Screening for sickle cell disease: focus on newborn investigations.

Drepanocytosis is a genetic disease relevant for its epidemiological, clinical and socio-economic aspects. In our country the prevalence is highly uneven with peaks in former malaria areas, but migration flows in recent years have led to significant changes. In this document we review the screening programs currently existing in Italy with particular emphasis on newborn screening, which in other countries around the world, including within Europe, is at most universal and mandatory. The essential laboratory issues are reviewed, from sampling aspects (cord blood or peripheral), to the analytical (analytical methods dedicated to neonatal screening and adult carrier detection) and post analytical (reporting, informative) ones. An economic analysis based on data collected in the province of Modena is also proposed, clearly showing that neonatal screening is also beneficial from an economic point of view.

Analysis of annual distributions of hemoglobin A2 values as a method to test for HbA2 standardization.

BACKGROUND AND AIMS: The monitoring of yearly distributions of HbA2 measured has been indicated as a reliable indicator of worldwide standardization. MATERIALS AND METHODS: Measurements/year of HbA2 have been collected over three consecutive years in 15 Italian laboratories each using the same analytical method over three years period. HbA2 distributions, cleaned of replicated measurements, were compared by the overlapping area of the raw probability density functions expressed by coefficient eta (η), and by comparing the reference intervals for the central part of each distribution estimated by the indirect method refineR using the R package "refineR". RESULTS: According to the overlapping areas analysis the distributions/year of the data provided by 4 centers able to perform at least 1000 measurements/year were similar in 2 consecutive years. Moreover, the reference intervals provided by 2 centers using the same analytical methods in two separate locations over the three consecutive years, were very similar. The highest overlap (99.7 %) was observed in one center over two consecutive years. The overlapping areas were very high (93.6-95.7%) in 8 out of 9 inter-comparisons. CONCLUSION: Despite the limitations of this study the yearly distribution of the HbA2 measured in various centers appears a reliable tool to test HbA2 standardization over different centers using different analytical methods.

Progressive Thinning of Retinal Nerve Fiber Layer/Ganglion Cell Layer (RNFL/GCL) as Biomarker and Pharmacological Target of Diabetic Retinopathy.

Diabetes-driven retinal neurodegeneration has recently been shown to be involved in the initial phases of diabetic retinopathy, raising the possibility of setting up a preventive strategy based on early retinal neuroprotection. To make this possible, it is crucial to identify a biomarker for early retinal neurodegeneration. To this end, in this study, we verified and confirmed that, in the Akita mouse model of diabetes, the thinning of the retinal nerve fiber layer/ganglion cell layer (the RNFL/GCL-the layer that contains the retinal ganglion cells) precedes the death of these same cells, suggesting that this dysfunction is a possible biomarker of retinal neurodegeneration. We then confirmed the validity of this assumption by starting a neuroprotective treatment (based on nerve growth factor eye drops) in concert with the first demonstration of RNFL/GCL thinning. In this way, it was possible not only to avoid the loss of retinal ganglion cells but also to prevent the subsequent development of the microvascular stage of diabetic retinopathy. In conclusion, in the case of diabetes, the thinning of the RNFL/GCL appears to be both a valid biomarker and a pharmacological target of diabetic retinopathy; it precedes the development of vascular dysfunctions and represents the ideal starting point for prevention.

Characterization of Maternal Circulating MicroRNAs in Obese Pregnancies and Gestational Diabetes Mellitus.

Maternal obesity (MO) is expanding worldwide, contributing to the onset of Gestational Diabetes Mellitus (GDM). MO and GDM are associated with adverse maternal and foetal outcomes, with short- and long-term complications. Growing evidence suggests that MO and GDM are characterized by epigenetic alterations contributing to the pathogenesis of metabolic diseases. In this pilot study, plasma microRNAs (miRNAs) of obese pregnant women with/without GDM were profiled at delivery. Nineteen women with spontaneous singleton pregnancies delivering by elective Caesarean section were enrolled: seven normal-weight (NW), six obese without comorbidities (OB/GDM(-)), and six obese with GDM (OB/GDM(+)). miRNA profiling with miRCURY LNA PCR Panel allowed the analysis of the 179 most expressed circulating miRNAs in humans. Data acquisition and statistics (GeneGlobe and SPSS software) and Pathway Enrichment Analysis (PEA) were performed. Data analysis highlighted patterns of significantly differentially expressed miRNAs between groups: OB/GDM(-) vs. NW: n = 4 miRNAs, OB/GDM(+) vs. NW: n = 1, and OB/GDM(+) vs. OB/GDM(-): n = 14. For each comparison, PEA revealed pathways associated with oxidative stress and inflammation, as well as with nutrients and hormones metabolism. Indeed, miRNAs analysis may help to shed light on the complex epigenetic network regulating metabolic pathways in both the mother and the foeto-placental unit. Future investigations are needed to deepen the pregnancy epigenetic landscape in MO and GDM.

