Handling of lipemic samples in the clinical laboratory.

Interferences in the clinical laboratory may lead physicians misinterpret results for some biological analytes. The most common analytical interferences in the clinical laboratory include hemolysis, icterus and lipemia. Lipemia is defined as turbidity in a sample caused by the accumulation of lipoproteins, mainly very-low density lipoproteins (VLDL) and chylomicrons. Several methods are available for the detection of lipemic samples, including the lipemic index, or triglyceride quantification in serum or plasma samples, or mean corpuscular hemoglobin (MCHC) concentration in blood samples. According to the European Directive 98/79/CE, it is the responsibility of clinical laboratories to monitor the presence of interfering substances that may affect the measurement of an analyte. There is an urgent need to standardize interference studies and the way interferences are reported by manufacturers. Several methods are currently available to remove interference from lipemia and enable accurate measurement of biological quantities. The clinical laboratory should establish a protocol for the handling of lipemic samples according to the biological quantity to be tested.

Low risk of viral hepatitis amongst patients with severe mental disorders.

BACKGROUND AND AIMS: Patients with severe mental disorders (SMD) have been classically considered as a particularly high-risk population for bloodborne virus infections. We performed a systematic screening of hepatitis B and C virus among the population with SMD in the area of influence of Hospital Clínic (Barcelona) in order to evaluate the real prevalence of these infections and achieve HCV microelimination in this subpopulation. METHODS: We screened two cohorts for anti-HCV and HBsAg: Cohort A (hospitalized patients with SMD, done systematically) and Cohort B (outpatients, mental health centre-CSMA, done voluntarily). Risk factors and socio-demographic variables were collected. In positive cases, telematic review was activated by Hepatology, calculation of FIB-4 and prescription of direct-acting agents (DAA) in HCV or follow-up in HBV. RESULTS: In Cohort A, 404 patients were screened. 3 HBV patients were detected (0.7%). In all of them, there was a history of drug use. 12 anti-HCV positive patients were detected (3%); 8 of them had a history of drug use. Among the HCV positive, only 2 patients were viraemic (received DAA, both achieving SVR) as most of them (n = 6) had already been cured with DAA. In cohort B, 305 patients were screened, after 542 (64% of the target population) declined to participate. No cases of HCV or HBV were detected. CONCLUSIONS: HCV/HBV prevalence among SMD population with no history of drug use does not seem to be different from the general population. These data may be of interest for defining health policies.

Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

Dynamic profiles and predictive values of some biochemical and haematological quantities in COVID-19 inpatients.

INTRODUCTION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in some hospitalized patients has shown some important alterations in laboratory tests. The aim of this study was to establish the most relevant quantities associated with the worst prognosis related to COVID-19. MATERIALS AND METHODS: This was a descriptive, longitudinal, observational and retrospective study, in a cohort of 845 adult inpatients from Bellvitge University Hospital (L'Hospitalet de Llobregat, Barcelona, Spain). A multivariate regression analysis was carried out in demographic, clinical and laboratory data, comparing survivors (SURV) and non-survivors (no-SURV). A receiver operating characteristic analysis was also carried out to establish the cut-off point for poor prognostic with better specificity and sensibility. Dynamic changes in clinical laboratory measurements were tracked from day 1 to day 28 after the onset of symptoms. RESULTS: During their hospital stay, 18% of the patients died. Age, kidney disease, creatinine (CREA), lactate-dehydrogenase (LD), C-reactive-protein (CRP) and lymphocyte (LYM) concentration showed the strongest independent associations with the risk of death in the multivariate regression analysis. Established cut-off values for poor prognosis for CREA, LD, CRP and LYM concentrations were 75.0 µmol /L, 320 U/L, 80.9 mg/L and 0.69 x109/L. Dynamic profile of laboratory findings, were in agreement with the consequences of organ damage and tissue destruction. CONCLUSIONS: Age, kidney disease, CREA, LD, CRP and LYM concentrations in COVID-19 patients from the southern region of Catalonia provide important information for their prognosis. Measurement of LD has demonstrated to be very good indicator of poor prognosis at initial evaluation because of its stability over time.

A high extra-virgin olive oil diet induces changes in metabolic pathways of experimental mammary tumors.

