Point: status of lipid and lipoprotein standardization.

Cholesterol and triglyceride standardization procedures have been used extensively and continuously since the 1950s. Definitive and Reference Methods, as well as primary and secondary standards, have been developed and maintained as the basis for evaluating the accuracy of results by various methods in many laboratories. But, although standardization efforts for apolipoprotein A-I and B measurements have been reported in detail in the scientific literature, much less has been reported in the area of total and lipoprotein cholesterol and triglyceride standardization efforts. Standardized cholesterol and triglyceride concentrations, determined in multiple large epidemiological and clinical studies, have been instrumental to the National Cholesterol Education Program panels that have assessed the lipoprotein values associated with risk of coronary disease, and have determined the cutpoints that are now used extensively by physicians to guide diagnosis and treatment of individual patients.

Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.

OBJECTIVES: To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. METHODS: From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. RESULTS: Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of screening. CONCLUSIONS: Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens.

Application of DNA analysis in a population-screening program for neonatal diagnosis of cystic fibrosis (CF): comparison of screening protocols.

We compare two protocols for newborn screening for cystic fibrosis (CF). The first uses the immunoreactive trypsinogen (IRT) assay with a cutoff of > or = 180 ng/ml and a sweat test to identify CF patients. The second uses the IRT assay with a 100 ng/ml cutoff in conjunction with direct analysis for the delta F508 CF transmembrane conductance regulator (CFTR) mutation in a two-tiered (i.e., IRT/DNA) protocol, followed by a sweat test. We screened 220,865 newborns from Wisconsin for CF, using the IRT protocol identifying 369 infants with an elevated IRT, of whom 46 were found to have CF. Another 7 CF patients were identified who had a false-negative IRT level. The CF incidence in the white population was 1 in 3,431 (carrier incidence of 1 in 30). The IRT protocol had a sensitivity of 87% and a positive predictive value of 12.5%. We subsequently used the IRT/DNA protocol to screen 21,258 infants. Of 518 infants with an IRT level > or = 100 ng/ml, 24 carried at least one copy of the delta F508 CFTR mutation, and 4 of these infants were found to have CF, yielding a positive predictive value for this protocol of 16.7%. Direct comparison of the positive predictive value of the two protocols is not valid, because of the different populations screened. However, had the IRT protocol been used on the IRT/DNA cohort, 50 infants, including the 4 with CF, would have received sweat tests, yielding a positive predictive value of 8%. Because of the small sample size, this positive predictive value is not significantly different from that obtained for the IRT/DNA test. However, from a practical point of view the IRT/DNA approach does decrease considerably the number of sweat tests that must be undertaken. The number of false positives for the IRT protocol (46 in 21,258) is increased significantly compared with that for the IRT/DNA approach (20 in 21,258; P < .001). The incidence of delta F508 carriers detected in cohorts with an elevated IRT level was increased compared with the incidence in the general population. The direct costs for the IRT/DNA approach (100 ng/ml) were $11,374 per CF patient detected, compared with $10,187 per CF patient detected for the IRT protocol. Therefore, we conclude that the IRT/DNA approach to CF newborn screening decreases the number of false-positive subjects contacted, without a significant increase in cost.

Comparison of plastic vs. glass evacuated serum-separator (SST) blood-drawing tubes for common clinical chemistry determinations.

We evaluated a plastic evacuated blood-drawing tube containing an integral serum-separating barrier gel, by direct comparison with a glass counterpart. The plastic tube demonstrated no differences when compared for common clinical chemistry analytes with multiple types of instruments and systems. A total of 260 such different combinations were studied with emphasis on tests sensitive to drawing and handling indexes such as lactate dehydrogenase and potassium. A total of six separate blood drawings were tested with no significant differences noted in these tests. The total study included subjective evaluations of the plastic tube's use as a blood-drawing device and objective studies based on quantitative test results from normal and hospitalized patients and use of the primary sampling tubes (both plastic and glass) for 48-h storage.

Limitations of proficiency testing under CLIA '67.

Proficiency testing (PT), recognized as a quality-assurance (QA) and quality-improvement tool, also has become the cornerstone of the Health Care Financing Administration's (HCFA) regulatory strategy under the revised Clinical Laboratory Improvement Act of 1967 (CLIA '67) and the proposed Clinical Laboratory Improvement Amendments of 1988 (CLIA '88). Use of PT as a regulatory tool corrupts it for things it can do better. PT as a primary regulatory strategy has severe limitations. We explore the nature of these limitations and their implications for clinical laboratories as they impact on the long-term success of HCFA's approved regulatory PT programs in 1991 and beyond, and CLIA '88 PT, which is to be implemented in 1994.

