FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

HE4 as a serum biomarker for the diagnosis of pelvic masses: a prospective, multicenter study in 965 patients.

BACKGROUND: To evaluate the diagnostic value of adding human epididymis protein 4 (HE4), cancer antigen 125 (CA125) and risk of malignancy algorithm (ROMA) to ultrasound for detecting ovarian cancer in patients with a pelvic mass. METHODS: This was a prospective, observational, multicenter study. Patients aged > 18 years who were scheduled to undergo surgery for a suspicious pelvic mass had CA125 and HE4 levels measured prior to surgery, in addition to a routine transvaginal ultrasound scan. The diagnostic performance of CA125, HE4 and ROMA for distinguishing between benign and malignant adnexal masses was assessed using receiver operating characteristic (ROC) analysis and the corresponding area under the curve (AUC). RESULTS: Of 965 evaluable patients, 804 were diagnosed with benign tumors and 161 were diagnosed with ovarian cancer. In late-stage ovarian cancer, CA125, HE4 and ROMA all had an excellent diagnostic performance (AUC > 0.92), whereas in stage I and II, diagnostic performance of all three biomarkers was less adequate (AUC < 0.77). In the differential diagnosis of ovarian cancer and endometriosis, ROMA and HE4 performed better than CA125 with 99 and 98.1% versus 75.0% sensitivity, respectively, at 75.4% specificity. CONCLUSIONS: ROMA and HE4 could be valuable biomarkers to help with the diagnosis of ovarian cancer in premenopausal patients in order to differentiate from endometriosis, whereas CA125 may be more adequate for postmenopausal patients.

Patients' subjective assessment as a decisive predictor of malignancy in pelvic masses: results of a multicentric, prospective pelvic mass study.

OBJECTIVE: The prognosis for ovarian cancer patients remains poor. A key to maximizing survival rates is early detection and treatment. This requires an accurate prediction of malignancy. Our study seeks to improve the accuracy of prediction by focusing on early subjective assessment of malignancy. We therefore investigated the assessment of patients themselves in comparison to the assessment of physicians. METHODS: One thousand three hundred and thirty patients participated in a prospective and multicenter study in six hospitals in Berlin. Using univariate analysis and multivariate logistic regression models, we measured the accuracy of the early subjective assessment in comparison to the final histological outcome. Moreover, we investigated factors related to the assessment of patients and physicians. RESULTS: The patients' assessment of malignancy is remarkably accurate. With a positive predictive value of 58%, the majority of patients correctly assessed a pelvic mass as malignant. With more information available, physicians achieved only a slightly more accurate prediction of 63%. CONCLUSIONS: For the first time, our study considered subjective factors in the diagnostic process of pelvic masses. This paper demonstrates that the patients' personal assessment should be taken seriously as it can provide a significant contribution to earlier diagnosis and thus improved therapy and overall prognosis.

Improved Diagnosis of Iron Deficiency Anemia in the Critically Ill via Fluorescence Flowcytometric Hemoglobin Biomarkers.

BACKGROUND: Iron deficiency anemia (IDA) is common in critically ill patients treated in the intensive care unit (ICU), and it can lead to severe consequences. Precise and immediate diagnostics are not available, but they are inevitably needed to administer adequate therapy. Serological parameters such as serum ferritin and transferrin saturation (TSAT) are heavily influenced by simultaneous inflammation reactions, resulting in the need for more suitable parameters. Reticulocyte biomarkers such as reticulocyte hemoglobin content (RET-He) and Delta-hemoglobin equivalent (Delta-He) determined by fluorescence flowcytometry are more specific for the diagnosis of IDA-based anemia and should be investigated for this purpose. METHODS: In a prospective cohort single-center study, serum ferritin and transferrin saturation (TSAT) were collected and compared to RET-He and Delta-He by performing a receiver operating curve (ROC) analysis. The sensitivity and specificity of a single variable or the combination of two variables, as well as cutoff values, for the diagnosis of IDA were calculated. A group comparison for IDA patients without IDA was performed for a control group. RESULTS: A total of 314 patients were enrolled from an interdisciplinary ICU. RET-He (area under the curve (AUC) 0.847) and Delta-He (AUC 0.807) did indicate iron-deficient anemia that was more specific and sensitive in comparison to serum ferritin (AUC 0.678) and TSAT (AUC 0.754). The detection of functional iron deficiency (FID) occurred in 28.3% of cases with anemia. CONCLUSIONS: Determination of RET-He and Delta-He allows for the increased precision and sensitivity of iron-deficient anemia in the ICU.

