Reference intervals for clinical biochemistry and haematology tests during normal pregnancy.

BACKGROUND: Pregnancy induces physiological changes which affect biochemical and haematological parameters. As the significance of laboratory test results change throughout pregnancy, the reference interval (RI) or key result interpretive guide should be specific to pregnancy. This study sought to establish trimester-specific-RIs for routine biochemical and haematological tests in healthy white European women with singleton pregnancies with comparison to RIs for non-pregnant European adults. METHODS: A retrospective analysis of a prospective longitudinal single-centre study of healthy pregnant women conducted between November 2018 and December 2020 in a tertiary academic hospital with approximately 3000 births annually. Inclusion criteria: signed informed consent, age ≥18 years, white European, body mass index (BMI) <25 kg/m2, blood pressure <140/90mmHg, non-smoker, no previous pathology or gestational diabetes. Trimester defined as T1: up to 13 weeks + 6 days, T2: 14-27 weeks + 6 days and T3: ≥28-41 weeks + 6 days. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 31 biochemical and 10 haematological ISO15189:2012 accredited tests were measured using Roche Cobas® and Sysmex XN-9100™ analysers, respectively. RIs were established according to the International Federation of Clinical Chemistry (IFCC) recommended method. RESULTS: Apparently healthy pregnant women (n = 124) with bio-banked serum samples in each trimester were recruited. At the booking visit, 49.2% (n = 61) of participants were nulliparous, with median age of 34.4 (IQR: 31.3-37.3) years, gestational age of 89 (IQR: 84-93) days, BMI of 22.5 (IQR: 21.0-23.7) kg/m2 and systolic and diastolic blood pressure of 116 (110-125) mmHg and 67 (61-75) mmHg, respectively. CONCLUSIONS: Normative trimester-specific biological intervals for routinely requested biochemical and haematological medical laboratory tests were established. These RIs will be invaluable to result interpretation and the management of pregnant women.

Remote capillary blood collection for HbA1c measurement during the COVID-19 pandemic: A laboratory and patient perspective.

AIMS: The purpose of this study was to assess the clinical performance and user acceptance of capillary blood samples prepared remotely using the MiniCollect® capillary blood collection device as an alternative to blood collection by venepuncture for glycated haemoglobin (HbA1c ) analysis. METHODS: Following written informed consent, a cross-sectional study was conducted in individuals aged ≥18 years with any type of diabetes who routinely self-monitor their blood glucose. Eligible participants recruited whilst attending their routine clinical appointments were required to provide a venous blood sample, prepare a capillary blood sample at home (remotely) and complete a bespoke questionnaire. HbA1c in whole blood collected in ethylenediaminetetraacetic acid was determined by capillary electrophoresis on the Sebia Capillary's 3 Tera analyser following standard operating procedure. RESULTS: HbA1c results from both venous and capillary collection demonstrated good agreement. Passing-Bablok regression: y = 0 + 1x (p = 0.18), Spearman correlation r = 0.986, p < 0.0001. The Bland-Altman difference plot provided a mean difference of 0.3 mmol/mol (2.2%). Over half of the participants found the MiniCollect device easy to use. The majority of participants were in favour of the remote capillary blood collection service and would use it if routinely available. CONCLUSION: The home collection of capillary blood for HbA1c determination is a valuable and convenient alternative to standard venous blood collection as it provides an opportunity to support routine HbA1c monitoring, whilst mitigating the transmission of SARS-CoV-2. This service would additionally allow individuals to attend clinic visits with a HbA1c value, ensuring optimal continuance of patient care for individuals with diabetes.

Reference intervals for commonly requested biochemical and haematological parameters in a healthy Irish adult Caucasian population.

