If manufacturers redesign their immunoassays to include preconjugation of biotinylated capture antibody to streptavidin, they could make them more accurate and robust against biotin interference (J Appl Lab Med 2022; doi: 10.1093/jalm/jfab169).

Streptavidin-to-biotin binding, among nature’s strongest noncovalent interactions, is incorporated into many immunoassays. It makes them susceptible to interference from free biotin in patient specimens and can produce inaccurate results.

Biotin interference has been particularly problematic in thyroid hormone assays in cases involving multiple sclerosis patients on 300 mg/day of biotin therapy. Some of these patients have been misdiagnosed with Graves’ disease because of falsely elevated thyroid hormones. Interference also has been a problem with parathyroid hormone and testosterone immunoassays, leading to misdiagnosed hypoparathyroidism and testosterone-secreting tumor, respectively, in patients taking 5−10 mg of biotin/day for hair and nail growth.

To prevent biotin interference, researchers developed a method to preconjugate biotinylated antibodies to the assay’s streptavidin solid surface before adding patient specimens. The researchers compared this technique to a biotin depletion protocol. The researchers established biotin interference in three manual ELISA assays and two automated immunoassays and evaluated mitigation of biotin interference by preincubation in each. The evaluation involved adding biotinylated antibody to the streptavidin-coated surface before adding biotin- or PBS-spiked serum.

In the presence of 400 μg/L biotin, the researchers found analyte detection reduced to 10%−15% of total in the ELISA assays. In the automated sandwich (thyroglobulin) immunoassay, analyte detection was reduced to 15.2% of total. In the automated competitive (free thyroxine) immunoassay, biotin caused increased detection of 551.6%. Preconjugation of the biotinylated capture antibody to the streptavidin surface in the ELISA assay resulted in 84% −99% activity recovery, compared to 84%−97% by a biotin depletion protocol. Automatic sandwich and competitive immunoassays got 97.1%−116.5%
recovery by preconjugation, compared with 95.6% and 100.3% via the depletion method, respectively.

RNA Profiles During Pregnancy Reveal Signatures of Disease

mRNA molecules in plasma cell-free RNA (cfRNA) sequenced from a single blood sample could predict preeclampsia—a major driver of maternal morbidity and mortality—months before symptoms appear (Nature 2022; doi: 10.1038/s41586-021-04249-w).

Researchers’ results came from comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data included 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks before delivery.

The researchers showed that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal, and fetal levels and can robustly predict preeclampsia, with a sensitivity of 75% and a positive predictive value (PPV) of 32.3%, compared with state-of-the-art methods that depend on clinical factors and have a PPV of 4.4%.

The cfRNA signatures of normal pregnancy progression and preeclampsia are independent of clinical factors, such as maternal age, body mass index, and race, which cumulatively account for less than 1% of model variance. Additionally, the cfRNA signature for preeclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of preeclampsia.

The researchers emphasized that given the study’s large sample size and diversity, it shows that race has a negligible effect on the expression patterns of gestational age estimates and preeclampsia risk evaluation. These findings allow for the development of personalized assessments for pregnancy, they added.

The researchers called for further research to identify drivers of the identified pathophysiological pathways and effective stratification of risk without the need for enrichment of pretest probabilities based on maternal sociodemographic characteristics. Understanding of the maternal-fetal-placental transcriptome may provide novel insights into maternal and neonatal morbidity and mortality, they wrote.

Equation Predicts End-Stage Kidney Disease

The Kidney Failure Risk Equation (KFRE) score better predicts 2-year end-stage kidney disease (ESKD) risk than estimated glomerular filtration rate (eGFR) alone, regardless of adjustment for race (Ann Intern Med 2022; doi:10.7326/M21-2928).

New eGFR equations from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) do not include a race adjustment, but little is known about the impact of removing race.

The researchers compared ESKD prediction performance of five different CKD-EPI eGFR equations in an observational prospective cohort study conducted at seven U.S. clinical centers involving 3,873 participants with CKD. The equations are based on serum creatinine and/or cystatin C, with or without adjustment for race. The researchers used KFRE to predict the 2-year risk
for ESKD. KFRE includes age, sex, eGFR, and urinary albumin-creatinine ratio. The researchers evaluated the prediction performance of eGFR equations and the KFRE score using
discrimination and calibration analyses.

During a maximum of 16 years of follow-up, 865 participants developed ESKD. Across all eGFR equations, the KFRE score worked better at predicting 2-year incidence of ESKD, compared with eGFR alone, based on area under the curve ranges from 0.945 to 0.954, versus 0.900 to 0.927. Prediction performance of KFRE scores using different eGFR equations was similar, but the creatinine equation without race adjustment improved calibration among Black participants. Among all participants, compared with an eGFR less than 20 mL/min/1.73 m2, a KFRE score greater than 20% had similar specificity for predicting 2-year ESKD risk, with a range of 0.94 to 0.97 versus 0.95 to 0.98. However, KFRE’s sensitivity was higher, with ranges of 0.68 to 0.78 versus 0.42 to 0.66.

Because a KFRE score greater than 20% showed similar specificity (approximately 95%) but higher sensitivity compared with an eGFR less than 20 mL/min/1.73 m2, a KFRE score greater than 20% could be used for preparing kidney replacement therapy, the researchers noted. “The four-variable KFRE showed better sensitivity and specificity in our study compared with eGFR alone, is easy to implement in routine clinical settings, and does not consider a patient’s race,” the researchers wrote.