Reticulocyte and Erythrocyte Hemoglobin Parameters for Iron Deficiency and Anemia Diagnostics in Patient Blood Management. A Narrative Review.

Anemia, iron deficiency and other hematinic deficiencies are a major cause of perioperative transfusion needs and are associated with increased morbidity and mortality. Anemia can be caused either by decreased production of hemoglobin or red blood cells or by increased consumption and blood loss. Decreased production can involve anything from erythropoietin or vitamin B12 insufficiency to absolute or functional lack of iron. Thus, to achieve the goal of patient blood management, anemia must be addressed by addressing its causes. The traditional parameters to diagnose anemia, despite offering elaborate options, are not ideally suited to giving a simple overview of the causes of anemia, e.g., iron status for erythropoiesis, especially during the acute phase of inflammation, acute blood loss or iron deficiency. Reticulocyte hemoglobin can thus help to uncover the cause of the anemia and to identify the main factors inhibiting erythropoiesis. Regardless of the cause of anemia, reticulocyte hemoglobin can also quickly track the success of therapy and, together with the regular full blood count it is measured alongside, help in clearing the patient for surgery.

Delta-Hemoglobin Equivalent and Granularity Index as Cell-Derived Biomarkers for the Detection of Bacterial Infections.

BACKGROUND: Prompt and precise detection of an infection in the blood is of great clinical importance in terms of early therapy initiation and the patient's prognosis. Infection-triggered inflammatory cellular and humoral signaling cascades offer great opportunities to redefine standard tests. However, while inexpensive and easy-to-use biomarkers for the detection of infections and the concomitant inflammatory processes exist, they are rarely used in clinical practice. We aimed to investigate the correlation of Granularity Index (GI) and Delta-hemoglobin equivalent (Delta-He) as inexpensive and easy-to-use cell-derived infection markers with established acute-phase parameters in a randomly selected patient. METHODS: We analyzed plasma concentrations of the established C-reactive protein (CRP) and procalcitonin (PCT) and leukocyte and thrombocyte counts in blood samples of 1,787 patients undergoing routine laboratory inflammation diagnostics. We also measured the Granularity Index (GI) and Delta-hemoglobin equivalent (Delta-He) in this cohort between February 2019 and February 2020. RESULTS: Delta-He and GI Index significantly correlated with CRP concentration (AUC 0.72, 95% CI 0.71 - 0.74; p < 0.001 for both analytes) and thrombocyte count (p < 0.001 for both analytes) but not with leukocyte count (AUC 0.54, 95% CI 0.50 - 0.59, p < 0.67). Furthermore, Delta-He significantly correlated with PCT (AUC 0.65, 95% CI 0.63 - 0.68, p < 0.001) while GI Index did not. Additionally, thrombocyte count significantly correlated with CRP (p < 0.001) and with PCT concentrations (p < 0.001). CONCLUSIONS: Delta-He and GI are two novel, inexpensive and easy-to-use cell-derived hematological biomarkers with the potential to be used as fully automated and highly standardized parameters. These biomarkers would be available on a 24 hours basis in the routine laboratory for the detection of bacterial infections by measuring a complete blood count (CBC) with differential and reticulocyte counts.

Nucleated red blood cells as predictors of mortality in patients with acute respiratory distress syndrome (ARDS): an observational study.

