BACKGROUND:Newborn screening for hemoglobinopathies is an effective tool for the early detection of clinically significant conditions, such as sickle cell disease. High-performance liquid chromatography (HPLC) is broadly used as it enables high-throughput automation and detection of clinically significant variants using minimal sample volumes. However, it may misidentify hemoglobin variants due to overlapping retention times. CASE REPORT:Abnormal sickle cell screening results in a newborn female, including a peak in the hemoglobin S window, prompted hemoglobinopathy investigation. Capillary electrophoresis results suggested a possible alpha chain variant. Genetic testing revealed four distinct alterations, including a hemizygous HBA2 c.224A > G (p.Asp75Gly) variant, known as Hb Chapel Hill, and a 3.7 kb alpha-globin gene deletion consistent with an alpha-thalassemia silent carrier state. CONCLUSION:This case represents the first documented occurrence of Hb Chapel Hill in an infant. Gamma globin production alongside Hb Chapel Hill results in a HbS zone peak on both HPLC and capillary electrophoresis, posing an interpretive challenge. Considering the complete pattern of peaks observed in hemoglobin fractionation methods can help distinguish clinically relevant conditions from likely benign profiles. Molecular analysis in complex cases is essential for confirmation of the suspected diagnosis.

BACKGROUND:Early detection of monoclonal gammopathies is challenging, as they often remain undiagnosed until morbidity develops. Monoclonal proteins (M-proteins), particularly IgM, can interfere with the lipemia index (L-index) on chemistry analyzers, producing elevations in visually clear samples. The L-index may allow incidental detection of M-proteins, but its clinical value is uncertain. METHODS:A retrospective chart review was conducted on 78 patients with visually clear serum samples with Siemens Atellica CH930 L-index ≥2 to assess diagnoses and follow-up of previously known and newly identified M-proteins. L-index was measured on Atellica, Roche cobas Pro c503, and Ortho VITROS XT3400 in 40 additional samples from patients with an IgM M-protein to assess L-index sensitivity for M-proteins. A survey of laboratories examined current practices for managing such samples. RESULTS:Sixty-three of 78 patients with visually clear serum samples with a reproducible Atellica L-index ≥2 had an M-protein(s) (34 previously known; 29 newly identified). Among these, IgM kappa predominated (47/63; 66%), and 5/29 newly identified cases were diagnosed with Waldenström macroglobulinemia. In 40 additional samples with known IgM M-proteins, only one had an elevated Atellica L-index, and none were elevated on cobas or Vitros. Survey results showed wide variation in managing these samples. CONCLUSIONS:An elevated L-index on the Atellica in visually clear serum samples, though insensitive, is predictive of the presence of an M-protein. These findings were inconsistent across platforms, highlighting the lack of L-index standardization. Protocol development of an elevated L-index in clear samples will help recognize and manage incidental M-proteins.

BACKGROUND:Capillary electrophoresis is a widely used method for hemoglobin (Hb) fraction separation and relative quantitation, where pre-defined Hb peaks (typically HbA and HbA2) act as reference points to "anchor" the electropherogram and define migration zones. In cases lacking HbA or involving variants with migration patterns similar to HbA or HbA2, mis-anchoring can occur-leading to incorrect zoning of Hb variants. This presents a diagnostic challenge, where follow-up investigations, often including molecular testing, are required to establish an accurate diagnosis. CASE REPORT:We report a case of a 43-year-old Thai female who underwent hemoglobinopathy investigation for microcytic anemia. Capillary electrophoresis showed peaks in the HbA (70.8%), HbA2 (24.4%), and HbC (2.9%) zones, as well as two small peaks in the Z11 (0.9%) and HbD (1.0%) zones. Gel electrophoresis at acid pH showed a band in the HbA position and one slightly anodal to the HbF position and at alkaline pH showed a band in the HbC/E position and another slightly anodal to the HbA position. HBB sequencing identified heterozygosity for the pathogenic HbE and clinically benign Hb Hope variants. HBA PCR detected a single alpha globin gene deletion (αα/α-3.7), consistent with alpha thalassemia silent carrier. Reinterpretation of the electropherogram showed that Hb Hope and HbE mis-anchored as HbA and HbA2, respectively, due to their similar migration deltas. CONCLUSION:This is the first documented case of compound heterozygosity for Hb Hope and HbE characterized by capillary electrophoresis. It highlights how beta chain variants with similar migration spacing to HbA and HbA2 can mis-anchor, emphasizing the need for molecular testing when results are unclear. Definitive testing helps avoid diagnostic misclassification and ensure accurate interpretation in complex hemoglobinopathy cases.

