Neonatal TSH Dried (Whole) Blood (EIA)

For the quantitative determination of TSH (thyroid stimulating hormone, thyrotropin) in blood dried on filter paper.

Screening for Congenital Hypothyroidism (CH) is important for the prevention of irreversible mental retardation.  Determination of TSH in neonatal blood dried on filter paper (the Guthrie Card) is used as a primary screening test or as a measure of iodine deficiency in adult populations.

The test is based on the "antibody sandwich" principle.  3mM discs punched from standard, control and patient blood filter paper are added to Anti-TSH Antibody-coated MicroPlate wells.  A buffer solution is added to effect elution of the TSH from the blood spot.  The TSH is bound to the antibody-coated wells.  This solid phase antibody is referred to as the "capture" antibody.  Following a wash, Peroxidase-conjugated monoclonal anti-TSH antibody is added which is specific for an epitope of TSH distal to that of the capture antibody.  When TSH is present, this two-sited interaction results in a bridge or sandwich with the two antibodies on the ends and TSH in the middle.

Following a final wash, the Color Reagent for peroxidase is added.  After an incubation the reaction is stopped and absorbance is measure using a MicroPlate spectrophotometer.  The concentration of TSH is directly proportional to the color (yellow).  The concentration of the unknown sample is determined from a standard dose-response curve.  back to top

SPOTCHECK Automation System

With the SPOTCHECK system, laboratories can offer same day turnaround of quantitative patient results. In most cases, results can be obtained before the early discharge of the newborn. This allows the physician to respond more effectively to a potentially critical condition. Designed for expansion and flexibility, the SPOTCHECK system maximizes the initial investment. Astoria-Pacific is committed to continuing the years of excellence into the future through its products, research and customer service.

Sample Handling
Efficient sample handling is optimized with the SPOTCHECK sampler. The large capacity 311 Sampler is computer controlled in an XYZ grid format to automatically introduce samples from a standard 96 well microtiter plate or test tube rack into the reagent stream. The carousel style 301 Sampler is an attractive option for those laboratories with smaller sample loads and different specimen processing requirements.

Methodology
The SPOTCHECK diagnostic reagent kits are manufactured under the strictest standards specifically for the SPOTCHECK system. Convenient, color coded kits provide 50 hours of operation and are available with a 2 year shelf life. Designed for use with proven methodologies, SPOTCHECK reagent kits provide reliable, low cost testing day after day.

Sample Analysis
The Analytical Cartridge provides the dialysis and incubation necessary to affect a reaction between sample and reagents for a specific test. Each sample is introduced to the analytical cartridge by way of the peristaltic pump. As each sample travels through the cartridge, it mixes with the appropriate reagents and a fluorescent product is formed. The sample continues to travel out of the cartridge and into a flow through fluorometer (321 Fluorometer) which measures the amount of fluorescence produced. The analog results can be sent to a strip chart recorder and/or to a computer for data reduction.

Microprocessing
The optional FASPac software provides system control, data acquisition, automatic calculations and reporting of patients' results. Peaks and analytical results are displayed on the CRT in real time as specimens are processed. For those labs with networking capabilities, the final report can be exported easily in Excel or Lotus file formats, or as an ASCII file in Standard text or Comma Delimited text format. back to top

SPOTCHECK System Tests 

SPOTCHECK Automation System can run six tests including  Phenylalanine, Total Galactose, Uridyltransferase, Tyrosine, Biotinidase Deficiency, and G6PD Deficiency. Each testing kit contains all necessary reagents needed for analysis and will provide approximately 50 hours of analyzer run time. Allowing for start up and calibrants, the approximate number of actual samples analyzed per kit is conservatively 2500-3000.

Phenylalanine

SPOTCHECK Phenylalanine test is used as an aid in screening for Phenylketonuria (PKU), an inherited disorder of phenylalanine metabolism caused by a decreased level of phenylalanine hydroxylase activity in newborns.

When phenylalanine is ingested by an affected newborn, the decrease or absence of phenylalanine hydroxylase activity, which converts phenylalanine to tyrosine, causes blood and urine levels of phenylalanine to become elevated. Serum phenylalanine measurements therefore can be used to diagnose and treat this disease.

