The term congenital adrenal hyperplasia (CAH) covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency.
Adrenal Glands ; Adrenal Hyperplasia, Congenital* ; Aldosterone ; Cholesterol ; Chromatography, Liquid ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluoroimmunoassay ; Gas Chromatography-Mass Spectrometry ; Genetic Counseling ; Humans ; Hydrocortisone ; Infant, Newborn ; Male ; Mass Screening ; Neonatal Screening ; Prognosis ; Radioimmunoassay ; Steroid 21-Hydroxylase* ; Tandem Mass Spectrometry

OBJECTIVETo investigate the correlation of gestational age and birth weight with 17α-hydroxyprogesterone (17α-OHP) levels, and with results of adrenal hyperplasia newborn screening.

METHODUsing time-resolved fluorescence immunoassay, the authors measured concentrations of heel blood 17α-OHP by newborn dried blood spots on filter paper which included 29 hospitals newborns of Wujiang, Taicang, Zhangjiagang, Kunshan, and Suzhou, where there were 118 050 infants in total who had accurate gestational age and birth weight (62 490 males, 55 560 females). According to the classification by gestational age, there were 4 693 premature infants, 113 300 term infants and 57 overdue infants. According to the classification by birth weight, there were 4 172 infants with weight < 2 500 g, and 113 878 infants weight ≥ 2 500 g. And, in all premature infants, gestational age of 189 infants was < 32 weeks, 2 277 infants less than 36 weeks but ≥ 32 weeks, and 2 227 infants less than 37 weeks but not less than 36 weeks. Neonatal heel blood concentration of 17α-OHP was measured by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), and the correlation between 17α-OHP and gestational age or birth weight was retrospectively analyzed by using Spearman test.

RESULTThe distribution of 17α-OHP levels was skew. The 17α-OHP levels decreased significantly from very preterm births, moderately preterm, later period preterm to term infants [19.21 (8.07, 24.00), 12.35 (6.81, 18.00), 8.58 (5.66, 13.80), 5.60 (3.57, 8.51) , 3.34 (2.58, 5.23) nmol/L; 479.42, 62.25, 36.24, 23.30, 13.73 nmol/L;P all = 0.000]. The 17α-OHP levels decreases from very low birth weight (VLBW), extremely low birth weight (ELBW), low birth weight (LBW), normal birth weight to macrosomia [5.24 (3.24, 8.96) , 11.30 (6.84, 22.95) , 8.50 (5.28, 14.90) , 5.66 (3.61, 8.62) , 5.38 (3.40, 8.11) nmol/L; 485.26, 125.18, 39.50, 23.80, 22.15 nmol/L; P = 0.000 for all comparison]. Neonatal 17α-OHP levels and gestational age, body weight was significantly negatively correlated respectively -16.40 and -10.10 (P both = 0.000) by using Spearman test. Neonatal 17α-OHP levels and gestational age, body weight were binomially distributed, and the formulae were y = 0.105 5x²-2.457 6x + 17.689, R² = 0.980 3 and y = 0.411x²-3.988x+14.75, R² = 0.983. Little preterm infants, preterm infants and term infants in low birth weight infants 17α-OHP levels were significantly higher than non-low birth weight infants [11.20 (6.01, 18.90) vs 9.05 (5.85, 14.90) nmol/L, 9.76 (4.32, 10.35) vs 5.59 (3.56, 8.48) nmol/L, P all = 0.000].

CONCLUSIONNeonatal 17α-OHP levels and gestational age, body weight was significantly negatively correlated; in order to improve the accuracy and sensitivity, cut-off value of neonatal 17α-OHP should be adjusted according to gestational age and weight.

17-alpha-Hydroxyprogesterone ; blood ; Adrenal Hyperplasia, Congenital ; blood ; diagnosis ; Birth Weight ; Female ; Fluoroimmunoassay ; Gestational Age ; Humans ; Infant, Newborn ; Infant, Premature ; blood ; Infant, Very Low Birth Weight ; blood ; Male ; Neonatal Screening ; Retrospective Studies ; Sensitivity and Specificity

OBJECTIVETo study the expression of the ID3 protein in prostate cancer and its clinicopathological significance.

METHODSWe detected the expression of the ID3 protein in PC-3M cells by indirect immunofluorescence, and that in 29 prostate cancer and 15 prostate hyperplasia specimens by immunohistochemistry. Then we analyzed the correlation between the expression level of ID3 and the clinicopathological parameters.

RESULTSThe ID3 protein was expressed predominantly in the nucleus of PC-3M cells. Its expression rate was 82.7% (24/29) in the prostate cancer specimens, significantly higher than 6.6% (1/15) in prostate hyperplasia (P < 0.05), and was positively correlated with the Gleason score of prostate cancer (P < 0.05).

CONCLUSIONThe ID3 protein is expressed in prostate cancer, and is elevated with the increase of Gleason score.

Aged ; Aged, 80 and over ; Fluoroimmunoassay ; Humans ; Immunohistochemistry ; Inhibitor of Differentiation Proteins ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).

METHODSThe CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.

RESULTSCFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.

CONCLUSIONThe reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.

Bacterial Proteins ; genetics ; standards ; Fluoroimmunoassay ; methods ; Gene Amplification ; Mycobacterium tuberculosis ; isolation & purification ; Reference Standards

OBJECTIVETo develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine.

METHODSA double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine.

RESULTSThe sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine.

CONCLUSIONThe TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.

Cross Reactions ; Fluoroimmunoassay ; methods ; Pertussis Toxin ; analysis ; Pertussis Vaccine ; chemistry ; standards ; Quality Control ; Sensitivity and Specificity

Biomarkers ; blood ; Enzyme-Linked Immunosorbent Assay ; Fluoroimmunoassay ; Hepatitis B ; blood ; Hepatitis B virus ; Humans ; Luminescent Measurements

OBJECTIVETo screen and diagnose Down's syndrome during mid-term pregnancy to reduce the number of babies with Down's syndrome.

METHODSWith the multi-level of stratified cluster sampling, twenty thousand and eight hundred and three women at 15-20 weeks gestation were screened by maternal serum AFP and beta-hCG using the time resolved fluoroimmunoassay (TRFIA). Then the screened high-risk women were diagnosed by amniocentesis, cell culture and chromosome analyses. The born children were diagnosed by follow-up and peripheral blood chromosome analyses.

RESULTSSix fetuses were diagnosed by serum screening and amniotic fluid chromosome analyses, and 3 born children were diagnosed by follow-up and peripheral blood chromosome analyses. Nine cases of Down's syndrome were detected in total, with the positive prenatal screen rate being 67% (6/9).

CONCLUSIONThe prenatal screening and diagnosis can reduce the birth of Down's syndrome patients and improve the population quality. However, the diagnosis accuracy still needs to be improved to further reduce the false negative rate and prevent misdiagnosis.

Adult ; Chorionic Gonadotropin, beta Subunit, Human ; blood ; Chromosome Aberrations ; Down Syndrome ; blood ; diagnosis ; genetics ; metabolism ; Female ; Fluoroimmunoassay ; Humans ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult ; alpha-Fetoproteins ; metabolism

OBJECTIVETo prepare a time-resolved fluoroimmunoassay (TRFIA) kit for clinical detection of IgM antibodies to hepatitis B core antigen (HBc).

METHODSImmunocapture method was used to develop the TRFIA kit for detection of the anti-HBc IgM antibodies, and the precision, cross-reactivity and sensitivity of the kit were tested with the clinical serum samples.

RESULTSThe intra- and inter-assay coefficients of variation of the TRFIA kit were 4.8%-7.2% and 7.5%-8.6%, respectively, and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found. Comparison of the results of the TRFIA kit and enzyme-linked immunosorbent assay (ELISA) demonstrated greater sensitivity of the kit than ELISA in detecting the anti-HBc IgM antibodies in 584 serum samples. According to the detection results in 300 serum samples from healthy donors, the cutoff value of the TRFIA kit was 4.5 times of the fluorescence value of the negative control.

CONCLUSIONThis TRFIA kit for detecting anti-HBc IgM antibodies meets the demand for clinical application and can replace the ELISA kits.

Fluoroimmunoassay ; methods ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Humans ; Immunoglobulin M ; blood ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

OBJECTIVETo establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors.

METHODSSpecific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-2) kinase activity was investigated.

RESULTSA homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/microl, 100 micromol/L and 1 micromol/L, respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC50 value of 86.7 nmol/L, which was close to the values tested using other methods.

CONCLUSIONThe homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.

Fluoroimmunoassay ; methods ; High-Throughput Screening Assays ; methods ; Indoles ; pharmacology ; Peptides ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Pyrroles ; pharmacology ; Time Factors ; Vascular Endothelial Growth Factor Receptor-2 ; antagonists & inhibitors ; metabolism

OBJECTIVE: To explore the expression of tryptase and chymase in human lung tissue of anaphylactic shock and its value for forensic medicine. METHODS: With ten carbon monoxide poisoning cases as control group, the levels of tryptase and chymase were observed by immunofluorescence and analyzed using the Image Analyze and the Image-pro plus 5.0.2. The positive mast cells were counted and the levels of the tryptase and chymase were calculated respectively. RESULTS: There was a statistically significant difference (P < 0.05) for the tryptase and chymase concentrations in the lung tissue between the anaphylactic shock group and the control group. CONCLUSION The levels of the tryptase and the chymase expression are greatly increased in human lung tissue of anaphylactic shock, which may provide the morphological evidence and reference for the diagnosis of anaphylactic shock in forensic practice.
Adolescent ; Adult ; Anaphylaxis/pathology* ; Cadaver ; Carbon Monoxide Poisoning/pathology* ; Child ; Child, Preschool ; Chymases/metabolism* ; Female ; Fluoroimmunoassay/methods* ; Forensic Pathology ; Humans ; Infant ; Lung/pathology* ; Male ; Mast Cells/enzymology* ; Middle Aged ; Staining and Labeling ; Tryptases/metabolism* ; Young Adult