Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper.

BACKGROUND: Clinical differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype. METHODS: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 degrees C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers. RESULTS: Intra- and interassay CVs were <9% and <15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 degrees C were < or =27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls. CONCLUSIONS: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.     

Quality control of reflectometric determinations of glucose in dried blood spots on filter paper

We evaluated the effect of sampling of capillary blood on filter paper on the later analysis for glucose. We found the method simple and reliable. Determination at the central laboratory of glucose in blood collected onto filter paper and comparison of the results with those obtained with test strips read in reflectometers at outpatient units is easy. Collecting duplicate samples on filter paper facilitates quality control by avoiding the complications that arise from using quality-control solutions that are not directly comparable with fresh blood, and it avoids the disturbances of test strip chemistry attributable to the glycolysis inhibitors added to blood samples intended for quality control.     

A simple quantitative micromethod of arginase assay in blood spots dried on filter paper

An accurate, precise and sensitive method has been developed for measurement of arginase activity in erythrocytes in dried blood spots. The assay is based on colorimetric measurement of ornithine produced by enzymatic hydrolysis of L-arginine. Only 10 microliter of capillary blood collected on filter paper are required. One disc of 3 mm in diameter punched from the dried blood spot is used for the arginase assay. The whole procedure is performed in one test-tube and does not need deproteinization. The second disc of the same diameter is used for hemoglobin (Hb) measurement. There was a good correlation between activities determined in dried blood spots and fresh erythrocytes of the same blood specimens taken from 101 healthy adults and 49 children (corre. coeff. 0.955 and 0.968, respectively). Arginase activity in dried blood specimens was 68.3 +/- 22.7 in adults and 62.7 +/- 15.7 U/g Hb in children. In 118 newborns, the activity was 101.9 +/- 29.2. In 1270 residents of nursing homes screened for hyperargininemia the activity was 21.5-171.2 U/g Hb. In screening for arginase deficiency, the method may be used as a simplified qualitative test without Hb assay.     

Oral testosterone administration detected by testosterone glucuronidation measured in blood spots dried on filter paper

BACKGROUND: Blood sampling is not a common practice for sports drug testing. Our aim was to investigate whether dried blood spots on filter paper could be an alternative to plasma samples for monitoring steroid profiles in dope testing. METHODS: We collected dried blood spots and plasma from six healthy Caucasian subjects after an oral 120-mg dose of testosterone undecanoate (TU). Nonconjugated testosterone, testosterone glucuronide (TG), androsterone glucuronide (AG), and etiocholanolone glucuronide (EtG) were measured by gas chromatography-mass spectrometry in both matrices. 17alpha-Hydroxyprogesterone (17alphaOHP) and luteinizing hormone (LH) also were measured in the plasma samples. For comparison, similar measurements were done on samples obtained from the same subjects given 25 mg of testosterone propionate (TP) plus 110 mg of testosterone enanthate (TE) intramuscularly after a wash-out period. RESULTS: After oral TU intake, TG, AG, and EtG increased sharply, whereas nonconjugated testosterone did not change significantly. Results on dried blood spots correlated well with those on plasma. The TG/testosterone ratio in blood or plasma was verified to be a sensitive and specific marker (significantly increased for up to 8 h after intake; P <0.05) for oral TU intake but not for intramuscular administration of TP plus TE. Little suppression of plasma LH and 17alphaOHP was observed after a single oral dose of TU. One subject did not show a significant increase of blood TG after oral TU intake. CONCLUSIONS: The measurement of glucuronide conjugates in blood and plasma samples is relevant for sports drug testing when analyzing the steroid profile. Dried blood spots collected on filter paper are a suitable alternative to plasma for detecting testosterone abuse.     

Fluorescence polarization immunoassay for theophylline modified for use with dried blood spots on filter paper

We used dried blood spots successfully as alternative specimens for quantifying concentrations of theophylline in the circulation by a modified fluorescence polarization immunoassay (FPIA) with the Abbott TDx instrument. The method described is simple, rapid, and acceptably precise. More importantly, it can provide results comparable with those of the conventional serum assay. Results for 64 pairs of dried blood spots (y) and serum (x) specimens analyzed by the respective FPIA methods yielded the following regression parameters: y = 0.94x + 0.71, r = 0.988, and Sxy = 0.92. A major advantage of FPIA is that it requires only basic laboratory skills. When coupled with the use of dried blood spots, this system can be effective in remote theophylline monitoring, particularly suited for domiciliary care.     

Gaucher and Niemann-Pick diseases--enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards

BACKGROUND: Gaucher disease (GD) and Niemann-Pick (NP) disease are caused by deficient activity of the lysosomal enzymes acid beta-D-glucosidase (ABG) and acid sphingomyelinase (ASM), respectively. For diagnosis, these enzymes are usually measured in the extracts of leukocytes or cultured fibroblasts. Chitotriosidase (CTE), a chitinolytic enzyme, is markedly increased in the plasma of Gaucher patients. We describe methods for the assay of acid beta-D-glucosidase, acid sphingomyelinase, chitotriosidase, and alpha-N-acetyl-galactosaminidase (NAGA) as a control enzyme in blood spots that were dried onto filter paper. METHODS: To tubes containing a 3 mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 degrees C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. We examined 80 healthy controls, 54 Gaucher patients, 8 Niemann-Pick patients, 27 obligate carriers, and the newborn-screening cards (NSC) from a case of Gaucher and a case of Niemann-Pick disease. RESULTS AND CONCLUSION: The described methodology is useful to identify Gaucher and Niemann-Pick patients and controls, using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases on a newborn-screening card was clearly established. The newborn-screening card has been added to the biological materials that allow the identification of patients with Gaucher and Niemann-Pick diseases.     

