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We describe the history and current implementation of an inexpensive thin layer chromatography (TLC) method, vertical sandwich‐type continuous/evaporative TLC with fixed mobile phase volume, that is convenient for detecting and identifying reducing sugars of clinical relevance in the paper‐borne blood and urine samples collected in neonatal screening programmes. This method facilitates screening by providing a considerable degree of standardization of chromatographic results. Among some 555,000 newborns to which it has been applied, it has detected 10 cases of classical galactosaemia, 7 cases of galactokinase deficiency, 2 cases of glucosuria, and 3 cases of transitory neonatal diabetes mellitus; the only false negatives we are aware of were two cases of galacto‐4‐epimerase deficiency detected by tandem mass spectrometry. Screening for sugars in urine has allowed the detection of galactosaemia when the accompanying blood sample was invalid because of transfusion or parenteral feeding. The conclusion is that this inexpensive procedure is very useful for the detection of relevant metabolopathies in circumstances where others fail. J. Clin. Lab. Anal. 24:106–112, 2010. © 2010 Wiley‐Liss, Inc.

Screening for hypothyroidism has been achieved by seeking raised concentrations of thyroid stimulating hormone (h-TSH) in dried blood samples collected on absorbent paper from some 1700 newborn babies each week using double antibody radioimmunoassay and 125I-label counting on a NE1600 gamma counter linked to a HP9815S desk top calculator. Specificity, accuracy, and quality control of the assay have been investigated. The calculator programme stores all count, time, and calculated data and uses log-log function formation and third-power homoscedastic polynomial fit to the calibration data followed, if necessary, by elimination of outliers set at p= 0·5 %. Branching in the calculation procedure provides the ability to process sample data in subgroups, completely to reprocess data (including presentation in a form suitable for making a histogram plot), and to reset without loss of data in case of instrument or main power failure. Variance analysis was used to select the minimum duration of count (50 s), and it was demonstrated that homoscedasticity pertains in the regression calculation.

We have recently developed an immunoassay that can measure thyroxine rapidly and accurately in the eluate of 40 mul of dried blood spotted on filter paper at the fifth day of life. The method is completely automated and by using the samples received by the Central Laboratory of the Quebec Network for Genetic Medicine and their follow-up facilities, we are now screening every newborn in the province of Quebec for neonatal hypothyroidism. To date, from 47,000 measurements, three newborn infants with abnormally low TBG and seven hypothyroid infants have been detected. From these data we conclude that the frequency of congenital hypothyroidism is about one in 7,000 births and that our method is effective in detecting thyroid hormone abnormalities with an acceptable percentage of false positive measurements; no false negative results have occurred to our knowledge.

Agar gel, cellulose acetate, and starch gel electrophoresis are all capable of diagnosing sickle-cell anaemia, sickle-cell haemoglobin C disease, and haemoglobin C disease in cord blood samples. Of these three electrophoretic techniques, agar gel is the easiest to interpret. Paper electrophoresis can reliably and rapidly detect sickle haemoglobin and haemoglobin C in cord blood samples. Being incapable of differentiating foetal and normal adult haemoglobin, the value of paper electrophoresis is limited to an initial screening procedure.

We describe a sensitive, simple, and rapid two-site immunoenzymometric assay for thyrotropin in dried blood samples on filter paper, for use in screening for neonatal primary hypothyroidism. In this method, two dried-blood spots of 3 mm diameter (equivalent to about 5.4 microliter of blood) are incubated overnight with anti-thyrotropin-beta-D-galactosidase complex in an anti-thyrotropin coated tube. Then the enzyme activity in the washed tube is determined fluorophotometrically. The range of thyrotropin measurable is 10 to 160 milli-int. units/L blood. Values for thyrotropin in dried blood samples determined by this method and those determined by radioimmunoassay correlated highly (r = 0.96).

A new sensitive radioimmunoassay method for measuring reverse triiodothyronine (rT 3 ) concentrations in dried blood samples, designed to screen newborn infants for congenital hypothyroidism, has been developed. Paper strips are impregnated with cord blood and dried. Duplicate 5-mm diameter discs are punched from the paper strips and added directly to the radioimmunoassay reaction mixture. After incubation, bound and free hormone are separated by dextran-coated charcoal. The disc remains in the solution throughout the procedure and the assay can be completed within 24 hr. Recovery of rT 3 is greater than 95% and coefficients of variation are 9.4% (intraassay) and 12.2% (interassay) at an rT 3 concentration of 220 ng/dl. At very low rT 3 concentrations (25 ng/dl), coefficients of variation are 14.2% (intraassay) and 18.7% (interassay). The method readily detects 12.5 ng/dl of rT 3 . With this paper disc method, rT 3 was measured in 38 newborns and compared with serum rT 3 measured in the same subjects by a standard radioimmunoassay method. The correlation between rT 3 values measured in dried blood disc and in serum was very high ( r = 0.918). The rT 3 in dried blood discs from the cord blood of 745 normal newborns was 228.9 ± 76.0 ng/dl (mean ± SD). In contrast, two infants with proven congenital hypothyroidism had rT 3 values of 35 and 75 ng/dl, respectively. This study indicates that rT 3 can be easily measured in dried blood discs and suggests that the described method may be a useful screening procedure in a program for the detection of neonatal hypothyroidism.

Filter paper discs saturated with dried blood can be used in the Immunotube solid-phase thyroxine radioimmunoassay. This assay utilizes polypropylene tubes to which antibody to thyroxine is covalently bound. The filter paper standards and samples are placed in the tubes, followed by an assay buffer that contains I125-labeled thyroxine and compounds to displace thyroxine from its binding proteins. After incubation, bound and free thyroxine are separated by aspirating or decanting the disc and buffer from the tube. The test can be used with 0.32 cm (1/8 inch) or 0.64 cm(1/4 inch) discs, and gives quantitative results that correlate well with those for serum samples. The intra-assay coefficient of variation is less than 10%. The assay may readily be mechanized with existing disc-punching equipment, and results of its use in mass screening programs are described.

We describe a microfluorometric method for determination of arginase activity in dried blood spots on filter paper. The arginase in discs punched from such dried blood specimens is activated by preincubation with Mn2+ at 37 degrees C. After incubation with substrate at the same temperature, urea is determined fluorometrically by oxidation of NADH to NAD+ in a coupled kinetic reaction. We compare the results of this method with those of a colorimetric method involving liquid blood samples, and assess the stability of the enzyme in dried blood on filter paper. The presence of serum has no effect on the activity. This method may be useful in the early detection of arginase deficiency and certain hematological disorders.

Experience with over 6000 Guthrie tests is presented. This test is a screening procedure for phenylketonuria using small amounts of blood spotted on a filter paper which are tested by a bacterial "inhibition assay". Certain technical aspects of the test (e.g. relation between the concentration of phenylalanine in the blood and extent of the bacterial growth zones produced, type of filter paper, size of the blood spot on the paper) were investigated. It was shown that the Guthrie test clearly distinguishes between subjects with normal plasma phenylalanine levels and patients with untreated phenylketonuria.Applications of the Guthrie test in screening a mental hospital population, admissions to a penitentiary and newborn babies are described.

The method of using automated analysis of galactose with galactose oxidase in blood absorbed on discs of filter paper is proposed. A galactose concentrations of 33–100 mg/100 ml the recovery was about 80 per cent the day, 60 per cent after 2 weeks storage at room temperature, and nearly 100 per cent after storage at −80° C for various lengths of time. Blood samples from 202 healthy infants five days old gave zero values for galactose. As yet, no infant with hereditary galactosaemia has been investigated.