Novel POC analysis for determination of total and 5-part differential WBC count among a US population, in comparison to Beckman Coulter LH750

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1 Novel POC analysis for determination of total and -part differential WBC count among a US population, in comparison to Beckman Coulter LH7 Reed, Jeremy*, Johnsson, Elin**, Hockum, Sandra** * Quest Diagnostics, Baltimore, MD, USA ** HemoCue AB, Ängelholm, Sweden Contact: Sandra.Hockum@hemocue.se Introduction: The purpose of the study was to evaluate possible effects of ethnical Scatter plot (least linear regression) for HemoCue WBC DIFF versus Beckman Coulter LH7 for total WBC count is presented in figure. and demographical differences on both total and differential WBC count when analyzed using the HemoCue WBC DIFF system. The study was performed at Quest Diagnostics hematology laboratory in Baltimore, MD, USA. different leftover blood samples were analyzed in one replicate on four different HemoCue WBC DIFF analyzers. Two different batches of microcuvettes were used. As reference method, all samples were analyzed in one replicate on a total of nine different Beckman Coulter LH7. The study was performed according to CLSI EP9-A. The study period ranged over five working days between -7- and All fresh samples received during the afternoon/evening were included in the study. Some older samples were selected to fulfill the suggested sample distribution (according to CLSI EP9-A). HemoCue WBC DIFF, total WBC (^9/L) y=x Beckman Coulter LH7, total WBC (^9/L) Figure. Scatter plot (least linear regression), total WBC 99. % of all samples are within a total analytical error of ±. x 9 /L or % (whichever is greater), in comparison to Beckman Coulter LH7 when determining total WBC. Scatter plots (orthogonal regression) for HemoCue WBC DIFF versus Materials and Methods: HemoCue WBC DIFF Analyzers different HemoCue WBC DIFF Microcuvette batches 9 Beckman Coulter LH7 different left over venous EDTA whole blood samples Table. Sample distribution WBC level (x 9 /L) No. of samples % of total Total: All samples were analyzed in single replicates on Beckman Coulter by Quest Diagnostics laboratory staff. All samples were then analyzed in single replicates using the HemoCue WBC DIFF system. Analysis on the HemoCue WBC DIFF system was performed by HemoCue staff at the site within two hours after analysis on Beckman Coulter. Results: A summary of results from regression analysis for the HemoCue WBC DIFF System in comparison to Beckman Coulter LH7 is presented in table. Basophils could not be evaluated due to very low absolute counts. Table. Summary of results from regression analysis Cell type No. of samples r Intercept (with 9 % CI) (x 9 /L) Slope (with 9 % CI) (x 9 /L) Total WBC.9 -. (-. to -.). (. to.) Neutrophils.9.7 (.9 to.).9 (.9 to.) Lymphocytes.9. (.7 to.).9 (.9 to.9) Monocytes.. (. to.). (. to.9) Eosinophils. -. (-. to.). (. to.) Beckman Coulter LH7 for neutrophils, lymphocytes, monocytes and eosinophils are presented in figure,, and respectively. HemoCue WBC DIFF, neutrophils (^9/L) y=x Beckman Coulter LH7, neutrophils (^9/L) Figure. Scatter plot (orthogonal regression), neutrophils HemoCue WBC DIFF, monocytes (^9/L) Beckman Coulter LH7, monocytes (^9/L) Figure. Scatter plot (orthogonal regression), monocytes Conclusion: HemoCue WBC DIFF, lymphocytes (^9/L) y=x Beckman Coulter LH7, lymphocytes (^9/L) Figure. Scatter plot (orthogonal regression), lymphocytes The novel HemoCue WBC DIFF system for total and -part differential WBC count correlates well to Beckman Coulter LH7 when analyzing samples from a US population. HemoCue WBC DIFF, eosinophils (^9/L) Beckman Coulter LH7, eosinophils (^9/L) Figure. Scatter plot (orthogonal regression), eosinophils References: Method, Comparison and Bias Estimation Using Patient Samples; Approved Guideline Second Edition (Interim Revision). CLSI document EP9-A-IR. Wayne, PA: Clinical and Laboratory Standards Institute; th International Symposium at the AACC CPOCT Division October -,, Prague, Czech Republic

