Oral glucose tolerance test to diagnose gestational diabetes mellitus: Impact of variations in specimen handling.

To improve birth outcomes, all pregnant women without known diabetes are recommended for an oral glucose tolerance test (OGTT) to screen for hyperglycaemia in pregnancy (diabetes in pregnancy or gestational diabetes mellitus (GDM)). This narrative review presents contemporary approaches to minimise preanalytical glycolysis in OGTT samples with a focus on GDM diagnosis using criteria derived from the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. The challenges of implementing each approach across a diverse Australian healthcare setting were explored. Many Australian sites currently collect and transport OGTT samples at ambient temperature in sodium fluoride (NaF) tubes which is likely to lead to missed diagnosis of GDM in a significant proportion of cases. Alternative preanalytical solutions should be pragmatic and tailored to individual settings and as close as possible to the preanalytical conditions of the HAPO study for correct interpretation of OGTT results. Rapid centrifugation of barrier tubes to separate plasma could be suitable in urban settings provided time to centrifugation is strictly controlled. Tubes containing NaF and citrate could be useful for remote or resource poor settings with long delays to analysis but the impact on the interpretation of OGTT results should be carefully considered. Testing venous blood glucose at the point-of-care bypasses the need for glycolytic inhibition but requires careful selection of devices with robust analytical performance. Studies to evaluate the potential error of each solution compared to the HAPO protocol are required to assess the magnitude of misdiagnosis and inform clinicians regarding the potential impact on patient safety and healthcare costs.

Real-world screening for diabetes in early pregnancy: Improved screening uptake using universal glycated haemoglobin.

AIMS: To improve perinatal outcomes, screening for hyperglycaemia using 75 g oral glucose tolerance test (OGTT) is recommended for all pregnant women at 24-28 weeks gestation (routine), and earlier if high-risk. Screening coverage for remote and Aboriginal Australian women is less than ideal. This study examined OGTT completion (early and routine) by women from rural and remote Western Australia compared with early glycated haemoglobin (HbA1c). METHODS: In 2015-2018, 27 primary health care sites recruited 600 (233 Aboriginal) women aged ≥16-years, without pre-existing diabetes, who delivered >30-weeks gestation. All women presenting <20-weeks gestation (541) were offered an early study HbA1c. Early OGTTs were requested at the discretion of the local clinician, with routine OGTT offered at 24-28 weeks. RESULTS: HbA1c uptake was high (85.7% Aboriginal, 86.4% non-Aboriginal); OGTT completion in Aboriginal women was low (early OGTT: 38.6% v 69.6% non-Aboriginal, P < 0.001; routine OGTT: 44.5% v 84.7% non-Aboriginal, P < 0.001). Aboriginal women with both early tests had HbA1c completed 3-weeks prior to OGTT (9.6 ± 3.5 v 12.5 ± 3.5 weeks gestation, P < 0.001). CONCLUSIONS: Universal early pregnancy HbA1c appears feasible as an early screening test for women at risk of hyperglycaemia in pregnancy and would expedite and increase screening in Aboriginal women compared to an early OGTT.

Prediabetes and pregnancy: Early pregnancy HbA1c identifies Australian Aboriginal women with high-risk of gestational diabetes mellitus and adverse perinatal outcomes.

AIMS: To assess whether early pregnancy HbA1c can predict gestational diabetes mellitus (GDM) and adverse birth outcomes in Australian women. METHODS: Prospective study of 466 women without diabetes, aged ≥16-years at first antenatal presentation. Recruitment was from 27 primary healthcare sites in rural and remote Australia from 9-January 2015 to 31-May 2018. HbA1c was measured with first antenatal investigations (<20-weeks gestation). Primary outcome measure was predictive value of HbA1c for GDM, by routine 75 g oral glucose tolerance test (OGTT; ≥24-weeks gestation), and for large-for-gestational-age (LGA) newborn. RESULTS: Of 396 (129 Aboriginal) women with routine OGTT, 28.8% had GDM (24.0% Aboriginal). HbA1c ≥5.6% (≥38 mmol/mol) was highly predictive (71.4%, 95% CI; 47.8-88.7%) for GDM in Aboriginal women, and in the total cohort increased risk for LGA newborn (RR 2.04, 95% CI; 1.03-4.01, P = 0.040). There were clear differences between Aboriginal and non-Aboriginal women: 16.3% v 5.2% (P < 0.001) had elevated HbA1c whereas 12.4% v 29.6% (P < 0.001) developed hyperglycemia during pregnancy. CONCLUSIONS: Early pregnancy HbA1c ≥5.6% (≥38 mmol/mol) identifies Aboriginal women with apparent prediabetes and elevated risk of having an LGA newborn. Universal HbA1c at first antenatal presentation could facilitate earlier management of hyperglycemia and improved perinatal outcome in this high-risk population.