Reference intervals for glycated albumin during physiological pregnancy of Europid women: Evidences from a prospective observational study.

BACKGROUND AND AIMS: Glycated albumin (GA) may assess glycometabolic control over a short period of time respect to HbA1c, and its use to screen for gestational diabetes in pregnancy has been suggested. To this regard few data on reference intervals (RI) for GA on Europid women have been collected, only from cross-sectional investigations. Aim of this work has been to collect trimester-specific RI for GA in physiological pregnancies, following a longitudinal prospective study. METHODS: Forty-five healthy pregnant Europid women have been enrolled for whom a GDM screening test was scheduled at 24-28 weeks, in 5 different Italian centers. Only those negative to the OGTT were included. The women had 4 successive visits at 6-10 weeks of gestation, at 16-18 weeks, at 24-28 weeks and at the end of pregnancy. ALT, AST, total bilirubin, C-reactive protein, cholinesterase, creatinine, GGT, glycated albumin, iron, total serum proteins, transferrin were measured in duplicate on aliquots of serum samples by a central laboratory. RESULTS: The RI (2.5-97.5 percentiles) for GA were 11.1-14.8 % (I visit), 10.9-15.6 % (II visit), 10.6-14.1 % (III visit) and 10.7-14.3 % (IV visit). The RI of other biomarkers confirmed previously published data. The RI for serum cholinesterase we present are novel, and were 5049-9906 U/L (Iv), 4212-8965 U/L (IIv), 3518-8470 U/L (IIIv) and 3945-8727 U/L (IVv). CONCLUSIONS: Trimester-specific RI are important for using GA and serum cholinesterase in pregnancy. However, considering the high inter-individual variability of both markers, the use of longitudinal interpretations of the individual variations of both proteins during pregnancy should be preferred.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Re-thinking diabetic nephropathy: Microalbuminuria is just a piece of the diagnostic puzzle.

The decline of the estimated glomerular filtration rate (eGFR) and the presence of albuminuria are the typical hallmarks of kidney disease arising as one of the most frequent diabetic complications over a long period of time, generally known as diabetic nephropathy or diabetes kidney disease (DKD). However, a decline in the renal function may occur in diabetic patients for other reasons unrelated to glycemic control, and this condition is known as non-diabetic kidney disease (NDKD). In this opinion paper we will review these conditions, and we outline the importance of other investigations, such as kidney biopsy and the measurement of novel biomarkers, in order to identify the disease progression early, and to allow a timely intervention. We will also focus on the actual limits of the quantitative measurements of albumin in urine, especially with regards to potential interferences due to the treatment of patients with statins.

Placental Antioxidant Defenses and Autophagy-Related Genes in Maternal Obesity and Gestational Diabetes Mellitus.

Maternal obesity and gestational diabetes mellitus (GDM) are increasing worldwide, representing risk factors for both mother and child short/long-term outcomes. Oxidative stress, lipotoxicity and altered autophagy have already been reported in obesity, but few studies have focused on obese pregnant women with GDM. Antioxidant and macro/chaperone-mediated autophagy (CMA)-related gene expressions were evaluated herein in obese and GDM placentas. A total of 47 women with singleton pregnancies delivered by elective cesarean section were enrolled: 16 normal weight (NW), 18 obese with no comorbidities (OB GDM(-)), 13 obese with GDM (OB GDM(+)). Placental gene expression was assessed by real-time PCR. Antioxidant gene expression (CAT, GPX1, GSS) decreased, the pro-autophagic ULK1 gene increased and the chaperone-mediated autophagy regulator PHLPP1 decreased in OB GDM(-) vs. NW. On the other hand, PHLPP1 expression increased in OB GDM(+) vs. OB GDM(-). When analyzing results in relation to fetal sex, we found sexual dimorphism for both antioxidant and CMA-related gene expressions. These preliminary results can pave the way for further analyses aimed at elucidating the placental autophagy role in metabolic pregnancy disorders and its potential targetability for the treatment of diabetes outcomes.

