The clinical value of fluorescence quantitative assay and G6PD/6PGD ratio method in the diagnosis of glucose-6-phosphate dehydrogenase deficiency and gene mutation / 中华检验医学杂志

Objective:To investigate the diagnostic value of fluorescence quantitative method and G6PD/6PGD ratio method in glucose-6-phosphate dehydrogenase (G6PD) deficiency and the type of gene mutation.Methods:A total of 1 201 patients (711 males and 490 females) with suspected G6PD deficiency in Shanghai Children′s Hospital were collected from June 2018 to March 2021. Fluorescence quantification method, G6PD/6PGD ratio method and multicolor melting curve were used to detects enzyme activity, ratio and gene mutation type. Comparison of each index and evaluation of its diagnostic efficiency were performed.Results:Among 1 201 suspicious samples, 163 cases (135 males and 28 females) were finally diagnosed. 156 cases were diagnosed by fluorescence quantitative method with a detection rate of 95.71%, and 140 cases were diagnosed by G6PD/6PGD ratio method with a detection rate of 85.89%. enzymatic activity of G6PD and ratio of G6PD/6PGD in male were significantly lower than female, and the differences were statistically significant ( U=642.5, 734.5, P<0.001). 112 cases received G6PD gene mutation detection and 92 cases were diagnosed, 74 were hemizygous mutations, 1 were homozygous mutations, 15 were heterozygous mutations, and 2 were compound heterozygous mutations. Among 15 cases of heterozygous mutations, 11 cases were diagnosed by fluorescence quantitative method, the diagnosed rate was 73.33%, 4 cases were diagnosed by G6PD/6PGD ratio method, and the diagnosed rate was 26.67%. A total of 7 mutation sites were detected and the proportions were c.1388G>A (32.22%), c.1376G>T (30.00%), c.871G>A (13.33%), c.1024C>T (11.11%). c.95A>G (7.78%), c.487G>A (4.44%), c.392G>T (1.11%). The enzymatic activities of c.1376G>T and c.1024C>T, c.487G>A were statistically significant ( P<0.001,0.015); the G6PD/6PGD ratios of c.1024C>T and c.1388G>A, c.1376G>T were statistically significant ( P=0.017,0.002,0.011,0.013). Fluorescence quantitative method had sensitivity of 100%, specificity of 95.65%, and the area under the curve (AUC) is 0.972. The sensitivity of the G6PD/6PGD ratio method was 100%, the specificity was 94.57%, and the AUC was 0.979. The sensitivity of fluorescence quantitative method combined with G6PD/6PGD ratio was 96.7%, the specificity was 100%, and the AUC was 0.992. Conclusions:Compared with fluorescence quantification, the G6PD/6PGD ratio method might not be able to diagnose female heterozygotes effectively; The panel of G6PD fluorescence quantification and G6PD/6PGD ratio was helpful to reduce the missed diagnosis. Combined with gene mutation analysis, it could improve the diagnosis rate of G6PD deficiency in the children.

Relación entre género y niveles de proteína C reactiva / Relation among gender and levels of C reactive protein

RESUMEN Fundamento: La proteína C reactiva es uno de los mejores marcadores para la valoración y seguimiento de enfermedades inflamatorias; los valores de referencias recomendados para su concentración en suero no están ajustados según género. Objetivo: Determinar si la concentración de proteína C reactiva difiere según el género. Metodología: Se realizó un estudio exploratorio en 3199 muestras de pacientes procedentes del Hospital General Provincial Camilo Cienfuegos y en un grupo control de 76 muestras de sueros de donantes de sangre para cada género, procedentes del Banco de Sangre de Sancti Spíritus. Los niveles se midieron a través un ensayo semicuantitativo de aglutinación con partículas de látex para la muestra supuestamente enferma y uno cuantitativo inmunoturbidimétrico para la supuestamente sana. Se compararon los niveles medios entre género en cada una mediante la prueba t de Student para muestras independientes. Resultados: La media de los niveles de proteína C reactiva en el género masculino y femenino de la muestra supuestamente enferma fue de 3.49 mg/L y 3.41 mg/L respectivamente. En el grupo control la comparación de medias de los niveles de proteína C reactiva entre género fue para los hombres de 1.38 mg/L y para las mujeres de 1.94 mg/L. Conclusión: No se encontraron diferencias significativas entre género en la muestra supuestamente enferma, ni en el grupo control.

