Impact of an Automated Population-Level Cirrhosis Screening Program Using Common Pathology Tests on Rates of Cirrhosis Diagnosis and Linkage to Specialist Care (CAPRISE): Protocol for a Pilot Prospective Single-Arm Intervention Study.

BACKGROUND: People with compensated cirrhosis receive the greatest benefit from risk factor modification and prevention programs to reduce liver decompensation and improve early liver cancer detection. Blood-based liver fibrosis algorithms such as the Aspartate Transaminase-to-Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4) index are calculated using routinely ordered blood tests and are effective screening tests to exclude cirrhosis in people with chronic liver disease, triaging the need for further investigations to confirm cirrhosis and linkage to specialist care. OBJECTIVE: This pilot study aims to evaluate the impact of a population screening program for liver cirrhosis (CAPRISE [Cirrhosis Automated APRI and FIB-4 Screening Evaluation]), which uses automated APRI and FIB-4 calculation and reporting on routinely ordered blood tests, on monthly rates of referral for transient elastography, cirrhosis diagnosis, and linkage to specialist care. METHODS: We have partnered with a large pathology service in Victoria, Australia, to pilot a population-level liver cirrhosis screening package, which comprises (1) automated calculation and reporting of APRI and FIB-4 on routinely ordered blood tests; (2) provision of brief information about liver cirrhosis; and (3) a web link for transient elastography referral. APRI and FIB-4 will be prospectively calculated on all community-ordered pathology results in adults attending a single pathology service. This single-center, prospective, single-arm, pre-post study will compare the monthly rates of transient elastography (FibroScan) referral, liver cirrhosis diagnosis, and the proportion linked to specialist care in the 6 months after intervention to the 6 months prior to the intervention. RESULTS: As of January 2024, in the preintervention phase of this study, a total of 120,972 tests were performed by the laboratory. Of these tests, 78,947 (65.3%) tests were excluded, with the remaining 42,025 (34.7%) tests on 37,872 individuals meeting inclusion criteria with APRI and FIB-4 being able to be calculated. Of these 42,025 tests, 1.3% (n=531) had elevated APRI>1 occurring in 446 individuals, and 2.3% (n=985) had elevated FIB-4>2.67 occurring in 816 individuals. Linking these data with FibroScan referral and appointment attendance is ongoing and will continue during the intervention phase, which is expected to commence on February 1, 2024. CONCLUSIONS: We will determine the feasibility and effectiveness of automated APRI and FIB-4 reporting on the monthly rate of transient elastography referrals, liver cirrhosis diagnosis, and linkage to specialist care. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12623000295640; https://tinyurl.com/58dv9ypp. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/56607.

Iron insufficiency among young Australian women: a population-based survey.

BACKGROUND: Although serum ferritin is considered a reliable indicator of iron stores, there are few data documenting the prevalence of low ferritin in representative samples of young women. AIMS: To estimate the prevalence of low ferritin and to identify factors associated with low ferritin in young Australian women. METHODS: Women, aged 18-39 years, living in the eastern states of Australia were recruited by email to a cross-sectional, online questionnaire-based study between November 2016 and July 2017. Participants not pregnant, breast feeding, taking hormonal contraception, using assisted reproduction or postmenopausal were invited to provide a blood sample. RESULTS: Of the 3689 invited participants, 761 (23.1%) provided a sample and 736 women, mean (SD) age 31.7 (±5.6) years, were included in the analyses. The overall prevalence of serum ferritin <30 µg/L was 34.8% (95% confidence interval (CI) 31.4-38.3%), with 41.4% (35.1-48.0%) in NSW, 31.5% (26.4-37.1%) in Victoria and 32.6% (26.8-39.0%) in Queensland. Serum ferritin <30 µg/L was positively associated with the reporting of >2 days of heavy menstrual bleeding (adjusted odds ratio (AOR) 1.73, 95% CI 1.15-2.59), living in New South Wales (AOR 1.57, 95% CI 1.07-2.30), not working outside home (AOR 1.58, 95% CI 1.01-2.49), and inversely associated with never experiencing heavy menses (AOR 0.46, 95% CI 0.23-0.93) and obesity (AOR 0.32, 95% CI 0.21-0.50). CONCLUSIONS: This study demonstrates that serum ferritin below 30 µg/L is common amongst young Australian women. Healthcare professionals should note the association between low ferritin and heavy bleeding.

Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays.

Background Biotin interference in streptavidin-based immunoassays causes widespread analytical distortions that may lead to clinical confusion, inappropriate patient management and, ultimately, adverse events. Its prevalence has increased recently due to the increased use of high-dose biotin therapy in specific patient groups (notably multiple sclerosis) and possibly the general community. Methods We have developed a method to deplete biotin from samples using the streptavidin-coated magnetic microparticles that are a component of most susceptible assays. Results We show that high concentrations of spiked biotin can be adequately depleted from serum using this approach, and that gross biochemical derangements can be restored to normality. We also show that biotin in samples derived from multiple sclerosis patients receiving 300 mg biotin daily can be adequately depleted to remove associated analytical interference and restore normal results. The method is applicable to competitive and sandwich immunoassays and importantly, because it does not change the volume of the sample, suitable for the measurement of free thyroid hormone assays. Application of the method does not significantly change the precision of measurement, and for the majority of analytes, the accuracy is not substantially altered. Conclusions Adopting this method enables laboratories to confirm biotin interference in the appropriate clinical setting. Moreover, it enables laboratories to remove the interference and report accurate and reliable results, without the need for patients to withhold beneficial therapy prior to blood tests. Until the biotin tolerance of susceptible assays is improved, our method gives laboratories a safe alternative for reporting results using streptavidin-based methods.