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.     

Carnitine in dried blood spots: a method suitable for neonatal screening

A method is described which enables the quantitative determination of both free and total carnitine levels in dried blood spots. This method is suitable for neonatal screening for either primary or secondary carnitine deficiency. The 95% confidence interval for free carnitine was 26-76 mumol/l (median = 44) and for total carnitine was 35-102 mumol/l (median = 60).     

Thyroxine binding globulin radioimmunoassay in dried blood spotted on filter paper

A method is described for the determination of thyroxine binding globulin (TBG) in dried blood spotted on filter paper using reagents from a test kit for the measurement of TBG in plasma. By minor modifications of the recommended procedure it was possible to improve precision, sensitivity and tracer displacement. Appropriate TBG standard samples were prepared in 'artificial blood' consisting of a suspension of erythrocytes in buffer with bovine serum albumin (50 milligrams). There is a good correlation between plasma TBG RIA results and blood spot TBG RIA results (r = 0.93). Attention must be paid to the stability of the TBG in blood: our experiments show a decrease of TBG content if filter paper cards with dried blood are stored longer than one month.     

Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper--a potential screening method for familial type II hypercholesterolaemia

In the search for an effective neonatal screening programme for familial type II hypercholesterolaemia, we have developed and validated a radial immunodiffusion assay for measuring, in dried blood spots, the concentration of apolipoprotein B. The method has advantages over previously described methods, being cheaper, more robust, and suitable for partial automation. In 34 adults there was a close linear relationship between capillary and venous blood spot apolipoprotein B concentrations (r = 0.94), between capillary blood spot and serum apolipoprotein B also measured by radial immunodiffusion (r = 0.90), and between capillary blood spot apolipoprotein B and LDL cholesterol measured by ultracentrifugation (r = 0.85). Apolipoprotein B levels in serum and LDL cholesterol also correlated closely (r = 0.95, n = 40). In 30 normal 3- to 5-day old babies, heel prick blood spot apolipoprotein B levels were 280 +/- 70 mg/l (mean +/- SD) and 790 mg/l in one presumed heterozygote for familial hypercholesterolaemia. We conclude that serum apolipoprotein B measured by radial immunodiffusion accurately reflects adult LDL cholesterol levels, and that the assay, using dried blood spots on filter paper should permit both neonatal and adult screening programmes to detect familial hypercholesterolaemia.     

Radioimmunoassay of somatomedin-C in filter paper discs containing dried blood

We describe a simple radioimmunoassay (RIA) for estimating concentrations of somatomedin-C (Sm-C) in dried blood on filter paper. A single 3.2-mm blood spot specimen on filter paper is eluted overnight into buffer containing antibody and 125I-labeled Sm-C. The following day, bound and free hormones are separated by addition of goat anti-rabbit gamma globulin in 60 g/L polyethylene glycol solution. The correlation between values obtained for such blood-spot discs and the corresponding wet plasma is highly significant (r = 0.90, p less than 0.001). The relative concentrations (arbitrary units) of Sm-C as determined for specimens on filter paper from mothers and infants, and for cord bloods, are similar to those reported by others using acidified serum.     

HPLC assay of phenylalanine and tyrosine in blood spots on filter paper

Blood spots on filter paper are used to screen for phenylketonuria. We have developed a high pressure liquid chromatographic assay of phenylalanine and tyrosine after elution of these amino acids from filter paper. The analysis time is 20 min. The amino acids are separated using an ion-exchange resin with post-column ortho-phthalaldehyde derivatization and subsequent fluorescence detection. Other common amino acids do not interfere. The assay is linear from 20 pmol to at least 2,000 pmol with a coefficient of variation of 3%. The assay is as accurate as the determination of phenylalanine by ion-exchange ninhydrin-reactive methods currently in use, but is 100 times more sensitive. The method has the advantages of avoiding venipuncture, is cost effective, has a high sensitivity, and a rapid turnaround.     

Enzyme immunoassay of free thyroxin in dried blood samples on filter paper

We describe a double-antibody enzyme immunoassay for determination of free thyroxin (FT4) in dried blood samples on filter paper, with use of a T4-beta-D-galactosidase complex. The measurable range of FT4 concentration in two 3-mm blood discs, each of which contained about 2.7 microL of blood, was 1.9 to 93 ng/L, as determined by comparison with concentrations of FT4 in known serum standards. FT4 in blood samples dried on filter paper was stable for at least four weeks when kept dry at -20 degrees C, room temperature, or 37 degrees C. The mean coefficients of variation were 7.6% (within assay) and 6.4% (between assays). Results for FT4 by this method correlated well with those for serum determined by radioimmunoassay (r = 0.98). The proposed method can be used to differentiate persons with hyper- and hypothyroidism from normal subjects and those with abnormal concentrations of thyroxin-binding globulin. The procedure seems suited for screening studies.     

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.