Quantitative Lipidomic Analysis of Serum Phospholipids Reveals Dissociable Markers of Alzheimer's Disease and Subcortical Cerebrovascular Disease.

BACKGROUND: Circulating phospholipid species have been shown to predict Alzheimer's disease (AD) prognosis but the link between phospholipid disturbances and subcortical small vessel cerebrovascular disease (CeVD) common in AD patients is not known. OBJECTIVE: Mass-spectrometry lipidomics was applied to quantify serum diacyl, alkenyl (ether), alkyl, and lyso phospholipid species in individuals with extensive CeVD (n = 29), AD with minimal CeVD (n = 16), and AD with extensive CeVD (n = 14), and compared them to age-matched controls (n = 27). Memory was assessed using the California Verbal Learning Test. 3.0T MRI was used to assess hippocampal volume, atrophy, and white matter hyperintensity (WMH) volumes as manifestations of CeVD. RESULTS: AD was associated with significantly higher concentrations of choline plasmalogen 18:0_18:1 and alkyl-phosphocholine 18:1. CeVD was associated with significantly lower lysophospholipids containing 16:0. Phospholipids containing arachidonic acid (AA) were associated with poorer memory in controls, whereas docosahexaenoic acid (DHA)-containing phospholipids were associated with better memory in individuals with AD+CeVD. In controls, DHA-containing phospholipids were associated with more atrophy, and phospholipids containing linoleic acid and AA were associated with less atrophy. Lysophospholipids containing 16:0, 18:0, and 18:1 were correlated with less atrophy in controls, and of these, alkyl-phosphocholine 18:1 was correlated with smaller WMH volumes. Conversely, 16:0_18:1 choline plasmalogen was correlated with greater WMH volumes in controls. CONCLUSION: This study demonstrates discernable differences in circulating phospholipids in individuals with AD and CeVD, as well as new associations between phospholipid species with memory and brain structure that were specific to contexts of commonly comorbid vascular and neurodegenerative pathologies.

Lot verification practices in Ontario clinical chemistry laboratories - Results of a patterns-of-practice survey.

Objectives: Verifying new reagent or calibrator lots is crucial for maintaining consistent test performance. The Institute for Quality Management in Healthcare (IQMH) conducted a patterns-of-practice survey and follow-up case study to collect information on lot verification practices in Ontario. Methods: The survey had 17 multiple-choice questions and was distributed to 183 licensed laboratories. Participants provided information on materials used and approval/rejection criteria for their lot verification procedures for eight classes of testing systems. The case study provided a set of lot comparison data and was distributed to 132 laboratories. Responses were reviewed by IQMH scientific committees. Results: Of the 175 laboratories that responded regarding reagent lot verifications, 74% verified all tests, 11% some, and 15% none. Of the 171 laboratories that responded regarding calibrator lot verifications, 39% verified all calibrators, 4% some, and 57% none. Reasons for not performing verifications ranged from difficulty performing parallel testing to high reagent cost. For automated chemistry assays and immunoassays, 23% of laboratories did not include patient-derived materials in reagent lot verifications and 42% included five to six patient materials; 58% of laboratories did not include patient-derived materials in calibrator lot verifications and 23% included five to six patient materials. Different combinations of test-specific rules were used for acceptance criteria. For a failed lot, 98% of laboratories would investigate further and take corrective actions. Forty-three percent of laboratories would accept the new reagent lot in the case study. Conclusion: Responses to the survey and case study demonstrated variability in lot verification practices among laboratories.

A Case of Clinical Confusion Due to Erroneous M-protein Quantifications: To Splice or Skim?

An M-protein identified on electrophoresis is conventionally quantified by integrating the M-spike from baseline (PD), invariably including some irrelevant/background proteins. The use of an alternative approach that skims the M-spike tangentially thereby excluding the background proteins (TS), however, has been scanty. We report herein a case in which PD overestimated the M-proteins inconsistently, leading to confusion over relapse in a multiple myeloma patient. At diagnosis, a 65-year old male had an IgG kappa M-spike of 44 g/L which decreased to 6 g/L (PD) following chemotherapy. Six weeks after autologous stem cell transplantation (ASCT), two M-spikes measuring respectively 10 and 5 g/L emerged. Together with decreases in hemoglobin and blood cell counts, a relapse was suspected. Bone marrow examinations, however, did not reveal any significant plasmacytosis or clonal restriction. Re-analyses by TS reduced the original M-protein estimations by 12% and 88% pre- and post-ASCT respectively, and corroborated the disease activity/status consistently.