ABSTRACT Background: C-reactive protein is one of the best marker for the assessment and monitoring of inflammatory diseases; the recommended reference values for its serum concentration are not gender-adjusted. Objective: To determine whether C-reactive protein concentration differs by gender. Methodology: An exploratory study was conducted on 3199 patient samples from Camilo Cienfuegos General Provincial Hospital and a control group of 76 serum samples from blood donors of each gender from the Sancti Spíritus Blood Bank. Levels were measured by a semi-quantitative latex particle agglutination assay for the presumed diseased sample and a quantitative immunoturbidimetric assay for the presumed healthy sample. Mean levels were compared between genders in each using Student's t-test for independent samples. Results: The mean of C-reactive protein levels in the male and female gender of the supposedly diseased sample was 3.49 mg/L and 3.41 mg/L respectively. In the control group the mean comparison of C-reactive protein levels between genders was 1.38 mg/L for males and 1.94 mg/L for females. Conclusion: No significant gender differences were found in the presumed ill sample, nor in the control group.

Cervicovaginal fluid beta human chorionic gonadotropin assay as an early predictor of spontaneous preterm delivery among antenatal patients

BACKGROUND: The aim of this study was to assess the usefulness of the beta subunit of hCG in cervicovaginal secretions as a biochemical predictor of spontaneous preterm delivery among pregnant women with and without preterm delivery risk.DESIGN: This was an eight-month prospective case control study of pregnant women with or without risk factors for preterm delivery. SETTING: Ifako- Ijaye General Hospital Lagos/ Lagos State University Teaching Hospital, Ikeja Lagos Nigeria. PARTICIPANTS: 150 pregnant women which consisted of 50 cases with preterm delivery risk and 100 controls without preterm delivery risk. INTERVENTIONS: A structured interviewer administered questionnaire which had been pretested, was used to collect data. Two cervicovaginal fluid samples at 26 weeks and 32 weeks were collected from each of the participants and it was quantitatively assayed using ELISA for presence of beta hCG. The participants were followed up till delivery. RESULTS: 15 participants out of the 50 cases delivered their babies preterm, while only 2 participants out of the 100 controls had preterm delivery. The 15 cases who delivered preterm had significant increase in their mean beta HCG value from 7.44±1.74 at 26 weeks to 32.6±1.32 at 32 weeks with p value<0.001. There was however no statistical difference in the mean beta HCG at 26 weeks and at 32 weeks for the control group. CONCLUSION: The concentration of beta HCG in the cervicovaginal fluid is a useful early predictor of preterm delivery especially among patients with risk factors.

Analysis on “Point-line-surface” Quality Standard of Yangjing Zhongyu Tang / 中国实验方剂学杂志

Objective: Based on the analysis of the total components of fingerprint and the determination methods of the existing components in the 2015 edition of Chinese Pharmacopoeia, the "point-line-surface" quality standard of Yangjing Zhongyu Tang was established by the "point" of each single component (morroniside, loganin, paeoniflorin, ferulic acid and verbascoside) to the "line" of multicomponent and the "face" of fingerprint of the whole component. Method: XB-C18 column (4.6 mm×250 mm, 5 μm) was used for gradient elution of 0.1% phosphoric acid aqueous solution-acetonitrile. The column temperature was 30℃, the injection volume was 10 μL, the flow rate was 1.0 mL·min-1, and the detection wavelengths were 240, 316, 230, 334 nm. The contents of these five components in Yangjing Zhongyu Tang were determined by three correction methods, external standard method and regression equation method. At the same time, the fingerprint of Yangjing Zhongyu Tang were analyzed by total component analysis and similarity evaluation. Result: With ferulic acid as reference, the relative correction factor (f) of morroniside, loganin, paeoniflorin and verbascoside were 0.392 1, 0.421 4, 0.261 7, 0.268 6 by multi-point correction method, and their f (slope correction method) were 0.385 4, 0.419 4, 0.255 9, 0.274 0, respectively. Twenty characteristic peaks of fingerprint were analyzed and the similarity was ≥ 0.999.There was no significant difference in the contents of these five components from Yangjing Zhongyu Tang determined by the quantitative assay of multi-components by single-marker (QAMS) correction method, the external standard method and the regression equation method. Conclusion: The comprehensive quality standard established by the total component analysis of fingerprint combined with various determination methods of existing components in the 2015 edition of Chinese Pharmacopoeia has been validated in famous classical formula of Yangjing Zhongyu Tang, which can provide ideas and methods for the quality control with quantitative determination and fingerprint of other famous classical formulas.