Characterization of the scope and magnitude of biotin interference in susceptible Roche Elecsys competitive and sandwich immunoassays.

Background Biotin interference is a significant problem to which at-risk laboratories must now be attuned. We sought to systematically characterize the nature of this interference in Roche immunoassays. Methods Known concentrations of biotin were titrated into serum samples and the effects on competitive and sandwich immunoassays were analysed. The maximum and minimum concentrations examined reflect those likely to be achieved in individuals on 5 to 10 mg supplements at the lower end, and 100 to 300 mg biotin at the high end. Results A high variability in biotin tolerance was observed. Some assays, such as troponin T, TSH and antithyroid antibodies, were extremely sensitive to the lower concentrations of biotin (15.6 and 31.3 ng/mL), whereas the majority of assays were relatively resistant. At concentrations ≥500 ng/mL, all assays showed significant interference from biotin but, again, the magnitude of the interference was variable. The more sensitive assays showed profound analytical bias at biotin concentrations that occur with high-dose therapy. Conclusion Our data demonstrate high variability in biotin tolerance across Roche immunoassays. The shape of the dose-response curves provides more detailed information than the single manufacturer-quoted figure for biotin tolerance. Accordingly, these data may be used by laboratories for more accurate risk assessment in predicting the effects of biotin. Our data may also be extrapolated to guide timing of blood tests in patients on high-dose biotin therapy: it demonstrates the number of half-lives required to withhold biotin in order to decrease its concentration to below a given assay tolerance.

Impact of High-Sensitivity Troponin I Testing with Sex-Specific Cutoffs on the Diagnosis of Acute Myocardial Infarction.

BACKGROUND: High-sensitivity cardiac troponin I (hs-cTnI) assays show sex-dependent differences in the 99th percentile of healthy populations, with concentrations in women approximately 50% lower. The adoption of sex-specific cutoffs seems appropriate, although it is not yet clear what effect these will have on acute myocardial infarction (AMI) diagnosis and management. METHODS: We conducted a retrospective pre- and postchangeover analysis of troponin I testing in the 6 months before and after moving from the contemporary Abbott Architect TnI assay (cTnI) to hs-cTnI at 2 tertiary centers in Australia and New Zealand. The cTnI cutoff was 30 ng/L for both sexes, whereas a female-specific cutoff of 16 ng/L was adopted upon changeover to hsTnI. RESULTS: Changeover from the cTnI assay to the hs-cTnI assay increased the number of female patients with increased troponin I concentrations at both sites (from 29.7% to 34.9% and from 22.4% to 30.8%; P < 0.001). There was no statistically significant change in the number of men with increased concentrations in the same time period (P = 0.09). The increased percentage of women with increased troponin I was not associated with an increase in the number of women with AMI diagnoses at either center. Angiographic data available from 1 center showed no change in the percentage of angiograms performed in women. CONCLUSIONS: Although increasing the proportion of women with increased troponin I, adopting sex-specific cutoffs with the hs-cTnI assay did not lead to an increase in AMI diagnoses in females, or in the number of women undergoing angiography.

A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin.

Mutations in the perforin gene have been found in patients with hemophagocytic lymphohistiocytosis (HLH), a rare autosomal recessive disease. We describe a patient expressing perforin with amino acid changes A91V and W374X. The ability of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to lyse target cells is greatly reduced. Here we demonstrate that perforin from this patient is not recognized using an antibody raised against native perforin (deltaG9), but is readily detected using an antibody raised against a peptide epitope (2d4), suggesting that the epitope recognized by deltaG9 is destroyed by the change at A91V. Immunoblotting reveals no protein corresponding to the truncated transcript encoded by W374X, revealing that only perforin with the A91V change is expressed in CTLs from the patient. Patient CTLs show bands corresponding to the immature and intermediate forms of perforin, but the mature active form of perforin is absent or barely detectable. The conformational changes and impaired cleavage of A91V perforin are likely to explain the reduced cytotoxicity in CTLs and NK cells from this patient and are likely to contribute to the pathogenesis of HLH.

Normal lytic granule secretion by cytotoxic T lymphocytes deficient in BLOC-1, -2 and -3 and myosins Va, VIIa and XV.

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.

Delivering the kiss of death.

Cytotoxic T lymphocytes and natural killer cells kill their targets by secreting specialized granules that contain potent cytotoxic molecules. Through the study of rare immunodeficiency diseases in which this granule pathway of killing is impaired, proteins such as Rab27a have been identified as components of the secretory machinery of these killer cells. Recent evidence suggests that the destruction of activated lymphocytes through granule-mediated killing may be an important mechanism of immunological homeostasis. Although the process by which this occurs is not yet known, it is possible that events taking place at the immunological synapse may render the killer cell susceptible to fratricidal attack by other killer cells.

The secretory synapse: the secrets of a serial killer.

Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.