The accuracy of mean corpuscular volume guided anaemia classification in primary care.

BACKGROUND: Anemia can be categorized into micro-, normo- or macrocytic anemia based on the mean corpuscular volume (MCV). This categorization might help to define the etiology of anemia. METHODS: The cohort consisted of patients newly diagnosed with anaemia in primary care. Seven aetiologies of anaemia were defined, based on an extensive laboratory protocol. Two assumptions were tested: (i) MCV <80 fl (microcytic) excludes vitamin B12 deficiency, folic acid deficiency, suspected haemolysis and suspected bone marrow disease as anaemia aetiology. (ii) MCV >100 fl (macrocytic) excludes iron deficiency anaemia, anaemia of chronic disease and renal anaemia as anaemia aetiology. RESULTS: Data of 4129 patients were analysed. One anaemia aetiology could be assigned to 2422 (59%) patients, more than one anaemia aetiology to 888 (22%) patients and uncertainty regarding the aetiology remained in 819 (20%) patients. MCV values were within the normal range in 3505 patients (85%). In 59 of 365 microcytic patients (16%), the anaemia aetiology was not in accordance with the first assumption. In 233 of 259 macrocytic patients (90%), the anaemia aetiology was not in accordance with the second assumption. CONCLUSIONS: Anaemia aetiologies might be ruled out incorrectly if MCV guided classification is used as a first step in the diagnostic work-up of anaemia. We recommend using a broader set of laboratory tests, independent of MCV.

Digital morphology analyzers in hematology: ICSH review and recommendations.

INTRODUCTION: Morphological assessment of the blood smear has been performed by conventional manual microscopy for many decades. Recently, rapid progress in digital imaging and information technology has led to the development of automated methods of digital morphological analysis of blood smears. METHODS: A panel of experts in laboratory hematology reviewed the literature on the use of digital imaging and other strategies for the morphological analysis of blood smears. The strengths and weaknesses of digital imaging were determined, and recommendations on improvement were proposed. RESULTS: By preclassifying cells using artificial intelligence algorithms, digital image analysis automates the blood smear review process and enables faster slide reviews. Digital image analyzers also allow remote networked laboratories to transfer images rapidly to a central laboratory for review, and facilitate a variety of essential work functions in laboratory hematology such as consultations, digital image archival, libraries, quality assurance, competency assessment, education, and training. Different instruments from several manufacturers are available, but there is a lack of standardization of staining methods, optical magnifications, color and display characteristics, hardware, software, and file formats. CONCLUSION: In order to realize the full potential of Digital Morphology Hematology Analyzers, pre-analytic, analytic, and postanalytic parameters should be standardized. Manufacturers of new instruments should focus on improving the accuracy of cell preclassifications, and the automated recognition and classification of pathological cell types. Cutoffs for grading morphological abnormalities should depend on clinical significance. With all current devices, a skilled morphologist remains essential for cell reclassification and diagnostic interpretation of the blood smear.

Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling.

Chronic lymphocytic leukemia (CLL) is characterized by an acquired immune dysfunction, which may underlie the hampered efficacy of cellular immunotherapy. Most data on dampened immune responses in CLL come from studies investigating CLL and T cell interactions. Natural killer (NK) cells may be an attractive alternative source of effector cells in immunotherapy in CLL, provided that functionality is retained within the CLL micro-environment. Despite their important role in anti-tumor responses, NK cells are not extensively characterized in CLL. Here, we studied the expression of activating and inhibitory receptors on CLL-derived and healthy control (HC) NK cells, and their functional response towards several stimuli. NK cells from CLL patients have an increased maturation stage, with an expansion of NKG2C+ NK cells in CMV seropositive individuals. The cytotoxicity receptor NKG2D is downregulated, and the killing capacity through this receptor was markedly reduced in CLL-derived NK cells. In contrast, activation via CD16 (FCγRIII) led to adequate activation and functional responses in CLL-derived NK cells. These findings indicate that NK cells in CLL are not intrinsically defect and still perform effector functions upon adequate activating signaling. Clinical relevance of this finding was shown by treatment with novel nanobody-Fc constructs, which induced cytotoxic responses in both CLL- and HC-derived NK cells via CD16. Our results show that NK cells, in contrast to the T cell compartment, retain their function within the CLL micro-environment, provided that they receive an adequate activating signal. These findings warrant future studies on NK cell mediated immunotherapeutic strategies in CLL.

Prevalence of potential underlying aetiology of macrocytic anaemia in Dutch general practice.