INTRODUCTION: In laboratory medicine, reference intervals (RIs) are key decision support tools used to guide the clinical interpretation of numerical test results. Best practice suggests each laboratory establishes RIs in the local population prior to introducing an assay into routine clinical practice. AIM: The aim of this study was to define RIs for frequently requested biochemical/haematological parameters in a healthy adult Irish Caucasian population. METHODS: A cross-sectional study of non-pregnant apparently healthy volunteers was conducted. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 37 commonly requested biochemical (serum, n = 26) and haematological (venous blood, n = 11) ISO15189:2012 accredited tests were analysed, using the Roche Cobas® Sebia Capillarys 3 Tera and Siemens Advia® 2120i platforms following standard operating procedures. RIs were defined according to the International Federation of Clinical Chemistry (IFCC) recommended method. RESULTS: Of 208 apparently healthy volunteers, 76 failed to meet the study inclusion criteria. The reference population comprised of 132 participants (males: n = 65, 49.2%) with a median age of 29.7 (18.1-62.2) years. RIs for the majority of biochemical/haematological parameters were broadly in accord with those provided by Pathology Harmony (UK)/Irish RI Harmonisation Project and the manufacturer Roche Diagnostics. However, the established RI defined for HbA1c: 27-37 mmol/mol was markedly different from that quoted nationally, HbA1c: 20-42 mmol/mol. CONCLUSION: Normative biological intervals established in a healthy adult Irish population for 37 commonly requested biochemical/haematological parameters will be a valuable aid to result interpretation in clinical laboratories after appropriate verification in accordance with ISO 15189: 2012.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis.

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature. This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum gp60 subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region of gp60. • Nested gp60 primers were designed to amplify a target region of <200 base pairs for effective HRM analysis • This method presents a rapid, sensitive, cost effective alternative to conventional sequencing. • This method is highly flexible and may be applied to other loci in order to facilitate multi-locus analysis and improve the discriminative abilities of the method.

Increased diversity and novel subtypes among clinical Cryptosporidium parvum and Cryptosporidium hominis isolates in Southern Ireland.

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.

Biallelic CYP24A1 variants presenting during pregnancy: clinical and biochemical phenotypes.

INTRODUCTION: Inactivating mutations in CYP24A1, encoding vitamin D-24-hydroxylase, can lead to an accumulation of active vitamin D metabolites and consequent hypercalcaemia. Patient (infantile and adult) presentation is varied and includes mild-severe hypercalcaemia, hypercalciuria, nephrocalcinosis and nephrolithiasis. This study aimed to characterize the clinical and biochemical phenotypes of a family with two CYP24A1 missense variants. METHODS: The proband and seven family members underwent detailed clinical and biochemical evaluation. Laboratory measurements included serum calcium, intact parathyroid hormone (iPTH), vitamin D metabolites and urine calcium and creatinine. RESULTS: The proband presented during the second trimester of a planned pregnancy with flu-like symptoms. Laboratory tests showed elevated adjusted calcium of 3.27 (upper reference limit (URL: 2.30) mmol/L), suppressed iPTH (<6 ng/L), elevated 25(OH)D (264 (URL: 55) nmol/L) and elevated 1,25(OH)D (293 (URL: <280) pmol/L). Ionized calcium was 1.55 (URL: 1.28) mmol/L. Sanger sequencing revealed two heterozygous missense variants in the CYP24A1: p.(Arg439Cys), R439C and p.(Trp275Arg), W275R. The proband's brother and sister had the same genotype. The brother had intermittent hypercalcaemia and hypervitaminosis D. Only the sister had a history of nephrolithiasis. The proband's daughter and two nephews were heterozygous for the R439C variant. The proband and her brother frequently had elevated 25(OH)D:24,25(OH)2D ratios (>50) during follow-up. CONCLUSIONS: W275R is a new pathogenic CYP24A1 mutation in compound heterozygotic form with R439C in this family.

Cryptosporidium spp surveillance and epidemiology in Ireland: a longitudinal cohort study employing duplex real-time PCR based speciation of clinical cases.

Cryptosporidium is a leading cause of gastroenteritis (cryptosporidiosis), with significant morbidity and mortality worldwide. Irish cryptosporidiosis incidence rates are consistently the highest reported in Europe. A retrospective, longitudinal study of clinical Cryptosporidium isolates was conducted from 2015 to 2018 in Cork, southern Ireland. Overall, 86.5% of cases were attributed to C. parvum, while the remaining 13.5% were caused by C. hominis Despite the widespread implications of this protozoan parasite in sporadic and outbreak-related illness in Ireland, the current dearth of species-level epidemiological surveillance and clinical studies needs to be addressed in order to elucidate the national impact of this enteric pathogen.

Vitamin D Status of Adults in the Community, in Outpatient Clinics, in Hospital, and in Nursing Homes in the West of Ireland.

