Preparation of the first national reference standard for determination of HEK293 cell DNA content / 中国生物制品学杂志

@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Preparation of the first national reference standard for determination of HEK293 cell DNA content / 中国生物制品学杂志

@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Preparation of national reference panel of HBV for nucleic acid testing donor screening / 中国生物制品学杂志

@#Objective To prepare the national reference panel of hepatitis B virus(HBV) for nucleic acid testing(NAT)donor screening.Methods A number of plasma samples from donors positive for HBV antibody and patients with HBV infection collected from blood centers,plasma stations and biological products companies in Shanghai,Gansu,Henan,Hunan,Hubei and other regions were tested for HBV DNA viral load agent,and negative and positive reference candidates were screened;The HBV DNA national standard was diluted to 10~3 IU/ml with human negative plasma,as a candidate for limit of detection(LOD).National negative and positive reference candidates of HBV for NAT donor screening and LOD reference to be calibrated were distributed to 8 enterprise laboratories for joint detection of HBVHCVHIV NAT donor screening.The homogeneity and stability of the national reference panel were investigated.Results A total of 8 negative samples with HBV viral load of 0 were screened as negative references and 9 positive samples with viral load of 10~3～10~4 IU/mL were used as positive references;One LOD reference was calibrated with WHO HBV DNA standard,and the virus content was 1.0 × 10~3 IU/ml.The national reference panel showed good stability and the homogeneity inspection met the requirements.Conclusion The national reference panel of HBV DNA for NAT donor screening was prepared,which provided a basis for the quality control and standardization of HBV DNA reagents for donor screening.

Preparation of Chinese national potency reference for hepatitis E vaccine / 中国生物制品学杂志

@#ObjectiveTo prepare the hepatitis E vaccine national potency reference for the quality control of hepatitis E vaccine.MethodsA batch of hepatitis E vaccine was selected as the row material,and the screened lyoprotectant was added. After aliquot and freeze-drying,it was prepared as candidate reference material,and the homogeneity and stability were investigated,which was distributed to four laboratories for collaborative calibration and applicability study.Results The 5% trehalose + 2% dextran was selected as the lyoprotectant to prepare the candidate reference material. The aliquot accuracy of candidate reference material was 0. 7%;The coefficient of variation(CV)of antigen content homogeneity was9. 0%;The CV of aluminum content homogeneity was 4. 0%. Moisture content was 1. 7%. The candidate reference material showed good acceleration stability and reconstituted stability. A total of 18 valid data were obtained from the collaborative calibration. The results showed that the average value of median effective dose(ED50)was 0. 15 μg(95% confidence interval of 0. 12 ～ 0. 18 μg)with the intra-laboratory CV of 30. 8% ～ 64. 2%,and the inter-laboratory CV of 32. 6%. Two hepatitis E vaccines produced by two laboratories themselves and the candidate reference material showed good dose-response relationship,of which the seroconversion rate decreased with the decrease of vaccine antigen content and the curve change trend was similar.ConclusionThe first Chinese national potency reference for hepatitis E vaccine(300031-201801)was prepared,which can be used for the quality control of potency ED50detection of hepatitis E vaccine.

Institutionalization of the Philippine Health Laboratory System (PHLS)

@#In 1966, Section 3 of Republic Act (R.A.) 4688, entitled “An Act Regulating the Operation and Maintenance of Clinical Laboratories and Requiring the Registration of the Same with the Department of Health, Providing Penalty for the Violation Thereof, and for Other Purposes,” provided for the establishment of the Bureau of Research and Laboratories (BRL) under the Department of Health (DOH). The BRL served as the central laboratory that governed the operation of regional public health laboratories. The BRL’s function was delegated to different offices in 2000 by Executive Order 102 s. 1999 entitled “Redirecting the Functions and Operations of the Department of Health,” which was premised in part with Section 78 of the General Provisions of R.A. 8522 (“General Appropriations Act of 1998”) authorizing the President to direct changes in organization and key positions of any department, bureau, or agency. Towards this direction, the function of the BRL was distributed to different agencies, and the Philippines designated six (6) national reference laboratories that catered to communicable and non-communicable diseases. Five (5) subnational reference laboratories for emerging and re-emerging infectious diseases were established in response to the 2009 Influenza AH1N1 pandemic. The DOH issued Administrative Order No 2012-0021 to establish a national and regional network of laboratories.