Screening of lignocellulolytic fungi for hydrolyzation of lignocellulosic materials in paddy straw for bioethanol production

Aims@#Paddy straw is known to have lignocellulosic materials such as cellulose and hemicellulose which can be readily converted into fermentable sugar for production of bioethanol via simultaneous saccharification and fermentation (SSF). In order to produce ethanol competently, the degradation of biomass by cellulase and highly ethanol-producing microorganism in fermentation process are necessarily needed. However, there is lacking in cellulose degrading organism in producing adequate amount of lignocellulosic enzyme. Therefore, the screening and selection for the best fungi to hydrolyze the lignocellulosic materials as well as forming consortium between two species of fungi has become the main focus. @*Methodology and results@#Thirteen strains of fast-growing fungi were tested qualitatively for cellulase (congo red staining) and polyphenol oxidase (Bavendamm test). All tested strains displayed lignocellulolytic fungi characteristics. The selection was narrowed down by quantitative assay on endoglucanase, exoglucanase, β-glucosidase and xylanase and the highest cellulases enzyme producer were Trichoderma asperellum B1581 (3.93 U/mL endoglucanase; 2.37 U/mL exoglucanase; 3.00 IU/mL β-glucosidase; 54.87 U/mL xylanase), followed by Aspergillus niger B2484 (5.60 U/mL endoglucanase; 1.08 U/mL exoglucanase; 1.57 IU/mL β-glucosidase; 56.85 U/mL xylanase). In compatibility test, both T. asperellum B1581 and A. niger B2484 were inoculated on the same Petri dish for 4 days and the interaction showed by the two species was mutual intermingling. @*Conclusions, significance and impact of study@#Both T. asperellum B1581 and A. niger B2484 produced the highest cellulase enzyme. Since both strains can co-exist and produce enzymes that complete each other, a fungal consortium was suggested to increase the yield of sugars in saccharification process.

Development and application of competitive light-initiated chemiluminescent assay for quantitation of total IgE in human serum / 临床检验杂志

Objective To develop a competitive immunoassay for quantitative determination of total immunoglobin E (tIgE) in human serum based on light-initiated chemiluminescent assay (LICA).Methods The LICA-tIgE assay was performed by incubating serum samples or calibrator with anti-human IgE antibody-coated chemiluminescet beads,biotinylated human IgE and streptavidin-coated sensitizer beads.The working conditions of this assay were optimized,analytical performance was detected and the correlation of tIgE results between LICA and Beckman Coulter IMMAGE 800 was evaluated.Results The precision of intra-assay and inter-assay (coefficient of variation) ranged from 5.50％ to 7.73％ and 6.45％ to 9.90％,respectively.The functional sensitivity of this assay was 12.65 IU/mL.The recovery rates measured by adding IgE calibrators to human sera with different IgE concentrations were ranged from 104.15％ to 109.37％.The disturbing rates measured by adding total bilirubin,hemoglobin and triacylglycerol to human sera with different IgE concentrations were ranged from-4.49％ to 8.46％.Also,the tIgE results of 111 patients measured by LICA correlated well with those by Beckman Coulter IMMAGE 800 (r2 =0.959).Conclusion LICA developed in this study for detecting tIgE of human serum showed effective perfomance and could meet the basic requirements of clinical diagnostic reagents.