A roadmap for the standardization of hemoglobin A2.

BACKGROUND: Standardization of laboratory tests can be a long process, and this is the case with regards to the methods used to measure hemoglobin A2 (HbA2), an important marker for beta-thalassemia and other thalassemic conditions. The IFCC standardization project started in 2004, and it took at least 15 years before developing a reference measurement procedure, defining and producing calibrators and certified reference materials. METHODS: A series of steps have to be undertaken in order to promote the standardization in the field, a process involving a number of stakeholders (manufacturers, scientific societies, national health bodies, laboratory professionals, clinicians). In this work we describe some possible process indicators, in order to assure that the standardization will have internal and external validity and be effective for a long time. These indicators concern the inter-method studies, elaboration of External Quality Assessment Schemes, and the evaluation of the yearly distributions of HbA2 measurements collected in selected laboratories. RESULTS: Preliminary results are reported concerning the yearly distributions of HbA2, collected in two different locations, and using different analytical methods. Median yearly values were found very constant over the years, but different between methods. On the other side, results obtained on the same specimens using two different techniques, proved that results by capillary electrophoresis in 2 out of the 3 years of observation, were significantly lower than those by HPLC. CONCLUSION: In this document we report what has been done so far, and what has to be done to achieve the standardization of the measurement of HbA2 worldwide.

Role of fructosamine-3-kinase in protecting against the onset of microvascular and macrovascular complications in patients with T2DM.

INTRODUCTION: Microangiopathic and macroangiopathic complications are the main cause of morbidity and mortality in the diabetic population. Numerous publications have highlighted the role of glycation in the onset of complications of diabetes. In this context, the detection of fructosamine-3-kinase (FN3K)-an enzyme capable of counteracting the effect of hyperglycemia by intervening in protein glycation-has attracted great interest. Several studies have linked FN3K genetic variability to its enzymatic activity and glycated hemoglobin (HbA1c) levels. Here, we investigated the role of FN3K polymorphisms in the development of microvascular and macrovascular complications of diabetes. RESEARCH DESIGN AND METHODS: The anthropometric and biochemical parameters, and any medical history of microangiopathic and macroangiopathic complications, were documented in a sample of 80 subjects with type 2 diabetes. All subjects were screened for FN3K gene and analyzed for the combination of three polymorphisms known to be associated with its enzymatic activity (rs3859206 and rs2256339 in the promoter region and rs1056534 in exon 6). RESULTS: The combination of allelic variants of FN3K polymorphisms resulted in 13 distinct genotypic variants within the cohort. Comparison between genotypes showed no significant differences in terms of demographic, anthropometric and biochemical parameters, risk markers and long-term complications, except for a higher age and vitamin E levels associated with the genotype presenting GG at position -385, TT at position -232, and CC at c.900 A. Evaluating the microangiopathic and macroangiopathic complications as a whole, we found that they appeared significantly less present in this genotype compared with all other genotypes (p=0.0306). CONCLUSIONS: The group of patients carrying the favorable allele for the three polymorphisms of the FN3K gene revealed less severe microangiopathy and macroangiopathy, suggesting a protective role of this genotype against the onset of the complications of diabetes.

The analytical performance of laboratory plasma glucose and HbA1c measurements are largely acceptable.

AIMS: Poor comparability between laboratory results may have a strong impact on clinical decisions. The aim of this study was to assess the quality of glucose and HbA1c measurements in a large cohort of laboratories in various countries, in order to evaluate whether the current state of these very basic laboratory examinations can be considered to be adequate with respect to the clinical needs in the management of glucose control in diabetic patients. METHODS: External quality assessment schemes and proficiency testing surveys performed in 2017 in several European and American laboratories were analyzed in order to estimate the percentage of laboratories reaching the desired quality criteria based on the allowable total error in accordance with various international recommendations. RESULTS: In 2017 more than 95% of laboratories met the allowable total error for measuring HbA1c, and 92-94% of the studied laboratories met the target for glucose measurement. CONCLUSIONS: The analytical quality for measuring glycated hemoglobin and glucose at laboratory level is generally acceptable, and accreditation to the ISO 15189:2012 standard is a robust guarantee that the laboratory meets the required criteria of acceptability. Several pre-analytical factors which may explain the discrepancies between the measured HbA1c and that estimated from other indicators of glucose control have to be taken into account, by focusing more on the pre-analytical than the analytical phase. In the case of glucose, special attention should be paid to the use of the correct anticoagulant, in order to avoid false negative results.