INTRODUCTION:Helicobacter pylori (H. pylori) colonization increases the risk of upper gastrointestinal disorders and can be detected by various tests, including the stool antigen test (HpSAT). DiaSorin recommends an HpSAT equivocal/indeterminate zone of 0.90-<1.10, but high variability observed in our laboratory prompted clinical implementation of a broader zone (0.60-<1.80). This study aimed to define an optimal HpSAT indeterminate zone using molecular as reference and urea breath test (UBT) for confirmation. MATERIALS AND METHODS:HpSAT and stool molecular results were available from 379 patients, of which 52 had follow-up UBTs. HpSAT was analyzed by the LIAISON HpSAT assay (DiaSorin), UBT by isotope ratio mass spectrometry, and molecular testing by qPCR targeting H. pylori DNA. Logistic regression modeled HpSAT index values against PCR positivity to define an optimal indeterminate zone, supported by clinical performance and flagging rates analyses. RESULTS:Logistic regression determined an HpSAT index of 0.79 (95 % CI: 0.34-1.14) had 90 % probability of a negative PCR result, and 4.99 (4.09-6.63) had 90 % probability of a positive result, rounded to an indeterminate zone of 0.80-<5.00. Lower thresholds assessed all had ≤ 2 % false negatives, while upper thresholds exhibited decreased false positive rates (22 % to 6 %) as thresholds increased (1.10 to 5.00), with minimal improvement beyond 3.00 (9 %). A modified zone of 0.80-<3.00 offered high accuracy with 12.9 % indeterminate results (DiaSorin's threshold: 2.8 %; laboratory's current threshold: 13.1 %). CONCLUSIONS:Our findings show that DiaSorin's HpSAT indeterminate zone is too narrow to reliably distinguish true positive and negative results in clinical practice. A modified broader zone (0.80-<3.00), derived via logistic regression using PCR as reference, improves diagnostic accuracy while minimizing indeterminate results.

Objectives: Multiple sclerosis is diagnosed based on clinical and laboratory findings, including cerebrospinal fluid (CSF) oligoclonal banding (OCB) analysis. The lack of updated CSF OCB laboratory guidelines in Canada has likely led to variation in processes and reporting across clinical laboratories. As a first step to developing harmonized laboratory recommendations, we examined current CSF OCB processes, reporting, and interpretation across all Canadian clinical laboratories currently performing this test. Design and methods: A survey of 39 questions was sent to clinical chemists at all 13 Canadian clinical laboratories performing CSF OCB analysis. The survey included questions regarding quality control processes, reporting practices for CSF gel electrophoresis pattern interpretation, and associated tests and calculated indices. Results: The survey response rate was 100%. Most (10/13) laboratories use >= 2 CSF-specific bands (2017 McDonald Criteria) as their CSF OCB positivity cut-off and only 2/13 report the number of bands with every report. Most (8/13 and 9/13) laboratories report an inflammatory response pattern and monoclonal gammopathy pattern, respectively. However, the process for reporting and/or confirming a monoclonal gammopathy varies widely. Variation was observed for reference intervals, units, and the panel of reported associated tests and calculated indices. The maximum acceptable time interval between paired CSF and serum collections varied from 24 h to no limit. Conclusions: Profound variation exists in processes, reporting, and interpretation of CSF OCB and associated tests and indices across Canadian clinical laboratories. Harmonization of CSF OCB analysis is required to ensure continuity and quality of patient care. Our detailed assessment of current practice variation highlights the need for clinical stakeholder engagement and further data analysis to support optimal interpretation and reporting practices, which will aid in developing harmonized laboratory recommendations.

