Urinary markers of type I collagen degradation in the dog.

Urinary assays for type I collagen metabolites provide a non invasive index of bone resorption in humans, and are widely used in the management of patients with metabolic bone diseases. The specific aims of this study were to investigate the feasibility of using commercial human assay kits for quantifying the urinary excretion of type I collagen metabolites in dogs of different ages. Urine and serum samples were collected from 35 beagle dogs in five age groups (0 to 1 years; 1 to 2 years; 2 to 3 years; 3 to 7 years; > 8 years old). Urinary concentrations of pyridinoline (Pyd), deoxypyridinoline (Dpd), and the carboxy- and amino-terminal cross-linked telopeptides of type I collagen (CTx and NTx, respectively) were measured with commercial enzyme-linked immunoassay kits. Serum concentrations of another type I collagen metabolite, the carboxy-terminal cross-linked teloptide of type I collagen (ICTP), were measured with a commercial radioimmunoassay. Dilutional studies indicated that the four urinary assays show specific cross-reactivity with canine urine. Age-related differences in urinary marker excretion were identified, with young dogs excreting the highest concentrations of Pyd, Dpd, NTx and CTx. The correlation between the individual urinary markers was excellent (r = 0.87 to 0.98), while the correlation between serum ICTP and individual urinary markers was weaker (r = 0.52 to 0.64). These results validate the usefulness of the commercial assay kits in monitoring type I collagen metabolism in dogs. Histomorphometric studies have confirmed the relationship between collagen degradation and bone resorption in humans, and similar studies are now needed in dogs.

A comparison of two techniques for the determination of serum bone-specific alkaline phosphatase activity in dogs.

Bone-specific alkaline phosphatase (BALP) shows potential as a marker of bone formation in the dog. Recent studies have indicated that serum BALP may provide a useful, non-invasive indicator of skeletal health in dogs, and as a diagnostic and prognostic marker in the management of dogs with musculoskeletal or metabolic disorders. Two assay techniques (one based on wheatgerm lectin precipitation followed by a simple enzymatic reaction, the second on a specific enzyme-linked immunoassay) were used to measure serum levels of BALP in 35 dogs of different ages. As expected, BALP concentrations decreased with age. For the enzymatic assay, mean (+/-SD) serum concentrations of BALP activities were 100.3 (+/-11.6) U/liter in dogs under 1 year of age, 25.3 (+/-6.8) U/L in dogs 1 to 2 years of age, 16.5 (+/-7.3) U/L in dogs 2 to 3 years of age, 14.3 (+/-5.6) U/L in dogs 3 to 7 years of age, and 12.3 (+/-4.8) U/L in dogs aged 8 years and older. Corresponding results from the immunoassay were 56.3 (+/-9.8) U/L, 10.7 (+/-4.5) U/L, 7.0 (+/-2.5) U/L, 6.7 (+/-3.6) U/L and 7.0 (+/-2.9) U/L. There was excellent correlation between the results from the two assay techniques (r = 0. 96; P < 0.0001). The correlation between BALP and total ALP activities was poor (r = 0.20 for enzymatic BALP, r = 0.31 for immunoreactive BALP), indicating that total ALP should be considered unreliable as an indicator of BALP activity in canine serum. The immunoassay demonstrated acceptable (13 per cent) cross-reactivity with the liver isoform of ALP. The commercial immunoassay kit is simple and provides fast results. Although the wheatgerm lectin/enzymatic technique is preferred in situations where the activities of all three isoforms of ALP are required, the immunoassay should be considered whenever the activity of BALP is the focus of interest.

Interference of IgM paraproteins in the Olympus AU800 uric acid assay.

INTRODUCTION: In the Olympus uric acid procedure, uric acid is converted by uricase to allantoin and hydrogen peroxide, which is reacted in a Trinder reaction to produce a chromophore read bichromatically at 520 and 660 nm. Repeated difficulty was encountered in obtaining uric acid results on samples from myeloma patients with known IgM paraproteins. Large absorbances in sample blanks were due to a visible precipitation observed in the reaction cuvettes. OBJECTIVE: To alter the Olympus method (OM) to eliminate the interference by IgM, and to verify the modified method (MM). METHODS: Dilution of the sample blank by saline was substituted for water in the MM, with small alterations in the reaction timing sequence necessary to accommodate the instrument requirements. RESULTS: A comparison of uric acid results obtained from nonmyeloma patient samples using the OM and the MM showed a good correlation (r = 0.970), and no statistical difference between the two means using a paired t-test. A similar comparison performed using the samples containing IgA and IgG paraproteins also revealed a good correlation (r = 0.981), and no statistical difference between the two means. Results on IgM containing specimens were assessed indirectly because the samples could not be assayed with the OM. First, removal of detectable levels of proteins using a 20% TCA solution did not affect the measurement of uric acid. Second, protein-free supernatants from IgM containing samples were measured by the OM and compared with the corresponding serum samples measured by the MM. There was good correlation between the two methods (r = 0.945), and no statistical difference between the means using a paired t-test. CONCLUSION: The modified method is satisfactory for routine analysis of samples, including those with IgM paraproteins.