BACKGROUND: Nucleated red blood cells (NRBCs) in critically ill patients are associated with increased mortality and poor outcome. The aim of the present study was to evaluate the predictive value of NRBCs in patients with acute respiratory distress syndrome (ARDS). METHODS: This observational study was conducted at an ARDS referral center and included patients from 2007 to 2014. Daily NRBC counts were assessed and the predictive validity of NRBCs on mortality was statistically evaluated. A cutoff for prediction of mortality based on NRBCs was evaluated using ROC analysis and specified according to Youden's method. Multivariate nonparametric analysis for longitudinal data was applied to prove for differences between groups over the whole time course. Independent predictors of mortality were identified with multiple logistic and Cox' regression analyses. Kaplan-Meier estimations visualized the survival; the corresponding curves were tested for differences with the log-rank test. RESULTS: A total of 404 critically ill ARDS patients were analyzed. NRBCs were found in 75.5% of the patients, which was associated with longer length of ICU stay [22 (11; 39) vs. 14 (7; 26) days; p < 0.05] and higher mortality rates (50.8 vs. 27.3%; p < 0.001). Logistic regression analysis with mortality as response showed NRBC positivity per se to be an independent risk factor for mortality in ARDS with a doubled risk for ICU death (OR 2.03; 95% CI 1.16-3.55; p < 0.05). Also, NRBC value at ICU admission was found to be an independent risk factor for mortality (OR 3.25; 95% CI 1.09-9.73, p = 0.035). A cutoff level of 220 NRBC/µl was associated with a more than tripled risk of ICU death (OR 3.2; 95% CI 1.93-5.35; p < 0.0001). ARDS patients below this threshold level had a significant survival advantage (median survival 85 days vs. 29 days; log rank p < 0.001). Presence of a severe ARDS was identified as independent risk factor for the occurrence of NRBCs > 220/µl (OR 1.81; 95% CI 1.1-2.97; p < 0.05). CONCLUSIONS: NRBCs may predict mortality in ARDS with high prognostic power. The presence of NRBCs in the blood might be regarded as a marker of disease severity indicating a higher risk of ICU death.

Delta-He, Ret-He and a New Diagnostic Plot for Differential Diagnosis and Therapy Monitoring of Patients Suffering from Various Disease-Specific Types of Anemia.

BACKGROUND: The present study was aimed to prove the usefulness of a new diagnostic plot (Hema-Plot), illustrating the relationship between the hemoglobin content of reticulocytes (Ret-He) as a marker of functional iron deficiency and the difference between the reticulocyte and erythrocyte hemoglobin content (Delta-He) as a marker of an impaired hemoglobinization of newly formed reticulocytes occurring during inflammatory processes, to differentiate between various disease-specific types of anemia. METHODS: A complete blood and reticulocyte count was performed on routine EDTA blood samples from 345 patients with and without various disease-specific types of anemia using the Sysmex XN-9000 hematology analyzer: blood healthy newborns (n = 23), blood healthy adults (n = 31), patients suffering from anemia of chronic disease (ACD) due to diverse oncological, chronic inflammatory, or autoimmune diseases (total n = 138) with (n = 65) and without therapy (n = 73), patients with thalassemia and/or hemoglobinopathy (n = 18), patients with iron deficiency anemia (IDA) (n = 35), patients with a combination of ACD and IDA (n = 17), as well as patients suffering from sepsis (total n = 83) with (n = 32) and without therapy (n = 51). The results for Ret-He, Delta-He, and C-reactive protein (CRP) were statistically compared (Mann-Whitney U Test) between the particular patient groups and the diagnostic plots were drawn. RESULTS: Delta-Hemoglobin showed a statistically significant difference between blood healthy newborns and blood healthy adults (p ≤ 0.05), while Ret-He and C-reactive protein did not. In addition, of all three biomarkers only Delta-He showed a statistically significant difference (p ≤ 0.05) between the ACD/IDA and IDA cohort. Delta-He, Ret-He, and CRP showed a statistically significant difference between patient cohorts with and without therapy suffering from ACD, ACD/IDA, and sepsis before and after medical therapy (p ≤ 0.05). The Hema-Plot illustrated the dynamic character of Ret-He and Delta-He, notably in inflammation-based types of anemia like ACD or ACD/ IDA. CONCLUSIONS: Delta-He is a new biomarker clearly distinguishing between inflammation-based types of anemia before and after medical therapy, as well as between ACD/IDA and IDA. The new Hema-Plot is a helpful tool for differential diagnosis and disease-monitoring in various types of disease-specific anemia, especially in ACD and ACD/IDA. The Hema-Plot can be used to identify non-adherent patients or an insufficient therapy.

Detection and quantification of hypo- and hypergranulated neutrophils on the new Sysmex XN hematology analyzer: establishment of an adapted reference interval for the neutrophil-granularity-intensity compared to XE-technology in adult patients.

