Abnormal chromatographic patterns of porphyrins in urine.

Urine is the most frequent specimen used in the initial evaluation of adult patients who present with photosensitivity. When urine porphyrins are abnormal, characterization of the chromatogram is facilitated by calculation of uroporphyrin-to-heptacarboxylate porphyrin (uro/hepta) and uroporphyrin-to-isocoproporphyrin (uro/iso) ratios. The most frequent abnormal pattern, and that most consistent with porphyria cutanea tarda (PCT), is an uro/hepta ratio < or =2.0 and an uro/iso ratio < or =18. When the uro/hepta or uro/iso ratios are less consistent with PCT, other less common porphyrin disorders should be considered. These include variegate porphyria, coproporphyria with manifestations of photosensitivity only, adult onset congenital porphyria, mixed porphyrias, and other less frequent porphyrin disorders. After initial evaluation, the diagnosis should ideally be confirmed by additional testing of blood and fecal specimens. Most attacks of the acute porphyrias are associated with a uro/hepta ratio >4, and can be confirmed by an elevated urine porphobilinogen concentration.

Maternal serum alpha-fetoprotein and altitude.

Maternal serum alpha-fetoprotein (MS-alphaFP) testing is widely used to screen for fetal defects. MS-alphaFP concentrations are affected by a number of variables such as gestational age, maternal weight, number of fetuses, race, and insulin-dependent diabetes. Undefined geographic factors may also influence MS-alphaFP. We have examined the effect of altitude in a sample of 1063 MS-alphaFP results selected to span a range of altitudes. The study sample was subjected to linear regression with and without a term for altitude, and multiple-of-the-median (MoM) values were calculated before and after adjusting for altitude. The median MS-alphaFP was found to decrease an average of 1 ng/mL for every 1100 ft increase in altitude, a change approximately equivalent to that seen with an increase in maternal weight of 6 lb. Adjusting for altitude resulted in the reclassification of 36 of 1063 patient results (3.4%), although the clinical utility of this adjustment remains unexamined.

Identifying delayed separation in plasma homocysteine specimens.

The delayed separation of plasma from the cellular components of blood can lead to falsely elevated homocysteine results. The incidence of delayed separation in patient specimens was examined by using the ratio of arginine to the sum of arginine plus ornithine [arg/(arg + orn)], a ratio <0.50 being consistent with delayed separation. Two groups were examined: 1) low homocysteine (<10 micromol/L, n = 10), and 2) elevated (>18 micromol/L, n = 12). Specimens in the low group showed an average ratio of 0.47, with 6 of 10 <0.50. In contrast, specimens in the elevated group showed an average ratio of 0.31, with 11 of 12 <0.50. Characteristics consistent with delayed separation occurred frequently, but were significantly more frequent (p = 0.009) in the elevated group. This suggests that many homocysteine results may be falsely elevated due to preanalytic collection problems.

Evaluating serum alpha-tocopherol (vitamin E) in terms of a lipid ratio.

It is well known that accurate assessment of serum alpha-tocopherol requires knowledge of the serum lipids also present. Patient specimens (n = 307) within the standard reference limit of 5-18 mg/L were used to determine the following 95 percent reference interval for a lipid ratio of alpha-tocopherol to the sum of cholesterol and triglycerides [E/(C + T)]: 1.4 (1.20-1.56) - 5.7 (5.51-6.91) mg/g (90 percent confidence interval). In terms of alpha-tocopherol status, patients with low results (< 5 mg/L) were normal on reevaluation with the lipid ratio in 47 percent of those examined (28 of 59), and elevated results (> 18 mg/L) were normal or low in 58 percent (26 of 4.5). Elevated triglycerides developed from non-fasting specimens were one common reason for misleading results when lipids were not considered. When measuring alpha-tocopherol in a patient population, evaluation of the lipid content is needed for accurate assessment in a significant number of cases.

Delayed separation and the plasma amino acids arginine and ornithine.

When collecting blood for amino acid testing, leaving plasma in contact with cells at room temperature lowers the concentration of arginine and raises that of ornithine. This is presumably due to the arginase content of red blood cells. In contrast, the sum of arginine and ornithine is constant over the first hour, and defines a reference interval of 74-148 mumol/L (mean +/- 2 SD, n = 20) which is more insensitive to delayed separation. The ratio of arginine to the sum of arginine plus ornithine [arg/(arg + orn)] can be used to estimate the number of specimens not separated promptly. A ratio of 0.74-0.50 (mean +/- 2 SD, n = 20) is characteristic of specimens placed on ice and separated promptly, where delayed separation produces lower ratios. Of 91 adult specimens received for plasma amino acid analysis over five months, 35 (38 percent) showed a ratio < 0.50 suggestive of delayed processing.

The incidence of elevations in urine 5-hydroxyindoleacetic acid.