Development of monoclonal antibodies against nucleoprotein of Newcastle disease virus and establishment of a quantitative double-antibody sandwich ELISA for NDV antigen / 中国人兽共患病学报

We developed the monoclonal antibodies against nucleoprotein (NP) of Newcastle disease virus (NDV),and established a double antibody sandwich ELISA method for quantitative determination of NP antigen of NDV (NDV NP ELISA).The recombination NP protein derived from strain F48E9 of NDV were prepared and used to immunize BLAB/c mice.The mouse splenic cells from immunized mice were fused with SP2/0 cells to generate monoclonal antibodies (mAb).The NDV NP specific mAbs were paired to establish a double antibody sandwich ELISA method.The performance of the NDV NP ELISA was evaluated,including specificity,sensitivity,precision,accuracy and linearity.The correlation between the ELISA and PFU virus titer was analyzed by regression analysis method.Two monoclonal antibodies 3C10 and 4E7 were selected to establish double antibody sandwich ELISA for NP antigen of NDV.The linearity and performance of the NDV NP ELISA was characterized.The detection linearity fell in the range of 0.015-0.250 μg/mL (R2 =0.997 4).The detection limit of the assay was 0.015 μg/mL.The recovery was between 88.4％ and 106.01％;the variation coefficient was below 3.4％.In testing of 50 NDV virus samples,this assay performed well and correlated comparably with PFU virus titer (R2 =0.920 9).The NDV NP ELISA for quantitative detection of NDV is a reliable quantifiable assay for detection of NDV NP protein;it provides a new approach for rapid and quantitative detection of Newcastle disease virus.

Establishment of the Quantitative Immunohistochemical Examination of DHBcAg in Duck Liver / 现代检验医学杂志

Objective To develop a quantitative immunohistochemistry assay for duck hepatitis B virus core antigen (DHB-cAg)in duck liver tissue.Methods By comparison with no repair antigen and repair antigen with high pressure,microwave and trypsin,the best solution of antigen retrieval was determined.By optimizing the parameter of image acquisition and de-ducting blank area,mean density of yellow areas was calculated using Image-Pro Plus 6.0 software.Using the assay devel-oped to determine the level of DHBcAg in liver tissue from duck infected by DHBV,anti-DHBV activity of DHBcMAb-TAT PTD conj ugate was examined.Results SABC method with no repair antigen was selected,which was better than other methods.DHBcAg expression in duck liver tissue could be objectively and accurately quantified by setting Image-Pro Plus 6.0 software parameters and calculating mean density of yellow areas.By comparison with the differences between mean densityat baseline of treatment and end of treatment,it was showed that DHBcMAb-TATPTD conjugate treatment dose-de-pendently reduced the levels of DHBcAg in liver tissue,which show that the assay developed could effectively evaluate the anti-DHBV activity of agent.Conclusion The immunohistochemistry assay developed in this study can objectively and accu-rately evaluate the level of DHBcAg in duck liver tissue.

Development of a quantitative CRP test kit with lateral lfow method / 中国生化药物杂志

Objective A kind of quantitative C reactive protein (CRP) test kit was developed with colloidal gold lateral flow method. Method The kit was prepared with double antibody sandwich technology, and by material optimization and strict process control to improve performance. Quantitative assay was realized by a specialized lateral flow reader. The kit performance was evaluated with series of tests and clinical trial. Results The kit was developed with functional sensitivity≤1 mg/L, linear range 1-200 mg/L, CV<15%and with stability of 12 months. 220 samples clinical trial showed 98.6%of coincidence rate. Pearson Correlation coefficient r is 0.987, which showed no significant difference in performance compare with control kit. Conclusion A quantitative CRP test kit was developed with easy to operating and good stability, Which can be used for point of care testing or laboratory testing.

