RESEARCH ON THE PRODUCTION OF HBsAg STANDARD SERUM FOR QUALITY CONTROL Prof. Dao Quang Minh PD.MD. Nguyen Minh Hiên MD. Nguyen Quang Hieu

PRESENT CONTENTS - Reason for study selection Objectives Some basic issues related to Study - Materials and methods - Results and discussion - Conclude

REASON FOR STUDY SELECTION

Vietnam is high HBV located Quality Assurance Standard Sample Vietnam is high HBV located HBsAg quantitative tests RESEARCH ON THE PRODUCTION OF HBsAg STANDARD SERUM FOR QUALITY CONTROL

OBJECTIVES 1. Product process of HBsAg Standard Serum 2. Evaluate the quality of HBsAg Standard Serum

SOME BASIC ISSUES RELATED TO STUDY

HEPATITIS B VIRUS Antigen Structure: Dane particles HBsAg HBcAg HBeAg HBsAg: Evidence of HBV infection

HBsAg DETECTION TECHNIQUES Direct: Finding Dane particle Indirec: + Elisa (Enzyme linked immunosorbent assay) + IEP (Immunoelectrophoresis) + Fluorescent Immunity (most popular method)

Internal Quality Control External quality control QUALITY ASSURANCE Internal Quality Control External quality control Standard Samples

STANDARD SAMPLES Certified Reference Materials (CRM) Standard Samples are produced in different forms for different purposes Requirement of Standard Samples: Ensure homogeneity Ensure stability over time

MANUFACTURING HBsAg STANDARD SERUM (HSS) CDC (USA): Producing and supplying the dry plasma for many countries. Standard laboratory (Australia, England, France, Korea, Thailand) produced HBsAg Standard Serum from serum or plasma. According to ISO/IEC 17043:2011 and TCVN 7366, TCVN 8245

MANUFACTURING HBsAg STANDARD In Vietnam: Mainly importing standard samples from Roche, Beckmancounter, Randox ... High cost Not be active because of dependence on imports (including the licences)

MATERIAL AND METHOD

MATERIAL OF STUDY * Plasma samples that HBsAg were positive at National Institute hematology and blood transfusion, Viet Duc Hospital The Selections: HBsAg is positive, The samples are not hemolytic, Precipitation. * The other name Manufacturer Made in HBsAg II Roche USA HBsAg QN Proclin Supelco Formalin Qingdao China CaCl2 anhydrous (NH4)2SO4 anhydrous

Time: From 3/2016 to 11/2016 Study Location: Quality control center for medical laboratory in HMU Biochemistry of Department in Thanh Nhan Hospital Method Production testing

BUILDING HBsAg STANDARD SAMPLES Samples have two levels: Level 1: [HBsAg]: During 7,500 – 11,500 IU/mL Level 2: [HBsAg] During 35 – 60 IU/mL Sample has been preserved two ways at-300C at 00C Time for preservation: 150 days

RESULT AND DISCUSSION

RESULTS OF COLLECTION Collections Volume [ HBsAg ] COI Sample 1 100 ml 3857 Sample 2 1631 Sample 3 2525 Sample 4 1143 Sample 5 1674 Total sample 500ml 4850

RESULT OF CHANGED FROM PLASMA TO SERUM * H.T.T.Hà (2014): 0,01M * Jin-Sook Wang (2012): 0,01M * WHO/UNAIDS (1996) Number Calcium concentration (M) Volume Before (ml) [HBsAG] (COI) after 1 0,1 10 4850 2,5 5072 2 0,05 5,0 5019 3 0,01 7,5 5025 Remain   470 352 5021

[ HBsAg]: CONCENTRATE AND DILUTION   Original Concentrate Dilution 200 times Result (IU/mL) 2101 11588 56,92

EVALUATION OF HOMOGENEITY HSS Samples Result (IU/mL) Level 1 Level 2 time 1th time 2th Sample 1 11337 11134 56,58 55,17 Sample 2 11521 11057 56,99 55,40 Sample 3 11255 11726 56,29 55,37 Sample 4 11163 11359 56,06 56,66 Sample 5 11227 11686 55,62 57,22 Sample 6 11652 11570 56,27 55,77 Sample 7 11498 11603 55,88 55,95 Sample 8 11320 11606 56,54 55,81 Sample 9 11117 11111 55,90 Sample 10 11187 11157 56,28 56,64 Mean 11327,7 11400,9 56,31 55,99 SE 55,6 83,8 0,124 0,207 SD 175,9 265,3 0,39 0,66 CV(%) 1,55 2,32 0,69 1,17 P (Ttest) 0,45 0,32 Roche Diagnotics: 5,6% Abbot Diagnotics: 6,7%

EVALUATION OF STABILITY OF LEVEL 1 HSS Time date Result (IU/mL) 0oC -30oC time 1th time 2th 1 29/6/2016 11078 11107 11274 11250 2 11/7/2016 11100 11190 11390 11500 3 20/7/2016 11140 11194 11494 11411 4 29/7/2016 11184 11128 11088 5 9/8/2016 11685 11439 11334 6 18/8/2016 11697 11653 11553 11519 7 28/8/2016 11299 11214 11114 11414 8 7/9/2016 11075 11023 9 17/9/2016 11186 11109 11409 11352 10 27/9/2016 11156 11197 11154 11 7/10/2016 11044 11138 12 17/10/2016 11592 11410 11289 11248 13 27/10/2016 11499 11562 11262 11322 14 6/11/2016 11364 11393 11493 11613 15 16/11/2016 11498 11464 11423 mean 11306,5 11281,4 11308,5 11329,1 SD 231,7 189,4 168,9 154,4 SE 59,8 48,9 43,6 39,9 CV(%) 2,05% 1,68% 1,49 1,36 P (Ttest) 0,23 0,37 Roche Diagnotics: 5,6% Abbot Diagnotics: 6,7%

EVALUATION OF STABILITY OF LEVEL 1 HSS AT -300 C VÀ 00C

EVALUATION OF STABILITY OF LEVEL 2 HSS Time date Result (IU/mL) 0oC -30oC time 1th time 2th 1 29/6/2016 55,08 55,63 53,51 54 2 11/7/2016 55,78 56,01 56,15 55,76 3 20/7/2016 55,31 54,93 4 29/7/2016 55,82 55,90 55,37 55,10 5 9/8/2016 54,99 55,29 55,49 55,07 6 18/8/2016 55,91 56,12 55,62 54,97 7 28/8/2016 53,96 54,78 55,71 8 7/9/2016 54,11 54,66 53,92 54,28 9 17/9/2016 55,67 55,73 56,63 55,99 10 27/9/2016 55,40 56,00 54,47 55,23 11 7/10/2016 55,09 54,95 56,5 56,19 12 17/10/2016 55,14 55,26 55 13 27/10/2016 55,83 55,11 14 6/11/2016 55,22 54,89 55,35 55,92 15 16/11/2016 54,61 55,00 55,96 55,13 Mean 55,44 55,32 SD 0,57 0,51 0,52 0,86 SE 0,148 0,132 0,133 0,22 CV(%) 1,03 1,04 0,93 1,56 p (T test) 0,34 0,11 BeckmanCounter: 8,3% Abbot Diagnotics: 7,8%

EVALUATION OF STABILITY OF LEVEL 2 HSS AT -300 C VÀ 00C

RESULT OF THE CULTURE Date At 370C At 220C tube 1 tube 2 tube 3 tube 4 (-) 7th 14th

CONCLUSION - HBsAg standard serum test has been developed . - Homogeneity and stability in 5 months at 0oC and -30oC According to the TCVN 8245, ISO 13528 Standard.