Soluble Epoxide Hydrolase-Derived Linoleic Acid Oxylipins in Serum Are Associated with Periventricular White Matter Hyperintensities and Vascular Cognitive Impairment.

White matter hyperintensities (WMH) are presumed to indicate subcortical ischemic vascular disease but their underlying pathobiology remains incompletely understood. The soluble epoxide hydrolase (sEH) enzyme converts anti-inflammatory and vasoactive cytochrome p450-derived polyunsaturated fatty acid epoxides into their less active corresponding diol species. Under the hypothesis that the activity of sEH might be associated with subcortical ischemic vascular disease and vascular cognitive impairment, this study aimed to compare the relative abundance of sEH substrates and products in peripheral blood between patients with extensive WMH (discovered due to transient ischemic attack; n = 29) and healthy elderly with minimal WMH (n = 25). The concentration of 12,13-DiHOME (a sEH-derived linoleic acid metabolite), and the ratio of 12,13-DiHOME to its sEH substrate, 12,13-EpOME, were elevated in the extensive WMH group (F1,53 = 5.9, p = 0.019), as was the 9,10-DiHOME/9,10-EpOME ratio (F1,53 = 5.4, p = 0.024). The 12,13-DiHOME/12,13-EpOME ratio was associated with poorer performance on a composite score derived from tests of psychomotor processing speed, attention, and executive function (ß = - 0.473, p = 0.001, adjusted r2 = 0.213), but not with a composite verbal memory score. In a mediation model, periventricular WMH (but not deep WMH), explained 37% of the effect of the 12,13-DiHOME/12,13-EpOME ratio on the speed/attention/executive function composite score (indirect effect = - 0.50, 95% bootstrap confidence interval [- 0.99, - 0.17] Z-score units). Serum oxylipin changes consistent with higher sEH activity were markers of vascular cognitive impairment, and this association was partly explained by injury to the periventricular subcortical white matter.

Diagnostic implications of enumerating and reporting beta fraction(s) for the detection of beta-migrating monoclonal immunoglobulins in serum protein electrophoresis.

BACKGROUND: Modern serum protein electrophoresis (SPE) resolves serum proteins in 6 major fractions, splitting beta (B) into beta1 (B1) and beta2 (B2). Beta-migrating monoclonal immunoglobulins (B-MC) commonly integrate into normal-looking peak shape(s) but may increase the fraction value, forming the basis for reflex testing. The objectives of this study were (1) to ascertain the value of beta fraction(s) reporting, and (2) to compare the diagnostic performance between different beta-flagging approaches, particularly ↑B versus ↑B1 and/or ↑B2. METHODS: We retrospectively studied 22,900 consecutive SPEs, and identified 3974 paired SPE and immunofixation electrophoresis (IFE) results obtained from the Sebia Capillarys™ 2 and Hydrasys™ electrophoresis systems respectively. ↑B, ↑B1 and ↑B2 were defined as fraction concentrations >11, 6 and 5 g/L respectively, and their respective diagnostic metrics calculated using IFE as the reference standard. RESULTS: 32 beta-gamma bridges were B-MC negative and thus excluded. Of 3942 cases, 142, 18, 285 and 300 had ↑B, ↑B1, ↑B2 and ↑B1 and/or ↑B2 respectively, while their corresponding sensitivities for B-MC were 0.38, 0.09, 0.45 and 0.54 respectively. Comparing ↑B and ↑B1 and/or ↑B2, ↑B had significantly lower sensitivity but higher specificity (0.98 Vs 0.95) and positive predictive value (0.47 Vs 0.31). All 4 beta fraction increases had odds ratios ranged from 14 to 118. CONCLUSION: Beta increases were associated with increased odds for B-MC, hence providing value and justification for their reporting and reflex testing. ↑B1 and/or ↑B2 detected more B-MC than ↑B, supporting separate reporting of B1 and B2.

Candidate recommendations for protein electrophoresis reporting from the Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group.

Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.

Peripheral lipid oxidative stress markers are related to vascular risk factors and subcortical small vessel disease.

