Ronald H. Laessig

Development of a routine newborn screening protocol for severe combined immunodeficiency

BACKGROUND Severe combined immunodeficiency (SCID) is characterized by the absence of functional T cells and B cells. Without early diagnosis and treatment, infants with SCID die from severe infections within the first year of life. OBJECTIVE To determined the feasibility of detecting SCID in newborns by quantitating T-cell receptor excision circles (TRECs) from dried blood spots (DBSs) on newborn screening (NBS) cards. METHODS DNA was extracted from DBSs on deidentified NBS cards, and real-time quantitative PCR (RT-qPCR) was used to determine the number of TRECs. Positive controls consisted of DBS from a 1-week-old T(-)B(-)NK(+) patient with SCID and whole blood specimens selectively depleted of naive T cells. RESULTS The mean and median numbers of TRECs from 5766 deidentified DBSs were 827 and 708, respectively, per 3.2-mm punch ( approximately 3 muL whole blood). Ten samples failed to amplify TRECs on initial analysis; all but 1 demonstrated normal TRECs and beta-actin amplification on retesting. No TRECs were detected in either the SCID or naive T-cell-depleted samples, despite the presence of normal levels of beta-actin. CONCLUSIONS The use of RT-qPCR to quantitate TRECs from DNA extracted from newborn DBSs is a highly sensitive and specific screening test for SCID. This assay is currently being used in Wisconsin for routine screening infants for SCID.

Improved precision of newborn screening for congenital adrenal hyperplasia using weight-adjusted criteria for 17-hydroxyprogesterone levels

OBJECTIVE To evaluate the efficacy and efficiency of weight-adjusted threshold levels for 17-hydroxyprogesterone (17-OHP) in screening newborn infants for 21 hydroxylase deficiency-congenital adrenal hyperplasia (21-OH-D-CAH). DESIGN Analysis of the number of false-positive reports and diagnoses in infants, of 21-OH-D-CAH with the use of two strategies. Before October 1993, separate criteria for definite abnormal 17-OHP levels were established and implemented for 41,846 infants on the basis of birth weight: either less than 2200 gm (17-OHP level, 90 ng/ml) or 2200 gm or more (40 ng/ml). To reduce the burden of follow-up testing in low birth weight infants, criteria for definite abnormal 17-OHP results were statistically determined for four, rather than two, birth weight divisions: 1299 gm or less (17-OHP level > or = 165 ng/ml), 1300 to 1600 gm (> or = 135 ng/ml), 1700 to 2200 gm (> or = 90 ng/ml), and more than 2200 gm (> or = 40 ng/ml). These criteria were applied to the next 149,684 infants screened, and rates of false-positive test results and of false-positive diagnoses of 21-OH-D-CAH were compared. RESULTS Before implementation of four-tiered weight-adjusted 17-OHP criteria, 205 definite abnormal reports yielded four confirmed cases of 21-OH-D-CAH (positive predictive value = 2%; incidence of 21-OH-D-CAH = 1 in 10,461). With the revised criteria, 61 of 149,684 infants had definite abnormal results and 14 cases of 21-OH-D-CAH were confirmed (positive predictive value, 20%; incidence of 21-OH-D-CAH, 1 in 10,692). No undetected severe cases of 21-OH-D-CAH have been subsequently reported. CONCLUSIONS Weight-adjusted criteria for 17-OHP levels in screening for 21 -OH-D-CAH markedly reduced the number of false-positive results requiring immediate follow-up testing, particularly among low birth weight infants. Increased specificity afforded by these criteria was not accompanied by diminished sensitivity in detecting severe cases. Long-term follow-up of this screened cohort will determine whether the goals of newborn screening for 21-OH-D-CAH are adequately and efficiently fulfilled by this approach.

Point-of-care testing, medical error, and patient safety: a 2007 assessment

Abstract Point-of-care testing (POCT) is the fastest growing segment of a US

Implementing Routine Testing for Severe Combined Immunodeficiency within Wisconsin's Newborn Screening Program

30 billion worldwide market. “Errors” in the testing process, as well as medical data interpretation and treatment associated with POCT, are recognized as leading to major compromises of patient safety. In todays environment, most testing errors (pre-analytical, analytical and post-analytical) can be virtually eliminated by proper design of testing systems. We cite examples of two systems that have made exceptional progress in this respect. It has been recently suggested that the basic errors associated with the testing process are amplified in the POC setting. Two of the amplifiers – incoherent regulations and failure of clinician/caregivers to respond appropriately to POCT results – lead us to recognize additional changes in todays POCT environment. The first is a willingness of manufacturers, not laboratories, to take responsibility for the quality of test results – an outgrowth of an industrial philosophy called autonomation. The second is a need to substantially modify the clinician/caregiver test utilization paradigm to take full advantage of POCT results, available on site in real time. Both have already begun to take place. Clin Chem Lab Med 2007;45:766–73.

Temporal Associations Among Energy Intake, Plasma Linoleic Acid, and Growth Improvement in Response to Treatment Initiation After Diagnosis of Cystic Fibrosis

Severe combined immunodeficiency (SCID) is the result of genetic defects that impair normal T-cell development. SCID babies typically appear normal at birth, but acquire multiple life-threatening infections within a few months. Early diagnosis and treatment with a bone-marrow transplant markedly improves long-term outcomes. On January 1, 2008, the newborn screening (NBS) program in Wisconsin became the first in the world to routinely test all newborns for SCID. A realtime quantitative polymerase chain reaction assay measures T-cell receptor excision circles (TRECs), which are formed during the maturation of normal T-cells. A lack or very low number of TRECs is consistent with T-cell lymphopenia. The development and validation of the TREC assay and the results of the first year of screening have been published. This article describes the process used to add SCID to the NBS panel, the establishment of follow-up capacity, and the integration of SCID screening into routine NBS workflows. The development of this expanded NBS program is described so that other states might benefit from the processes used in Wisconsin.