A 24-hour urine collection for 5-hydroxyindoleacetic acid (HIAA) is commonly performed to evaluate patients with suspected carcinoid syndrome. However, carcinoids are rare, and elevated results are common even when using an analytically specific method. To characterize this problem, the incidence of elevated results was examined in a population of 947 patient specimens received in a clinical reference laboratory setting. Using a reference limit of 15 mg/d identified 7.9 percent of the results as elevated, with 3 percent > 100 mg/d, and about 1 percent > 350 mg/d. Males showed 14 percent > 15 mg/d compared to 5.2 percent for females. Characterization of incomplete and excess 24-hr urine collections is facilitated by use of a creatinine ratio, with a reference limit of 14 mg/g creatinine equivalent to 15 mg/d. Given the frequency of elevated results, HIAA should be used to support the diagnoses of carcinoid only when consistent with other objective findings.

Effect of storage on serum vitamin B12 and folate stability.

To facilitate transport from remote locations, the stability of vitamin B12 and folate was investigated in serum specimens. Serum vitamin B12 proved to be highly unstable, emphasizing that specimens should be frozen if not analyzed immediately. Light protection is necessary if the sample cannot be analyzed within 4 hours. In contrast, folate is a more robust analyte. In refrigerated serum specimens, folate was stable up to 7 days of storage. In situations where specimen stability is important, vitamin B12 status is better assessed with serum or urine methylmalonic acid measurements. Although folate status can be assessed in a similar fashion with homocysteine, specimen stability indicates that direct measurement of folate is a better strategy.

The adequacy of capillary specimens for determining whole blood lead.

In response to demands for reliable alternatives to collection of venous specimens for determination of whole blood lead levels in children, the Centers for Disease Control has called for increased research into capillary methodologies. In this study, a three tiered approach was developed to assess the adequacy of capillary specimens for determining whole blood lead. Patient blood lead results from capillary and venous specimens were compared for obvious differences. Next, follow-up specimens for patients with elevated lead levels were compared with the initial results. In addition, experiments were conducted to determine whether or not handwashing eliminates gross contamination. Although the differences are not clinically important, the mean, 3.83 micrograms/dL for 5,100 venous specimens, was significantly lower (p < 0.005) then the mean of 4.6 micrograms/dL for 1,100 capillary specimens. Gross contamination was rare. Lead levels in follow-up specimens on patients whose initial screens were elevated were generally low. Handwashing greatly reduced the amount of external lead contamination. It is concluded that capillary specimens are an acceptable alternative to venous specimens for whole blood screening programs provided the patient and collector meticulously follow the prescribed collection protocol. Nevertheless, all elevated whole blood lead screening results, venous or capillary, should be confirmed with a venous collection before follow-up action is taken.

Serum succinate by capillary zone electrophoresis: marker candidate for hypoxia.

Serum succinate may offer an alternate analyte to lactate for the evaluation of hypoxia. To evaluate the potential uses of succinate, a relatively rapid capillary zone electrophoresis assay was developed for use in the clinical laboratory setting. Employing a simple indirect ultraviolet detection method with commercially available instrumentation, the limit of detection for serum succinate was determined to be 0.1 mumol/L, the upper limit of linearity 100 mumol/L, and the between-run coefficient of variation about 15 percent. Based on specimens from 202 apparently healthy adults, the non-parametric reference interval was 1.0 to 9.2 mumol/L. Preliminary studies in stored blood show succinate increased 2-fold while lactate increased 11-fold, suggesting that succinate may be a clinically useful marker for hypoxia in patients after blood transfusion. This assay provides a practical tool for the investigation of the clinical applications of succinate.

Testing for inborn errors of metabolism in the acutely ill newborn.

The differential diagnosis of the acutely ill newborn should include inborn errors of metabolism along with much more common conditions such as sepsis and hypoxemia. Testing recommendations, which can take place simultaneously with other studies, are presented for the evaluation of inborn errors in acutely ill newborns. Initial hospital-based tests include arterial blood gases, glucose, electrolytes, urinalysis, complete blood count, and cultures. The results from these initial tests are used to categorize the clinical presentation into one of several categories, and each category in turn serves to direct further testing in an efficient manner. Definitive diagnosis often requires referral to a pediatric specialist, as well as testing available only through reference and research laboratories. Transfer to a tertiary care facility may be advisable when evidence for an inborn error is found.

Suspected lead poisoning in a public school.

Reports of lead exposure can generate considerable public concern, particularly when children are involved. In December, 1996, a public elementary school in rural Utah was found to have elevated concentrations of lead in its drinking water. The local public health department responded by instituting remediation of the water supply and by warning parents of the possible danger to their children. Subsequent blood lead testing in 116 of the approximately 300 children involved showed an average lead concentration in the range expected for the U.S. population at large. One of the 116 specimens was marginally elevated and was probably unrelated to the school drinking water. Reducing lead exposure is an important public health concern which sometimes generates a response out of proportion to the danger involved.