Breast cancer is the most common malignancy in women worldwide, and environmental factors, especially diet, have a role in the etiology of this disease. This work aimed to investigate the influence of high fat diets (rich in corn oil or extra virgin olive oil -EVOO-) and the timing of dietary intervention (from weaning or after induction) on tumor metabolism in a seven,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer model in rat. The effects of lipids (oils and fatty acids) have also been investigated in MCF-7 cells. The results have confirmed different effects on tumor progression depending on the type of lipid. Molecular analysis at mRNA, protein and activity level of enzymes of the main metabolic pathways have also shown differences among groups. Thus, the animals fed with the EVOO-enriched diet developed tumors with less degree of clinical and morphological malignancy and showed modified glucose and mitochondrial metabolism when compared to the animals fed with the corn oil-enriched diet. Paradoxically, no clear influence on lipid metabolism by the high fat diets was observed. Considering previous studies on proliferation and apoptosis in the same samples, the results suggest that metabolic changes have a role in the molecular context that results in the modulation of different signaling pathways. Moreover, metabolic characteristics, without the context of other pathways, may not reflect tumor malignancy. The time of dietary intervention plays also a role, suggesting the importance of metabolic plasticity and the relation with mammary gland status when the tumor is induced.

Estimation of change limits (deltacheck) in clinical laboratory.

Objectives: Change limits, more commonly called delta check, are those in which a change in a patient's measured result in relation to their corresponding preceding measurement is suspected of being erroneous and should be considered as a doubtful result. The aim of this study was to provide change limits for some biochemical and haematological quantities to detect doubtful measured results and to assess its effectiveness to detect erroneous results for their application in and the standardization of the plausibility control. Methods: Change limits have been estimated for 13 biochemical and 6 haematological quantities. For each quantity, relative differences (D), expressed as a percentage between the two consecutive measured results from the same patient (from scheduled laboratory requests), were calculated. From these differences (D), the p5 and p95 percentiles of the data distribution were calculated. To assess the effectiveness of the change limits to detect laboratory errors, 43 erroneous laboratory reports, containing different biochemical and haematological quantities, were obtained from the standard laboratory plausibility control procedure. Results: From the 43 erroneous laboratory reports, 31 (72%) were due to endovenous administration errors and 12 (28%) were due to mislabeling errors. All erroneous laboratory reports were detected when the change limits of the quantities were combined and applied together. Conclusions: The best combination of quantities, which detect all the erroneous reports in the same specimen were: potassium, albumin, creatinine, glucose and haemoglobin.

Biomaker evaluation for major adverse cardiovascular event development in patients undergoing cardiac Surgery.

Objectives: The postoperative period of cardiac surgery (CS) is associated with the development of major adverse cardiovascular events (MACEs). However, the evaluation of MACE after CS by means of biomarkers is poorly developed. We aimed to evaluate postoperative biomarkers that could be associated with MACE. Methods: Two Hundred and ten patients who underwent CS were enrolled during the study period. The diagnosis of MACE was defined as the presence of at least one of the following complications: acute myocardial infarction, heart failure, stroke presented during intensive care unit (ICU) stay, and 30-day mortality after CS. High-sensitive troponin T (hs-TnT), C-reactive protein, procalcitonin, interleukin-6, and immature platelet fraction (IPF) were measured on ICU admission and after 24 h. The difference between both measurements (Δ) was calculated to assess their association with MACE. Early infected patients (n=13) after CS were excluded from final analysis. Results: The most frequent surgery was single-valve surgery (n=83; 38%), followed by coronary artery bypass graft (n=72; 34%). Postoperative MACE was diagnosed in 31 (14.8%) patients. Biomarker dynamics showed elevated values at 24 h compared with those at ICU admission in patients with MACE versus no-MACE. Multivariate analysis showed that ΔIPF (OR: 1.47; 95% CI: 1.110-1.960; p=0.008) and Δhs-TnT (OR: 1.001; 95% CI: 1.0002-1.001; p=0.008) were independently associated with MACE. Conclusions: These findings suggest that postoperative ΔIPF and Δhs-TnT may be useful biomarkers for the identification of patients at risk of MACE development.

Evaluation of the delta of immature platelet fraction as a predictive biomarker of inflammatory response after cardiac surgery.

AIMS: Cardiac surgery (CS) can induce an inflammatory response (IR) that is associated with poorer outcomes. Immature platelets are among the factors that may be associated with IR development. We aimed to evaluate whether immature platelet fraction (IPF) could be a predictive biomarker for IR and whether IPF could improve the prognosis assessment of IR for Acute Physiologic and Chronic Health Evaluation (APACHE II) and Sequential Organ Failure Assessment (SOFA) following CS. METHODS: Three-hundred and twenty-seven (327) patients who underwent CS were enrolled during the study period. IR was defined according to the need for vasopressor support (>48 hours). Perioperative variables and outcomes were registered in our database. IPF was measured immediately following CS and at 24 hours by Sysmex XN analyzer and the difference between both measurements (ΔIPF) was calculated. To assess the relationship between ΔIPF and IR, univariate and multivariate logistic regression were performed. To analyse the additive value of ΔIPF in APACHE II and SOFA scores in predicting IR, an area under the receiver operating characteristic (AUROC) curve was calculated. RESULTS: Among 327 patients included, 60 patients (18.3%) developed IR. Multivariate analysis showed ΔIPF was significantly associated with IR (OR: 1.26; 95% CI: 1.01 to 1.56; p=0.038). The combination of ΔIPF with scores improved the AUROC for IR prediction: 0.629 vs 0.728 (p=0.010) for APACHE II and 0.676 vs 0.715 (p=0.106) for SOFA. CONCLUSION: These findings suggested that ΔIPF may be a useful and low-cost biomarker for the early identification of patients at risk of IR development.

