ALBERT

Description: Abstract 4737 Objective: Microscopic examination of peripheral blood cells is an important diagnostic tool. We evaluated the CellaVision DM96 (CellaVision AB, Lund, Sweden), an automated image analysis system for digital peripheral blood cell analysis, comparing the results with direct manual microscopy. The system obtains digital images of the blood cells at high magnification and these images are analyzed using a neural network based on a large database of cells. Material and Methods: We analyzed 234 PB films stained with May-Grünwald-Giemsa from patients of the Hospital Clínic of Barcelona. Leukocyte values were from 1.12 to 282 × 109/L (Advia 2120, Siemens Healthcare Diagnostics SL). 177 of the PB films were from patients with hematological diseases: lymphoid neoplasias: 83, acute leukemias: 52, chronic myeloproliferative diseases: 20, myelodisplastic syndromes: 18, paroxysmal nocturnal hemoglobinuria: 2, hemoglobin S: 1 and thrombotic thrombocytopenic purpura: 1. Manual differentials were performed using standard microscopy. After the microscopic analysis the slides were loaded into the CellaVision DM96 obtaining digital images of preclassified cells, which were verified or corrected when necessary by the hematologist (DM96POST). WBC differentials were abnormal in 120 cases. Statistical analysis was performed using correlation (Pearson) and concordance (Lin) tests. Results: Correlation coefficients between results obtained from the CellaVision DM96 preclassification and by conventional direct microscopy were excellent for segmented neutrophils, lymphocytes, monocytes and blasts (r〉0.87

Description: Abstract 1347 Poster Board I-369 Introduction Sepsis is among the top 10 causes of death, but improvements in the diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last years. Neutrophil CD64 expression increases rapidly in the presence of inflammation mediators and in response to infection and tissue damage. We have evaluated changes in the expression of neutrophil CD64 in infected patients in comparison with other markers of infection and sepsis. Methods Prospective analysis of 56 blood samples from patients from the intensive care unit at our institution was performed for neutrophil CD64 expression, C-reactive protein (CRP), automated absolute neuthophil count (ANC), and complete manual leucocyte formula including % of bands (BANDS), and % of metamyelocytes and myelocytes (IG). Neutrophil CD64 expression was measured by flow cytometry using a quantitative method (Leuko64TM, Trillium Diagnostics, LLC). Patients were categorized into 5 groups (CLINIC) based on the clinical history and the degree of a systemic inflammatory response, from 1 (no inflammation) to 5 (septic shock). Statistics were performed using linear regression, correlation coefficient, and Passing-Bablock (P-B) regression. Sensitivity (S), specificity (SP), efficiency (E), and positive and negative predictive values (PPV and NPV respectively) were analyzed for all the parameters measured. Results Our results showed a correlation with CLINIC of 0.417, 0.552, 0.268, and 0.136 for CD64, CRP, BANDS, and ANC, respectively. P-B regression was only good for CD64, with a slope of 1.03 (0.6-1.4). Percentages (%) of S, SP, E, PPV, and NPV for CD64 were of 81%, 72%, 71%, 46% and 92%, respectively for groups 4 and 5. For CRP, S was of 93% with SP of 20%, E of 38%, PPV of 27%, and NPV of 91%. The remaining parameters showed deficient correlation with CLINIC. Correlations between CD64 and CRP, BANDS, and ANC were of 0.435, 0.342, and 0.01, respectively. Conclusions Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice. CD64 expression showed a better PPV than CRP, and an acceptable NPV. CRP showed deficient SP and E. BANDS, GI, and ANC showed no correlation with CLINIC. CD64 is a new indicator of infection that deserves consideration to be introduced in the daily hematology laboratory analysis. Disclosures No relevant conflicts of interest to declare.

