Trefor N. Higgins

Measurement of Hba1C in patients with chronic renal failure

BACKGROUNDnCarbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA(1c) in diabetic patients with chronic renal failure (CRF). There is also concern that HbA1c may give low results in these patients due to shortened erythrocyte survival.nnnMETHODSnWe evaluated the effect of carbHb on HbA(1c) measurements and compared HbA(1c) with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA(1c) results by using 11 assay methods. Subjects included those with and without renal failure and diabetes. Each subjects estimated glomerular filtration rate (eGFR) was used to determine the presence and degree of the renal disease. A multiple regression model was used to determine if the relationship between HbA(1c) results obtained from each test method and the comparative method was significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance by using the difference between the eGRF quartiles of >7% at 6 or 9% HbA(1c). The relationship between HbA(1c) and glycated albumin (GA) in patients with and without renal failure was also compared.nnnRESULTSnSome methods showed small but statistically significant effects of eGFR; none of these differences were clinically significant. If GA is assumed to better reflect glycemic control, then HbA(1c) was approximately 1.5% HbA(1c) lower in patients with renal failure.nnnCONCLUSIONSnAlthough most methods can measure HbA(1c) accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due to the propensity for shortened erythrocyte survival in renal failure.

Effects of hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by twelve methods

BACKGROUNDnHemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 12 current assay methods.nnnMETHODSnHemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on one enzymatic, nine ion-exchange HPLC and two Capillary Electrophoresis methods. Trinity ultra(2) boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >7% difference from HbAA at 6 or 9% HbA1c compared to ultra(2) using Deming regression.nnnRESULTSnAll methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G8 variant mode and GX (all variants), GX V1.22 (all but HbAE) and G11 variant mode (HbAC). All other methods (Abbott Architect c Enzymatic, Bio-Rad D-100, Variant II NU and Variant II Turbo 2.0, Menarini HA-8180T thalassemia mode and HA-8180V variant mode, Sebia Capillarys 2 and Capillarys 3) showed no clinically significant differences.nnnCONCLUSIONSnSeveral methods showed clinically significant interference with HbA1c results from one or more variants which could adversely affect patient care.

Analytical Evaluation of the Diazyme Glycated Serum Protein Assay on the Siemens ADVIA 1800: Comparison of Results Against HbA1c for Diagnosis and Management of Diabetes

Background: Hemoglobin A1c (HbA1c) is considered the gold standard for assessment of glycemic control in diabetic patients. HbA1c is inadequate in individuals homozygous or compound heterozygous for hemoglobin variants or in conditions with an altered red blood cell turnover. In these cases glycated albumin (GA) is proposed as an alternative assay. We aimed to evaluate the analytical performance of the Diazyme glycated serum protein (GSP) assay on an automated analyzer, to establish a reference interval (RI), and to compare from a clinical perspective, GSP/GA with glycated Hb (glyHb) results. Methods: Validation studies followed the CLSI guidelines and included precision, linearity, interferences, concordance of results with glyHb, and RI calculation. GSP was analyzed on representative samples with previously ordered HbA1c and albumin from the Dyna LIFE DX laboratory. Samples from patients with bisalbuminemia, hemoglobinopathies, and multiple myeloma were also included. Results: Within-run and total imprecision was <3.0% at both levels of control, analytical sensitivity was 5.31 μmol/L, and linearity was verified from 10 to 1150 μmol/L (total allowable error of 5%). Clinical concordance between %GA and glyHb was substantial (n = 175, R2 = .91, kappa = .78, P = .167). GSP RI was 160 to 340 μmol/L or if expressed as %GA 10.5 to 17.5%. Conclusion: Analytical performance of the Diazyme GSP assay on the Siemens ADVIA 1800 is acceptable for clinical use. The RI obtained was higher than that suggested by the manufacturer.

Identification of Hb Wayne and its effects on HbA1c measurement by 5 methods.

