Metabolic profiling of CD19+ cells in chronic lymphocytic leukemia by single-cell mass spectrometry imaging.

BACKGROUND AND AIMS: Modern mass spectrometry imaging (MSI) enables single cells' metabolism exploration. Aims of this study were development of the single-cell MSI of human CD19+ lymphocytes and metabolic profiling of chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: Blood donor (BD) samples were used for the optimization of CD19+ lymphocyte isolation and single-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) MSI. Independent set of 200 CD19+ lymphocytes coming from 5 CLL patients and 5 BD was used for the CD19+ lymphocytes classification assessment and the untargeted metabolic profiling. CLL vs BD lymphocyte classification was performed using partial least squares-discriminant analysis (PLS-DA) using normalized single-cell mass spectra recorded in 300-600 and 600-950 Da ranges was applied. RESULTS: Accuracy assessed by 10-fold cross-validation of CD19+ lymphocyte PLS-DA classification reached >90.0 %. Volcano plots showed 106 significantly altered m/z signals in CLL of which 9 were tentatively annotated. Among tentatively annotated m/z signals formaldehyde and glutathione metabolites and tetrahydrofolate stand out. CONCLUSION: A method for single-cell MALDI TOF MSI of CD19+ lymphocytes was successfully developed. The method confirmed the significance of oxidative stress and single-carbon metabolism, pyruvate and fatty acid metabolism and apoptosis in CLL and it provided metabolic candidates for diagnostic applications.

MALDI TOF Mass Spectrometry Imaging of Blood Smear: Method Development and Evaluation.

The aim of this study was to develop and evaluate matrix assisted LASER desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry imaging (MSI) of blood smear. Integrated light microscope and MALDI IT-TOF mass spectrometer, together with a matrix sublimation device, were used for analysis of blood smears coming from healthy male donors. Different blood plasma removal, matrix deposition, and instrumental settings were evaluated using the negative and positive ionization modes while agreement between the light microscopy images and the lateral distributions of cellular marker compounds served as the MSI quality indicator. Red and white blood cells chemical composition was analyzed using the differential m/z expression. Five seconds of exposure to ethanol followed by the 5 min of 9-aminoacridine or α-cyano-4-hydroxycinnamic acid deposition, together with two sets of instrumental settings, were selected for the MALDI TOF MSI experiments. Application of the thin and transparent matrix layers assured good correspondence between the LASER footprints and the preselected regions of interest. Cellular marker m/z signals coincided well with the appropriate cells. A metabolite databases search using the differentially expressed m/z produced hits which were consistent with the respective cell types. This study sets the foundations for application of blood smear MALDI TOF MSI in clinical diagnostics and research.

False positive immunoassay for heparin-induced thrombocytopenia in the presence of monoclonal gammopathy: a case report.

Heparin induced thrombocytopenia (HIT) is a life-threatening disorder which diagnosis depends on laboratory evaluation. The objective of this report is to present the impact of different laboratory methods for HIT detection on the diagnostic evaluation process. In this case, a 78-year old female patient previously diagnosed with monoclonal gammopathy of undetermined significance (MGUS) was administered with heparin for pulmonary embolism treatment. Patient's initial diagnostic work-up (determination of platelet count and prothrombin time measurement for monitoring of pharmacotherapy) was followed by the clinical estimation of HIT likelihood by "4Ts" score, two immunoassays (ID-PaGIA Heparin/PF4 Antibody Test and ELISA PF4 IgG assay) and one functional test called high-performance liquid chromatography serotonin release assay (HPLC-SRA). The result of "4Ts" score indicated a low likelihood of HIT but persistent thrombocytopenia that appeared days after discontinuation of heparin therapy suggested delayed-onset HIT. Both immunoassays were positive for presence of HIT-autoantibodies, while the functional HPLC-SRA was negative. Since different methods gave opposing results, their interpretation required great attention. In comparison to the HPLC-SRA, immunoassays are prone to the analytical interferences associated with the presence of non-specific antibodies, which may lead to false positive results. In this case, where the patient is known to produce antibodies of undetermined significance, HIT was ruled out as the possible cause of persistent thrombocytopenia primarily due to the negative result of HPLC-SRA, which is not prone to this type of interferences, but also due to the low "4Ts" clinical score.

[Croatian Society for Haematology and Transfusion Medicine Guidelines on the diagnosis and management of heparin induced thrombocytopenia (HIT)]. / Smjernice Hrvatskog drustva za hematologiju i transfuzijsku medicinu u dijagnosticko-terapijskom postupku za trombocitopeniju izazvanu heparinom (HIT).

Heparin induced thrombocytopenia (HIT) is a serious complication of heparin administration. In the last decade, this clinical syndrome has come into the focus of interest, primarily because of the severe thromboembolic complications that may lead to lethal outcome. In addition, great improvements have been made in the treatment with direct thrombin inhibitors and in laboratory diagnosis of HIT. As guidelines for diagnostic and management of HIT upgrade the quality of patient treatment, activities for their development have been launched in the Republic of Croatia. Based on British Committee for Standards in Haematology (BCSH) recommendations on diagnostic and treatment of HIT from 2006, activities for the introduction of new assays for anti-heparin antibodies were launched in 2008 and 2009, including algorithm of laboratory testing for HIT, sheet for clinical assessment of HIT (4T score), and education oftransfusiologists and clinicians. Upon evaluation of the results collected during one-year period, the Croatian Society of Haematology and Transfusion Medicine nominated a task force for the development of guidelines for HIT in January 2010. Following wide-ranging discussion, the guidelines were adopted in May 2011.