Effects of different anticoagulants on glycated albumin quantification.

INTRODUCTION: In the last 20 years glycated albumin (GA) measurement has been demonstrated to be a reliable glycation marker and recently as the most innovative one in western countries. Glycated albumin has been already adopted by some Asian countries due to its usefulness in diabetes screening. The aim of the present study was to investigate for the first time the effects of different anticoagulants on GA assay. MATERIALS AND METHODS: From each of 60 patients a serum tube and K3EDTA, Li-Heparin and NaF-EDTA containing tubes were collected. All tubes were from Sarstedt (Verona, Italy). Glycated albumin was measured in duplicate in each sample tube in a single analytical run with quantILab glycated albumin (Instrumentation Laboratory SpA - A Werfen Company, Milan, Italy) on Architect c8000 analyser (Abbott SRL, Rome, Italy). Comparison of GA% in evaluated tubes was made by paired Wilcoxon test. RESULTS: Median and interquartile range GA% concentrations were 15.4% (13.2 - 19.1) in serum, 15.7% (13.6 - 19.9) in K3EDTA, 15.6% (13.3 - 19.7) in Li-heparin and 15.5% (13.1 - 19.3) in NaF-EDTA samples, respectively. Glycated albumin mean relative bias respect to serum was within desirable bias derived from biological variation studies (± 2.9%) when K3EDTA (+ 2.8%), Li-heparin (+ 0.9%) or NaF-EDTA (+ 0.1%), were used as anticoagulants. CONCLUSIONS: Our results demonstrate that the GA% assay is not affected by relevant interferences when K3EDTA, Li-heparin or NaF-EDTA are used as anticoagulants, so they can be used interchangeably without a relevant impact on the clinical use of the test.

Standardization of the HbA2 Assay.

BACKGROUND: A project for the standardization of HbA2 was launched by the IFCC back in 2004. MATERIALS AND METHODS: In this work we report on the state-of-the-art of the project on standardization of HbA2. Data obtained from various EQAS studies, and from previous experimental evaluations, are presented. RESULTS: We have proven that biases between various commercial methods are still currently significant. We have also shown that calibration by commutable control materials may halve the inter-method variability. CONCLUSIONS: The foundation of the reference system for HbA2, together with a brief preliminary presentation of the proposed primary reference measurement procedure based on ID-MS are outlined.

Determination of HbA2 by quantitative bottom-up proteomics and isotope dilution mass spectrometry.

BACKGROUND: Poor comparability between laboratories is often observed in the measurement of HbA2. A measurement procedure of higher metrological order is needed for value assignment to a reference material that shall be used as primary calibrator. METHOD: A reference measurement procedure has been developed based on isotope dilution mass spectrometry (IDMS). The α- and δ- subunits are quantified by signature peptides released by tryptic digestion of a 25 µL-blood sample. Full length U-15N-labeled HbA0 and HbA2 are used as internal standards and added to the sample at concentrations closely matching the levels of the natural forms in blood. By this, an improvement in precision could be achieved with respect to previous mass-spectrometry based methods. RESULTS: Recovery of HbA2 added to a blood sample was within 102.6-105.2%. Repeatability and within-laboratory imprecision was <2.0% for two blood samples containing HbA2 at a low and a high fraction. Total combined measurement uncertainty is estimated as 5.5%. Good agreement (r = 0.998) of results was obtained in a comparison of two laboratories using the described IDMS procedure. There is good correlation between commercial analytical systems and IDMS (r = 0.975-0.989). Some of the platforms provide significantly biased results, however, which potentially could be mitigated by reference to IDMS. CONCLUSION: IDMS holds a promise to be suitable as a reference measurement procedure for standardization of HbA2-measurements in laboratory medicine.

Sources and performance criteria of uncertainty of reference measurement procedures.

