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/CD14‐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 ≥ 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 ≥ 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. © 1996 Wiley‐Liss, Inc.

Flow cytometric enumeration of monocytes stained with fluorescence-labelled monoclonal antibodies has been proposed as a possible reference method for monocyte counting. We compared precision and accuracy of monocyte counting of the Coulter STKS, the Cobas Argos 5 Diff, the 800-cell manual differential, and the Coulter Epics Profile II flow cytometer using double-staining with fluorescence-labelled monoclonal antibodies (CD45-F1TC and CD14-PE).Precision: STKS, Argos and Profile II achieved a precision analogous to a 3423-, 1298-, and 11089-cell differential, respectively, confirming the superiority of automated methods. Accuracy (136 normal and abnormal samples): Correlation of automated methods with the manual differential was good (STKS: r = 0.934, Argos 5 Diff: r = 0.808, Profile : r = 0.924; Spearman's rank correlation coefficient). The mean relative STKS monocyte result was 0.52 ± 1.63% (mean i SD) higher than the manual differential, whereas the Argos 5 Diff results were 1.22 ± 2.51% lower (p < 0.001). Profile II results showed a small bias against the manual differential (-0.18 ± 1.44%, p < 0.05).Analysing 135 healthy adult subjects on the Profile II, males were found to have a higher mean monocyte count (relative count: 6.95 ± 1.43% vs. 5.86 ± 0.98%; absolute count: 0.48 ± 0.15 X 10 9 /1 vs. 0.39 ± 0.11 X 10 9 /1, p < 0.001) and a higher and wider normal range than females (relative count: 4.97 to 9.78% vs. 4.26 to 7.81%, absolute count: 0.30 to 0.84 X 10 9 /1 vs. 0.25 to 0.65 X 10 9 /1).Flow cytometry based on fluorescence-labelled monoclonal antibodies for monocyte enumeration seems an efficient tool to evaluate the monocyte counting performance of haematology analysers and an ideal successor to the manual differential as reference method for monocyte counting. IntroductionEvaluations of the differential leukocyte count of haem-cannot be the main reason for this, as the less frequent atology analysers have often yielded satisfactory results eosinophils usually showed good results (1-8, 11). The for neutrophils, lymphocytes, and eosinophils, whereas morphological variety of monocytes definitely poses the performance of monocyte counting has been disap-problems for automated differentiating techniques, anpointing (1^8), even when studying only normal sam-other serious problem being lack of an appropriate referples (9, 10). The correlation with the reference method ence method. The value of the manual 400-cell difwas frequently poor and both accuracy and precision ferential, which is still used as reference in monocyte worse than for other leukocyte classes. Although mono-counting (12), is diminished by subjectivity of the examcytes represent a relatively small leukocyte class, this iner (13) and a low precision for smaller cell populations Eur J Clin Chem Clin Biochem 1995; 33 (No 11) Brought to you by | University of Arizona Authenticated Download Date | 7/20/15 8:35 PM

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The shortcomings of current methods of basophil enumeration detract from the clinical value of the basophil count. Moreover, sophisticated and costly techniques of automated basophil counting hardly can be validated for lack of a suitable reference method. We investigated whether a flow cytometric technique using double staining with fluorescence-labelled monoclonal antibodies (mAb) CD45-FITC and CD14-PE on a Coulter Epics Profile II could be used to evaluate basophil counting performance of hematology analyzers. The technique was compared with the 800-cell manual differential, the Coulter STKS, and the Cobas Argos 5 Diff. Precision: STKS, Argos and Profile II showed a precision analogous to a 2,173, 2,250-, and 14,705-cell differential, respectively, illustrating the superiority of automated methods. Accuracy (150 normal and abnormal samples): Using the Profile II as reference the STKS showed a notably weaker correlation than the Argos (r = 0.581 and 0.718, respectively), although this difference was nearly concealed when the imprecise manual differential served as reference (r = 0.517 and 0.562, respectively). The Profile II correlated relatively well with the manual differential (r = 0.730). Analyzing 137 healthy adult subjects, we obtained a reference range of 0.33 to 1.35% (0.020 to 0.102 x 10(9) basophils/L) for the mAb-based method. These data would recommend mAb-based basophil counting as a valuable tool for instrument evaluation. However, an observed bias of 0.09% against the manual differential suggests that modifications are necessary before this technique can be considered as new reference method.