2 Novel Technology for -Part Differentiation of Leukocytes Point-of-Care Jönsson, Inger*, Nilsson, Magnus*, Wahlström, Saga**, Johnsson, Elin ***, Jonasson-Bjäräng, Tomas ***, Lindberg, Stellan*** * Växjö Central Hospital Clin ChemDept, Växjö, Sweden, ** Malmö University Hospital, Clin Chem Dept, Malmö, Sweden *** HemoCue AB, Ängelholm, Sweden Contact: Stellan.Lindberg@hemocue.se Introduction and technology White blood cell count (WBC) and diff are two of the most ordered tests within and outside hospitals. A POCT system would improve the availability of rapid test results facilitating better decisions in emergency care. A new hematology system has recently been introduced by HemoCue. The system uses state-of-the-art imaging analysis techniques to count the white blood cells and perform a -part differentiation. A microcuvette serves as a pipette, sample container and reaction chamber. A blood sample of approximately µl is drawn into a cavity by capillary action. The blood dissolves the dry content in the microcuvette, the erythrocytes are hemolyzed and the leukocytes are stained with methylene blue. No dilution is required. The fixed volume used in the test is defined by the depth of the cavity in the microcuvette and the size of the image (no. of pixels). A camera moved by a high precision motor to achieve an exact and repeatable movement is used to capture images of the stained white blood cells. As the camera moves throughout the cavity of the microcuvette, it takes more than images of each cell. Imaging technology is used to decide when a cell is in focus, and all focused cells are merged into one final image. The cells are classified as neutrophils, lymphocytes, monocytes, eosinophils, basophils, pathological white blood cells (blasts and immature granulocytes) and others. The analyzer will automatically flag all samples containing pathological white blood cells. Cavity depth µm Mount the focused cells into one image. Fill the microcuvette with µl blood. Push the button for patient test and place the microcuvette in the cuvette holder. The results will be displayed within five minutes Figure : Technology overview Volume appr.. µl Disposable cuvette (chamber) with a fixed thickness and a digital photo given a fixed volume for enumerations of dyed cells The camera lens moves in small steps taking several images through the cavity of the cuvette All cells in all images will be cut out Identifying when each cell is in focus Count number of leukocytes and differentiate on this final image HemoCue has in the development of the algorithms used state-of-the-art imaging technology. More than different features (size, shape, texture, granules etc.) have been identified for each cell type, translated into a mathematical algorithm and implemented in the analyzer. The system is factory calibrated and needs no further calibration by the user. The measuring range is WBC. x 9 /L. An advanced inbuilt Quality Control (QC) system will check for correct filling of the microcuvette, dirt, improper light, blurred cells, reagent stability etc. An error code will be displayed if the QC system fails. Materials and results A Multicenter study has been carried out at three sites using venous EDTA left-over samples in order to check how the system correlates with different available cell counters. The two Clinical Chemistry Laboratory sites in hospitals; Växjö Central Hospital and Malmö University Hospital were comparing Siemens ADVIA respectively Beckman Coulter LH7. At the third site tests were performed with untrained users on three different doctor s offices compared to Sysmex XS-i. All samples were analyzed in dublicate using the HemoCue analyzer. The comparison data of the first replicate are presented in Fig. Most of the comparisons shown excellent correlations. The ADVIA gave slightly higher results than the HemoCue WBC DIFF on total leukocytes and neutrophils. Monocytes on the HemoCue WBC DIFF correlates best with ADVIA. Figure : Comparison plots for the first replicate HemoCue WBC DIFF results (y) versus Siemens ADVIA, Beckman Coulter LH7 and Sysmex XS-i. The precision between the double samples for WBC were SD=. in the low range (< x 9 /L; n=9),. in the normal range (n=) and (n=9) in the high range. The precision between the double samples for neutrophils were SD=. in the low range (< x 9 /L; n=),. in the normal range ( - 7 x 9 /L; n=) and. in the high range (n=7). ) Saga Wahlström has sadly passed away since the study was done, the data has been published by permission from her clinic in Malmö Conclusion The novel POCT HemoCue WBC DIFF technology is built on state-of-the art imaging technology. The results from the system are accurate and precise correlating well to laboratory cell counters both in the hands of lab technicians and with untrained users. A white blood cell count including a -part diff at the point of care will increase the availability of already well established and frequently used lab parameters. Rapid and easy access will be a valuable tool for physicians in making direct and more well based decisions in several clinical situations. Eosinophils Monocytes Lymphocytes Neutrophils Total Leukocytes x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF Siemens ADVIA,,,, y =.9x -.7 r =.99 n = 9 Leukocytes x 9 /L Siemens ADVIA y =.7x +. r =.99 n = Neutrophils x 9 /L ADVIA y =.9x +. r =.9 n = Lymphocytes x 9 /L ADVIA y =.x -. r =.9 n = Monocytes x 9 /L ADVIA y =.9x +. r =.7 n =,,,, Eosinophils x 9 /L ADVIA x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF Beckman Coulter LH7 x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF,,,, y =.9x -. r =.99 n = Leukocytes x 9 /L Beckman Coulter LH7 y =.9x +. r =.99 n = Neutrophils x 9 /L Beckman Coulter LH7 y =.99x +. r =.9 n = Lymphocytes x 9 /L Beckman Coulter LH7 y =.x +. r =. n = Monocytes x 9 /L Beckman Coulter LH7 y =.x +, r =. n =,,,, Eosinophils x 9 /L Beckman Coulter LH7 th International Symposium at the AACC CPOCT Division October -,, Prague, Czech Republic HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF x9/l HemoCue WBC DIFF x 9 /L HemoCue WBC DIFF,,, Sysmex XS-i y =.97x -. r =.997 n = Leukocytes x 9 /L Sysmex XS-i y =.9x +. r =.99 n = 9 Neutrophils x 9 /L Sysmex XS-i y =.x -. r =.9 n = 9 Lymphocytes x 9 /L Sysmex XS-i y =.x +. r =.7 n = 9 Monocytes x9/l Sysmex XS-i y =.x -. r =.9 n = 9,,, Eosinophils x 9 /L Sysmex XS-i

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5 Unique built-in QC in a POCT WBC and -Part Diff system Lindberg, Stellan* HemoCue AB, Ängelholm, Sweden Contact: Stellan.Lindberg@hemocue.se Background: Anewpoint-of-carehematologysystemhasrecentlybeen introduced by HemoCue.µLwholebloodissampledfromthe finger or from a venous sample directly into a microcuvette. The measurement starts automatically when inserting the microcuvette into the analyzer (figure ). The novel system uses state-of-the-art imaging technology to count the white blood cells and perform a -part differentiation. The analyzer will flag all samples containing pathological white blood cells (blasts and immature granulocytes). The system is factory calibrated and needs no further calibration by the user. The HemoCue WBC DIFF system has an advanced built-in QC system. At start-up it will automatically control the electronics, software and the optics. Between every measurement the system will check the optical system. It also has a unique human factor/system check during each measurement. Using the imaging technology, the system will check for correct filling, correct position of the microcuvette, reagent functionality, dirt, blurred cells, non-homogenous sample, small coagula, etc. An error code will be displayed if any of these QC checks fails. The checks are described in figure in more detail. At power up (the Self test ): Test of electronics and software Blanking test (Quality of light, check for dirt in the optics) Between samples: Blanking test For each sample: Human factor/system test Possible cause of error (human factor/system error) Normal image with no error Measurement area soiled by blood, scratches etc. Blood sample on the outside of the microcuvette. Cuvette without blood inserted into the analyzer. Microcuvette not completely filled. Detection Bad area detection Err, Err Condensation on optical parts. Out of focus detection Incorrect positioning of cuvette Err inside