Evaluation of the Atellica® UAS 800: a new member of the automated urine sediment analyzer family.

BACKGROUND: In 2017 the Atellica® UAS 800 urine sediment analyzer was introduced by Siemens Healthineers. We investigated its applicability in the standardization and automation of the laboratory urinalysis workflow, including the prediction of urine culture outcome and glomerular pathology. METHODS: We evaluated the performance characteristics of the Atellica® UAS 800 and its correlation with the iQ200 (Beckman Coulter). In addition, we studied the agreement between Atellica® UAS 800 and CLINITEK Novus® and determined the predictive value of bacteria and leukocyte counts for urine culture outcome. Furthermore, we investigated the ability of Atellica® UAS 800 to identify pathological casts and dysmorphic erythrocytes in comparison to manual microscopy. RESULTS: Erythrocyte and leukocyte analyses indicated high intra- and inter-run precisions and good correlations with the iQ200. We found that the Atellica® UAS 800 detects bacteria with higher sensitivity than the iQ200. The Atellica® UAS 800 and CLINITEK Novus® showed a high degree of conformity. We determined seven combinations of clinical cut-off values of bacteria and leukocytes for predicting urine culture outcome with sensitivity, specificity, and negative predictive values of 95%, 52%, and 93%, respectively. Using the Atellica® UAS 800, hyaline casts, erythrocyte casts, leukocyte casts, and dysmorphic erythrocytes were correctly recognized in 76%, 22%, 2%, and 39% of the samples, respectively. CONCLUSIONS: The Atellica® UAS 800 is a robust, fast, and user-friendly analyzer, which accurately quantifies erythrocytes, leukocytes, bacteria and squamous epithelial cells, and may be utilized for predicting positive urine cultures. The detection of clinically important pathological casts and dysmorphic erythrocytes proved insufficient.

Current state of the morphological assessment of urinary erythrocytes in The Netherlands: a nation-wide questionnaire.

Background The morphological assessment of urinary erythrocytes (uRBC) is a convenient screening tool for the differentiation of nephrological (dysmorphic) and urological (isomorphic) causes of hematuria. Considering the morphological heterogeneity, this analysis is often perceived as difficult. There is no clear (inter)national consensus and there is a lack of external quality assessment programs. To gain insight into the heterogeneity within and between laboratories, we scrutinized the current state of this analysis in Dutch medical laboratories. Methods The laboratories, affiliated with the Dutch Foundation for Quality Assessment in Medical Laboratories, were invited to participate in a web-based survey, consisting of two questionnaires. The first one provided information about the institution and laboratory organization, and the second explored the variability in the morphological analysis of uRBC on the basis of categorization of 160 uRBC images. Statistical analysis was premised on binomial significance testing and principal component analysis. Results Nearly one third of the Dutch medical laboratories (65/191) with 167 staff members participated in the survey. Most of these laboratories (83%) were an integral part of secondary care. The statistical analysis of the evaluations of the participants in comparison to the consensus (three experts from two different medical laboratories) suggested a great degree of heterogeneity in the agreement. Nearly half of the participants consciously disagreed with the consensus, whereas one fifth demonstrated a random relationship with it. Conclusions In Dutch medical laboratories, results from morphological analysis of uRBC are heterogeneous, which point out the necessity for standardization and harmonization.

Is there a suitable point-of-care glucose meter for tight glycemic control? Evaluation of one home-use and four hospital-use meters in an intensive care unit.