Comparison of the Ames, Randox and Roche methods with the Synermed method for the determination of serum iron concentrations on nondialysis and dialysis specimens.

OBJECTIVES: To evaluate the Ames, Randox, Roche, and Synermed methods for the measurement of serum iron and to investigate patterns of possibly discrepant results in dialysis patients. METHODS: Assays were performed on the Cobas Fara II analyzer. Precision and accuracy studies were conducted; recovery studies were done by adding pooled serum from dialysis patients to an assayed human serum-based control. Patient comparisons included over 150 nondialysis patients and 30 dialysis patients. RESULTS: For the Ames, Randox, Roche and Synermed methods, the between-run precision was less than 2.80% with the normal aqueous iron standard; 2.00, 2.70, 0.80, and 2.00% for the four methods with the high serum iron control, respectively, and less than 2.30% with the serum pool. Using a pooled serum from dialysis patients, between-run precision was higher with all four methods. With an abnormal assayed human serum-based control, accuracy was over 98% for the four methods. Recoveries were 121% for the Ames and Randox methods and 104-105% for the Roche and Synermed methods. Accuracy as assessed with Murex EQAS specimens ranged from 71 to 80%, 71 to 96%, 98 to 99.5%, and 42 to 50% for the four methods, respectively. For comparisons of the Ames, Randox, and Roche methods with the Synermed method, difference analyses revealed biases (SD) for nondialysis patients of 1.9 (2.7), 1.5 (3.3), and 1.8 (2.2) mumol/L, respectively; and for dialysis patients of 8.2 (13.3), 5.1 (5.4), and 1.4 (1.7) mumol/L. Standard linear regression analyses and correlation coefficients are also provided. CONCLUSIONS: The Roche method was slightly more precise than the other methods. Using an abnormal assayed serum-based control, all methods showed good accuracy. Recovery studies with pooled serum from dialysis patients showed interferences with the Ames and Randox methods and good recovery with the Roche and Synermed methods. With the bovine serum-based Murex samples, all but the Roche method yielded some low results; the Synermed method has been reported to suffer from matrix problems with bovine serum albumin. Based on recovery studies and difference analyses, the Ames and Randox methods revealed discrepancies in iron results for samples from dialysis patients. The Roche and Synermed methods appeared to be suitable for measurement of serum iron in dialysis patients.

Prostate-specific antigen in amniotic fluid of normal and abnormal pregnancies.

OBJECTIVE: To examine if prostate-specific antigen (PSA) is present in amniotic fluid or maternal serum during pregnancy and if its presence is associated with fetal abnormalities. METHODS: Samples tested included amniotic fluids from 853 pregnant women for whom amniocentesis was performed; 312 nonpregnant women who donated blood; 259 pregnant women who donated blood at various gestational ages. Amniotic fluid or serum PSA was measured with an ultrasensitive time-resolved immunofluorometric procedure. 372 pregnancies were studied for the presence of genotypic or phenotypic fetal abnormalities. RESULTS: PSA was present in most amniotic fluids; the median PSA concentration increased from gestational week 11 to 22 and stabilized thereafter until delivery. The most prominent PSA concentration change occurred during gestational weeks 13-14. Pregnant women had significantly higher serum PSA concentrations than nonpregnant women; the pattern of serum PSA concentration change during pregnancy was similar to that of amniotic fluid; however, serum PSA concentrations were lower by a factor of 20-40. No association existed between amniotic fluid PSA and maternal age, gender of fetus, or length of abstinence of mother from sexual intercourse. After gestational week 15, fetuses with trisomy 21 or 18, anencephaly, or renal disorders were associated with low amniotic fluid PSA levels. CONCLUSION: Our data suggest that PSA may play a role in fetal development, especially at gestational ages between 13-20 weeks. The diagnostic usefulness of PSA in identifying fetal abnormalities remains to be determined.

Postdoctoral training in clinical chemistry: laboratory training aspects.