Orotic acid in clinical urine specimens by capillary zone electrophoresis using polyvinyl alcohol coated capillaries.

Orotic acid is an important compound in the investigation of several inherited metabolic diseases, and capillary zone electrophoresis provides an effective assay method for this analyte in the clinical laboratory setting. Capillaries with a polyvinyl alcohol (PVA) coating performed well in this application. However, it was necessary to employ several specimen preparation steps to achieve good assay precision.

Reference intervals for 24-hour and random urine porphyrins.

Urine porphyrin analysis is an important part in evaluation of photosensitivity. Since porphyrin excretion is variable throughout the day, analysis is traditionally based on 24-hour collections. To facilitate the use of random specimens, as well as poorly collected 24-hour specimens, reference limits based on the porphyrin to creatinine ratio have been developed. Based on 1,171 adult specimens, it is estimated that the 95 percent reference limit (90 percent confidence interval) is < or = 3.9 (3.5-5.7) mumol/mol of creatinine for uroporphyrin and < or = 22 (19-34) mumol/mol for coproporphyrin. These values apply to both 24-hour and random specimens, although random specimens show a higher degree of variability. Modest differences exist between males and females, but they are not significant given the degree of uncertainty in the confidence intervals. In terms of more traditional 24-hour units, reference limits correspond to < or = 37 (32-63) nmol/day for uroporphyrin and < or = 221 (195-320) nmol/day for coproporphyrin.

Serum methylmalonic acid by capillary zone electrophoresis using electrokinetic injection and indirect photometric detection.

Elevated methylmalonic acid (MMA) is a superior marker for cobalamin (vitamin B12) deficiency. A rapid assay for serum MMA suitable for clinical specimens has been developed. The specimen is injected electrokinetically after a simple extraction. MMA is measured using indirect photometric detection at 210 nm, based on the displacement of phthalic acid in the running electrolyte; no derivatization is needed. The detection limit is 0.2 mumol/L, well below 0.4 mumol/L, which defines elevated concentrations. The assay is linear up to 100 mumol/L, although specimens above 10 mumol/L are diluted to maximize resolution. The CV is < 10% at 0.30 mumol/L and 0.80 mumol/L. The assay gives results that are comparable to more traditional methods.

Oral dimercaptosuccinic acid and ongoing exposure to lead: effects on heme synthesis and lead distribution in a rat model.

Lead (Pb) exposure and subsequent toxicity continues to be a significant problem in the United States. Treatment with meso-2,3-dimercaptosuccinic acid (DMSA) has been reported to be effective in reducing the body's Pb burden, with fewer adverse side effects than other chelating agents. The oral availability and relative safety of DMSA presents the controversial option of treating patients with Pb poisoning on an outpatient basis. Despite recommendations that children be removed from the Pb contaminated environment, some children will inevitably be exposed to environmental Pb while receiving oral DMSA therapy. The study hypothesized that oral DMSA chelation therapy is beneficial even when faced with continued dietary Pb. Sprague-Dawley rats were exposed to Pb in water for 35 days and then placed in various treatment groups, including groups administered oral DMSA with and without concurrent Pb exposure. The concentration of Pb in blood and critical organs and Pb diuresis were measured. The effect of Pb on heme synthesis was determined by assaying the urinary delta-aminolevulinic acid (delta-ALA), and blood zinc protoporphyrin (ZPP). DMSA reversed the hematological effects of Pb, decreased the blood, brain, bone, kidney, and liver Pb concentration, and produced a marked Pb diuresis, even when challenged with ongoing Pb exposure. In conclusion, even though DMSA treatment without exposure to Pb is optimal, oral DMSA could be beneficial even when challenged with ongoing Pb exposure.

Inductively coupled plasma mass spectrometry for trace element analysis in the clinical laboratory.

Inductively coupled plasma mass spectrometry (ICP-MS) is a relatively new technique for trace element analysis. The basic operating principles of ICP-MS are described and our experience with this technique in a clinical setting is discussed for the analysis of serum, whole blood, and urine. Advantages to ICP-MS include the favorable detection limits (0.01 to 0.1 micrograms/L for many elements), simple specimen preparation, high throughput (about 40 specimens per hour), and the ability to measure more than one element simultaneously. A major disadvantage is the high capital cost of the instrumentation. Heavier elements, such as lead, are well-suited for ICP-MS analysis, whereas lighter elements are prone to more interferences. Lighter elements which are not amenable to assay by ICP-MS include chromium and iron. The ability to measure isotopes is a major advantage for mass spectrometry methods and has the potential to expand the usefulness of trace element analysis.