BACKGROUNDnThe World Health Organization and the American and Canadian Diabetes Associations approved HbA1c >6.5% as diagnostic for type 2 diabetes mellitus (T2DM). Hb variants and/or their chemically modified species can interfere with HbA1c measurements. We recently described a patient with Hb Wayne trait who was misdiagnosed with T2DM based on falsely elevated HbA1c. Hb Wayne is a clinically silent variant that exists as two isoforms: Hb Wayne I (Asn 139) and Hb Wayne II (Asp 139).nnnMETHODSnHemoglobinopathy investigation was performed by HPLC (Bio-Rad VARIANT-II), alkaline and acid electrophoresis (Sebia Hydrasis2), capillary zone electrophoresis (Sebia CAPILLARYS2™) and DNA sequencing. HbA1c was measured by five methods.nnnRESULTSnHb Wayne eluted as two small fractions with retention times of 1.0 and 1.46min on the HPLC (Bio-Rad VARIANT-II). Alkaline gel and capillary electrophoresis showed two small bands migrating faster than HbA. Hb Wayne generated spuriously high results on the Bio-Rad VARIANT-II Turbo 2.0, no results on the Tosoh G8, and did not interfere with either the Sebia CAPILLARYS2™ or immunoassays from Roche (tinaquant) and Siemens (Bayer DCA2000+). Based on the Hb Wayne HPLC profile of 3 patients, an algorithm was developed to facilitate its detection, which identified 9 additional patients with Hb Wayne trait.nnnCONCLUSIONSnWe characterize Hb Wayne by chromatographic and electrophoretic techniques and show the effect of Hb Wayne on five common HbA1c methodologies. We developed a quality assurance tool to assist in detecting Hb Wayne trait during HbA1c analysis on the Bio-Rad VARIANT-II™ Turbo 2.0.

A novel double heterozygous Hb Fontainebleau/HbD Punjab hemoglobinopathy.

OBJECTIVESnTo report the finding of a novel double heterozygous hemoglobinopathy, the coinheritance of Hb Fontainebleau (α-chain variant) with HbD-Punjab (β-chain variant) discovered upon investigation of unexplained microcytosis in an infant.nnnDESIGN AND METHODSnHemoglobinopathy investigation was performed by high performance liquid chromatography (HPLC) using the β-thalassemia Short Program on the Bio-Rad Variant II(TM) followed by gel electrophoresis at alkaline and acid pH (Sebia Hydrasys 2 Electrophoresis System) and molecular diagnostic testing. This study complied with our institutional board ethics requirements.nnnRESULTSnHPLC and electrophoresis suggested a complex α- and β-chain hemoglobinopathy with presumptive identification of the beta Hb variant as Hb D-Punjab. DNA sequencing analysis revealed the presence of a double heterozygous status for Hb Fontainebleau/Hb D-Punjab.nnnCONCLUSIONSnIn this paper we report the coinheritance of Hb Fontainebleau with Hb D-Punjab.

Stability of specific IgE antibodies to common food and inhalant allergens

OBJECTIVEnTo determine the optimum storage temperature for serum allergen specific IgE antibodies (sIgE) to common food and inhalant allergens.nnnMETHODSnPatient sera with sIgE concentrations ≥0.7kIUA/l were pooled accordingly: pool 1-peanut and hazelnut, pool 2-egg white, cows milk and cod fish, pool 3-soy, wheat and shrimp and pool 4-dust mite Dermatophagoides farinae, dog dander, cat dander, Timothy grass pollen, and silver birch pollen. Aliquots stored frozen, refrigerated and at room temperature were tested in duplicate (Phadia ImmunoCAP® 250) over two weeks. The relative difference was calculated for each sIgE as a percentage of the initial value and compared to the analytical reference change value.nnnRESULTSnMinimal effects on specimen stability were noted for all sIgE analyzed under the three storage conditions tested in this study. All changes observed in sIgE concentrations were related to the assay variability and not to sample deterioration.nnnCONCLUSIONnSerum allergen specific IgE concentrations are stable at all temperatures studied for up to 17days.

MALDI-TOF MS/microwave-assisted acid hydrolysis identification of HbG Coushatta.

OBJECTIVEnTo evaluate the effectiveness of microwave-assisted acid hydrolysis of proteins combined with MALDI-TOF MS to characterize hemoglobin G Coushatta.nnnDESIGN AND METHODnFollowing separation of the globin chains, the purified beta chain fractions were hydrolyzed by acid hydrolysis prior to MALDI-TOF MS.nnnRESULTSnThe method correctly identified the abnormal amino acid in HbG Coushatta. The results of this method were confirmed by an established de novo sequencing method.nnnCONCLUSIONnThe correct amino acid sequence for HbG Coushatta was found. The proposed method is an alternative to MS/MS methods available for identification of abnormal hemoglobin variants.