OBJECTIVE: To provide a general outline for a 2-year postdoctoral training program in clinical chemistry, and a detailed outline of the first year laboratory training program. METHODS & RESULTS: Essential elements of the 2-year Postdoctoral Training Program in Clinical Chemistry at the University of Toronto are its didactic courses and a comprehensive, structured laboratory rotation in the first year. Residents rotate in hospital laboratories in both years of the Program. The hospital laboratory rotation in first year includes a 36-week laboratory rotation based on the Laboratory Training Program Manual. In the second year, they consolidate the basic knowledge acquired in first year and gain experience in pediatric testing and other specialty areas. In both years, residents attend teaching and ward rounds on a regular basis, investigate unusual test requests and patient results, and make regular presentations at case presentation and journal club sessions. They undertake research and development projects which lead to presentations at scientific meetings and to publication. Residents attend departmental management meetings, arrange discussions on management topics, and attend a short course on key management topics. Approaches for strengthening the knowledge and skills of residents in the areas of hematology, microbiology and pathology are being developed. CONCLUSION: The program outline described should provide a useful framework for other such programs both nationally and internationally.

Pregnancy outcome after gestational exposure to amiodarone in Canada.

OBJECTIVE: Our purpose was to quantitate the risk of perinatal thyroid dysfunction and other amiodarone-induced adverse effects among infants exposed in utero to amiodarone. STUDY DESIGN: A historic cohort study of gestational exposure to amiodarone was conducted by contacting Canadian cardiac electrophysiologists. RESULTS: Twelve cases were identified. Of six with first-trimester exposure, one child had congenital nystagmus with synchronous head titubation. There was one case each of transient neonatal hypothyroidism (9%) and hyperthyroidism (9%). A fourth child, exposed to amiodarone from 20 weeks' gestation, had developmental delay, hypotonia, hypertelorism, and micrognathia. Four small-for-gestational-age infants were also exposed to beta-blockers, which in addition to maternal cardiac disease, have been recognized to cause growth restriction. beta-Blockers may also have contributed to bradycardia in one of the three fetuses in whom this was observed. CONCLUSIONS: Gestational exposure to amiodarone may be complicated by perinatal hypothyroidism or hyperthyroidism and possibly neurologic abnormalities, intrauterine growth retardation or fetal bradycardia. Concomitant beta-blocker therapy should probably be avoided. Full neonatal thyroid function tests and developmental follow-up are recommended.

Accelerated gallstone dissolution in methyl tert-butyl ether by sonication. An in vitro study.

OBJECTIVES: The authors tested the effect of 195 KHz therapeutic ultrasound energy on gallstone dissolution in concert with methyl tert-butyl ether (MTBE) in vitro. METHODS: Sixteen sets of three gallstones matched for weight and appearance were selected from 16 surgically resected human gallbladders. One stone from each set was analyzed for its density pattern by computed tomography (CT) and biochemically for cholesterol content. Based on CT appearance, the stones were classified into eight noncalcified, four partially calcified, and four heavily calcified sets. The three stones were subjected to dissolution with MTBE: one with simultaneous sonication via an experimental ultrasound unit, one with manual stirring, and one acted as control without added treatment. RESULTS: Sonication reduced the dissolution time of noncalcified stones by 96% (range, 94%-98%; standard deviation [SD], 2%) relative to controls, and it was three to four times more effective than manual stirring. It was similarly effective in helping to dissolve partially calcified stones, but not heavily calcified stones. CONCLUSIONS: This study demonstrates the positive effect of sonication in accelerating gallstone dissolution with MTBE in vitro for stones without heavy calcification.

Gallstone dissolution in methyl tert-butyl ether after mechanical fragmentation: in vitro study.

Twenty sets of three gallstones matched for weight and appearance were selected from 20 surgically resected human gallbladders to test the effect of intracorporeal mechanical fragmentation on gallstone dissolution with methyl tert-butyl ether in vitro. One stone from each set was fragmented by a mechanical lithotriptor and then treated with methyl tert-butyl ether, and one was used as control and was treated intact. The third stone was analyzed for its density pattern on CT and biochemically for its cholesterol and calcium content. On the basis of CT appearance, the stones were classified as noncalcified, partially calcified, or heavily calcified. Mechanical fragmentation reduced dissolution time by 25-69% (mean +/- SD, 44 +/- 16%) for the noncalcified stones and by 20-42% (mean +/- SD, 30 +/- 8%) for the partially calcified stones. No significant reduction was observed for the heavily calcified stones. The degree of reduction was inversely related to maximal stone density (r = -.72) and was independent of its pattern of calcification. This study shows that mechanical fragmentation is effective in accelerating gallstone chemolysis by methyl tert-butyl ether for noncalcified and partially calcified but not for heavily calcified stones.