Underestimation of risk for large babies in rural and remote Australia: Time to change plasma glucose collection protocols.

AIMS: Preanalytical glycolysis in oral glucose tolerance tests (OGTT) leads to substantial underestimation of gestational diabetes mellitus (GDM) and hence risk for large-for-gestational-age (LGA) babies. This paper quantified the impact of glycolysis on identification of LGA risk in a prospective rural and remote Australian cohort. METHODS: For 495 women, OGTT results from room temperature fluoride-oxalate (FLOX) tubes were algorithmically corrected for estimated glycolysis compared to 1) the Hyperglycaemia and Adverse Pregnancy Outcomes (HAPO) study protocol (FLOX tubes in ice-slurry); and 2) room temperature fluoride-citrate (FC) tubes. GDM was defined by International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria. Unadjusted and corrected OGTT were related to LGA outcome. RESULTS: Correction for FC tubes increased GDM incidence from 9.7% to 44.6%. After correction for HAPO protocol, GDM incidence was 27.7% and prediction of LGA risk (RR 1.82, [1.11-2.99]) improved compared to unadjusted rates (RR 1.12, [0.51-2.47]). To provide similar results for FC tube correction (29.3% GDM; RR 1.81, [1.11-2.96]) required + 0.2 mmol/L adjustment of IADPSG criteria. CONCLUSIONS: FC tubes present a practical alternative to the HAPO protocol in remote settings but give + 0.2 mmol/L higher glucose readings. Modification of IADPSG criteria would reduce perceived 'overdiagnosis' and improve LGA risk-assessment.

Real-World Gestational Diabetes Screening: Problems with the Oral Glucose Tolerance Test in Rural and Remote Australia.

Gestational diabetes mellitus (GDM) is the most common antenatal complication in Australia. All pregnant women are recommended for screening by 75 g oral glucose tolerance test (OGTT). As part of a study to improve screening, 694 women from 27 regional, rural and remote clinics were recruited from 2015-2018 into the Optimisation of Rural Clinical and Haematological Indicators for Diabetes in pregnancy (ORCHID) study. Most routine OGTT samples were analysed more than four hours post fasting collection (median 5.0 h, range 2.3 to 124 h), potentially reducing glucose levels due to glycolysis. In 2019, to assess pre-analytical plasma glucose (PG) instability over time, we evaluated alternative sample handling protocols in a sample of participants. Four extra samples were collected alongside routine room temperature (RT) fluoride-oxalate samples (FLOXRT): study FLOXRT; ice slurry (FLOXICE); RT fluoride-citrate-EDTA (FC Mix), and RT lithium-heparin plasma separation tubes (PST). Time course glucose measurements were then used to estimate glycolysis from ORCHID participants who completed routine OGTT after 24 weeks gestation (n = 501). Adjusting for glycolysis using FLOXICE measurements estimated 62% under-diagnosis of GDM (FLOXRT 10.8% v FLOXICE 28.5% (95% CI, 20.8-29.5%), p < 0.001). FC Mix tubes provided excellent glucose stability but gave slightly higher results (Fasting PG: +0.20 ± 0.05 mmol/L). While providing a realistic alternative to the impractical FLOXICE protocol, direct substitution of FC Mix tubes in clinical practice may require revision of GDM diagnostic thresholds.

Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death.

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.