Review of actual proficiency-testing performance under CLIA '67 (March 14, 1990) rules: perspective from the first year's data.

Under the Clinical Laboratory Improvement Act of 1967 the Health Care Financing Administration's proficiency-testing requirement applies to approximately 12,000 hospital, reference, and large-clinic laboratories in the United States. The Wisconsin State Laboratory of Hygiene is approved by the Health Care Financing Administration to provide proficiency testing in all specialties and subspecialties. The focus of the program is to provide highly specialized service and support to a limited number of participants in order to assess intralaboratory performance correctly. We report the findings over the four proficiency-testing events in 1991 for the subspecialty of routine chemistry, which serves approximately 470 participants. Failure rates for individual analytes on single proficiency testing events ranged from 0% to 13%. After four events or one year, if the mandated evaluation criteria and failure rules were strictly applied, as many as 11% of the laboratories could have found themselves involuntarily suspended from offering all routine chemistry testing.

Performance of the Kodak DT-60 physician's office analyzer as measured by CLIA-88 proposed evaluation criteria. Assessment of demographic and quality assurance factors influencing performance.

The 1988 Clinical Laboratories Improvement Act (CLIA-88) proposes to mandate universal proficiency testing and internal quality assurance practices for all laboratories, including those in physician offices. For 3 years, we have provided an independent voluntary proficiency testing program to more than 400 physician office laboratories using Kodak DT-60 analyzers (Eastman Kodak, Rochester, NY). This unique data set enables us to evaluate, using the CLIA-88 proposed grading criteria, the ability of these laboratories to meet the proposed regulatory standards. Using the equivalent of a year's participation under the CLIA proficiency testing format (20 challenges per analyte), at least 88% would "pass," ie, achieve acceptable performance. We investigated the relationship between proficiency testing performance and several internal quality assurance practices as well as other factors commonly associated with quality performance, including analyst's professional background, monthly test volume, number of physicians served, and source of training on the instrument. The best indicator of successful performance in proficiency testing was on-site training provided by the manufacturer, as opposed to training provided by distributor personnel. We conclude that with proper on-site training and retraining, physician office laboratories will be able to meet the mandatory CLIA-88 proficiency testing requirements.

Current issues in neonatal screening for cystic fibrosis and implications of the CF gene discovery.

Many questions remain regarding the efficacy, toxicity, and costs of CF neonatal screening. It would be premature, in our opinion, to implement mass population screening of newborns for CF until the benefits and risks have been fully defined, and an adequate and logistically feasible testing system developed and/or highly effective therapy for CF lung disease becomes available. In addition, the ethical issues described herein need to be resolved. This pertains not only to the CF patient but also the heterozygote carrier. These reservations notwithstanding, the discovery of the CF gene should have a favorable impact both directly and indirectly on neonatal screening for the disease. Mutation analysis coupled to IRT testing seems most attractive at this time, at least on a research basis, but primary molecular diagnostic procedures might supervene in the future, particularly if they are financially feasible.

Laboratory quality control issues related to screening newborns for cystic fibrosis using immunoreactive trypsin.

We have incorporated the IRT assay for CF to our newborn screening program, relying heavily on electronic data processing to optimize the test results in order to provide the most reliable data possible from the specimen at hand. We have established an internal cut-off of 100 ng/mL and an external referral of 180 ng/mL; this virtually eliminates the possibility that analytical imprecision will result in misidentifying a positive patient specimen. The relationship between IRT levels and various mutant forms of CF are not well established, and it is possible that various forms of CF may exhibit different levels of IRT in the first few days of life. We believe that IRT screening for CF could be a useful procedure for early identification of potential CF. However, by comparison with other newborn screening tests, its sensitivity, 90%, presents a concern. The expectation for PKU, hypothyroidism, MSUD, and galactosemia screening is 100% sensitivity. A false-negative usually results in litigation. The use of IRT in routine newborn screening will require considerable education of the general public and physicians receiving test results. Our program, along with many others, is anxiously watching the developments in the area of gene testing. We feel the relatively inexpensive IRT, used for mass screening can be successfully coupled with the more definitive (and expensive) gene test on a selected population to identify CF at the earliest possible age in a more effective manner. It is possible that in the near future gene probe tests will be applied in a cost effective manner to the initial filter paper specimen.

Newborn screening for cystic fibrosis is complicated by age-related decline in immunoreactive trypsinogen levels.