The Astoria-Pacific SPOTCHECK Analyzer automates the measurement of phenylalanine in the newborn's sample by first diluting and then dialyzing the sample to remove interfering substances. The phenylalanine in the sample then reacts with ninhydrin in the presence of a dipeptide to form a fluorescent end product.

The fluorescent end product formed is measured quantitatively with a fluorometer equipped with a specially designed flow-through flowcell. The amount of fluorescence, excited at 405 nm and emitted at 480 nm, is proportional to the phenylalanine concentration in the sample.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 1/8 inch spot but may be adapted to alternative punch protocols with appropriate validation.

Other tests, such as Total Galactose or Uridyltransferase, can be run with the Phenylalanine analysis simultaneously from the same extracted sample. Sample throughput is 90 per hour.  back to top

Total Galactose

The Astoria-Pacific SPOTCHECK Galactose test is used as an aid in screening for Galactosemia, an inherited disorder of galactose metabolism caused by a decreased level of galactose-1-phosphate uridyltransferase enzyme activity in newborns.

When galactose is ingested by an affected newborn, the galactose-1-phosphate accumulates intracellularly and blood and urine levels of galactose become elevated. Serum galactose measurements therefore can be used to diagnose and treat this disease.

The Astoria-Pacific SPOTCHECK Analyzer automates the measurement of galactose in the newborn's sample by first hydrolyzing galactose-1-phosphate to simple galactose using alkaline phosphatase. The total galactose is then oxidized by NAD in the presence of galactose dehydrogenase to form galactonolactone and NADH.

The NADH produced in each sample is measured quantitatively with a fluorometer equipped with a specially designed flow-through flowcell. The resulting fluorescence at 465 nm, generated by excitation at 365 nm, is proportional to the total galactose concentration.

Simple galactose can also be measured quantitatively with this test. By removing the hydrolysis step, only simple galactose in the sample is oxidized by NAD with subsequent reduction to NADH. The resulting fluorescence is also measured at 465 nm and is proportional to the simple galactose concentration.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 1/8 inch spot but may be adapted to alternative punch protocols with appropriate validation.

Other tests, such as Phenylalanine or Uridyltransferase, can be run with the Galactose analysis simultaneously from the same extracted sample. Sample throughput is 90 per hour.  back to top

Uridyltransferase

The Astoria-Pacific SPOTCHECK Galactose-1-phosphate uridyltransferase (GALT) test is used as an aid in screening for Galactosemia due to decreased levels of GALT enzyme activity in newborns.

Galactose -1- phosphate uridyltransferase (GALT) activity is determined by measuring its reaction products over time. GALT catalyzes the conversion of galactose -1- phosphate to glucose -1- phosphate. Further reactions with glucose -1- phosphate result in the reduction of NADP+ to NADPH. The amount of fluorescent NADPH produced is proportional to the GALT enzyme activity.

The Astoria-Pacific SPOTCHECK Analyzer measures the NADPH produced in each sample with a fluorometer (active channel). A second fluorometer (blank channel) is used to subtract any endogenous fluorescence that may be present in the sample thereby eliminating any false negatives. In addition, the intermediary enzymes phosphoglucomutase (PGluM) and G6PDH are added to ensure any lack of fluorescence is not due to inactivity of these enzymes in the sample. This helps eliminate any possible false positives. A sample integrity test can be performed on any samples that appear deficient in GALT by substituting PGluM substrate containing glucose -1- phosphate for the GALT substrate.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 3/16 inch spot or two 1/8 inch spots but may be adapted to alternative punch protocols with appropriate validation.

