Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

The gap effect and express saccades in the auditory modality

Latencies of eye movements to peripheral targets are reduced when there is a short delay (typically 200 ms) between the offset of a central visual fixation point and the target onset. This has been termed the gap effect. In addition, some subjects, usually with practice, exhibit a separate population of very short latency saccades, called express saccades. Both these phenomena have been attributed to disengagement of visual attention when the fixation point is extinguished. A competing theory of the gap effect attributes it to disengagement of oculomotor fixation during the temporal gap. It is known that auditory targets are effective in eliciting saccadic eye movements, and also that covert attention operates in the auditory modality. If the gap effect and express saccades are due to disengagement of spatial attention, both should persist in the auditory modality. However, fixation of gaze is largely under visual control. If the gap effect results from disengagement of fixation, then at least a reduced effect should be seen in the auditory modality. Human subjects performed the gap task and a control task in the dark, using auditory fixation points and saccadic targets, on five successive days. Despite this practice, express saccades were not observed. There was a reliable gap effect, but the reduction in saccadic latency was only 17 ms, compared with 32 ms for the same subjects in the visual modality. This suggests that about half the gap effect is due to disengagement of visual fixation. The remainder was not due to non-specific warning effects and could be attributed to offset of the auditory fixation stimulus. Received: 1 March 1996 / Accepted: 11 July 1997     

Routine HLA-B27 typing by flow cytometry: differentiation of the products of HLA-B*2702, B*2705 and B*2708

Patient HLA-B27 typing is widely performed as an aid to the diagnosis of several diseases, particularly ankylosing spondylitis. Typing by flow cytometry, using monoclonal antibodies, has been shown to be a potentially useful alternative to classical serology on account of its speed, simplicity and economy. However, we required a flow cytometry typing procedure that would accurately differentiate HLA-B27 (Bw4) from B2708 (Bw6) and not be confounded by other HLA-B7/B27 cross-reactive group antigens. Accordingly, we evaluated the simultaneous use of two monoclonal antibody preparations, ABC-m3-FITC (anti-B27 + weak B7)/BB7.1-PE (anti-B7) and FD705-FITC (anti-B27), by testing a highly selected panel of 62 reference lymphocytes containing examples of all HLA-B7/B27 cross-reactive group antigens, including: HLA-B42, B47, B48, B73, B703, B2702, B2705 and B2708. In addition, 268 whole blood samples from routine patient requests for B27-associated disease typing were tested in parallel with HLA-B typing using the standard complement-dependent microlymphocytoxicity test. The detailed specificity of the three monoclonal antibodies was established and the products of HLA-B*2702, B*2705 and B*2708 were found to be readily differentiated from each other and all other HLA-B7/B27 cross-reactive HLA-B antigens.