the analyzer. Bad staining of cells. Abrupt movement of analyzer. Incorrect filling of microcuvette. Microcuvette not completely filled. Incorrect storage of microcuvette Deterioration of the detergent Optical parts dirty or scratched. The LEDs are malfunctioning. Not hemolyzed sample poor function of hemolyzing agent Very high number of leukocytes. A non-homogenous sample is measured. Due to air bubble, concentration of cells is not evenly distributed. Air bubble detection Err Poor light intensity detection Err, Err Bad cell distribution detection Err Figure : Detailed description of the built-in QC system Technical description When the background color approximates the color of stained cells, the area is eliminated from counting. If the eliminated area is too big, the sample is rejected and an error code is displayed. When the sharpness of the cells is not clear enough, the area is eliminated from counting. If the eliminated area is too big, the sample is rejected and an error code is displayed. When an air bubble is detected, the bubble and the area around it are eliminated from counting due to erroneous distribution of cells. If the eliminated area is too big, the sample is rejected and an error code is displayed. When the measuring area is so dark that the light adjustment cannot compensate enough to detect the cells correctly; the sample is rejected and an error code is displayed. The image is divided into sub-parts and when the counted number of cells in any of the parts differs significantly, the sample is rejected and an error code is displayed. Figure : Description on how to run a test Method: The built-in QC system has been validated in different user error cases identified by an FMEA risk analysis. EDTA venous blood samples were used to test the different failure mode cases. Acceptable results is either a correct result or an error code. Case.Fill the microcuvette with µl blood.push the button for patient test and place the microcuvette in the cuvette holder.the results will be displayed within five minutes Results: Totally 7 blood samples were analysed on the different cases identified in the FMEA risk analysis. The identified cases were blood outside instead of inside the cuvette, air bubbles in the cavity of the cuvette, moisture on the optics of the analyzer, finger prints, grease, blood smears, scratch, powder or reagent placed on the measuring eye on outside surface of the cuvette, measuring with an empty cuvette or with no cuvette, measuring with a partly filled cuvette and measuring with a cuvette filled in two steps. The results are presented in table. Either correct results or error codes from the built-in QC system were displayed in all samples. No incorrect results were obtained. Number of tested samples Correct result Incorrect result Number of samples with error codes Blood on the surface of the cuvette None None instead of blood in the cavity of the cuvette Air bubbles in the cuvette None 9 Moisture on the optics of the analyzer None None Finger prints or grease on the 9 None measuring eye of the outside of cuvette Blood smear placed on the measuring None eye outside of the cuvette Scratch from the single package 9 None Powder placed on the measuring eye outside of the cuvette Reagent placed on the measuring eye on the outside of the cuvette Measuring with an empty cuvette or with no cuvette Measuring with partly filled cuvette ( µl) Measuring with partly filled cuvette ( µl) Measuring with a cuvette filled in two steps None 7 None None None 7 None None 9 None 9 Table : Results from the studies on different cases of identified user errors. Conclusions: The novel POCT HemoCue WBC DIFF technology is based on state-of-the art imaging technology. The unique and advanced built-in QC system will secure that results from the system will not be influenced by user or instrument errors. Awhitebloodcellcountincludinga-partdiffatthepointofcarewill increase the availability of already well established and frequently used lab parameters. Rapid and easy access will be a valuable tool for physicians in making direct and more well based decisions in several clinical situations.