Subcortical white matter hyperintensities (WMH), presumed to indicate small vessel ischemic vascular disease, are found commonly in elderly individuals with and without Alzheimer's disease (AD). Oxidative stress may instigate or accelerate the development of vascular disease, and oxidative stress markers are elevated in AD. Here, we assess independent relationships between three serum lipid peroxidation markers (lipid hydroperoxides [LPH], 8-isoprostane, and 4-hydroxynonenal) and the presence of extensive subcortical WMH and/or AD. Patients were recruited from memory and stroke prevention clinics into four groups: minimal WMH, extensive WMH, AD with minimal WMH, and AD with extensive WMH. Extensive WMH, but not AD, was associated with higher serum concentrations of 8-isoprostane and LPH. Peripheral LPH concentrations mediated the effect of hypertension on deep, but not periventricular, WMH volumes. 4-hydroxynonenal was associated with hyperlipidemia and cerebral microbleeds, but not with extensive WMH or AD. We conclude that lipid peroxidation mediates hypertensive injury to the deep subcortical white matter and that peripheral blood lipid peroxidation markers indicate subcortical small vessel disease regardless of an AD diagnosis.

A Challenging Diagnosis of IgG4-Related Disease When Understanding Limitations of Laboratory Testing Was Pivotal.

A 76-year-old man was incidentally found on a CT scan to have lymphadenopathy and bilateral kidney enlargement suggestive of infiltrative renal disease. He was largely asymptomatic but had bilateral salivary and lacrimal gland enlargement. A grossly elevated serum IgG (>70 g/L) with concomitant suppression of other immunoglobulins, a small IgG restriction, and a parotid biopsy revealing lymphoplasmacytic infiltrate with slight kappa light chain excess all suggested a lymphoproliferative disorder (LPD). The diagnostic workup was further confounded by a normal serum IgG4 concentration. Moreover, bone marrow and renal biopsies did not reveal evidence of LPD. Discussion with the laboratory not only clarified that the markedly increased total IgG could not be accounted for by the small IgG restriction, but also identified a discrepancy in the IgG4 measurement. Repeat analysis of a follow-up sample revealed an elevated IgG4 of 5.94 (reference interval: 0.039-0.864) g/L, which prompted a repeat parotid biopsy that showed predominant IgG4+ lymphocytic infiltrates. Despite the deluding presentations, a final diagnosis of IgG4-related disease (IgG4-RD) was made based on elevated serum IgG4 concentrations and histopathological findings. This case highlights the importance of recognizing limitations of laboratory testing and the benefit of close communications among clinical subspecialties and the laboratory.

Extended Stability of Sodium Phosphate Solutions in Polyvinyl Chloride Bags.

BACKGROUND: Sodium phosphate injection is used to treat moderate to severe hypophosphatemia. There have been no published reports documenting the physical compatibility or chemical stability of sodium phosphate injection in IV solutions. OBJECTIVE: To evaluate the physical compatibility and chemical stability of 30 and 150 mmol/L solutions of phosphate, prepared from sodium phosphate injection, in 5% dextrose in water (D5W) and in 0.9% sodium chloride (normal saline [NS]) and stored in polyvinyl chloride (PVC) bags at 23°C or 4°C over 63 days. METHODS: On study day 0, solutions of phosphate 30 and 150 mmol/L in D5W or NS were prepared in PVC bags and stored at 4°C and 23°C. On prespecified days during the 63-day study period, the concentrations of sodium and phosphate were determined, and admixture weight was checked to assess moisture loss during storage without a plastic overwrap. Chemical stability was calculated from the intersection of the lower 95% confidence limit of the degradation rate and the lower limit of acceptability (90%) for concentration remaining. RESULTS: The analytical methods for both sodium and phosphate were found to be precise (coefficient of variation averaging less than 1% for pre-study validation samples). Both sodium and phosphate retained more than 94% of the initial concentration over the 63-day study period. With 95% confidence, the time to achieve 90% of the initial concentration of both sodium and phosphate approached or exceeded the 63-day study period, regardless of temperature, concentration, or base solution. CONCLUSIONS: Sodium phosphate solutions at a phosphate concentration of 30 or 150 mmol/L in either NS or D5W retained more than 94% of the initial concentration of both sodium and phosphate over 63 days when stored at 23°C or 4°C. In compliance with United States Pharmacopeia General Chapter <797> recommendations, a beyond-use date of 14 days (with refrigeration) or 48 h (room temperature) may be applied. Extending the beyond-use date beyond these limits may be considered, if a validated sterility test is performed.