BACKGROUND: Since the recent introduction of Sysmex hematology analyzers of the XN-series it can be expected that the values of individual hematological parameters might differ between the new XN and the well-established XE platform. One such parameter is called Neutrophil-Granularity-Intensity or NEUT-GI on the XN-series and NEUT-X on the XE-series. Both parameters are used by clinicians to calculate the Granularity-Index (GI-Index), an important tool to detect hypo- or hypergranulated neutrophils occurring during myelodysplasia or inflammation. The aims of this study were to determine if previously reported reference intervals for NEUT-X can be used for NEUT-GI as well and if the GI-Indices on both analyzer platforms correlate with each other. METHODS: NEUT-GI and NEUT-X were assessed in a set of 789 blood samples (n = 543 samples from adult intensive care units and n = 246 samples from adult "blood-healthy" control patients) and the corresponding Granularity-Indices were calculated for all samples using data obtained from XE-5000 and XN-1000 hematology analyzers. RESULTS: NEUT-GI and NEUT-X correlated significantly with each other (r2: 0.6512; p < 0.0001) with statistically significant higher values for NEUT-GI compared to NEUT-X in the control group (p < 0.0001) as well as in the ICU patients (p < 0.0001). This indicated that previously established reference intervals for NEUT-X cannot be used for NEUT-GI. In contrast, the GI-Indices showed no statistically significant difference between the analyzers in both groups. The GI-Indices were higher in the ICU patients compared to the control group on both analyzer platforms (p < 0.0001), as would be expected. CONCLUSIONS: Our study revealed the emphatic need for a new reference interval for NEUT-GI on the XN platform. The resulting 95% reference intervals were 140.91 - 160.46 channels for NEUT-GI and 129.20 - 142.33 channels for NEUT-X. The GI-Indices showed no significant statistical difference between the XN- and XE-series in both cohorts.

Intensive Care Infection Score--A new approach to distinguish between infectious and noninfectious processes in intensive care and medicosurgical patients.

OBJECTIVES: Clinicians regularly encounter substantial time delays in diagnosing sepsis and administering appropriate antibiotic treatment. This study investigated the ability of the Intensive Care Infection Score (ICIS) to distinguish between infectious and noninfectious processes, and to assess the justified commencement of antibiotic therapy retrospectively, in line with hospital actual best practice and applied laboratory parameters. METHODS: Intensive-care unit (ICU) patients were enrolled in this retrospective, observational study. Clinical data and laboratory parameters were determined daily. The cohort was divided into infected and noninfected patient groups. RESULTS: Out of 172 ICU patients, including 72 postoperative patients, the predictive value for infection throughout the first 5 days in 'all patients' and the 'postoperative patient' group was highest for ICIS. An ICIS cut-off value of three could predict infection in postoperative patients with 82.9% sensitivity and 75.1% specificity. ICIS showed the lowest rate of potentially 'falsely encouraged' and 'discouraged' antibiotic therapies for noninfected and for septic postoperative patients, respectively, compared with C-reactive protein, procalcitonin and white blood cell levels. CONCLUSIONS: In the ICU, particularly for postoperative patients, ICIS is a reliable marker for the timely identification of infection. ICIS may qualify as a new decision support tool for antibiotic therapy, when interpreted within the clinical context.

Diagnostic testing for a high-grade inflammation: parameter dynamics and novel markers.

BACKGROUND: The timely detection of severe inflammatory conditions is of great importance for early therapy initiation and the patient's prognosis. The complex humoral and cellular processes involved in inflammation offer many opportunities for diagnostic testing, which are still unused in clinical practice. We investigated the dynamics of four established and two novel potential markers during the onset and resolution of a high-grade inflammation. METHODS: We retrospectively analyzed C-reactive protein and procalcitonin concentrations, leukocyte and thrombocyte counts, granularity index, and δ-hemoglobin measured in peripheral blood samples of patients undergoing inflammation diagnostics between September 2010 and November 2010. Data from a consecutive sample of 53,968 patients were available. RESULTS: Trajectories for the parameters' dynamics during the onset and resolution of a high-grade inflammation were calculated with a locally weighted scatter plot smoothing method. The leukocyte count trajectories did not exceed the reference range. CONCLUSIONS: We were able to elucidate the parameter dynamics with time coordinates rounded to the nearest hour and a follow-up of 168 h.