Field Performance of the Intoxilyzer 5000: A Comparison of Blood- and Breath-Alcohol Results in Wisconsin Drivers

OBJECTIVE. It is unclear why some patients with cystic fibrosis (CF) succeed (“responders”) in recovering from malnutrition and growth faltering after treatment initiation whereas others fail to do so (“nonresponders”). We conducted a study to test the hypothesis that sustained high energy intake (↑EN) and normal plasma essential fatty acid status are critical determinants of treatment responsiveness within 2 years after diagnosis of CF. METHODS. A total of 71 CF children who had pancreatic insufficiency but not meconium ileus and were enrolled in the Wisconsin CF Neonatal Screening Project were studied. Responders were defined by having achieved adequate weight gain, as indicated by a recovery of weight z score (Wtz) comparable to Wtz at birth (WtzBR) within 2 years of diagnosis. ↑EN and sustained normal plasma linoleic acid level (↑pLA) were defined by achieving energy intake ≥120% of estimated requirement for ≥75% of the time and maintaining plasma LA ≥26% of total fatty acids for ≥75% of the time, respectively. RESULTS. Thirty-two (68%) screened patients and 13 (54%) patients whose CF was diagnosed conventionally recovered WtzBR within 2 years of diagnosis. Screened patients responded at significantly younger ages (mean/median: 6.3/4.3 months) than patients whose CF was diagnosed conventionally (mean/median: 15.8/11.8 months). Proportionately fewer screened patients (33%) achieved ↑EN compared with patients whose CF was diagnosed conventionally (73%). However, more screened patients responded to ↑EN and recovered WtzBR (91%) than patients whose CF was diagnosed conventionally (56%), although this difference was of borderline significance. Compared with having neither ↑EN nor ↑pLA, the likelihood of being a responder was greatest with combined ↑EN and ↑pLA, followed by ↑EN only. The positive associations between ↑EN and ↑pLA to treatment responsiveness remained significant after adjustment for neonatal screening status, baseline height and weight status, and indices of pulmonary disease severity. CONCLUSION. ↑EN and ↑pLA are critical in promoting adequate weight gain in children with newly diagnosed CF.

The effect of dentures and denture adhesives on mouth alcohol retention.

Intoxilyzer 5000 and blood-alcohol results from drivers arrested for operating a motor vehicle while intoxicated and for related offenses were compared during a two-year period. Three hundred and ninety-five pairs of results were studied. The breath- and blood-alcohol specimens in this study were collected within 1 h of each other. The mean blood-alcohol concentration obtained was 0.180 g/dL, with a range from zero to 0.338 g/dL. By comparison, the mean Intoxilyzer 5000 result was 0.16 g/210 L with a range from zero to 0.32 g/210 L. Compared with the blood-alcohol result, Intoxilyzer 5000 results were lower by more than 0.01 g/210 L 67% of the time, within 0.01 g/210 L 31% of the time, and higher by more than 0.01 g/210 L 2% of the time.

Simultaneous submicrodetermination of glucose and uric acid

A total of 24 alcohol-free, denture-wearing subjects were tested for mouth-alcohol retention times with an Intoxilyzer 5000. The subjects were given 30 mL doses of 80 proof brandy to swish in their mouths without swallowing for 2 min prior to expectorating the dose. Subjects were tested under three conditions: 1) with dentures removed, 2) with dentures held loosely in place without an adhesive, and 3) with dentures plus an adhesive. Beyond 20 min following expectoration, mouth alcohol made no significant contribution to the apparent breath alcohol concentration (BrAC), with trace (less than or equal to 0.01 g/210 L) readings found in only two of the subjects. Denture use, both with and without the concurrent use of adhesives does not significantly affect BrAC as long as a pretest alcohol deprivation period of 20 min is observed.

Simultaneous automated submicrodetermination of glucose, blood urea nitrogen, and uric acid on 25 microliters of capillary blood

Abstract An automated submicro procedure for the simultaneous determination of glucose and uric acid from a single 25 microliter capillary blood specimen is presented. The system is based on the Technicon AutoAnalyzer analytical system and the Unopette submicro sampling system. The colorimetric determinations for glucose and uric acid, respectively are the alkaline ferricyanide and phosphotungstic acid procedures. The proposed method is suitable for routine use as well as for population screening programs. Accuracy and precision are evaluated by comparison to standard macro procedures.

Randomized Neonatal Screening for Cystic Fibrosis (CF) Reveals Significant Nutritional Benefits over 11 Years

Abstract The current technique is both accurate and precise. The usefulness on pediatric cases is obvious. Further use of Unopette systems in screening programs, particularly those currently using the Unopette in a program of diabetic detection, could triple the value of the program with very nominal increase in costs. Most programs find the greatest expenditure of funds and effort are placed in the area of collection and reports. These functions of a program are nearly the same for one test or three. The increase in laboratory expense is incidental for the usual routine laboratory.