BACKGROUND: Implementation of tight glycemic control (TGC) and avoidance of hypoglycemia in intensive care unit (ICU) patients require frequent analysis of blood glucose. This can be achieved by accurate point-of-care (POC) hospital-use glucose meters. In this study one home-use and four different hospital-use POC glucose meters were evaluated in critically ill ICU patients. METHODS: All patients (n = 80) requiring TGC were included in this study. For each patient three to six glucose measurements (n = 390) were performed. Blood glucose was determined by four hospital-use POC glucose meters, Roche Accu-Check Inform II System, HemoCue Glu201DM, Nova StatStrip, Abbott Precision Xceed Pro, and one home-use POC glucose meter, Menarini GlucoCard Memory PC. The criteria described in ISO 15197, Dutch TNO quality guideline and in NACB/ADA-2011 were applied in the comparisons. RESULTS: According to the ISO 15197, the percentages of the measured values that fulfilled the criterion were 99.5% by Roche, 95.1% by HemoCue, 91.0% by Nova, 96.6% by Abbott, and 63.3% by Menarini. According to the TNO quality guideline these percentages were 96.1% , 91.0% , 81.8% , 94.2% , and 47.7% , respectively. Application of the NACB/ADA guideline resulted in percentages of 95.6%, 89.2%, 77.9%, 93.4%, and 45.4%, respectively. CONCLUSIONS: When ISO 15197 was applied, Roche, HemoCue and Abbott fulfilled the criterion in this patient population, whereas Nova and Menarini did not. However, when TNO quality guideline and NACB/ADA 2011 guideline were applied only Roche fulfilled the criteria.

[An asymptomatic patient with lactate acidosis and hypercapnia]. / Een asymptomatische patiënt met lactaatacidose en hypercapnie.

BACKGROUND: Blood gas analyses are used to identify acid-base and respiratory disturbances. Blood gas abnormalities can be first signs of serious underlying disease. CASE DESCRIPTION: An 82-year old women with chronic kidney disease (eGFR 35 ml/min/1.73m2) and rheumatoid arthritis was admitted to the geriatric ward due to a urinary tract infection and bloody diarrhea. She repeatedly had a low oxygen saturation, although without symptoms. Capillary blood gas analysis was evidently disturbed (pH 6.96, pCO2 9.3 kPa, lactate 8.4 mmol/l), and led to consultation of the intensivist. Careful physical examination demonstrated that the patients' hands had blue discoloration, typical for Raynaud's phenomenon. Arterial blood gas demonstrated normal results. CONCLUSION: Capillary blood gas analysis is less reliable when peripheral circulation is disturbed. When the results are aberrant, it is important to distinguish between peripheral vascular disease and diminished systemic circulation. Arterial blood gas analysis can be used to support the diagnosis.

Performance of commercially-available cholesterol self-tests.

BACKGROUND: Hypercholesterolemia (plasma cholesterol concentration ≥5.2 mmol/L) is a risk factor for cardiovascular disease and stroke. Many different cholesterol self-tests are readily available at general stores, pharmacies and web shops. However, there is limited information on their analytical and diagnostic performance. METHODS: We included 62 adult patients who required a lipid panel measurement (cholesterol, high-density lipoprotein (HDL), triglycerides and LDLcalc) for routine care. The performance of five different cholesterol self-tests, three quantitative meters (Roche Accutrend Plus, Mission 3-in-1 and Qucare) and two semi-quantitative strip tests (Veroval and Mylan MyTest), was assessed according to the manufacturers' protocol. RESULTS: The average plasma cholesterol concentration was 5.2 ± 1.2 mmol/L. The mean absolute relative difference (MARD) of the five cholesterol self-tests ranged from 6 ± 5% (Accutrend Plus) to 20 ± 12% (Mylan Mytest). The Accutrend Plus cholesterol meter showed the best diagnostic performance with a 92% sensitivity and 89% specificity. The Qucare and Mission 3-in-1 are able to measure HDL concentrations and can thus provide a cholesterol:HDL ratio. The Passing-Bablok regression analyses for the ratio showed poor performance in both self-tests (Mission 3-in-1: y = 1.62x-1.20; Qucare: y = 0.61x + 1.75). The Accutrend Plus is unable to measure the plasma high-density lipoprotein concentration.Conclusions/interpretation: The Accutrend Plus cholesterol meter (Roche) had excellent diagnostic and analytic performance. However, several of the commercially-available self-tests had considerably poor accuracy and diagnostic performance and therefore do not meet the required qualifications, potentially leading to erroneous results. Better regulation, standardization and harmonization of cholesterol self-tests is warranted.