6 Evaluation of HemoCue WBC DIFF system versus Sysmex XE in blood samples from pediatric patients Anette Friis Larsen; Ole Halfdan Larsen, M.D., Ph.D., Tore Forsingdal Hardlei, Ph.D.; Anne Dorthe Møller; and Søren Andreas Ladefoged, M.D., Ph.D., D.M.Sc. Department of Clinical Biochemistry, Aarhus University Hospital, Denmark Contact: INTRODUCTION The HemoCue WBC DIFF has been demonstrated as a useful point-of-care device performing white blood cell (leucocyte) differentiation in venous or capillary blood. However, data from pediatric patients are still lacking. OBJECTIVES This study aimed to compare the performance of the HemoCue WBC DIFF system to the Aarhus University Hospital laboratory method, Sysmex XE-/ in pediatric patients. METHODS Venous blood samples were collected in EDTA from pediatric patients with a median age of years (range: - years). Whole blood differential including leucocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils were analyzed using the HemoCue WBC DIFF (HemoCue, Ängelholm, Sweden) and the Sysmex XE-/ (Sysmex, Kobe, Japan). Ten samples were analyzed in duplicate on the HemoCue WBC DIFF. RESULTS Figur. Total leucocyte count Figur. Neutrophils Figur. Lymphocytes The patients had a median total leucocyte count of.9 x 9 /l (range:.7-9.). The HemoCue WBC DIFF displayed acceptable slope (9% CI:.-.) and correlation for leucocytes The patients had a median neutrophil count of. x 9 /l (range:. -.). The HemoCue WBC DIFF displayed acceptable slope (9% CI:.9-.) and correlation for neutrophils. The patients had a median lymphocyte count of. x 9 /l ( range:. - 9.). The HemoCue WBC DIFF displayed acceptable slope (9% CI:.9-.) and correlation for lymphocytes. Figur. Monocytes (panel A), eosinophils (panel B), and basophils (panel C) Variation in double measurements on the HemoCue WBC Diff was.9% (leucocytes),.% (neutrophils), 7.% (lymphocytes).7% (monocytes),.% (eosinophils) and.% (basophils). The total number of flags or errors were using the HemoCue WBC diff and using the Sysmex of which was overlapping. Evaluation of monocytes, eosinophils, and basophils was challenged by a limited number of samples above the low normal range. The patients had a median monocyte count of. x 9 /l (range:.7 -.), a median eosinophil count of. x 9 /l (range:.), and a median basophil count of. x 9 /l (range:.). The data suggested a proportional bias for monocytes (slope 9% CI:.-.9). Funding HemoCue AB, Ängelholm, Sweden delivered laboratory reagents and utensils for this study free of charge. CONCLUSION The HemoCue WBC DIFF displays acceptable performance in pediatric blood samples compared to the Sysmex XE-/ in the evaluation of leucocytes, neutrophils and lymphocytes. Evaluating performance on monocytes, eosinophils, and basophils was challenged due to low ranges in the investigated patient group. Overall, the data demonstrate that the HemoCue WBC DIFF is a useful point-of-care device for pediatric samples.

7 Counting leucocytes in a point of care setting: a comparison to routine hematology analyzers DR. M. NOORDEGRAAF, DR. P. VAN T SANT, DR. J. LEUVENINK Laboratorium Klinische Chemie en Hematologie, Jeroen Bosch Ziekenhuis, s-hertogenbosch. Background Results The use of Point of care testing (POCT) in hospitals and physicians practice is expanding rapidly. Leukocyte count and differentiation is an important tool in diagnosing infections. Recently Hemocue launched a POCT analyzer, to WBC (x 9/l) Sysmex (. +.x) neut abs Sysmex (-. +.x) lym abs Sysmex (-. +.x) count white blood cells in finger prick blood. Aim of this study was to evaluate its WBC (x 9/l) Hemocue neut abs Hemocue lym abs Hemocue performance compared to routine hematology analyzers. Figure. Passing and Bablok regression lines for White blood cell counts, neutrophil and lymphocytes. Comparison between XE- and Hemocue WBC diff analyzer 7 Methods The Hemocue WBC diff analyzer is able to count and differentiate WBC using a microcuvette. Blood is drawn into the cuvette. In the cuvette WBC (x 9/l) Advia (. +.7x) neut abs Advia (-. +.x) lym abs Advia ( x) red blood cells are lysed and white blood cells are stained. Cells are counted and 7 WBC (x 9/l) Hemocue neut abs Hemocue lym abs Hemocue analyzed by using image analysis. Samples from patients were first counted on routine hematology analyzers. Sysmex-XE- (Sysmex) and Advia i. (Siemens) Data was compared by using Analyse-it or Excel.. Figure. Passing and Bablok regression lines for White blood cell counts, neutrophil and lymphocytes. Comparison between Advia i and Hemocue WBC diff analyzer Conclusion Measurements on a Hemocue WBC diff analyzer correlate well with the XE- and Adviai. The analyzer could be of use in a POCT setting in a physicians practice. Therefore we are currently investigating the predictive value of neutrophil lymphocyte count ratio compared to CRP for bacterial infections.