CONTEXTE: Le phosphate de sodium injectable est employé pour traiter l'hypophosphatémie modérée et grave. À ce jour, aucun rapport portant sur la compatibilité physique ou la stabilité chimique du phosphate de sodium injectable contenu dans les solutions intraveineuses n'a été publié. OBJECTIF: Évaluer la compatibilité physique et la stabilité chimique de solutions de phosphate à des concentrations de 30 et de 150 mmol/L préparées à partir de phosphate de sodium injectable dilué dans du dextrose à 5 % dans l'eau (D5E) ou du chlorure de sodium à 0,9 % (solution physiologique salée [SP]) puis rangées dans des sacs de polychlorure de vinyle (PVC) à des températures de 4 °C ou de 23 °C pendant 63 jours. MÉTHODES: Au jour 0 de l'étude, les solutions de phosphate à des concentrations de 30 et de 150 mmol/L ont été préparées avec du D5E ou de la SP dans des sacs de PVC, puis entreposées à des températures de 4 °C ou de 23 °C. À des jours donnés pendant la période de 63 jours de l'étude, on a évalué les concentrations de sodium et de phosphate et l'on a pesé les mélanges pour vérifier la perte d'humidité pendant un entre-posage n'utilisant pas de suremballage de plastique. La stabilité chimique était calculée au point d'intersection entre la limite inférieure de confiance à 95 % du taux de dégradation et la limite inférieure d'acceptabilité (90 %) de la concentration restante. RÉSULTATS: Les méthodes analytiques employées pour évaluer le sodium et le phosphate se sont révélées précises (coefficient de variation moyen inférieur à 1 % pour les échantillons aux fins de validation avant l'étude). Le sodium et le phosphate conservaient chacun plus de 94 % de leurs concentrations initiales pendant la période d'étude de 63 jours. Avec un niveau de confiance de 95 %, le temps nécessaire pour atteindre 90 % de la concentration initiale pour le sodium et pour le phosphate approchait ou dépassait les 63 jours de la période d'étude, peu importe la température, la concentration ou la solution de base. CONCLUSIONS: Les solutions de phosphate de sodium dont la concentration en phosphate est de 30 ou de 150 mmol/L, qu'elles soient à base de D5E ou de SP, conservaient plus de 94 % des concentrations initiales de sodium et de phosphate pendant 63 jours, qu'elles soient entreposées à des températures de 4 °C ou de 23 °C. Conformément aux recommandations contenues dans le chapitre <797> de la United States Pharmacopeia, une date limite d'utilisation de 14 jours (sous réfrigération) ou de 48 heures (à température ambiante) peut être utilisée. Allonger la date limite d'utilisation au-delà des bornes fixées par l'organisme américain peut être envisageable si une épreuve validée de stérilité est réalisée.

Value of reflex testing based on hypogammaglobulinemia as demonstrated in serum protein electrophoresis.

OBJECTIVES: Hypo-gammaglobulinemia (hypoGG) in serum protein electrophoresis (SPE) reflects, variably, reduced serum immunoglobulin (Ig) concentrations, which may be caused by hematological neoplasms, among other causes. HypoGG in the absence of a discernible M-spike (MC) has been the basis of reflexive testing e.g., by immunofixation electrophoresis (IFE). However, the utility of this practice has not been fully evaluated. Thus, we aimed to (1) determine the predictive power of hypoGG for reduced Ig levels, (2) compare the IFE positive rates and sensitivity between hypoGG and non-hypoGG patients, and (3) examine the M-protein isotype distributions. METHODS: We retrospectively analyzed 3974 matched SPE and IFE results at the Sunnybrook Health Sciences Centre from January 2010 to June 2013. SPE and IFE were performed on the Sebia Capillarys™ 2 and Hydrasys™ systems respectively. RESULTS: 2723/3974 (68.5%) patients were SPE negative, 246/2723 (9%) had hypoGG and 192 (7.1%) were IFE positive. HypoGG predicted 93.1% cases with at least one Ig reduction. Among SPE-negative patients, the IFE positive rate and sensitivity in hypoGG were 12.2% and 15.6% respectively, compared to 6.4% and 77.1% in normo-GG. Proportion of non-IgG isotypes in both groups were comparable but fewer MC were detected in hypoGG. CONCLUSIONS: Both the 7% false negative rate of SPE and the poor sensitivity (16%) of reflex testing based on hypoGG should be taken into consideration when devising a screening strategy based on SPE.

Measurement uncertainty for serum free light chain assays: estimation and implication on result interpretation.