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors.

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Interaction of acute lymphopblastic leukemia cells with C-type lectins DC-SIGN and L-SIGN.

The C-type lectins DC-SIGN (CD209) and L-SIGN (CD299) recognize defined carbohydrates expressed on pathogens and cells. Those lectins are expressed on dendritic cells (DC) and/or on liver-sinusoidal endothelial cells. Both cell types modulate immune responses. In acute lymphoblastic leukemia (ALL), aberrant glycosylation of blast cells can alter their interaction with the C-type lectins DC-SIGN and L-SIGN, thereby affecting their immunological elimination. We investigated whether recombinant DC-SIGN and L-SIGN bind to blood or bone marrow cells from B- and T-ALL patients and compared that with binding of peripheral blood lymphocytes from healthy donors. It was found that increased binding of ALL cells to DC-SIGN and L-SIGN was observed compared to cells from healthy donors. Furthermore, L-SIGN bound a higher percentage of leukemic and normal cells than DC-SIGN. B-ALL bone marrow cells showed the highest binding to L-SIGN. DC-SIGN bound equally well to B-ALL and T-ALL cells. Within ALL subtypes, DC-SIGN binding was higher with mature T-ALL. Interestingly, our data demonstrate that increased binding of DC-SIGN and L-SIGN to peripheral leukemic cells from B-ALL patients is associated with poor survival. These data demonstrate that high binding of B-ALL peripheral blood cells to DC-SIGN and L-SIGN correlates with poor prognosis. Apparently, when B-ALL cells enter the blood circulation and are able to interact with DC-SIGN and L-SIGN the immune response is shifted toward tolerance. Additional studies are necessary to ascertain the possible role of these results in terms of disease pathogenesis and their potential as target to eradicate leukemic cells.

Relevance of DC-SIGN in DC-induced T cell proliferation.

The role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in DC-T cell communication was assessed by analyzing the effect of DC-SIGN-blocking mAb in MLR. The results show that the degree of inhibition by DC-SIGN and LFA-1 mAb depends on the magnitude of the MLR and the maturation status of the DC. Addition of DC-SIGN mAb at several time-points during MLR showed that DC-SIGN is involved early on in DC-T cell contacts. This initial role is masked by strong adhesive and costimulatory mechanisms, indicating a short-lived effect of DC-SIGN in DC-T cell interactions. To examine this concept in more detail, the percentage of PBL capable of binding DC-SIGN was determined. Analysis of several donors revealed that 1-20% PBL bind to beads coated with recombinant DC-SIGN, and the DC-SIGN-binding cells comprised all major cell subsets found in blood. PBL isolated from a donor with high DC-SIGN-binding capacity were more prone to blocking by DC-SIGN mAb in MLR than PBL from a donor with low DC-SIGN-binding capacity. This study indicates an initial and transient role for DC-SIGN in T cell proliferation, which becomes apparent when T cell proliferation is low and when the percentage of DC-SIGN binding PBL is high.

Binding of the adhesion and pathogen receptor DC-SIGN by monocytes is regulated by the density of Lewis X molecules.

Soluble DC-SIGN (CD209) bind unsialylated Lewis X epitopes that are abundantly expressed on neutrophils. Due to the low expression of unsialylated Lewis X epitopes on monocytes, no binding of soluble DC-SIGN molecules was seen. In contrast, beads coated with multiple DC-SIGN molecules show a high percentage of binding to monocytes. The increased number of DC-SIGN molecules present on the beads enable multivalent interactions between the DC-SIGN molecules and the scarce Lewis X epitopes present on monocytes. Increased expression of unsialylated Lewis X epitopes on monocytes after neuraminidase treatment coincided with enhanced binding to soluble DC-SIGN. Multiple unsialylated Lewis X epitopes in close proximity of each other are now able to interact multivalently to soluble DC-SIGN. From these findings, we conclude that firm interactions between DC-SIGN and monocytes can be established by either increasing the density of DC-SIGN molecules at the cell surface or by increasing the number of Lewis X epitopes. Regulating the number of ligands endows monocytes with the capacity to modulate binding to DC-SIGN. This may result in a bi-directional cross-talk between DC and monocytes, to modulate innate and/or adaptive immune responses.