OBJECTIVE: This article wants to focus on the today available Reference Measurement Procedures (RMPs) for the determination of various analytes in Laboratory Medicine and the possible tools to evaluate their performance in the laboratories who are currently using them. METHODS: A brief review on the RMPs has been performed by investigating the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. In order to evaluate their performances, we have checked the organization of three international ring trials, i.e. those regularly performed by the IFCC External Quality assessment scheme for Reference Laboratories in Laboratory Medicine (RELA), by the Center for Disease Control and Prevention (CDC) cholesterol network and by the IFCC Network for HbA1c. RESULTS: Several RMPs are available through the JCTLM database, but the best way to collect information about the RMPs and their uncertainties is to look at the reference measurement service providers (RMS). This part of the database and the background on how to listed in the database is very helpful for the assessment of expanded uncertainty (MU) and performance in general of RMPs. Worldwide, 17 RMS are listed in the database, and for most of the measurands more than one RMS is able to run the relative RMPs, with similar expanded uncertainties. As an example, for a-amylase, 4 SP offer their services with MU between 1.6 and 3.3%. In other cases (such as total cholesterol, the U may span over a broader range, i.e. from 0.02 to 3.6%). With regard to the performance evaluation, the approach is often heterogenous, and it is difficult to compare the performance of laboratories running the same RMP for the same measurand if involved in more than one EQAS. CONCLUSIONS: The reference measurement services have been created to help laboratory professionals and manufacturers to implement the correct metrological traceability, and the JCTLM database is the only correct way to retrieve all the necessary important information to this end.

Calibration by commutable control materials is able to reduce inter-method differences of current high-performance methods for HbA2.

BACKGROUND: Most of the current methods used for the determination of HbA2 seem not well aligned. A comparison among the best performing techniques and the commutability of some control materials currently available and under development has been evaluated. METHODS: Forty blood samples were analyzed in duplicate over two separate days by different HPLC and capillary electrophoresis systems. The commutabilities of different control materials (NIBSC WHO reagent, Bio-Rad Lyphochek, and home prepared lyophilized controls RP1-3) have been assessed by analyzing the controls in quadruplicate over two consecutive days together with the blood samples. RESULTS: The mean within-run imprecision of HbA2 measurement on blood samples (CV, %) was between 0.6% and 10.1% for HbA2 values <3.5%, and between 1.1 and 3.1 for HbA2≥3.5%. The different methods were highly correlated (r between 0.9941 and 0.9995) although biased each other. The NIBSC WHO reagent was found not commutable in 15 over 28 comparisons, the Lyphochek 2 in 18/28, and RP3 in 4/28. Recalibration of all methods by RP1 and RP2 materials was able to reduce the overall variability from 6.8% to 3.4% at HbA2≤3.0% and from 6.7% to 3.0% at HbA2≥4.6%. CONCLUSION: The use of adequate commutable control materials as calibrators may reduce the inter-method variability of routine methods to an extent closer to the current analytical goals of bias based on biological variability.

Multicenter evaluation of an enzymatic method for glycated albumin.

BACKGROUND: The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA1c test is not reliable. METHODS: We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). RESULTS: The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. CONCLUSION: The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use.

The role of socio-economic and clinical factors on HbA1c in children and adolescents with type 1 diabetes: an Italian multicentre survey.

OBJECTIVE: To identify the role of the family's socio-economic and clinical characteristics on metabolic control in children and adolescents with type 1 diabetes. METHODS: In this cross-sectional, multicentre study, 768 subjects with type 1 diabetes under 18 years of age were consecutively recruited from January 2008 to February 2009. Target condition was considered for HbA1c values <7.5% (<58 mmol/mol). A multiple correspondence analysis (MCA) was performed to analyze the association between the socio-economic and clinical characteristics of the participants. A logistic regression analysis was performed to identify factors associated with the subjects metabolic control. In both analyses, the family's socio-economic status was represented, measured by the Hollingshead Four-Factor Index of Social Status (SES) or by parental years of education. RESULTS: A total of 28.1% of subjects reached target HbA1c values. The MCA identified a strong association between at-target condition and several factors: high levels of SES or high levels of parental education, the use of the carbohydrate counting system, the use of insulin pumps, the use of the insulin delivery system over a short period of time, a normal body mass index. The logistic regression analysis showed that SES and the mother's years of education were significantly associated with the target condition [odds ratio (OR): 1.01, 95% confidence interval (CI): 1.01-1.03, p = 0.029; OR: 1.05, 95% CI: 1.01-1.10, p = 0.027, respectively). CONCLUSIONS: Personal, clinical, and family characteristics were found to be associated with HbA1c target. Their identification can be crucial in addressing strategies to optimize metabolic control and improve diabetes management.

Developing a reference system for the IFCC standardization of HbA2.

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of ß-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.