Detection of elevated levels of immunoreactive trypsinogen (IRT) in dried neonatal blood spots has been used as a screening test for cystic fibrosis. In other cystic fibrosis newborn-screening studies, a sweat chloride test is generally performed only if an infant has a persistent IRT level above a selected cutoff value on both the initial and subsequent specimens. Neither the timing of the second specimen nor the value of the cutoff point for the second specimen has been comprehensively evaluated. In this randomized, controlled study, 145,024 infants were screened in the neonatal period for cystic fibrosis using the 99.8 percentile (180 ng/mL) as the neonatal cutoff point. A total of 129 infants had elevated neonatal IRT levels and had negative results on sweat tests (false-positive by IRT screening). A total of 54 children with cystic fibrosis were identified in the screened and comparison groups. Excluding patients with meconium ileus, 4 infants with cystic fibrosis had neonatal IRT values less than 180 ng/mL, and an additional 9 infants with cystic fibrosis had values decline to less than 180 ng/mL within the first 2 1/2 months of age. The IRT values of infants with and without cystic fibrosis overlapped considerably beyond 30 days of age. These findings suggest that further refinement of cystic fibrosis screening methodology will be necessary to achieve an acceptable sensitivity and specificity.

Neonatal screening for cystic fibrosis in Wisconsin.

Primary care physicians have been very cooperative in referring screened patients to the two designated CF centers in Wisconsin--the University of Wisconsin Cystic Fibrosis Center, and the center at the Medical College of Wisconsin in Milwaukee--and their help has made this study possible. By 1990, we anticipate that meaningful clinical comparisons between the screened and control groups will be possible, and at that time we can begin to obtain some definitive answers concerning the benefits and potential risks of neonatal screening for cystic fibrosis. At this time, it would be premature to make a decision concerning the efficacy of screening for cystic fibrosis for the State of Wisconsin. It is very important that the study go to completion before making conclusive recommendations. We are eager to meticulously document the natural history of CF by following study patients for a long time. Answers to questions concerning rate of decline of the IRT value in true positives, psychosocial risks of screening to true positives, effect on future reproductive plans, and the cost effectiveness of the screening program will not be available for at least two more years. False positive IRT results seem to be related to perinatal asphyxia. We postulate the mechanism is ischemia in the pancreas related to hypoxia during the perinatal period leading to transient release of trypsin from the pancreas into the bloodstream. Decline of the IRT result over time is of great interest because a repeat blood sampling approach would hopefully eliminate several false positives.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive trypsinogen screening for cystic fibrosis: characterization of infants with a false-positive screening test.

Blood immunoreactive trypsinogen (IRT) is elevated in newborns with cystic fibrosis (CF) and has been used as a neonatal screening test. However, not only is the benefit of early diagnosis unknown, but also the sensitivity, specificity, and time related decline of IRT values have yet to be comprehensively evaluated. This report describes the characteristics of infants with a false-positive IRT in our experience with CF screening of 87,000 infants. The IRT value was elevated in 92 newborns; 13 had a confirmed diagnosis of CF by quantitative pilocarpine iontophoresis sweat testing, and 79 infants did not have CF and were therefore classified as false positives by IRT screening. In order to test the hypothesis that perinatal stress factors are associated with high neonatal IRT values, we evaluated Apgar scores at 1 and 5 minutes. We found that the scores of false-positive infants were significantly lower (P = 0.0004 and P = 0.0102 at 1 and 5 minutes, respectively), compared with infants in the general population. While perinatal asphyxia as reflected by low Apgar scores is an associated factor accounting for an elevated IRT value, the majority of non-CF newborns with an elevated IRT have normal Apgar scores.

Testing cholesterol accuracy. Performance of several common laboratory instruments.

The national emphasis on assessing coronary risk by cholesterol testing mandates that analytical determinations be as accurate and precise as is medically necessary. One goal of the National Cholesterol Education Program is to improve the quality of laboratory tests. Currently, 5% limits of imprecision and 5% of bias vs the nationally accepted Abell-Kendall reference method is recommended, with a goal of reducing these limits to 3% by 1992. Clinicians must be aware of how the cholesterol values are obtained, and especially whether the laboratory's method meets the performance goals. We assayed patients' serum samples for cholesterol using several commercially available, routinely used enzymatic methods and by the Abell-Kendall reference method. Precision of these methods was also assessed using serum-based controls. All instruments were operated precisely according to the manufacturers' instructions. Performance was objectively judged based on the National Cholesterol Education Program goals and on medically allowable total error. In all cases, the DuPont aca, the Kodak Ektachem, the Hitachi 737, and the Cobas FARA determined cholesterol levels acceptably. The precision and accuracy goals of 3% are achievable by these methods.

Screening programs for congenital hypothyroidism. How can they be improved?