Activated Protein C Anticoagulant System Dysfunction and Thrombophilia in Asia

Thrombophilia that is common among Caucasians is caused by genetic polymorphisms of coagulation factor V Leiden (R506Q) and prothrombin G20210A. Unlike that in Caucasians, thrombophilia that is common in the Japanese and Chinese involve dysfunction of the activated protein C (APC) anticoagulant system caused by abnormal protein S and protein C molecules. Approximately 50% of Japanese and Chinese individuals who develop venous thrombosis have reduced activities of protein S. The abnormal sites causing the protein S molecule abnormalities are distributed throughout the protein S gene, PROS1. One of the most common abnormalities is protein S Tokushima (K155E), which accounts for about 30% of the protein S molecule abnormalities in the Japanese. Whether APC dysfunction occurs in other Asian countries is an important aspect of mapping thrombophilia among Asians. International surveys using an accurate assay system are needed to determine this.

Performance Evaluation of 3 Kinds of HBsAg Qualitative Assays and 2 Kinds of Quantitative Assays

BACKGROUND: Currently used techniques for quantitation of HBsAg often yield discordant results; therefore, development of quantitation techniques that can detect HBsAg with high accuracy has become very important. Recent advances have led to the development of several HBsAg detection systems. Here, we evaluated the performance of 3 newly developed detection systems, which can detect HBsAg both qualitatively and quantitavely, and determined the concordance among their results. METHODS: Four hundred and thirty two samples assigned to 4 groups-patient group, dilution group, weakly reactive group, and linearity group- were subjected to qualitative and quantitative detection of HBsAg by using the 3 systems developed by 3 major manufacturers; Abbott Architect, Roche E170 and Siemens Centaur XP. RESULTS: The results for the qualitative analyses were closely concordant among the three systems (98.3%) for all 432 samples. In 123 samples that were determined as HBsAg-negative, E170 (76%) distributed frequently at the upper half level (0.5-1.0) of negative reference range, compared with Architect (11%) and Centaur XP (22%). In particular, in 65 samples that were diluted from the strongly positive samples to obtain weakly positive samples, the average index values obtained using Architect (3.6 S/CO), E170 (4.2 COI) and Centaur XP (11.4 index value) differed significantly (P<0.0001). In the antiviral treatment group and the post-liver transplantation group, no inconsistency was observed among the results of the qualitative and quantitative assays. In the 18-fold serially diluted samples, no linearity was observed. CONCLUSIONS: Because of the possibility of false-positive detection in the HBsAg-negative samples, regular management of equipment and appropriate selection of reagents are very important. In weakly positive samples, quantitative assay has not to be replaced for qualitative assay. Therefore, the qualitative assays should be used for screening the samples, whereas the quantitative assays should be used for monitoring the Hepatitis B virus (HBV) load in the samples determined as HBsAg-positive. The qualitative index value should not be interpreted as a quantitative measure of HBV load.

Glucose-6-phosphate dehydrogenase screening of babies born in a tertiary care hospital in West Bengal.

About 400 million individuals worldwide have been affected by the inherited disorder of glucose-6-phosphate dehydrogenase (G6PD) deficiency that predisposes individuals to neonatal jaundice or hemolytic crisis due to drugs or infections. A descriptive observational study with longitudinal design was undertaken among 109 live newborns, delivered in labor room of IPGME and R, Kolkata during the period from June to August 2009. An objective of the study was to estimate the occurrence of G6PD deficiency among newborns and its association with different socio-demographic, clinical and gestational characteristics. 14.68% newborns were found G6PD deficient. This occurrence was not significantly related to gender, religion and ethnicity, consanguineous marriage of the parents, gestational age and birth weight of the baby. Development of severe jaundice (total serum bilirubin >15 mg/dl) was found 23.8% among G6PD deficient babies and 12.5% among non-G6PD deficient. This difference was statistically not significant.

In vitro transcription of HIV-1 RNA for standard RNA.

Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The important step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1 RNA transcription to produce the standard HIV-1 RNA. Methods: The HIV-1 DNA was amplified from pNL43 by PCR using a primer pair that was specific for conserved region of HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted with EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performed simultaneously for confirmation of synthesized RNA fragment. Results: A 115 bp DNA of HIV-1 Gag gene has been cloned into pBluescript II SK with the exact true orientation. The reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showed the accuracy of the transcripts. Conclusion: We successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, and the HIV-1 RNA has been transcribed in vitro as well.