BACKGROUND: Macrocytic anaemia (MCV ≥ 100 fL) is a relatively common finding in general practice. However, literature on the prevalence of the different causes in this population is limited. The prevalence of macrocytic anaemia and its underlying aetiology were analysed in a general practice population. The potential effect of the different aetiology on survival was also evaluated. METHODS: Between the 1st of February 2007 and the 1st of February 2015, patients aged 50 years or older and presenting to their general practitioner with a newly diagnosed anaemia, were included in the study. Anaemia was defined as haemoglobin level below 13.7 g/dL in men and below 12.1 g/dL in women. A broad range of laboratory tests was performed for each patient. The causes of anaemia were consequently determined by two independent observers based on the laboratory results. RESULTS: Of the 3324 included patients, 249 (7.5 %) displayed a macrocytic anaemia and were subsequently analysed. An underlying explanation could be established in 204 patients (81.9 %) with 27 patients (13.2 %) displaying multiple causes. Classic aetiology (i.e. alcohol abuse, vitamin B12/folic acid deficiency, haemolysis and possible bone marrow disease) was found in 115 patients. Alternative causes (i.e. anaemia of chronic disease, iron deficiency, renal anaemia and other causes) were encountered in 101 patients. In addition, a notable finding was the median gamma GT of 277 U/L in patients diagnosed with alcohol abuse (N = 24, IQR 118.0-925.5) and 23 U/L in the remaining cohort (N = 138, IQR 14.0-61.0). The distribution of gamma GT values was statistically different (P < 0.001). Five year survival rates were determined for six categories of causes, ranging from 39.9 % (95 % CI 12.9-66.9) for renal anaemia to 76.2 % (95 % CI 49.4-103.0) for the category multiple causes. CONCLUSION: In addition to classic explanations for macrocytosis, alternative causes are frequently encountered in patients with macrocytic anaemia in general practice.

Interlaboratory Reproducibility of Blood Morphology Using the Digital Microscope.

Differential counting of peripheral blood cells is an important diagnostic tool. However, manual morphological analysis using the microscope is time-consuming and requires highly trained personnel. The digital microscope is capable of performing an automated peripheral blood cell differential, which is as reliable as manual classification by experienced laboratory technicians. To date, information concerning the interlaboratory variation and quality of cell classification by independently operated digital microscopy systems is limited. We compared four independently operated digital microscope systems for their ability in classifying the five main peripheral blood cell classes and detection of blast cells in 200 randomly selected samples. Set against the averaged results, the R(2) values for neutrophils ranged between 0.90 and 0.96, for lymphocytes between 0.83 and 0.94, for monocytes between 0.77 and 0.82, for eosinophils between 0.70 and 0.78, and for blast cells between 0.94 and 0.99. The R(2) values for the basophils were between 0.28 and 0.34. This study shows that independently operated digital microscopy systems yield reproducible preclassification results when determining the percentages of neutrophils, eosinophils, lymphocytes, monocytes, and blast cells in a peripheral blood smear. Detection of basophils was hampered by the low incidence of this cell class in the samples.

[A woman with low hemoglobin values]. / Een vrouw met een lage hemoglobinewaarde.

A 48-year-old woman presented with a low hemoglobin count and multiple lytic skull lesions caused by multiple myeloma.

Automated morphological analysis of cells in body fluids by the digital microscopy system DM96.

BACKGROUND: Differential counting and morphological analysis of nucleated cells in body fluids (eg, cerebrospinal fluid and pleural fluid) are of great diagnostic importance to the clinician. A recent development in this field was the introduction of an application for an automated microscopy system, the DM96 Body Fluid module, enabling the automated analysis of body fluid samples. This computerised system provides an automated morphological analysis of body fluids, including an automated classification of all nucleated cells. AIMS: To investigate the ability of the digital microscopy system, DM96, to automatically classify cells in different types of body fluids. METHODS: A total of 177 body fluids (including cerebrospinal fluid, abdominal fluid and continuous ambulant peritoneal dialysis fluid) were analysed on the DM96, and results were compared with the manual microscopy method. RESULTS: A study in 177 samples demonstrates an overall preclassification accuracy of 90% in spinal fluid and 83% in other body fluids using the automated system. Correlation coefficients for postclassification as compared with manual review range from 0.92 to 0.99 for spinal fluid sample analyses and from 0.83 to 0.98 for other body fluids. The within-run variation of automated classification is less than 6% for all cell categories (4% excluding macrophages). CONCLUSION: The DM96 has proven to be reliable and efficient, contributing to overall quality improvement in morphological analysis and automated cell classification of peripheral blood and other body-fluid samples.

Automated quantification of cellular traffic in living cells.

Cellular traffic is a central aspect of cell function in health and disease. It is highly dynamic, and can be investigated at increasingly finer temporal and spatial resolution due to new imaging techniques and probes. Manual tracking of these data is labor-intensive and observer-biased and existing automation is only semi-automatic and requires near-perfect object detection and high-contrast images. Here, we describe a novel automated technique for quantifying cellular traffic. Using local intrinsic information from adjacent images in a sequence and a model for object characteristics, our approach detects and tracks multiple objects in living cells via Multiple Hypothesis Tracking and handles several confounds (merge/split, birth/death, and clutters), as reliable as expert observers. By replacing the related component (e.g. using a different appearance model) the method can be easily adapted for quantitative analysis of other biological samples.

Down-regulation of Rap1 activity is involved in ephrinB1-induced cell contraction.

Ephrins are cell surface ligands that activate Eph receptor tyrosine kinases. This ligand-receptor interaction plays a central role in the sorting of cells. We have previously shown that the ephrinB-EphB signalling pathway is also involved in the migration of intestinal precursor cells along the crypts. Using the colon cell line DLD1 expressing the EphB2 receptor, we showed that stimulation of these cells with soluble ephrinB1 results in a rapid retraction of cell extensions and a detachment of cells. On ephrinB1 stimulation, the small GTPases Rho and Ras are activated and Rap1 is inactivated. Importantly, when a constitutively active Rap1 mutant was introduced into these cells, ephrinB1-induced retraction was inhibited. From these results, we conclude that down-regulation of Rap1 is a prerequisite for ephrin-induced cell retraction in colon cells.

Binding of protein kinase B to the plakin family member periplakin.

The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.