An enzymatic method for oxalate automated with the Cobas Fara centrifugal analyzer.

Measurement of oxalate in urine has been automated for use with the Cobas Fara centrifugal analyzer. No sample pretreatment other than (a critical) pH adjustment is required. Between-run CVs were less than 4%. Results were linearly related to oxalate concentration to 1000 mumol/L. Ascorbic acid ingestion, up to 5 g of ascorbate daily, caused no demonstrable interference with the assay. This practical, automated method for assaying urinary oxalate is substantially faster than other chemical or enzymatic methods.

Maternal serum alpha-fetoprotein screening: report of a Canadian pilot project.

A pilot project of maternal serum alpha-fetoprotein (MSAFP) screening was carried out in Ontario from 1982 to 1985 to examine the feasibility and acceptability of screening a prenatal population for open fetal neural tube defects. A total of 8140 patients at low genetic risk were screened. Patient acceptance was excellent. Blood samples were taken at 16 to 18 weeks' gestation. If the MSAFP level was elevated, the assay was repeated and an ultrasound examination performed. Amniocentesis was offered to 67 women with unexplained persistently elevated levels. The outcome of pregnancy was known in 7473 patients (91.8%). Seven of nine known open fetal neural tube defects were detected. All were confirmed, and no unaffected fetuses were aborted on the basis of the screening results. The rates of perinatal death (6.7%), intrauterine growth retardation (11.7%) and prematurity (23.3%) were significantly higher among the patients with unexplained elevated MSAFP levels than among those with normal levels (p less than 0.001). Of 20 patients with unexplained low levels, 10 subsequently had spontaneous abortions and 10 gave birth to term appropriate-for-gestational-age infants. Seven of nine patients who gave birth to infants with autosomal trisomy had MSAFP values below the median. The findings indicate that MSAFP screening is feasible, accurate and acceptable in a low-risk area.

Assessment of four enzyme immunoassay kits for serum beta-hCG screening.

This pilot study evaluates four qualitative serum beta-human chorionic gonadotropin (beta-hCG) enzyme immunoassay kits for both routine pregnancy testing and screening for ectopic pregnancy. We found that the Tandem-E HCG assay had the lowest limit of detection, and longest turnaround time. Tandem ICON HCG assay had the shortest turnaround time. The Vis-Con II was the most economical batch test. The Quest assay kit was both fast and simple and had the lowest cost for stat testing.

Daily maintenance dose of a long-acting theophylline from a single theophylline serum level.

A single point method has recently been described whereby the daily maintenance dose of theophylline required to achieve a desired steady state serum concentration can be established from a nomogram with the aid of a single blood sample drawn after a test dose. This approach has been validated for intravenous administration and for a rapidly absorbed elixir (coefficient of absorption ka 2.3 h-1) given to children. In order to study whether this method could be applied to a slowly absorbed preparation (ka 0.44 h-1), we gave theophylline (Theo-Dur) to nine adults as a single dose and for one week. In applying a nomogram, we found that a sample taken nine hours after a test dose would more accurately predict the daily dose required to achieve a desired steady state concentration than a sample taken at six hours as has previously been recommended (error nine hours -16.8 percent to +29 percent, six hours -59.7 percent to +29.4 percent).

Effects of hemoglobin variants on Hb A1c determinations using Bio-Rad columns.

In the column chromatographic determination of Hb A1c, hemoglobin variants affect Hb A1c results and column patterns. Samples from several patients with hemoglobin variants were run on Bio-Rad Hb A1c columns to demonstrate these patterns. Columns should be examined during the run to detect the presence of Hb F which migrates very rapidly, and after the run to detect abnormal column patterns. Hb A1c levels in patients heterozygous for Hb S or C are low but may be interpreted in relation to the patient's level of Hb A. On the other hand, Hb A1c results on patients with homozygous Hb S, Hb C, or high levels of Hb F cannot be interpreted.

An automated "high-pressure" liquid-chromatographic assay for hemoglobin A1c.

An automated "high-pressure" liquid-chromatographic assay for hemoglobin A1c is described. We use a 45-min incubation in acetate buffer (pH 5.5) to eliminate labile glycated hemoglobins. In this automated system conventional modules are used but it incorporates a solvent-switching valve to select either of two buffers, which differ in pH and NaCl concentration. The chromatographic column contains "polyCAT" (a weak cation-exchanger, polyaspartic acid linked to silica). Run time is 14 min per sample. The method is precise and results correlate well with those by other ion-exchange procedures.