Establishment of National Reference Standards of Sulfadimidine Impurities / 中国药学杂志

OBJECTIVE: To establish the national reference standards of sulfadimidine impurities thus to provide guarantee for improving the standard of quality control of sulfadiazine in China. METHODS: First, the structures of sulfadimidine impurities A and E were validated by infrared spectrocopy, mass spectrum and nuclear magnetic resonance method. Then, the purities of impurities A and E were determined using the method of related substance test for sulphadiazine in the European Pharmacopoeia (version 9.0),their water content and residue on ignition were determined as well. The contents of sulfadimidine impurities A and E were determined by using mass balance method. Meanwhile, external standard method and nuclear magnetic quantitative method were used to calculate the content, which were mutually verified with the mass balance method. Finally, the correction factors of sulfadiazine impurities A and E at 241 nm were determined using standard curve method. RESULTS: The structures of sulfadimidine impurities A and E were confirmed, and the contents of impurities A and E were 99.1% and 98.7%, respectively, which were calculated by mass balance method. The results were consistent with those obtained from external standard method and nuclear magnetic quantification method. The correction factors of impurities A and E to sulfadimidine were 0.97 and 0.63, respectively. CONCLUSION: The first batch of national standard substances of sulfadimidine impurities A and E were established successfully.

Establishment of national reference standard of fluconazole impurity h / 中国药学杂志

OBJECTIVE: To introduce the procedure of developing reference standards of fluconazole impurities using fluconazole impurity H as an example and reveal a special problem for establishment of national reference standards. METHODS: Firstly, the structure of fluconazole impurity H was validated by infrared, mass spectrum and nuclear magnetic resonance method. Secondly, its purity was determined using the related substances test of fluconazole in European Pharmacopoeia 8.0 (EP8.0) and Chinese Pharmacopeia (2010 version, Volume 2, ChP2010). Then the major impurities determined by the above two HPLC systems were analyzed by LC-MS method. Finally, the content of fluconazole impurity H, its water content and inorganic impurities were determined by quantitative nuclear magnetic resonance (qNMR) method, Karl Fischer titrimetry method and residue on ignition method respectively. RESULTS: The structure of fluconazole impurity H was identical with that in EP 8.0. The contents of water and inorganic impurities were 0.05% and 0.04%, respectively. It was found that fluconazole impurity H would be partially degraded into fluconazole impurity G in water solution, which resulted in the inaccuracy of the related substances test. The content of impurity H was 99.5% by qNMR method. CONCLUSION: Due to the structural characteristics of fluconazole impurity H, the mass balance method, which is the routine method for determination of the content of reference standards, is not suitable for fluconazole impurity H. In the circumstances, qNMR method can be used as a complementary method for the content determination of reference standards.

Long-Term Stability of HBV, HCV, and HIV-1 National Reference Standards for in vitro Diagnostic Medical Devices Intended to Be Used for the Nucleic Acid Amplification Test / 대한수혈학회지

BACKGROUND: National reference standards are essential to the quality assessment and regulatory approval of in vitro diagnostic medical devices. However, the long-term stability of national reference standards has not been comprehensively secured. This study was performed to assessment on the long-term stability of the hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus-1 (HIV-1) national reference standards intended to be used for the nucleic acid amplification test (NAT). METHODS: The viral loads of the MFDS (Korea Ministry of Food and Drug Safety) working standard and recombinant DNA for HBV, HCV, and HIV-1 were measured before and after storage at −70℃ for up to 72 months using Cobas Ampliprep/Cobas Taqman assays (Roche Molecular System, Inc., Branchburg, USA) at defined time points. RESULTS: The viral loads of national reference standards for in vitro diagnostic medical devices of HBV, HCV, and HIV-1 stored at −70℃ for up to 72 months did not differ significantly from the baseline viral load. The changes in viral load of national reference standards of HBV, HCV, and HIV-1 tested after storage at −70℃ for up to 72 months ranged from −0.36 to 0.16 log10 IU/mL and did not exceed 0.5 log10, which is the estimated intra-assay variation of molecular tests. CONCLUSION: The HBV, HCV, and HIV-1 national reference standards for in vitro diagnostic medical devices intended to be used for the NAT were relatively stable after long-term storage at −70℃ for up to 72 months, regardless of the initial titer.