Analytical sensitivity and diagnostic performance of serum protein electrophoresis on the HYDRAGEL 30 PROTEIN(E) β1-β2 Sebia Hydrasys system

BACKGROUNDnSerum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are used in the diagnosis and monitoring of plasma cell dyscrasias. IFE is considered the most sensitive method for the detection of monoclonal proteins (M-proteins), but it is not quantitative. The goal of this study was to establish the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2.nnnMETHODOLOGYnPatient sera with a previously identified M-protein (IgG, IgA or IgM) were serially diluted with a normal serum pool and electrophoresed on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2. The SPE gels were individually interpreted by five independent observers and IFE was performed on selected samples. Limit of detection was determined as the lowest concentration of M-protein band visible on the gel. SPE diagnostic performance was evaluated against the gold standard IFE according CLSI EP12-A2 guidelines.nnnRESULTSnDetection limit was comparable among all M-proteins migrating in the gamma region, IgG-κ (0.18±0.08g/L; n=6), IgG-λ (0.36±0.25g/L; n=8), IgA-κ (0.40±0.13g/L; n=7), IgA-λ (0.37±0.23g/L; n=4), IgM-κ (0.47±0.20g/L; n=13) and IgM-λ (0.29±0.24g/L; n=6). Percentage agreement with IFE for IgG and IgA in the gamma region ranged from 65% to 100%, whereas IgM migrating in the gamma region and immunoglobulins co-migrating with alpha or beta globulins, showed poor (0-38%) agreement.nnnCONCLUSIONSnThis study evaluates the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) β1-β2. There was acceptable agreement between SPE and IFE for IgG-κ/λ and IgA-κ/λ migrating in the gamma region, suggesting that repeating IFE for samples with these isotypes, when the previous IFE and second SPE are both negative, may not be necessary.

Erroneous Diabetes Diagnosis: A Case of HbA1c Interference

A 66-year-old Caucasian female was referred for specialist follow-up of her treatment-refractory type 2 diabetes. The diagnosis was made based on two consecutive HbA1c results >6.5% (48 mmol/mol) in accordance with the Canadian Diabetes Association and the American Diabetes Association guidelines (1,2). Fasting glucose was normal and hyperglycemic symptoms were absent at diagnosis. The patient’s hematological indices were normal and she was unaware of any family history of hemoglobinopathy. Her glycemic control proved difficult to manage, with persistently elevated HbA1c (10.8–11.2% [95–99 mmol/mol]) despite treatment with metformin and, eventually, insulin glargine. Further, with treatment, the patient began to experience symptoms of episodic hypoglycemia.nnA fasting glucose of 84.6 mg/dL (4.8 mmol/L) obtained at the same time as an HbA1c of 11.2% (99 mmol/mol) triggered suspicion of interference. As all of the HbA …

Incidence of hemoglobinopathies and thalassemias in Northern Alberta. Establishment of reference intervals for HbF and HbA2

OBJECTIVESnThe aims of this study were to identify the incidence of hemoglobinopathies and thalassemias in Northern Alberta and calculate the reference intervals (RI) for hemoglobin (Hb) HbF and HbA2.nnnMETHODSnA retrospective ad-hoc analysis of the structural Hb variants and thalassemias identified on patients who had a hemoglobinopathy/thalassemia investigation performed between February 1 to December 31, 2013. Results were extracted from the Laboratory Information System. Statistical analysis was performed using MedCalc® version 11.4.2.0 for Windows software.nnnRESULTSn6616 hemoglobinopathy/thalassemia investigations and HbS screens were physician requested and 602 Hb variants were fortuitously found during HbA1c analysis. 3438 were interpreted as normal and 532 were classified as iron deficient. 3306 individuals, with age ranging from 3 to 92 years were included in the RI calculation. HbA2 RI was 2.3% to 3.4% and HbF 0.0% to 1.8%. 524 and 423 α and β thalassemia traits respectively were identified. Additionally ten δβ thalassemia traits and twelve cases of HbH disease were identified. Regarding hemoglobinopathies, 7% were classified as α-chain variants and 93% as β-chain variants with HbS (46%), HbE (16%), HbD Punjab (8%) and HbC (7%) traits being the most prevalent. We also documented 20 homozygous hemoglobinopathies and 36 compound/double heterozygous hemoglobinopathies.nnnCONCLUSIONnA wide diversity of hemoglobinopathies is found in the Northern Alberta population, 80% of the hemoglobinopathies were found as a reflex to HbA1c testing. Reference intervals for HbF and HbA2 were established.