High-sensitivity neonatal hypothyroid screening tests are used throughout the country. Because of low specificity, primary care physicians are faced with an abundance of false-positive results that challenge the interpreting physician with clinical, economic, and medicolegal considerations. We surveyed 154 physicians caring for Wisconsin-born infants with the highest newborn-screen thyrotropin values in a two-year period. Our results indicated that (1) confirmation of thyroid normalcy is often delayed beyond 6 weeks of age; (2) there is wide variation among physicians regarding therapeutic goals if hypothyroidism is confirmed; and (3) physicians prefer autonomy in the management of congenital hypothyroidism. Modifications in hypothyroid screening programs may include confirmatory tests by a central laboratory (that distributes filter paper with all abnormal results), provision of a management decision tree for primary care physicians, and a one-time subsidy for a visit to a pediatric endocrinologist. We suggest that these modifications may improve the long-term outcome of hypothyroid infants identified by the screening program.

Quality control and quality assurance.

The office laboratory's need for quality is no different from that of any other clinical laboratory. If patients are to receive the benefit of physician's office testing, reliable, high-quality laboratory results are essential. To achieve this, the physician's office laboratory must have an adequate quality assurance program. Several fundamental components of such a program have been addressed in this article: procedure manuals, record-keeping, maintenance logs, quality control charts, participation in proficiency testing, and laboratory inspection. If your state's regulations do not yet require these activities in the physician's office laboratory, they soon will! A successful laboratory's quality assurance program will provide the following assurances. (1) Quality practices are established and followed by all personnel involved with the testing in the laboratory. (2) The technologist performing the test will know when systems and instruments are working properly and the patients' results are reliable. (3) High-quality information needed by the physician interpreting or evaluating patient laboratory results will be generated. (4) A set of written records is available demonstrating to the inspector that uniform and acceptable protocols have been established and are practiced in the laboratory. One short article cannot provide all the specifics for a laboratory's quality assurance program. The manufacturers and suppliers of instruments and reagents should be able to provide support in the following areas: calibration, type of controls to be used, development of a control chart, required maintenance procedures, establishment of accuracy and precision, and troubleshooting. If they cannot or will not, your laboratory should, perhaps, consider an alternative vendor to supply instrumentation and/or reagents. Additionally, resources such as the professional organizations, consultants, other clinical laboratories, and the inspectors or certifying agencies should also be considered in developing a comprehensive quality assurance program.

Dual-channel continuous-flow system for determination of phenylalanine and galactose: application to newborn screening.

We describe a dual-channel AutoAnalyzer (Technicon) system for the simultaneous measurement of phenylalanine and galactose from blood specimens on filter-paper. Using a single 1/4-in.-diameter (6-mm) specimen, we measure both components fluorometrically at a rate of 70/h. Analytical recovery with the method and the linearity of measurements vs sample concentration are excellent through the ranges of interest, 0-200 mg/L for phenylalanine and 0-800 mg/L for galactose. Carryover at the critical values during screening, 40 mg/L for phenylalanine and 100 mg/L for galactose, is essentially zero. The dual-channel system provides the means to incorporate a low-incidence test, i.e., galactosemia (incidence 1/70 000), into an existing program for phenylalanine analysis, for which the higher rates (phenylketonuria, incidence 1/11 500) easily justify the cost of mass screening.

A comparison of hard and soft glass blood-drawing tubes.

The authors examined both hard and soft glass evacuated blood-drawing tubes for possible effects on clinical chemistry measurements. Using routine laboratory procedures, no clinically or statistically significant difference could be detected in 34 analytes using 66 different methods. A special high-precision study utilizing an adaption of the NBS round-robin procedures for calcium, magnesium, sodium, and potassium detected no difference between paired sera when drawn or stored for 72 hours, or both, in the two types of glass. The authors conclude that the type of glass used in production of the evacuated blood-drawing tubes does not affect the clinical chemistry results obtained.

The effects of 0.1 and 1.0 per cent erythrocytes and hemolysis on serum chemistry values.

Changes in serum chemistry values as a result of incomplete removal of erythrocytes and in vitro hemolysis during the preparative process have been studied. Two levels of contamination, corresponding to removal of 99% and 99.9% of the erythrocytes, were used to examine the effects of both hemolyzed and intact cells. Forty chemical procedures and methods were considered. Serum LDH values were most strongly affected by hemolyzed erythrocytes. Potassium, creatine phosphokinase, aspartate aminotransferase, alanine aminotransferase, and iron showed smaller but significant effects due to the presence of 1% hemolyzed cells, with lesser effects observed at the 0.1% level. The presence of non-hemolyzed cells at either level did not significantly alter chemistry results.