C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous glycoconjugates.

Human C-type lectin receptors (CLRs) characteristically bind glycosylated ligands in a Ca(2+)-dependent way via their carbohydrate recognition domain (CRD). Their carbohydrate preference is dependent on the amino acid sequence in the CRD domain and on the ability and flexibility of the CRD domain to accommodate sugar moieties that are located at different distances from each other in the glycoconjugate. Although microbial and vertebrate cells are able to produce similar polysaccharide chains, the density of carbohydrates on microbes is much higher compared to vertebrate cells. Despite this difference, carbohydrates present on both cell types can be recognized by the CLRs. These receptors are predominantly expressed by antigen presenting cells such as dendritic cells. In addition to the Toll-like receptor family, CLRs function as pattern recognition receptors by recognizing glycosylated patterns on pathogens. This usually results in internalization of the pathogen, lysosomal degradation and subsequent loading of pathogen-derived peptides into major histocompatibility complex molecules for antigen presentation. However, several pathogens have developed ways to exploit the CLRs to evade immune eradication by for example escaping from the lysosomal degradation pathway or by inducing anti-inflammatory cytokines. When CLRs bind endogenous glycosylated ligands they mediate several processes like cell-cell adhesion and clearance of aberrant cells like tumor cells or apoptotic cells.

Human dendritic cells are less potent at killing Candida albicans than both monocytes and macrophages.

Dendritic cells (DC) function as professional phagocytes to kill Candida albicans and subsequently present it to the adaptive immune system. Monocytes, macrophages and DC were generated from five individual donors and their Candida-killing capacity and cytokine release were assessed. Compared to monocytes and macrophages, DC from healthy volunteers were significantly less effective in C. albicans--stimulated cytokine release, killing of C. albicans blastoconidia and damaging of C. albicans hyphae. In conclusion, while important as antigen-presenting cells and initiators of the adaptive immune system, DC are poor in both intracellular killing and damaging of C. albicans hyphae. Effective handling of large numbers of C. albicans is the prime task of the innate immune system consisting of large numbers of neutrophils and monocytes.

HI responses induced by seasonal influenza vaccination are associated with clinical protection and with seroprotection against non-homologous strains.

Vaccination against influenza induces homologous as well as cross-specific hemagglutination inhibiting (HI) responses. Induction of cross-specific HI responses may be essential when the influenza strain does not match the vaccine strain, or even to confer a basic immune response against a pandemic influenza virus. We carried out a clinical study to evaluate the immunological responses after seasonal vaccination in healthy adults 18-60 years of age, receiving the yearly voluntary vaccination during the influenza season 2006/2007. Vaccinees of different age groups were followed for laboratory confirmed influenza (LCI) and homologous HI responses as well as cross-specific HI responses against the seasonal H1N1 strain of 2008 and pandemic H1N1 virus of 2009 (H1N1pdm09) were determined. Homologous HI titers that are generally associated with protection (i.e. seroprotective HI titers ≥40) were found in more than 70% of vaccinees. In contrast, low HI titers before and after vaccination were significantly associated with seasonal LCI. Cross-specific HI titers ≥40 against drifted seasonal H1N1 were found in 69% of vaccinees. Cross-specific HI titers ≥40 against H1N1pdm09 were also significantly induced, especially in the youngest age group. More specifically, cross-specific HI titers ≥40 against H1N1pdm09 were inversely correlated with age. We did not find a correlation between the subtype of influenza which was circulating at the age of birth of the vaccinees and cross-specific HI response against H1N1pdm09. These data indicate that the HI titers before and after vaccination determine the vaccination efficacy. In addition, in healthy adults between 18 and 60 years of age, young adults appear to be best able to mount a cross-protective HI response against H1N1pdm09 or drifted seasonal influenza after seasonal vaccination.