Angiotensin converting enzyme intron 16 insertion/deletion genotype is associated with plasma C-reactive protein concentration in uteroplacental dysfunction.

INTRODUCTION: Disturbance of the uteroplacental circulation (UPC) and the renin-angiotensin system are involved in the pathogenesis of preeclampsia. In women with history of preeclampsia persistently elevated C-reactive protein (CRP) levels have been described. The angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) genotype is associated with ACE activity and assumed to be a risk factor for preeclampsia. As ACE generates proinflammatory angiotensin II, we analysed, whether ACE intron 16 I/D genotype is associated with CRP and whether this association exhibited a relation to uteroplacental dysfunction. MATERIALS AND METHODS: A total of 639 women have been followed during pregnancy with repeated measurements of CRP levels (observations: n=2333). ACE intron 16 I/D genotype was determined, and its association with CRP was assessed with adjustment for non-independent observations. RESULTS: CRP levels of ACE D allele carriers were significantly higher than those of the ACE II (wild-type) genotype (p=0.0003, p(adj)=0.04). This relation was allele-dose dependent (p<10(-4), p(adj)<0.02). Association between ACE I/D and CRP was significantly restricted to patients presenting with impaired UPC in univariate (p<0.04) and multivariate analyses (p=0.01). CONCLUSIONS: The ACE I/D genotype is significantly associated with CRP elevations during pregnancies complicated by disturbed UPC. Whether this effect on CRP is involved in pathogenesis of preeclampsia has to be elucidated.

Improving compliance to colorectal cancer screening using blood and stool based tests in patients refusing screening colonoscopy in Germany.

BACKGROUND: Despite strong recommendations for colorectal cancer (CRC) screening, participation rates are low. Understanding factors that affect screening choices is essential to developing future screening strategies. Therefore, this study assessed patient willingness to use non-invasive stool or blood based screening tests after refusing colonoscopy. METHODS: Participants were recruited during regular consultations. Demographic, health, psychological and socioeconomic factors were recorded. All subjects were advised to undergo screening by colonoscopy. Subjects who refused colonoscopy were offered a choice of non-invasive tests. Subjects who selected stool testing received a collection kit and instructions; subjects who selected plasma testing had a blood draw during the office visit. Stool samples were tested with the Hb/Hp Complex Elisa test, and blood samples were tested with the Epi proColon® 2.0 test. Patients who were positive for either were advised to have a diagnostic colonoscopy. RESULTS: 63 of 172 subjects were compliant to screening colonoscopy (37%). 106 of the 109 subjects who refused colonoscopy accepted an alternative non-invasive method (97%). 90 selected the Septin9 blood test (83%), 16 selected a stool test (15%) and 3 refused any test (3%). Reasons for blood test preference included convenience of an office draw, overall convenience and less time consuming procedure. CONCLUSIONS: 97% of subjects refusing colonoscopy accepted a non-invasive screening test of which 83% chose the Septin9 blood test. The observation that participation can be increased by offering non-invasive tests, and that a blood test is the preferred option should be validated in a prospective trial in the screening setting.

The fraction of varicella zoster virus-specific antibodies among all intrathecally-produced antibodies discriminates between patients with varicella zoster virus reactivation and multiple sclerosis.

BACKGROUND: Primary infection with or reactivation of varicella zoster virus (VZV) can cause neurologic complications, which typically result in an intrathecal production of VZV-specific antibodies. Intrathecal antibodies to VZV are detectable by an elevated antibody index (AI). However, elevated VZV AIs are also found in more than half of patients with multiple sclerosis (MS), where they are thought to be part of a polyspecific intrathecal immune response. Determination of the fraction of intrathecally-produced virus-specific antibodies among all intrathecally produced antibodies may discriminate between virus-specific and polyspecific intrathecal immune responses, but the fraction of intrathecally-produced VZV-specific immunoglobulin (Ig)G of the total intrathecally produced IgG (FS anti-VZV) in patients with MS and VZV reactivation has hitherto not been compared. FINDINGS: FS anti-VZV was calculated in patients with a clinically isolated syndrome suggestive of multiple sclerosis (MS) or MS (n = 20) and in patients with VZV reactivation (7 samples from 5 patients), which all had elevated VZV AIs. The median FS anti-VZV was 35-fold higher in patients with VZV reactivation (45.1%, range 13.5-73%) than in patients with CIS/MS (1.3%, range 0.3-5.3%; p = 0.0001). While there was thus no overlap of FS anti-VZV values between groups, VZV AIs completely overlapped in patients with CIS/MS (1.6-14.8) and VZV reactivation (2.1-8.1). CONCLUSIONS: The fraction of intrathecally-produced VZV-specific IgG of the total intrathecally produced IgG discriminates between patients with VZV reactivation and MS. Our results provide further evidence that intrathecally-produced VZV antibodies are part of the polyspecific immune response in patients with MS.