BACKGROUND: Approximately 1 billion people worldwide have Vitamin D deficiency. The aim of this study was to compare Vitamin D status and serum 25-hydroxyvitamin D (25(OH)D) concentrations among adults sampled in the community, in outpatient clinics, as hospital inpatients and in nursing homes in the West of Ireland. The secondary aim was to determine the associations between length of hospital stay (inpatients) at the time of serum 25(OH)D sampling and Vitamin D status. METHODS: A cross-sectional study was carried out. Patients who had serum 25(OH)D analysis carried out in Galway University Hospitals (January 2011-December 2015) were identified following interrogation of the electronic laboratory data system. Baseline demographics, location, and date of sample collection were recorded. Vitamin D deficiency was defined as a serum 25(OH)D concentration <25 nmol/L. RESULTS: In total, 24,302 patient samples were eligible for inclusion: community 15,319; outpatient clinics 6,371; inpatients 2,339; and nursing home residents 273. Vitamin D deficiency was more common in nursing home residents than inpatients, or those sampled in outpatient clinics or in the community (42% vs 37% vs 17% vs 13%; p < .001). Inpatients sampled further into their hospital stay (≥3 days) had greater Vitamin D deficiency than inpatients sampled on 0-2 days (p = .007). Season (p < .001), sex (p < .001), and age (p < .001) were associated with 25(OH)D concentrations. Vitamin D deficiency was more common in Winter/Spring, in males, and in those aged ≥80 years. CONCLUSIONS: Nursing home residents and inpatients are at the highest risk for Vitamin D deficiency. Season, sex, age, and day of hospital stay on which serum 25(OH)D concentrations were sampled were associated with Vitamin D status.

Higher risk of vitamin D insufficiency/deficiency for rural than urban dwellers.

There are many risk factors for Vitamin D deficiency. This study aimed to compare the Vitamin D status and serum 25(OH)D concentrations of adults living in an urban area to adults living in a rural area in the West of Ireland (latitude 53.27° North). A cross-sectional retrospective analysis of clinical records was performed. Following interrogation of the electronic laboratory information system, individuals who had serum 25(OH)D concentrations measured at Galway University Hospitals between January 2011 and December 2015 were identified. Clinical demographics, setting and date of sampling were recorded. In total, 17,590 patients (urban n = 4,824; rural n = 12,766) were eligible for inclusion. Serum 25(OH)D concentrations were lower among rural compared to urban dwellers irrespective of season (spring p < 0.001, summer p = 0.009, autumn p = 0.002, winter p < 0.001). There was a significant difference in Vitamin D status between urban and rural dwellers in three of the four seasons: spring- deficiency: 16%-v-23%, insufficiency: 39%-v-43%, sufficiency: 45%-v-35% (p < 0.001); autumn- deficiency: 11%-v-10%, insufficiency: 30%-v-35%, sufficiency: 59%-v-56% (p = 0.01); winter- deficiency: 23%-v-25%, insufficiency: 35%-v-42%, sufficiency: 41%-v-33% (p < 0.001). Serum 25(OH)D concentrations were higher and the prevalence of deficiency lower in urban/rural females compared to urban/rural males (p < 0.001). Serum 25(OH)D concentrations increased sequentially from the 18-39 year age group to the 60-69 year age group in both urban (p < 0.001) and rural (p < 0.001) dwellers and then decreased progressively as age increased to ≥90 years. The odds of Vitamin D deficiency increased with age, lower daily sunshine hours, male gender, rural address and season.

Effect of Sodium Glucose Co-Transporter-2 Inhibition on the Aldosterone/Renin Ratio in Type 2 Diabetes Mellitus.