Background: LDL-C, a cardiovascular disease risk assessment biomarker, is commonly calculated using the Friedewald equation. The NIH equation overcomes several limitations of the Friedewald equation. Consistent with the Canadian Society of Clinical Chemists (CSCC) lipid reporting recommendations, we assessed the NIH LDL-C equation in Alberta prior to its provincial implementation.Methods: 1-year (01/01/2021-12/31/2021) of lipid results (n = 1,486,584 after data cleaning) were obtained from five analytical instrument groups used across Alberta. Analyses were performed on all data and after separating by age, analytical instrument group, and fasting status. The correlation between Friedewald- and NIHcalculated LDL-C and between Friedewald- and NIH-calculated LDL-C difference and each lipid parameter, was determined. The frequency of unreportable/inaccurate LDL-C results was compared between the two equations. The concordance between the two equations and with non-HDL-C was determined at LDL-C thresholds. Lastly, LDL-C calculated by Friedewald, NIH, and Martin-Hopkins equations was compared to density-gradient ultracentrifugation. Results: Friedewald- and NIH-calculated LDL-C exhibit the strongest correlation when triglycerides <= 4.52 mmol/ L. The difference between Friedewald- and NIH-calculated LDL-C increases with decreasing LDL-C concentration. The NIH equation yields fewer inaccurate results (0.35 % vs. 22.0 %). The percent agreement between equations was > 96 % at all LDL-C thresholds, suggesting most patients will not require treatment changes. NIH-calculated LDL-C exhibited better agreement with non-HDL-C when triglycerides <= 9.04 mmol/L and better correlated with LDL-C measured by ultracentrifugation (r(2) = 0.926 vs. 0.775 (Friedewald) and 0.863 (Martin-Hopkins)). Results were consistent across age, analytical instrument group, and fasting status.Conclusions: Our findings demonstrate the benefits of implementing the NIH equation across Alberta.

Background: Hemoglobinopathies include thalassemia syndromes, where production of one or more globin subunits of hemoglobin (Hb) is reduced, and structural Hb variants. Over 1000 disorders of Hb synthesis and/or structure have been identified and characterized, with phenotypes ranging from having severe clinical manifestations to clinically silent. Various analytical methods are used to phenotypically detect Hb variants. However, molecular genetic analysis is a more definitive method for Hb variant identification.Case report: Here, we report a case of a 23-month-old male with results from capillary electrophoresis, gel electrophoresis (acid and alkaline), and high-performance liquid chromatography most consistent with HbS trait. Specifically, capillary electrophoresis showed slightly elevated HbF and HbA2, HbA of 39.4% and HbS of 48.5%. The HbS percentage was consistently higher than expected (typically 30-40%) for HbS trait with no concurrent thalassemic indices. The patient has not experienced any clinical complications due to the hemoglobinopathy and he is thriving.Conclusion: Molecular genetic analysis revealed the presence of compound heterozygosity for HbS and Hb Olupona. Hb Olupona is an extremely rare beta-chain variant that appears as HbA on all three common methods used for phenotypic Hb analysis. When the fractional concentration of Hb variants is unusual, more definitive methods should be used, such as mass spectrometry or molecular genetic testing. In this case, incorrectly reporting this result as HbS trait is unlikely to have a significant clinical impact, as current evidence suggests Hb Olupona is not a clinically significant variant.

BACKGROUND:The diagnosis of alpha-1-antitrypsin (A1AT) deficiency has been hindered by obscurity concerning the testing process and treatment implications. In this study, we aimed to identify regional differences in the diagnostic rates for A1AT deficiency in the western Canadian provinces of British Columbia (BC) and Alberta (AB).METHODS:The number of A1AT deficiency variant genotype (ZZ, SZ, MZ, SS, and MS) diagnoses were reviewed for BC and AB. The regional diagnostic rates for A1AT deficiency variants in these two provinces, normalized for the predicted population prevalence of each variant genotype, was defined as the annual provincial diagnostic rate (APDR) for a given variant genotype. Sex specific variations in the mean age at diagnosis for the five variant genotypes were compared both within and between provinces.RESULTS:The SZ and MZ genotype APDRs were significantly increased in the AB population compared to the BC population. The SS and MS APDRs were similar between AB and BC. There was a significantly decreased mean age of diagnosis for AB males, as compared to BC males (for the SZ, MS, and MZ genotypes) and as compared to AB females (for the MS, MZ, and SS genotypes). There were no significant differences in the mean age of diagnosis between the females and males in BC, or between females in AB and BC, for any genotype.CONCLUSION:The notably higher APDR for more severe A1AT deficiency genotypes, and lower mean age of diagnosis for most variant genotypes in AB males, deserves further investigation to determine the explanation(s) for these differences.