Other tests, such as Phenylalanine or Total Galactose, can be run with the GALT analysis simultaneously from the same extracted sample. For the GALT assay, two fluorometric detectors (active and blank channels) and two cartridges (active and blank) are required. The active cartridge includes an on-line incubator to enhance enzymatic response. Sample throughput is 90 per hour after an initial dwell time of approximately 80 minutes.  back to top

Tyrosine

The Astoria-Pacific SPOTCHECK Tyrosine test is used as an aid in screening for hypertyrosinemia due to decreased levels of p-hydroxyphenylpyruvate oxidase (p-HPPA oxidase) activity in newborns. This is most frequently seen as a transient low enzyme activity due to late maturation of the enzyme in newborn infants resulting in the temporary elevation of both tyrosine and phenylalanine in the blood. Occurring less frequently are the rare inherited conditions causing hypertyrosinemia, which cause neurological, liver/kidney disorders.

Tyrosine in a sample reacts with 1-nitros-2-naphthol and the fluorescent compound formed is then measured quantitatively using SPOTCHECK's flow through fluorometer.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 1/8 inch spot but may be adapted to alternative punch protocols with appropriate validation.

Other tests, such as Phenylalanine or Total Galactose, can be run with the Tyrosine analysis simultaneously from the same extracted sample. Sample throughput is 90 per hour.  back to top

Biotinidase Deficiency

The Astoria-Pacific SPOTCHECK biotinidase determination is used as an aid in screening for biotinidase deficiency in newborns using dried whole blood spots.

The Astoria-Pacific SPOTCHECK biotinidase determination is used as an aid in screening for biotinidase deficiency in newborns using dried whole blood spots.

Biotinidase activity is determined colorimetrically by measuring the amount of p-aminobenzoic acid (PABA) released from biotinyl-p-aminobenzoate (Biotin-PAB). Samples are eluted in water and then incubated on-line for 90 minutes with Biotin-PAB in a pH 6 buffer. On-line dialysis separates the released PABA from other proteins in the sample. The PABA is then diazotized and coupled to a napthol derivative to form an azo dye by the addition of sodium nitrite, acidic ammonium sulfamate and N-1-naphthylethylenediamine dihydrochloride (NED). The chromophore produced is measured at 550 nm.

The intensity of color produced in each sample is directly proportional to the amount of biotinidase activity. Samples with biotinidase activity develop a purple color. Samples deficient in biotinidase remain straw-colored. A standard curve prepared from a stock PABA solution is used to quantitate the results.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 3/16 inch spot or two 1/8 inch spots but may be adapted to alternative punch protocols with appropriate validation.

Uridyltransferase (GALT) can be run with the biotinidase analysis simultaneously from the same extracted sample. For the GALT assay, two fluorometric detectors (active and blank channels) and two cartridges (active and blank) are required. The active cartridge includes an on-line incubator to enhance enzymatic response. Sample throughput for both tests is 90 per hour after an initial dwell time of approximately 90 minutes. back to top

G6PD Deficiency

The Astoria-Pacific SPOTCHECK G6PD test is used as an aid in screening for glucose-6-phophate dehydrogenase deficiency in newborns. Early detection of this enzyme deficiency can aid in the diagnosis and treatment of congenital nonspherocytic hemolytic anemia or drug induced hemolytic anemia associated with a G6PD deficiency.

Glucose-6-phosphate dehydrogenase is the initial enzyme in the hexose monophosphate pathway of glucose metabolism. In the SPOTCHECK method, enzyme activity is measured by observing the fluorescence produced after NADP+ is reduced to NADPH when glucose-6-phosphate is present as a substrate. As G6PD catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate, NADP+ is reduced to NADPH, which is measured by a fluorometer. The amount of NADPH produced is proportional to the G6PD enzyme activity in the sample. Maleimide, an inhibitor of 6-phosphogluconate dehydrogenase activity, is added to prevent the conversion of 6-phosphogluconate to ribulose-5-phosphate and additional production of NADPH.

Whole blood, spotted on standardized filter paper, S&S 903 or equivalent is suitable for analysis. The procedure is designed for use with one 1/8 inch spot but may be adapted to alternative punch protocols with appropriate validation. Plasma or serum may also be analyzed.

Other tests, such as Phenylalanine or Total Galactose, can be run with the G6PD analysis simultaneously from the same extracted sample. The cartridge includes an on-line incubator to enhance enzymatic activity, and a dialyzer to filter out interferences. Sample throughput is 90 per hour or 4-5 microtiter trays per day.  back to top