Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results.

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.

Lamellar body counts compared with traditional phospholipid analysis as an assay for evaluating fetal lung maturity.

OBJECTIVE: To compare lamellar body counts with the lecithin/sphingomyelin ratio and phosphatidylglycerol analysis in terms of assessment of risk of respiratory distress syndrome (RDS). METHODS: Lamellar body counts, lecithin-sphingomyelin ratios (L/Ss), and phosphatidylglycerol levels were assessed in 1611 amniotic fluid samples obtained at four clinical sites from pregnant women whose fetuses were at risk for RDS. Cases in which delivery occurred within 72 hours of sample collection (n = 833) were analyzed. Specific cutoffs for predicting the likelihood of RDS for both the lamellar body count and the L/S had been derived previously at each of the clinical sites based on receiver operating characteristic curves using unrelated samples, whereas phosphatidylglycerol was reported as either mature (present) or immature (absent). Standard clinical and radiographic criteria were used to diagnose RDS, and the diagnosis was confirmed by review of newborn records. RESULTS: One hundred (12.0%) of the 833 infants delivered within 72 hours of sample collection developed RDS. The negative predictive value of the lamellar body count (97.7%) was similar to that of the L/S (96.8%) and slightly better than that of phosphatidylglycerol analysis (94.7%) (P =.048). The lamellar body count performed as well as phospholipid analysis irrespective of gestational age or patient population. CONCLUSION: The lamellar body count compares favorably with traditional phospholipid analysis as an assay for assessment of fetal lung maturity. Lamellar body counts are preferable because they are faster, more objective, less labor intensive, less technique dependent, and less expensive and because they can be performed with equipment available in every hospital laboratory.

Lamellar body counts: a consensus on protocol.

Lamellar bodies, concentrically layered "packages" of phospholipid that represent the storage form of surfactant, can be counted in the platelet channel of most electronic cell counters. The lamellar body count has been used for more than a decade and performs as well as traditional phospholipid analysis as an assay for evaluating fetal lung maturity. It is preferable to phospholipid analysis because it is rapid, objective, and inexpensive and can be performed in any hospital laboratory. The current methodologies for specimen preparation vary widely among laboratories, most notably with respect to centrifugation, resulting in differences in maturity cutoffs used. Our goal was to establish a consensus regarding a standardized methodology for the lamellar body count. Institutions that previously had published their results with lamellar body counts were invited to contribute. The consensus of the four participating institutions includes the following: centrifugation is not a necessary step and should be abandoned, maturity is suggested by a count of 50,000/microL or greater, and immaturity is suggested by a count of 15,000/microL or lower. As the lamellar body count gains wider acceptance as a primary assay for assessing fetal lung maturity, the test must be performed uniformly and accurately, given the implications of acting on a falsely negative test resulting from improper methodology.

Performance characteristics of the COBAS AMPLICOR hepatitis C virus MONITOR Test, version 2.0.

We evaluated the performance characteristics of the COBAS AMPLICOR Hepatitis C Virus (HCV) MONITOR Test, version 2.0. Dilution studies using patient specimens demonstrated a lower limit of detection of 1,000 copies per milliliter. The assay was linear from 1,000 to 1 million HCV RNA copies per milliliter. Within-run precision and between-run precision were acceptable (approximately 0.100 and 0.14 SD for log10 [copies per milliliter]). A comparison of this version of the test (y), with the manual AMPLICOR HCV MONITOR Test, version 1.0 (x), yielded the following Deming regression equation: y = 1.004(+/- 0.04)x + 0.654(+/- 0.22); Sy/x¿D = 0.336; n = 92; r2 = 0.846; r = 0.920. Further comparison of the COBAS version 2.0 assay (x) with the QUANTIPLEX HCV bDNA Test (y) yielded the following Deming regression equation: y = 0.943 (+/- 0.130)x + 0.473 (+/- 0.717); Sy/x¿D = 0.194; n = 26; r2 = 0.600; r = 0.774. Version 2.0 detected the spectrum of HCV genotypes better than version 1.0.

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.

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.

Standards of laboratory practice: evaluation of fetal lung maturity. National Academy of Clinical Biochemistry.

In this standard of laboratory practice I recommend guidelines for fetal lung maturity (FLM) testing. If possible, obtain a 10-mL uncontaminated sample by amniocentesis. Keep the amniotic fluid at 4 degrees C and mix well before testing. If centrifugation is required, strictly adhere to the protocol. Most laboratories should offer a rapid test, such as fluorescence polarization, phosphatidylglycerol, or foam stability index, daily on both a routine and emergency basis. Requests for lecithin/sphingomyelin ratio may be referred to a reference laboratory. Communicate immediately the results of any FLM test to the ordering location. The report should contain the result, sample contamination, and reference information. Separate reference intervals for diabetic patients are not recommended.

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.

Enzymatic measurement of phosphatidylglycerol in amniotic fluid.

We describe an enzymatic method for measuring phosphatidylglycerol (PG) at concentrations as low as 0.2 mumol/L in amniotic fluid. Amniotic fluid (1.5 mL) is centrifuged at 10,000g for 20 min to obtain a lamellar body pellet, which is reconstituted with 0.5 mL of buffer. The PG is measured by a two-step enzymatic scheme. Recovery studies demonstrated that the pellet contains > 97% of the PG present in amniotic fluid. Between-run CVs were 28%, 5.7%, and 2.6% for amniotic fluid controls with means of 0.32, 3.9, and 10.7 mumol/L, respectively (n = 20). The enzymatic procedure was not significantly affected by blood, meconium, bilirubin, or other phospholipids. Lecithin/sphingomyelin ratio (n = 101) and fluorescence polarization (n = 127) compared with log(PG) showed correlation coefficients of 0.832 and -0.866, respectively. This test's ability to detect low concentrations of PG in amniotic fluid may make it a better predictor of fetal lung immaturity than previous methods.