OBJECTIVES: To estimate the measurement uncertainty (MU) and reference change value (RCV) for serum free light chain assays (sFLC), and to review their implications on result interpretation. DESIGN AND METHODS: Data from 6 to 9months of internal QC and up to 3.5years of EQA results were collected retrospectively, on the Roche Modular P analyzer and Dade Behring BN II nephelometer from two independent laboratories. MU was estimated from its components related to imprecision and bias determinations, while RCV was calculated from the estimated MU and published values on biological variation. RESULTS AND CONCLUSION: The standard uncertainty related to imprecision for the FLC were similar between two instruments, ranging from ~4% to ~8%, so were the uncertainty related to bias, ranging between 24% and 44%. The overall MU with 95% coverage for Fκ, Fλ and their ratio (rFLC) were 55%, 87% and 103% (Modular P), and 49%, 76% and 91% (BNII) respectively. The RCVs with biological variation for Fκ, Fλ and rFLC were comparable between two instruments and averaged 106%, 138% and 173% respectively. The relatively method-independent MU reflected the sFLC limitations. For monoclonal gammopathy detection, the universal rFLC reference limits ±MU gave an effective range of 0.02-3.15 (BNII) (as compared to 0.26-1.65), revealing substantial diagnostic grey zones on both ends. When monitoring disease activity, a minimum change (RCV) of ~100% in FLC concentrations or ~170% in rFLC would be considered significant. Applying MU when interpreting sFLC results puts current diagnostic cut-offs into perspective, and highlights the importance of collating other clinical findings.

Validating urinary measurement of beta-2-microglobulin with a Roche reagent kit designed for serum measurements.

OBJECTIVE: To validate the use of a Roche serum beta-2-microglobulin (B2MG) kit for urinary B2MG measurements, and to establish reference limits for urinary B2MG/creatinine ratio from healthy individuals. DESIGN AND METHODS: The Roche B2MG Tina-Quant serum kit was used to measure urinary B2MG immunoturbidimetrically. RESULTS: Using human urine as a diluent, the B2MG method was linear from 73-2156 µg/L. The imprecision on a commercially available urine QC was 4.4% at a concentration of 380 µg/L. Limit of quantification at <20% CV was 40 µg/L. Method comparison with Immulite 2000 (Siemens) yielded slope=1.180 (95% CI 1.14-1.22), intercept=11.5 (95% CI -3.6-26.6), SEE=27.6 and r=0.99 (n=26) by the Deming regression analysis. The upper reference limit of B2MG/creatinine ratio determined from 195 healthy adults was 29 µg/mmol (97.5th centile). CONCLUSIONS: The serum B2MG Tina Quant reagent kit is acceptable to measure urinary B2MG.

Evaluation of a point-of-care glucose meter for general use in complex tertiary care facilities.

OBJECTIVE: To evaluate the performance of the StatStrip glucose meter from Nova Biomedical for use in complex tertiary care facilities. METHODS: Performance evaluation was conducted in 6 clinical locations involving nurse end-users. Imprecision (CV)<5% was considered acceptable. Ten substances were examined for potential analytical interferences at two levels and 3 glucose concentrations. Inaccuracy was determined by 386 paired glucose differences between glucose meter and laboratory reference analyses. Acceptability was based on CSLI/ISO15197 criteria. Potential clinical impact was classified under a 5-level severity scheme. RESULTS: Imprecision varied from CV 2.4 to 4.9%. Significant interference was observed only for free hemoglobin at 10 g/L. 97% of paired glucose meter-laboratory differences met CSLI/ISO15197 criteria. All discrepant results had low severity scores. CONCLUSION: The StatStrip glucose meter demonstrated acceptable imprecision and inaccuracy, and was relatively free from common interferences. It should be a good candidate for general use in complex tertiary care facilities.

Update on parathyroid hormone: new tests and new challenges for external quality assessment.

It is now 43 years since Berson and Yalow published the first radio-immunoassay (RIA) for parathyroid hormone (PTH) [S.A. Berson, R.S. Yalow, G.D. Aurbach, J.T. Potts, Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci U S A 49 (1963) 613-617] [1]. Since then, there have been marked advances in our understanding of this peptide hormone, its mechanism of action and biological regulation [J.T. Potts, Parathyroid hormone: past and present. J. Endocrinol. 187 (2005) 311-325] [2]. PTH has become a routine assay in tertiary care hospitals and is an essential element in the management of chronic kidney disease, parathyroid disorders and the investigation of abnormalities in calcium homeostasis. Despite continuing technological advances in PTH measurement, analyte heterogeneity remains a problem, while improved turnaround time and better precision are constantly escalating clinical demands. This mini-review begins with a brief update of current knowledge on PTH, followed by a summary of a recent Ontario-wide External Quality Assurance (EQA) survey, and concludes with comments on utilization trends, current and future.