Measurement of Hyaluronic Acid Amounts Using ELISA from Cultured Human Skin Fibroblasts / 대한피부과학회지

BACKGROUND: Assay of glycosaminoglycans or proteoglycans from skin is complicated due to individual methods where the measurements are highly specialized. The results from these different methods are not able to be compared and there is a large variance. OBJECTIVE: It seems reasonable that a major glycosaminoglycan in the skin, hyaluronic acid, might be ideal as a representative instead of the whole components of glycosaminoglycan. To develop a simple and reliable assay method, the in vitro cell culture system was selected to reduce time and variety of data. The usefulness of the ELISA method, using hyaluronic acid binding protein (HA-ELISA), was evaluated. METHODS: The amount of hyaluronic acid synthesis was measured under a standardized protocol for cultured human skin fibroblasts from the elderly and neonates, as well as the NIH 3T3 mouse fibroblast cell line. To see whether this screening method (HA-ELISA) could be time-saving and reliable under in vitro conditions, some well-known stimulants for glycosaminoglycan synthesis such as retinol, retinyl palmitate, polyethoxyretinide retinamide and hydroxyproline were treated. RESULTS: The production of hyaluronic acid was influenced by both culture condition and source of fibroblasts. The level of quantity showed different patterns due to factors such as culture period, serum in the medium and cell proliferation rate. We found that stable levels of hyaluronic acid assay from culture supernatant were obtained by delaying the sampling time after 24 hours of treatment with stimulants. CONCLUSION: For a reliable quantitative assay, either NIH 3T3 mouse fibroblasts or neonate fibroblasts were suitable. The culture condition and time of harvest should be determined first to estimate the stable kinetics of hyaluronic acid synthesis. This in vitro test protocol can be used as an additional evaluation system towards a potential agent for dermal connective tissue, while further efforts are still mandatory to correlate the confounding factors of in vitro and in vivo.

A High-throughput and Quantitative Assay Based on Fluorescence Intensity for Detection of Apoptosis / 中国生物工程杂志

Based on the different permeability of DNA-intercalant dyes YO-PRO-1(YP) and propidium iodide (PI) to the membrane of viable, apoptotic and necrotic cells, cell samples were stained with 4?mol/L YP and 4?g/ml PI for 10 min, and the fluorescence intensity of both YP and PI were measured by fluorometer at Ex/Em wavelength of 485/538nm and 530/590nm, respectively. The correlation between YP fluorescence intensity and the apoptotic cell number was confirmed by fluorescence microscope and linear regression(r=0.999,P

The expression of cyclooxygenase-2(COX-2) in human colorectal cancer and its clinical significance / 中国癌症杂志

Purpose:To evaluate the role of COX 2 in the carcinogenesis and progression of human colorectal neoplasms. Methods:The expression level of COX 2 in 122 colorectal neoplasm tissues(including 35 colorectal adenomas, 67 colorectal carcinoma and 23 colorectal cancer with synchoronous hepatic metastasis) was assayed by immunohistochemical methods. All specimens was analyzed by Conformation quantitative assay system, their stain strength was calculated.Results:Conformation quantitative assay showed the mean stain strength is 704.5 131.8 in colorectal adenoma, 1197.2 204.3 in colorectal cancer without hepatic metastasis and 1901.2 324.8 in colorectal cancer with synchoronous hepatic metastasis, there are significant differences among them statistically. According to the Dukes stages, mean stain strength is 1145.3 187.0 in B stage,1237.0?298.7 in C stage,1901.2 in D stage. There is statistic difference between Dukes stage D and B, C. Also our study indicated there was no relationship between age, sex, tumor location, tumor differentiation or tumor size with the level of COX 2 protein expression.Conclusions:In our test,higher COX 2 expression is seen in colorectal cancer than colorectal adenomas, colorectal cancer with synchoronous hepatic metastasis than colorectal cancer without hepatic metastasis,and Dukes D stage than the B?C. Thus COX 2 responses is important in the carcinogenesis and progression of colorectal cancer. NSAIDs or the others may play a role in the chemoprevention strategies of colorectal neoplasm as the COX 2 inhibitor.