Description: More aggressive therapies used for treatment of oncohematological malignancies or control of immune responses are resulting in an increased frequency of platelet counts below the 50 x 109/L limit. The recommended reference method for platelet counts was tedious and showed low reproducibility until now. In the last 2 years, flow cytometry based techniques combined with specific monoclonal antibodies (MoAbs) have been accepted as reference method. We have evaluated the accuracy for low platelet counts of several hematologic analyzers currently used in our laboratories. The new reference method approved by ISLH, ICSH y NCCLS is based on double labeling of platelets using MoAbs directed to CD41 and CD61 followed by flow cytometry analysis. Absolute platelet counts are calculated using a ratio with red blod cell (RBC) counts provided by the hematological analyzers. In our studies, 50 blood samples with platelet counts ranging from 1.5 to 39.4 x109/L were processed in duplicate through 1 Advia 2120 (Bayer Diagnostics), 2 Advia 120 (Bayer Diagnostics) and 2 Pentra 120 DX (Horiba-ABX Diagnostics). Advia analyzers use laser-based technology while Pentra analyzers use impedance one for cell counting. All samples were also processed through the reference flow cytometric method, being platelets identified by their double labeling for CD41 and CD61. The minimal number of platelets acquired in the platelet region was established at 1000. Absolute platelet counts were calculated using RBC counts provided by the respective analyzers. All blood samples were processed within 6 hours from phlebotomy. Statistical methods applied included: coefficient of variation (%CV), coefficient of correlation (r ), linear regression, Passing-Bablock (P-B) regression and Bland-Altman test. Precision of each analyzer was obtained by processing in 10 times different blood samples with counts from 4 to 39 x 109/L. Global results were evaluated, though special attention was paid to subgroups of results below or above 20 x 109/L. Correlation between reference values and counts provided by the Advia 2120 was 0.945 with a linear regression of 0.987x+2.9. P-B correlation was good and the average difference was 2.7 x 109/L. In the subgroup of samples with counts below 20 x 109/L correlation was 0.874 with 1.00x+2.7. P-B was correct and the average difference was 2.8 x 109/L. Results with Advia 120 were always similar to those calculated with the Advia 2120, though the average difference was slightly lower with a value of 1.7 x 109/L. Precision (CV) was 16% for platelet count levels at 4 x 109/L, 12% for those at 13 x 109/L and 4% for those at 39 x 109/L. Correlation with Pentra 120 Dx was 0.937 with a linear regression of 0.894x+2.7, the P-B was acceptable with an average difference of 1.2 x 109/L. Correlation index was 0.824 with a linear regression of 0.88x+2.8 for platelet counts below 20 x 109/L, average difference was of 1.4 x 109/L and a correct P-B. Precision (CV) ranged from 26% at 4 x 109/L and 8% at 20 x 109/L platelet counts. Our data demonstrate that hematological analyzers evaluated provided very reliable results at low platelet counts. Advia and Pentra analyzers investigated tend to over calculate the number of platelets (2.5 and 1.4 x109/L respectively). Correlation scattering was slightly superior with the Pentra analyzer. Overall reproducibility for low platelet counts was excellent for both laser and impedance technologies tested.

Description: Detection of platelet activation through conventional blood analyzers appears as a promising approach for a wide population of patients subjected to elevated cardiovascular risk. The mean platelet component (MPC) parameter calculated by the new generation of blood cell analyzers (ADVIA 120 and ADVIA 2120) provides direct information on density and granularity of platelets. Recent studies suggest that this parameter could correlate with the platelet activation state and become a potential predictive parameter for acute ischemic complications or thrombotic risk. Currently, the MPC parameter is largely affected by time and storage conditions. Standard anticoagulants based on K3EDTA are known to posses a deleterious effects on platelet morphology and granule content. It would be desirable that the stability of the MPC in blood specimens should be improved, to facilitate a more standardized use in different laboratories. Blood from healthy controls was collected into K3EDTA plus fixatives, additives, and stored at different conditions that could prolong the stability of the MPC. MPC and the mean platelet volume (MPV) were assessed 30 min and at 1, 3, 6 and 24 hours after blood drawing on the ADVIA 2120 system. Flow cytometry techniques were used to identify platelet-activation proteins. Impact of these strategies on ultrastructural morphology of platelets was morphometrically evaluated using electron microscopy (EM). Strategies based on addition of low concentrations of fixatives (paraformaldehyde or glutaraldehyde) that have proved useful for flow cytometry were unsuccessful to guarantee the stability of MPC. A solution based on K3EDTA containing wortmanin and tyrphostin (ED-WORTY) provided good stability for most of the parameters tested up to 6 hours at room temperature. Ultrastructural morphometry revealed a progressive statistically significant reduction in the number of alpha-granules per platelet section in samples stored in EDTA (from 12.3±6.07 to 6.4±2.3 and to 3.5±1.4; baseline, 6 and 24 h respectively, p