Separation and size distribution of red blood cells of diverse size, shape, and origin by flow/hyperlayer field-flow fractionation.

Red blood cells (RBCs) from human, equine, canine, feline, and bovine whole blood samples have been separated and characterized by high-speed flow/hyperlayer field-flow fractionation (Fl/HyFFF). The elution-based separation of RBCs by this method is based mainly on the size and shape of the cell particles. The typical separation time for RBCs is less than 3 min. Size distributions can be derived from the fractograms of cell samples using a calibration plot based on retention data for uniform polystyrene beads. The method is shown to be effective both to separate and to characterize cell populations, including those with cells of abnormal shape and size. In order to investigate differences in cell morphology, shape and size changes induced by 500,000 Da Dextran were monitored. The changes in the Fl/HyFFF elution profiles indicate that the RBCs decrease in size but become partially aggregated in the presence of the dextran. These changes were found to depend on polymer concentration and specific blood samples. Some of the results from Fl/HyFFF were compared with those from the Coulter counter and from microscopy.

Lamellar body counts for rapid fetal lung maturity testing.

OBJECTIVE: To evaluate amniotic fluid lamellar body counting as a fetal lung maturity test. Lamellar body particles can be rapidly counted using the platelet channel of most blood cell analyzers. METHODS: We conducted a 3-year prospective clinical outcome study. During the interval under study, outcomes of 247 neonates were used to evaluate the test; 28 neonates developed respiratory distress syndrome (RDS). Lecithin-sphingomyelin ratio (L/S) was available for 187 cases. RESULTS: All cases of RDS had lamellar body counts of 55,000/microL or less and L/S of 2.2 or less; 59% of cases with no RDS had counts greater than 55,000/microL and 70% of normal cases had L/S higher than 2.2. CONCLUSION: Use of lamellar body counts is justified as a rapid screening test to predict fetal lung maturity. Immature results should be followed by a more specific test such as L/S.

Assessment of fetal lung maturity by fluorescence polarization in high-risk pregnancies.

Although the validity of amniotic fluid fluorescence polarization (FPOL) has been documented in normal pregnancies, data are lacking on the predictive value of this method in high-risk pregnancies where biochemical maturation of the fetal lung may be altered. In this study, amniotic fluid was obtained from 86 women with pregnancies complicated by insulin-dependent diabetes (42), twin gestation (22), Rh sensitization (13) and known fetal anomalies (9). In all groups, when FPOL was > .280 (immature), phosphatidylglycerol (PG) was always absent and lecithin and sphingomyelin ratio (L/S) was < 2.5:1. When FPOL values were < .260 (mature), PG was always present and L/S was > 2.5 in 45/50 samples. When FPOL values ranged between .260-.280 (intermediate), L/S and PG values varied and were inconsistent. We conclude that FPOL assessment of fetal lung maturity in pregnancies complicated by diabetes, Rh sensitization, twin gestation and fetal anomalies is as accurate a procedure as is fetal lung maturity testing by L/S and PG. In addition, the testing procedure is quicker, more reproducible and, possibly, more reliable.

Rapid fetal lung maturity testing: commercial versus NBD-phosphatidylcholine assay.

We compared the TDx Fetal Lung Maturity test and the fluorescence polarization method using 1-palmitoyl-2(6-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]caproyl)phosphatidylcholine (NBD-phosphatidylcholine). Using 76 paired human amniotic fluid samples, the fluorescence polarization values of the two methods were found to have a strong nonlinear correlation (r2 = 0.946). Both assays can be completed in less than 1 hour, have excellent precision (between-day variation less than 2%), and indicate the amount of surfactant phospholipid relative to albumin. The FLM assay is calibrated with surfactant/albumin standards; therefore, the reported results (in mg/g) correlate inversely with polarization of NBD-phosphatidylcholine. Strong correlations were seen for both assays with the lecithin-sphingomyelin ratio and phosphatidylglycerol. The correlations indicate that the recommended reference range for FLM will have more false predictions of immaturity than the NBD-phosphatidylcholine assay.

Using 16O35Cl to correct for chloride interference improves accuracy of urine arsenic determinations by inductively coupled plasma mass spectrometry.

We have observed inaccurate urine arsenic values with the method of isobaric fractionation, which was designed to correct for the 40Ar35Cl interference with 75As quantitation by inductively coupled plasma mass spectrometry. Isobaric fractionation, which is based on ion intensities at m/z 77 and 82, consistently underestimates the 40Ar35Cl interference and overestimates urine arsenic. We present an improved method for identifying the argon-chloride interference. We observed that signal intensities for the species 16O35Cl and 40Ar35Cl are proportional (I75 = 0.0295 x I51 - 14.7, r2 = 0.998; where Ix is the normalized ion intensity at m/z X) in water and urine, over a broad range of chloride concentrations (0-800 mmol/L). The proportionality constant is remarkably stable within a run (mean and SD, 0.0295 +/- 0.0023, based on 10 replicates of five chloride calibrators, 0, 100, 200, 400, and 800 mmol/L). Increased sensitivity (50-fold) for detecting the 40Ar35Cl interference provides improved accuracy for urine arsenic quantitation as demonstrated by a split-sample comparison with graphite-furnace atomic absorption spectrophotometry.