Establishment of national reference panel for methamphetamine Kits / 中国药学杂志

OBJECTIVE: To establish the reference panel of methamphetamine (MET) Kits. METHODS: After determining the cut-off value of MET kits, selecting the raw material, and determining the component of reference material, MET kits reference material was prepared. The reference material was characterized by verified LC-MS method, and also tested for the stability. RESULTS: The national reference material of MET kits consists of one positive sample, 12 negative samples, three LOD samples, and one repeatability sample. This reference material passed the co-characterization of five companies. CONCLUSION: The developed national reference material of MET kits meets the corresponding requirement. The establishment of the national reference material of MET kits may help regulate the development and production process of the drug kits.

Establishment and Multicenter Evaluation of a National Reference Panel for Syphilis Antibodies in Korea

BACKGROUND: Establishment of a national reference panel for syphilis antibodies is necessary to evaluate the performance of in-vitro diagnostic tests for syphilis and to verify test quality. This study aimed to establish a national reference panel for syphilis antibodies, to assess the suitability of a panel for non-treponemal and treponemal testing, and to assess the reactivity of the various tests currently in use. METHODS: Treponemal pallidum particle agglutination (TPPA)-positive and -negative fresh frozen plasma samples were obtained. After the fresh frozen plasma was converted to serum by defibrination, the samples were pooled. Two candidate reference standards containing no syphilis antibodies and 10 candidate reference standards containing syphilis antibodies were prepared on the basis of reactivity in the TPPA assay. Candidate reference standards were tested by three laboratories using five non-treponemal tests and four treponemal tests. RESULTS: All three laboratories reported positive non-treponemal test results for the mixed-titer performance panel (MP)/6-MP/12. MP/1, MP/2, and MP/3 were negative for non-treponemal tests. MP/4 and MP/5 were reported either as positive or negative according to the laboratories. All laboratories reported positive TPPA results for MP/3-MP/12 and negative results for MP/1 and MP/2. No significant difference was detected among the treponemal testing results in three laboratories. CONCLUSIONS: We established 12 candidate national reference standards containing various concentrations of syphilis antibodies. A collaborative study using nine tests demonstrated that 12 candidate national reference standards presented consistent results, except a few assays with low sensitivity, and thus could be used as a national reference panel for syphilis antibody testing.

Caracterización de las principales variables clínicas del glaucoma en pacientes con retinosis pigmentaria / Main clinical variables in characterization of patients with retinitis pigmentosa and glaucoma

Se realizó un estudio epidemiológico, descriptivo y transversal de 85 pacientes con alguna de las formas clínicas de glaucoma, dispensarizados en el Centro Nacional de Referencia de Retinosis Pigmentaria durante el primer semestre del 2010. Entre las variables analizadas de acuerdo con la clasificación de la Escuela Cubana de Retinosis Pigmentaria, figuraron: agudeza visual, alteraciones campimétricas, herencia de retinosis y consanguinidad, hipertensión ocular y espesor corneal, según los tipos de dichas afecciones. En la serie, 32,9 % tenía visión entre 0,1 y 0,3; 47,05 %, reducción concéntrica del campo visual (5 y 10 °) y patrón autosómico recesivo; 17,64 %, antecedentes de consanguinidad y 38,8 %, comienzo juvenil. Se halló hipertensión ocular en 82,35 %, con predominio en la retinosis típica de grado IV (40,0 %), así como disminución del espesor corneal en 34,1% de la casuística. La letalidad visual por la asociación de ambas oftalmopatías exige que sea investigada detenidamente. Hasta el momento, la medición adecuada de la presión intraocular (aplanación con ajuste a los valores del espesor corneal) constituye la herramienta más útil para diagnosticar la enfermedad y evaluar su evolución en estos pacientes.

