Blood Spot Assay Research Articles

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

Fluorometric method for phenylalanine microplate assay adapted for phenylketonuria screening.

We adapted the method of McCaman and Robins for fluorometry of phenylalanine to a microplate assay for routine phenylketonuria screening. Sensitivity is 15 mumol/L for the plasma assay and 30 mumol/L for the dried blood-spot assay, with CV less than 10% for both assays. Results for human plasma by microplate assay correlated well (r = 0.99) with results of amino acid analyzer determination of phenylalanine. When measurements are performed in an automated reader, the microplate assay has considerable advantages over conventional measurements in cuvettes: smaller volumes of reagents and automation enabling high throughput and convenience. Because the described method is a quantitative one, we can postulate that, compared with the semiquantitative Guthrie inhibition bioassay, this microplate assay is more reliable, easier to perform, and about twofold less costly.

Thyrotropin Enzyme-Immunoassay in Dried Blood Spots: A Spectrophotometric Method for Neonatal Thyroid Screening

We describe an enzyme-immunoassay with photometric endpoint determination, suitable for the measurement of thyrotropin (TSH) in dried blood spotted on filter paper. Using reagents of a commercially available test kit provided for the measurement of thyrotropin in 200 microliter serum, we have adapted the method to the determination of thyrotropin in blood spots containing ca. 10 microliter blood. This was achieved by prolongation of the assay time from 3 to 20 hours, and by increasing the amount of enzyme-antibody complex. Precision and sensitivity of the blood spot assay are comparable to those of our in-house thyrotropin RIA, and the RIA/EIA correlation coefficient is 0.987 (n = 150). The advantages of EIA are the simplicity of the photometric end point determination (although strict time control has to be observed in order to avoid drifts in results), the long stability of reagents, and the non-isotopic label. The method therefore appears to be a suitable alternative to thyrotropin RIA for the determination of thyrotropin in neonatal thyroid screening.

118 CONGENITAL ADRENAL HYPERPLASIA (CAH) MONITORED BY SERIAL 170H-PROGESTERONE (17P) DETERMINED IN 3 MATRICES

A blood spot (BS) assay was developed for use in infants to supplement plasma/saliva 17P assays used to monitor older CAH children. 17P recovery from a 6 mm blood disc immersed in ethenol, eluted with buffer and solvent extracted was >95%. A magnetised solid-phase antiserum and 125I-tracer was used as in the routine 17P assay. Sensitivity was 1.5 pg/tube (0.5 nmol/L). Simultaneous plasma, saliva and BS samples were collected 1-2 hrly for 12-24hr from treated patients. Profiles of 17P in all 3 matrices was identical, whatever the control (r=0.99). Weekend profiles of BS and saliva 17P in samples collected at home are shown in the Table (means of 2 daily profiles). BS and saliva 17P values indicated the degree of control, identified over-treatment and again showed a diurnal rhythm in the under-treated. Selective use of plasma, BS and saliva 17P measurement now permits detailed monitoring of CAH at all ages.

Serum pancreatic lipase as a screening test for cystic fibrosis.

Pancreatic lipase catalyses the hydrolysis of emulsified triglycerides to form a transparent solution of monoglycerides and fatty acids. Levels of serum pancreatic lipase were measured in neonates known to have cystic fibrosis and compared with levels in control infants. During the first weeks of life infants with cystic fibrosis had raised serum pancreatic lipase values in parallel with raised serum trypsin values. A simple and specific turbidimetric dried blood spot assay for serum pancreatic lipase was used as a screening test fo cystic fibrosis in the neonate.

Neonatal screening for hypothyroidism in Scotland: results of a pilot study.

We have assessed the feasibility of screening for neonatal hypothyroidism based on thyroxine (T4) or thyrotrophin (TSH) assays on dried blood spots collected for the phenylketonuria (PKU) screening programme in Scotland. The TSH assay was both more sensitive and specific than T4 in detecting primary hypothyroidism and was sufficiently rugged and rapid for routine application. Using a labelled antibody assay for TSH, 99.7 per cent of the first 30,000 babies tested had values less than 25 mU/l, and 0.24 per cent had values between 25 and 50 mU/l. Of the 11 suspected positives (TSH greater than 50 mU/l), nine were confirmed on serum specimens as primary hypothyroidism (prevalence 1:3,300) and the two false positives (0.007% of total) were explicable by inappropriate time of sampling. No false negatives have so far been identified. The median time to detection by blood spot assay was 16 days (range 6 to 22 days). Five of the nine cases were not suspected clinically at the time of screening. It is concluded that TSH fulfils most of the criteria for an ideal screening test for primary hypothyroidism and a screening programme based on blood spot TSH will allow therapy to be commenced within one month of birth.

Blood-spot thyrotropin radioimmunoassay in a screening program for congenital hypothyroidism.

We describe a highly sensitive and precise radioimmunoassay for thyrotropin in dried blood spots on filter paper cards. In a screening program for congenital hypothyroidism, blood-spot thyrotropin concentrations are measured in infants whose blood-spot thyroxine concentrations are in the lower 10%, and this strategy has reduced the recall rate from 1.7% (thyroxine assay alone) to 0.17%. Thyrotropin assay samples consist of discs 4.5-mm in diameter, containing about 6 microL of blood, punched from blood spots. By appropriate attention to assay conditions, a mean least-detectable thyrotropin concentration equivalent to 2.5 milliunits/L plasma has been achieved. Concomitant measurement of thyrotropin by plasma and blood-spot assays in 91 subjects yielded a Spearman rank correlation coefficient of 0.9732. An analysis of variance of the distribution volume of thyrotropin in blood spots and a covariance analysis of factors affecting blood-spot thyroxine results are presented.

DRIED-BLOOD SPOT SCREENING FOR CYSTIC FIBROSIS IN THE NEWBORN

Serum-immunoreactive-trypsin (I.R.T.) was measured in children with cystic fibrosis (C.F.) and a variety of controls. In the first few months of life all C.F. children had a raised serum-I.R.T. A dried blood-spot assay for I.R.T. was established and has potential as a screening test for C.F. in the newborn.