Background: Hemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 15 current assay methods. Methods: Hemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on 2 enzymatic, 4 ion-exchange HPLC and 9 immunoassay methods. Trinity Premier Hb9210 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 >6% difference from HbAA at 6 or 9% HbA1c compared to Premier Hb9210 using Deming regression. Results: All methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G11 variant mode (HbAD), Roche b 101 (HbAC and HbAE) and Siemens DCA Vantage (HbAE and HbAS). All other methods (Beckman Coulter B93009 and B00389 on DxC700AU, and Unicel DxC, Ortho Clinical Vitros 5.1, Roche cobas c 513, Siemens Dimension RxL and Vista, and Enzymatic on Advia and Atellica, Tosoh G8 5.24 and 5.28, and GX) showed no clinically significant differences. Conclusions: A few methods showed interference from one or more variants. Laboratories need to be aware of potential HbA1c assay interferences.

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.

Background: alpha(1)-Antitrypsin (A1AT) deficiency predisposes patients to pulmonary disease due to inadequate protection against human neutrophil elastase released during inflammatory responses. A1AT deficiency is caused by homozygosity or compound heterozygosity for A1AT variants; individuals with A1AT deficiency most commonly have at least one Z variant allele (c.1096G > A (Glu366Lys)). Null variants that result in complete absence of A1AT in the plasma are much rarer. With one recent exception, all reported A1AT variants are characterized by a single pathogenic variant. Case: An 8 years old patient from Edmonton, Alberta, Canada, was investigated for A1AT deficiency. His A1AT phenotype was determined to be M (wild type)/Null by isoelectric focusing (IEF) but M/Z by targeted genotyping. Gene sequencing revealed two heterozygous variants: Z and Ile100Asn (c.299 T > A). The Ile100Asn substitution is predicted to disrupt the secondary structure of an a-helix in which it resides and the neighbouring tertiary structure, resulting in intracellular degradation of A1AT prior to hepatocyte secretion. Methods: Family testing was conducted to verify potential inheritance of an A1AT allele carrying the two mutations in cis, as this arrangement of the mutations would explain "Z" detection by genotyping but not by IEF. Molecular modeling was used to assess the effect of the variants on A1AT structure and stability. Discussion: Carrier status for a novel variant Null(Canada) with in cis mutations (c.[299 T > A;1096G > A], p. [(Ileu100Asn;Glu366Lys)]) was confirmed. A sibling was identified as having A1AT deficiency on the basis of compound heterozygosity for two alleles: Null(Canada) and the common Z allele. A separate pedigree from the Maritimes was subsequently recognized as carrying Null(Canada). Conclusion: In cis mutations such as Null(Canada) may be more common than previously described due to failure to detect such mutations using historical testing methods. Combined approaches that include gene sequencing and segregation studies allow recognition of rare A1AT variants, including in cis mutations.

•Describes prevalence of A1AT variants at four testing centres in Canada.•Examines a period from 2011 to 2018.•Reflects the testing strategies used at these centres.•Provides basis of devising the optimum testing strategy.

BACKGROUND:Cardiac troponin I (cTnI) 99th percentile cutoffs, used in the diagnosis of acute myocardial infarction, are not standardized across cTnI assays. We compared 3 point-of-care (POC) and 1 central laboratory contemporary cTnI assays against the Abbott high-sensitivity (hs) cTnI to evaluate the analytical concordance and the feasibility of using a single cutoff value for all assays.METHODS:Fresh blood samples collected from 102 inpatients in the coronary care unit were measured on central laboratory instruments (Beckman Coulter DxI AccuTnI+3 TnI, Abbott Architect hs-TnI) and cTnI POC analyzers (Alere Triage Troponin I, Radiometer AQT90, Abbott i-STAT). Agreement and correlation between the contemporary cTnI assays and hs-cTnI assay were assessed using regression analysis. Proportional bias was assessed using Bland-Altman plots. Concordance between the contemporary cTnI and hs-cTnI assays was determined by diagnostic contingency tables at specific cutoffs.RESULTS:Most POC cTnI assays had excellent correlation with the Abbott hs-cTnI method (r 2 = 0.955-0.970) except for Alere Triage (r 2 = 0.617), while proportional bias is evident between all cTnI assays. Overall concordance between POC contemporary cTnI assays and hs-cTnI assay was 80% to 90% at their respective 99th percentile cutoffs. The concordance increased to 90% to 95% when a fixed cutoff of 0.03 to 0.05 ng/mL was used across the assays.CONCLUSIONS:This study demonstrates poor analytical concordance between cTnI assays at the 99th percentile and supports the notion of a single clinical decision limit for cTnI and consequently standardization of diagnostic protocols despite the analytical differences among these assays.