Granularity Index of the SYSMEX XE-5000 hematology analyzer as a replacement for manual microscopy of toxic granulation neutrophils in patients with inflammatory diseases.

BACKGROUND: When certain inflammatory processes occur, toxic granulation neutrophils (TGNs) appear in the blood showing prominent cytoplasmic granules. Currently, the granularity of TGNs is analyzed by manual microscopy of blood smears. The SYSMEX XE-5000 is an automated hematology analyzer, which can measure toxic granulation of TGNs by calculating the Granularity (GI) Index. In this study we investigated if the GI-Index is suitable as a parameter for the TGN granularity in inflammatory diseases. METHODS: An evaluation of the toxic granulation neutrophil (TGN) granularity by manual microscopy, the GI-Index and the C-reactive protein (CRP) concentrations of 158 patients were determined. Blood samples from 40 healthy individuals were incubated with lipopolysaccharide (LPS) for in vitro kinetic measurements of the GI-Index. Furthermore, time course measurements of the GI-Index and CRP concentrations of 100 intensive care unit patients were performed. RESULTS: The GI-Index correlated with the microscopic rating of TGNs (n=158; r(s)=0.839; p<0.0001). When incubating the blood samples with LPS, the neutrophils displayed hypogranulation 30 min after incubation and a hypergranulation after 90 min. In vivo, the GI-Index indicated changes of the bacterial infection status 1 day earlier than the CRP concentration. The correlation of CRP and GI-Index varied between the patient cohorts (n=158; r(s)=0.836) (n=100; r=0.177), depending on the cause and extent of inflammation. CONCLUSIONS: The GI-Index is suited to quantify the granularity of TGNs. The GI-Index is an automated, standardized parameter available on a 24 h basis. We suggest that it replace the time-consuming, subjective and semiquantitative microscopic procedure.

Evaluation of the iChem® Velocity™ urine chemistry analyzer in a hospital routine laboratory.

BACKGROUND: The novel urine chemistry analyzer iChem Velocity (IRIS Diagnostics) offers improved urinalysis automation options through integration with the well-established iQ200 urine microscopy analyzer. In the course of optimizing the workflow in our hospital routine laboratory, we evaluated the performance of the iChem Velocity. METHODS: A total of 257 random urine samples were analyzed with the iChem Velocity, iQ200, Clinitek Atlas (Siemens Healthcare Diagnostics) and by manual microscopy. RESULTS: Depending on the parameter, 93% (hemoglobin) to 100% (urobilinogen), the iChem Velocity and Clinitek Atlas results agreed within the same rank or within one level of difference. The Clinitek Atlas featured a higher sensitivity for hemoglobin (area under the curve 0.86) and leukocyte esterase (area under the curve 0.85) compared with the iChem Velocity (area under the curve for hemoglobin 0.73, leukocytes 0.78). Imprecision was highest for hemoglobin and leukocytes in a pathological sample pool. While the precision of the Clinitek Atlas for hemoglobin measurements was superior, the iChem Velocity was more precise in analyzing protein and pH. CONCLUSIONS: Through urinalysis automation with the iChem Velocity and iQ200, we achieved a reduction of hands-on time by 89%. The sensitivity of this new system should be further improved through ongoing development.

U373-MG cells express PepT2 and accumulate the fluorescently tagged dipeptide-derivative ß-Ala-Lys-N(ε)-AMCA.

