Electron imaging with Medipix2 hybrid pixel detector.

The electron imaging performance of Medipix2 is described. Medipix2 is a hybrid pixel detector composed of two layers. It has a sensor layer and a layer of readout electronics, in which each 55 microm x 55 microm pixel has upper and lower energy discrimination and MHz rate counting. The sensor layer consists of a 300 microm slab of pixellated monolithic silicon and this is bonded to the readout chip. Experimental measurement of the detective quantum efficiency, DQE(0) at 120 keV shows that it can reach approximately 85% independent of electron exposure, since the detector has zero noise, and the DQE(Nyquist) can reach approximately 35% of that expected for a perfect detector (4/pi(2)). Experimental measurement of the modulation transfer function (MTF) at Nyquist resolution for 120 keV electrons using a 60 keV lower energy threshold, yields a value that is 50% of that expected for a perfect detector (2/pi). Finally, Monte Carlo simulations of electron tracks and energy deposited in adjacent pixels have been performed and used to calculate expected values for the MTF and DQE as a function of the threshold energy. The good agreement between theory and experiment allows suggestions for further improvements to be made with confidence. The present detector is already very useful for experiments that require a high DQE at very low doses.

Use of precision profiles to evaluate precision of the automated leukocyte differential.

The commonly used methods of assessing the precision of the automated leukocyte differential have certain drawbacks that affect the validity and comparability of results. In the present report, we introduce a procedure based on building precision profiles from a large number of within-run imprecision experiments. The profiles are fitted to the function for the CV of proportions, which yields the number of theoretically differentiated leukocytes. Differences between fitted curves are evaluated for statistical significance by the F-test. As an example, we compared the precision of two hematology analyzers, a flow-cytometric technique involving fluorescence-labeled monoclonal antibodies, and the manual differential. We were able to establish definite differences in precision between different analyzers and different leukocyte classes. Our data also indicated that conventional within-run imprecision studies may completely misjudge analyzer precision. Furthermore, we could demonstrate that the precision of analyzers that analyze a fixed amount of blood rather than a fixed number of leukocytes is strongly influenced by the leukocyte count of the sample, leading to high imprecision for leukopenic samples. We believe the proposed procedure is a useful addition to currently used protocols; it yields clear results and creates a statistical basis of comparison between various instruments and techniques of differentiation.

Proposed reference method for peripheral-blood monocyte counting using fluorescence-labelled monoclonal antibodies.

Flow cytometry using fluorescence-labelled monoclonal antibodies has been proposed as a possible new reference method to evaluate the monocyte counting performance of automated hematology analyzers. Since in previous studies only one such technique was applied, we investigated how different flow cytometric techniques compared to the manual differential and a hematology analyzer. Relative monocyte counts of 60 samples of the daily routine were determined on a Coulter Profile II flow cytometer after incubation with two different CD45-FITC/CD 14-PE antibody combinations and subsequent preparation with two whole-blood lysis techniques, including one no-wash technique. Results were compared to those of a 600-cell manual differential and to those of the Coulter STKS hematology analyzer. All flow cytometric methods correlated very well with the manual differential (r > or = 0.925) and none showed a significant bias. The Coulter STKS relative monocyte counts were slightly higher than those of the manual differential (8.76% vs. 8.18%). The correlations between the methods employing monoclonal antibodies were excellent (r > or = 0.995) and the mean monocyte counts identical although a small, non-systematic influence of sample preparation techniques was noted. An influence of the antibody clones was not observed. The precision of the Profile II results was far superior to that of the manual differential and the STKS. Our data show that flow cytometry employing fluorescence-labelled monoclonal antibodies is a potentially ideal new reference method for monocyte counting. However, they also show that establishing a new reference method will require extensive investigation and exact definition of the sample preparation procedure to be used.

Precision and accuracy of monocyte counting. Comparison of two hematology analyzers, the manual differential and flow cytometry.

The Coulter STKS (Coulter, Hialeah, FL), the Abbott CD3500 (Abbott Diagnostics, Abbott Park, IL), a 400-cell manual differential, and flow cytometry using double-staining with fluorescence-labelled monoclonal antibodies (CD45-FITC and CD14-PE) on a Coulter Epics Profile II were evaluated for precision and accuracy in relative monocyte counting. STKS, CD3500, and Profile II achieved a precision analogous to a 3,542-, 1,835-, and 11,998-cell differential, respectively, demonstrating the superiority of automated methods. Analysis of 156 normal and abnormal samples revealed that the mean relative monocyte counts of the manual differential, CD3500 and Profile II were not significantly different. Only the STKS results showed a positive bias (0.79% +/- 1.65), which was increased in lymphocytic samples. Linear regression between the Profile II as independent viable, and the other techniques yielded acceptable correlation coefficients (STKS: 0.861, CD3500: 0.844, manual differential:0.833). Profile II results were also compared to those of a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA), which yielded an excellent correlation (r = 0.991) but a slightly smaller relative monocyte count (bias-0.39% +/- 0.60) of the latter. On the basis of these data, the authors recommend the use of monoclonal antibodies as a new reference method, but also indicate the need for further methodological investigations.