All-trans-retinoic acid activates the pro-invasive Src-YAP-Interleukin 6 axis in triple-negative MDA-MB-231 breast cancer cells while cerivastatin reverses this action.

All-trans-retinoic acid (RA), the active metabolite of vitamin A, can reduce the malignant phenotype in some types of cancer and paradoxically also can promote cancer growth and invasion in others. For instance, it has been reported that RA induces tumor suppression in tumor xenografts of MDA-MB-468 breast cancer cells while increasing tumor growth and metastases in xenografts of MDA-MB-231 breast cancer cells. The signaling pathways involved in the pro-invasive action of retinoic acid remain mostly unknown. We show here that RA activates the pro-invasive axis Src-YAP-Interleukin 6 (Src-YAP-IL6) in triple negative MDA-MB-231 breast cancer cells, yielding to increased invasion of these cells. On the contrary, RA inhibits the Src-YAP-IL6 axis of triple-negative MDA-MB-468 cells, which results in decreased invasion phenotype. In both types of cells, inhibition of the Src-YAP-IL6 axis by the Src inhibitor PP2 drastically reduces migration and invasion. Src inhibition also downregulates the expression of a pro-invasive isoform of VEGFR1 in MDA-MB-231 breast cancer cells. Furthermore, interference of YAP nuclear translocation using the statin cerivastatin reverses the upregulation of Interleukin 6 (IL-6) and the pro-invasive effect of RA on MDA-MB-231 breast cancer cells and also decreases invasion and viability of MDA-MB-468 breast cancer cells. These results altogether suggest that RA induces pro-invasive or anti-invasive actions in two triple-negative breast cancer cell lines due to its ability to activate or inhibit the Src-YAP-IL6 axis in different cancer cells. The pro-invasive effect of RA can be reversed by the statin cerivastatin.

Reference intervals for a complete blood count on an automated haematology analyser Sysmex XN in healthy adults from the southern metropolitan area of Barcelona.

BACKGROUND: The examination of peripheral blood is routinely used as a basic test in daily medical practice. Reliable reference intervals are necessary to avoid misdiagnoses, and the establishment of those intervals is an important task for clinical laboratories.The aim of the present study was to establish the reference intervals for complete blood count (CBC) on a Sysmex XN haematology analyser in healthy adults from the southern metropolitan area of Barcelona (Spain). METHODS: A total of 213 apparently healthy adults who received a general health examination at Hospital Universitari de Bellvitge were recruited to this study. Blood samples collected in K3EDTA tubes were analysed on a Sysmex XN. Statistically relevant gender based partition was assessed, outliers removed, and the reference intervals calculated in concordance with Clinical and Laboratory Standards Institute (CLSI) EP28-A3C guidelines. RESULTS: The CBC reference intervals were established in 191 adults (64 men and 127 women) who fulfilled all of the inclusion criteria. Significant gender-dependent differences in red blood cells, haematocrit, haemoglobin and platelets were found. The rest of the CBC reference intervals were obtained from the overall data. CONCLUSIONS: We report CBC reference intervals established on a Sysmex XN analyser, a widely used automated analyser for which reference intervals were previously lacking in the literature. However, these reference intervals we recommend should be validated by individual laboratories for the local population as recommended by CLSI.

Reference interval for immature platelet fraction on Sysmex XN haematology analyser in adult population.