An epidemiological, descriptive and cross-sectional study was carried out in 85 patients with some clinical forms of glaucoma, attended and monitored at the National Reference Center of Retinitis Pigmentosa during the first semester of 2010. Among the analyzed variables according to the classification of the Cuban School of Retinitis Pigmentosa were visual acuity, visual field defects, retinitis inheritance and consanguinity, ocular hypertension and corneal thickness according to the types of these conditions. In the series 32,9 % had vision between 0,1 and 0,3; concentric reduction of vision field (5 and 10 °) and autosomal recessive pattern in 47,05 %; history of consanguinity in 17,4 % and juvenile onset in 38.8%. Ocular hypertension was found in 82,35 % with predominance in grade IV typical retinitis (40,0 %), as well as reduction of corneal thickness in 34,1 % of all cases. The visual letality caused by association of both eye diseases requires to be studied carefully. So far, proper measurement of intraocular pressure (applanation adjusted to the values of corneal thickness) is the most useful tool to diagnose disease and to evaluate its progress in these patients.

Study on preparation and standardization of reference standard of chondroitin substance sodium / 中国药学杂志

OBJECTIVE: to establish a national reference substance for chondroitin sulfate sodium. METHODS: Identification of chondroitin sulfate sodium was carried out by infrared(IR) and high performance liquid chromatography(HPLC). A high performance size exclusion chromatography in conjunction with refractive index detector and multi-angle laser light scatter(MALLS) was used to determine the molecular weight and molecular weight distribution of chondroitin sulfate sodium. The content of chondroitin sulfate sodium was calculated by mass balance method. RESULTS: The content of chondroitin sulfate sodium reference substance was 99.1%. CONCLUSION: The establishment of national reference substance of chondroitin sulfate sodium can effectively control the quality of the product. Copyright 2012 by the Chinese Pharmaceutical Association.

A Proposal for Developing a National Quality Assurance Program for Donor Bloodassays / 대한수혈학회지

BACKGROUND: Viral screening assays performed for blood donors are required to have high sensitivity because false negative results can lead to transfusion-transmitted infections. To minimize the number of false negative cases, a systematic quality assurance program is required to verify donor screening tests. METHODS: The current status of quality assurance (QA) for blood donor screening tests in Korea and other countries was reviewed. A quality assurance program using the national standards of the Korea Food and Drug Administration (KFDA) was done as a pilot study to evaluate both the need for such a program and the feasibility of such a program. RESULTS: Singapore had a national quality assurance programs for the anti-HIVdonor screening tests. In the United Kingdom, all laboratories use the NIBSC working standards as QA materials for the donors screening. Ninety-five % (84/80) of blood centers replied that they would participate in a national quality assessment program and 92% (84/77) of the blood centers also felt that an independent organization should be designated to operate the program. Quality control materials with a weak reactivity should be included in a quality assessment program for donor screening. CONCLUSION: We propose 2 models for a National Quality Assurance Program (NQAP). In the first model, an independent national reference laboratory (NRL) needs to be established that operates the national quality assurance program. The second model involves the integration of the national quality assurance program for donor screening into the External Quality Assurance Survey run by the Korean Association of Clinical Assurance for Clinical Laboratory (KAQACL) using the national standards.

Establishment of the first national reference pertussis vaccine

Quality of RP5 pertussis vaccines for National Reference was tested and its potency was also calibrated in comparison with the International Standard of Pertussis Vaccine. The results showed that the candidate for National reference RP5 pertussis vaccine lot was met to the quality requirements after being freeze dried. The homogenicity in dry weight, residual moisture, potency and stability of its potency make it became as the first National Reference standard pertussis.