Aim of this study was to examine the dipeptide transport of ß-Ala-Lys-N(ɛ)-AMCA in the human glioma cell line U373-MG and its potential regulation by diverse hormones and culture media. A mixed glial primary cell culture of the newborn rat served as reference cell system. ß-Ala-Lys-N(ɛ)-AMCA (ß-Ala-Lys-N(ɛ)-7-amino-4-methyl-coumarin-3-acetic acid) is a highly specific reporter substrate to investigate the dipeptide transport system PepT2. We were able to demonstrate that U373-MG cells express PepT2-mRNA and translocate ß-Ala-Lys-N(ɛ)-AMCA via PepT2 into the cytoplasm. Previous results demonstrated that ß-Ala-Lys-N(ɛ)-AMCA specifically accumulates in differentiated and dedifferentiated astrocytes but neither in differentiated nor dedifferentiated oligodendrocytes and in neurons. U373-MG cells were incubated with estradiol, testosterone, thyronine, dexamethasone, dibutyryl cyclic adenosine monophosphate and tetradecanoylphorbol acetate in order to detect potential substance-dependent changes in dipeptide uptake. There was no significant increase or decrease of ß-Ala-Lys-N(ɛ)-AMCA-uptake after stimulation. Northern blot analyses confirmed that PepT2-mRNA is expressed in U373-MG and glial cells but showed no regulation of PepT2-mRNA expression in both cell types. Future investigations might offer the opportunity of an anti-tumor therapy with cytotoxic agents linked to a dipeptide-derivative such as ß-Ala-Lys.

Comparison of six immunohistochemical markers for the histologic diagnosis of neoplasia in Barrett's esophagus.

In esophageal neoplasms, the histopathologic differentiation between Barrett's esophagus with or without intraepithelial neoplasia and adenocarcinoma is often challenging. Immunohistochemistry might help to differentiate between these lesions. The expression of CDX2, LI-cadherin, mucin 2 (MUC2), blood group 8 (BG8, Lewis(y)), claudin-2, and villin was investigated in normal gastroesophageal (n = 23) and in Barrett's (n = 17) mucosa, in low-grade (n = 12) and high-grade (n = 9) intraepithelial neoplasia (IEN) as well as in esophageal adenocarcinoma (n = 16), using immunohistochemistry. For CDX2 and LI-cadherin, the immunoreactivity score was highest in IEN while for MUC2, BG8, and villin, it dropped gradually from Barrett's via IEN to adenocarcinoma, and expression of Claudin-2 was only weak and focal in all lesions. The expression of MUC2 and LI-cadherin differed significantly between all examined lesions except between low-grade and high-grade IEN. MUC2 and LI-cadherin are useful immunohistochemical markers for the differentiation between normal glandular mucosa, Barrett's mucosa, IEN, and invasive carcinoma of the esophagus; however, none of the examined markers was helpful for the differentiation between low-grade and high-grade IEN.

CDX2 and LI-cadherin expression in esophageal mucosa: use of both markers can facilitate the histologic diagnosis of Barrett's esophagus and carcinoma.

BACKGROUND: Barrett's mucosa is a risk factor for esophageal adenocarcinoma and should be detected at an early stage. CDX2 and liver-intestine (LI)-cadherin are intestine-specific markers. Aberrant CDX2 expression has been demonstrated in Barrett's metaplasia, esophagitis, and intestinal metaplasia of the stomach. METHODS: The relationship between CDX2 and LI-cadherin expression was investigated in normal gastroesophageal (n = 24) and in Barrett's (n = 20) mucosa, in low-grade (n = 15) and high-grade (n = 13) intraepithelial neoplasia (IEN) as well as in esophageal adenocarcinoma (n = 16), using immunohistochemistry. RESULTS: Nuclear positivity for CDX2 coupled with membranous expression of LI-cadherin was observed in about 70% of the epithelial cells of Barrett's mucosa. The intensity of staining and the percentage of positive cells increased within the sequential steps of low-grade to high-grade IEN, whereas the normal cylindric epithelium lacked the expression of both. In adenocarcinoma, the expression of LI-cadherin and CDX2 was significantly weaker or absent. CONCLUSIONS: CDX2 and LI-cadherin are sensitive markers of intestinal metaplasia with or without dysplasia in the upper gastrointestinal tract. Both can be helpful for the early histologic diagnosis of Barrett's esophagus and its subsequent lesions; however, they do not significantly discern between different grades of dysplasia.