INTRODUCTION: The Sysmex XN-series haematology analyser has newly adopted a fluorescent channel to measure immature platelet fraction (IPF). To promote the clinical utility of this promising parameter, establishing a reliable reference interval is mandatory. According to previous studies, IPF values may be affected by the employed analyser and the ethnic background of the individual, but no differences seem to be found between individuals' genders. Therefore, this study aimed to define the reference interval for IPF in a Spanish population following Clinical and Laboratory Standard Institute (CLSI) guidelines. MATERIALS AND METHODS: A total of 153 healthy Caucasian adults from Spain met the inclusion criteria. IPF measurement was performed by means of a Sysmex XN-2000 haematology analyser. A non-parametric percentile method was used to calculate the reference intervals in accordance with CLSI guidelines. RESULTS: The obtained reference interval for IPF on the Sysmex XN-2000 was 1.6-9.6% (90% confidence intervals (CIs) were 1.5-1.8 and 9.3-11.5, respectively). No significant gender difference in IPF reference intervals was observed (P = 0.101). CONCLUSIONS: This study provides, for the first time, a reference interval for IPF using a Sysmex XN-2000 in a Spanish population, ranging from 1.6 to 9.6%. These data are needed to evaluate platelet production in several conditions such as thrombocytopenia, inflammatory states and cardiovascular diseases, as well as for future research.

Estimation of Alert and Change Limits of Haematological Quantities and its Application in the Plausibility Control.

INTRODUCTION: In the process of quality assurance of the measured values of the clinical laboratory, one of the purposes is to perform the validation of patients' measured values in the most objective way. This validation process is called plausibility control which may be defined as the set of procedures used to decide if a patient's measured value is valid according to established clinical and biological criteria. The aim of this study is to propose a model to estimate alert and change limits of measured values of the blood cell count, to be applied to detect doubtful patients' measured values. METHODS: Some alert and change limits were estimated from the emergency laboratory database of the year 2010 using different percentiles. A verification of the suitability of the proposed model was also performed. RESULTS: Most of the fractions of the measured values excluded by the alert and change limits were according to the theoretical expected. The overall fraction of the number of doubtful clinical laboratory reports ranged between 0.6 and 47.6 %. CONCLUSIONS: The proposed model helps, improves and standardizes the process of detection of doubtful measured values since they are produced objectively. These limits can also be configured in a laboratory information system letting the clinical laboratory professional staff to save time and efforts.

A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates.

Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.

Switching on cytotoxicity by a single mutation at the heavy chain variable region of an anti-ganglioside antibody.

Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E(99)→R mutant antibody did not exclusively depend on ganglioside expression on tumor cells.

Papular epidermal nevus with "skyline" basal cell layer (PENS).

BACKGROUND: Several types of epidermal keratinocytic nevus are recognized. OBJECTIVE: We sought to describe a previously unreported keratinocytic nevus with distinctive clinical and histopathologic features in 5 patients. METHODS: We performed a clinical and photographic review, and obtained skin biopsy samples for histopathologic examination from each patient. Genetic analysis to screen for fibroblast growth factor receptor 3 and phosphatidylinositol 3-kinase, catalytic, alpha hotspot mutations was performed on lesional skin from two patients. RESULTS: Five infants (2 male, 3 female) had from 1 to 11 lesions present since birth. These consisted of 1- to 7-mm hyperkeratotic papules with a rough, flat surface and a round, commalike, rectangular, or polygonal shape. Histopathologic examination showed acanthosis with broad and rectangular rete ridges, and strikingly arranged basal cells with palisaded nuclei. Genetic testing on paraffin-embedded specimens from two patients ruled out hotspot mutations in the fibroblast growth factor receptor 3 and phosphatidylinositol 3-kinase, catalytic, alpha genes. LIMITATIONS: A small number of patients are presented. CONCLUSION: We propose the name "papular epidermal nevus with 'skyline' basal cell layer" (PENS) for this newly recognized condition.

Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity.

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid-containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4(+) T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab')(2) but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.

B7 and 34B7 monoclonal antibodies: a theoretical approach to the molecular basis of immunoglobulin cross-reactive antibodies.

Neonatal natural antibodies (NAbs) are characterized by their high degree of idiotypic cross reactivity, together with some restrictions in the genetic mechanisms of variable region diversity. We report here the immunogenetic analysis of two anti-idiotype antibodies (B7 and 34B7 monoclonal antibodies [MAbs]), which are also polyreactive as NAbs. Evidence of a process of somatic mutations were found for heavy and light chain variable regions of both antibodies. A phylogenetic analysis of the V(H)J558 family showed that the immunoglobulin cross-reactivity displayed by B7 and 34B7 MAbs is not restricted to a particular subgroup of this family. Moreover, we identified amino acid motifs in the CDR H1 and H2 of B7 and 34B7 MAbs that are also present in high proportion in immunoglobulin cross-reactive antibodies (ICRA) reported in the Kabat database. We propose that these regions are involved in ICRA activity.

Insights into the immunogenetic basis of two ganglioside-associated idiotypic networks.

The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund's adjuvant, only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigens which are not present in the normal tissues of this animal species. This phenomenon could be due to the lack of idiotypic connectivity of 14F7MAb.