Impaired production of TNF-α by dendritic cells of older adults leads to a lower CD8+ T cell response against influenza.

Seasonal influenza causes more morbidity and mortality in older adults than in young adults, apparently because of a decline in immune function with increasing age, known as immunosenescence. In this study, we compared the capacity of dendritic cells (DCs) from healthy older adults (≥65 years) with DCs from healthy young adults (20-40 years) to initiate a T cell response against influenza. DCs from older adults were impaired in the induction of influenza-specific CD8+ T cells as compared to DCs from young adults, which was demonstrated by a decreased proliferation, an impaired production of IFN-γ and a reduced expression of the degranulation marker CD107a by CD8+ T cells. Importantly, DCs from older adults produced significantly less TNF-α, showed a decreased expression of HLA class I and had a lower maturation state after influenza virus infection. Supplementing TNF-α increased the expression of HLA class I and of maturation markers and enhanced the induction of the influenza-specific CD8+ T cell response. Together, these findings indicate that the impaired influenza-specific CD8+ T cell response in older adults is associated with a reduced production of TNF-α and with a lower DC maturation. We suggest that the production of TNF-α is a determining factor in the DC-mediated CD8+ T cell response against influenza.

Standardization and validation of assays determining cellular immune responses against influenza.

Influenza vaccine efficacy does not always correlate with humoral immune responses. Recent reports indicate that the cellular immune response also contributes to protection, however robust assays are lacking. We standardized and validated assays for detection of human influenza-specific cellular responses in four international laboratories. The production of granzyme B as marker of T cell-mediated cytotoxicity and release of Th1 and Th2 cytokines were evaluated. The granzyme B and cytokine assays were specific, accurate, precise, and robust. Replicate stimulations with PBMC from the same donors showed an intra-laboratory robustness (coefficient of variation) for quantitation of granzyme B of 33% and for cytokines - including IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4, IL-13, GM-CSF and including the log IFN-gamma/IL-10 ratio - of 52%. The inter-laboratory robustness for detection of granzyme B was 29% and for detection of all cytokines was 49%. The assays can now be used for determining cell-mediated immunity and explored as correlates of protection. Moreover, the precision and robustness of these cellular assays allow the reliable detection of cellular responses even in small study populations.

Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLH-pulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1-KLH-targeted DCs induced proliferation of naive T cells recognizing KLH epitopes in the context of major histocompatibility complex (MHC) classes I and II. We conclude that antibody-mediated targeting of antigen to DCs via DC-SIGN effectively induces antigen-specific naive as well as recall T-cell responses. This identifies DC-SIGN as a promising target molecule for DC-based vaccination strategies.

The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells.

Dendritic cells (DC) that express the type II C-type lectin DC-SIGN (CD209) are located in the submucosa of tissues, where they mediate HIV-1 entry. Interestingly, the pathogen Candida albicans, the major cause of hospital-acquired fungal infections, penetrates at similar submucosal sites. Here we demonstrate that DC-SIGN is able to bind C. albicans both in DC-SIGN-transfected cell lines and in human monocyte-derived DC. The binding was shown to be time- as well as concentration-dependent, and live as well as heat-inactivated C. albicans were bound to the same extent. Moreover, in immature DC, DC-SIGN was able to internalize C. albicans in specific DC-SIGN-enriched vesicles, distinct from those containing the mannose receptor, the other known C. albicans receptor expressed by DC. Together, these results demonstrate that DC-SIGN is an exquisite pathogen-uptake receptor that captures not only viruses but also fungi.