool Divide the plasma pool 20 eppendorf tubes 20 glass vials V = mL V = 0.5 mL Prepare frozen sample, Prepare lyophilized sam- ples, store at -200C store at -20 C Randomly select 10 samples, Randomly select 10 sam- run repeats per sample ples, reconstitute, run repeats per sample Perform homogeneity test Compare Take sample every Take sample every days, run 10 repeats days, run 10 repeats Perform stability test JMR 116 E3 (7) - 2018 Perform homogeneity test Compare Perform stability test ... harmonized V CONCLUSION protocol for the proficiency testing of analytical The procedure for production of lyophilized quality control material for several clinical chemistry tests showed initial success... ophilized samples were selected for testing within normal reportable range These samples were then repeatedly tested Exclusion criteria for this study were as follows: samples that test positive for. .. performance was consistent throughout the lot JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH IV DISCUSSION Procedure for production of plasmabased lyophilized QC material for clinical chemistry