The aldosterone to renin ratio (ARR) is recommended for case detection of primary aldosteronism (PA). Several factors including medications can undermine its diagnostic accuracy. The objective was to explore the effect of Sodium Glucose Co-Transporter-2 Inhibition on the ARR in patients with type 2 diabetes mellitus (T2DM) who were prescribed a Sodium Glucose Co-Transporter-2 Inhibitor (SGLT-2i) as part of routine clinical care. The primary outcomes were intra-individual changes in aldosterone, renin and ARR. Participants were recruited at routine diabetes outpatient visits as part of a prospective longitudinal study. Eligible participants were prescribed standard doses of empagliflozin and sampled at baseline (pre-SGLT-2i) and at their next routine outpatient visit (post-SGLT-2i). After a mean of 198 (±87) days on SGLT-2i treatment (n=20), there was a significant reduction in HbA1c, BMI, eGFR and serum triglycerides and a significant increase in serum creatinine and sodium. Compared with baseline, there was a significant increase in median direct renin concentration (mIU/l) [40.3 (6.2-249.5) vs. 70.2 (7.0, 551.0) (p=0.005)] and no significant change in median plasma aldosterone concentration (pmol/l) [296 (101, 685) vs. 273 (101, 794) (p=0.541)] with a significant reduction in median ARR (pmol/mIU) [6.9 (0.6-70.7) vs. 5.3 (0.2-39.3) (p=0.007)]. The proportion of participants with a screen positive ARR decreased from 20% (pre-SGLT-2i) to 5% (post-SGLT-2i) (p=0.248). Although performed in a relatively small cohort of medically complex patients, the study indicates that SGLT-2i therapy has the potential to cause false-negative screening for PA in the setting of T2DM. Future confirmatory studies should include patients with confirmed PA.

Improved detection of bacterial pathogens in patients presenting with gastroenteritis by use of the EntericBio real-time Gastro Panel I assay.

In this study, we evaluated the use of EntericBio real-time Gastro Panel I (Serosep, Limerick, Ireland) for routine use in a clinical microbiology laboratory for simultaneous detection of Campylobacter jejuni, coli, and lari, Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Shigella spp. in feces. This system differs from its predecessor (the EntericBio Panel II system, Serosep) in that it allows real-time detection of pathogens directly from feces, without pre-enrichment. It also specifically detects Campylobacter jejuni, coli, and lari rather than all Campylobacter species, as is the case with the previous system. A total of 528 samples from patients presenting with acute gastroenteritis were screened prospectively with this assay, and results were compared with those of the current method, which combines screening the samples with a molecular assay (the EntericBio Panel II assay) and retrospective culture of the specimens in which the target was detected. Discrepancy analysis was conducted using culture and molecular methods. The real-time assay produced 84 positive results, specifically, Campylobacter spp. (n=44); Stx1 and/or Stx2 (n=35); Shigella spp. (n=3); and Salmonella spp. (n=6). Of these, 4 samples represented coinfections with Campylobacter spp. and STEC. The real-time assay showed an increased detection rate for pathogens, apart from Salmonella spp. Four Campylobacter-positive and 6 Stx-positive results remained unconfirmed by any other method used. The isolation rates for PCR-positive samples were as follows: Campylobacter spp., 80%; STEC, 45.7%; Salmonella spp., 100%; and Shigella spp., 66.7%. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were 100%, 97.8%, 88.1%, 100%, and 98.1%, respectively.

Introduction of an NT-proBNP assay to an acute admission unit--a 2-year audit.

BACKGROUND: The differential diagnosis of dyspnoea is difficult due to the low predictive value of clinical and laboratory parameters. The elevated levels of NT-proBNP in congestive heart failure may improve diagnostic accuracy. We have evaluated the effect of the introduction of an NT-proBNP assay on hospital length of stay (LOS) and mortality. METHODS: There were 11,853 AMAU patient episodes in the 22 months study period (March 2005-Dec 2006). An NT-proBNP assay was requested in 657 (5.5%) of these. Comparison between categorical variables such as diagnosis, NT-proBNP testing, LOS, and in-hospital mortality was made using Chi-square tests. Literature review suggested that an NT-proBNP cut-off >or=5000 ng/L should predict acute in-patient mortality. Logistic regression analysis was used to examine the association between such an elevated NT-proBNP level and outcomes. RESULTS: Of the 396 patients with NT-proBNP <5000 ng/L, 8.1% died compared with 22.5% of the 178 patients dying with values >or=5000 ng/L (p<0.0001). An NT-proBNP >or=5000 ng/L was predictive of both LOS >or=9 days (odds ratios (OR) 1.54 (95% CI 1.06, 2.24: p=0.02) and LOS >or=14 days (OR=1.87 (95% CI 1.29, 2.71: p=0.0009). NT-proBNP requests increased over time, from 2.6% to 8.2% of all patients; the result fell in the diagnostic range for CHF in 60% of requests. CONCLUSION: The introduction of an NT-proBNP was reflected in an appropriate but rapidly increasing pattern of requests from clinicians. High NT-proBNP levels predicted in-hospital mortality and longer LOS in an acute medical population.