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Chapter 11.16 : Neutrophil Assays
Category: Clinical Microbiology
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Whole-blood flow cytometry assay for the screening diagnosis of LAD-1. These plots are representative of the results from the LAD-1 assay performed on the whole blood of a healthy control volunteer. In the diagram at the top is a dot plot of the events generated by a lysed whole-blood sample represented as forward angle light scatter (FSC-Height) on the x axis and right angle light scatter (side scatter) on the y axis. An electronic analysis gate is drawn around the neutrophil cluster (R1), and the level of fluorescence (FL2-Height) generated by these events is displayed in each of the tubes treated as described in the protocol. Below that diagram is a histogram for tube 1, which is stimulated (as described in the text) and labeled with the isotype control antibody, IgG2a-PE. Note that the mean level of fluorescence (mean fluorescent channel [MFC] = 4.8) is within the first decade of a four-decade log scale. The second histogram represents the events within the neutrophil gate of tube 2. These events correspond to the level of CD11b-PE expressed on the surface of neutrophils which have not been stimulated. Note that the level of fluorescence (MFC = 77.4) is greater than 10-fold higher than the level represented by the isotype control antibody. The last histogram represents the neutrophils within the analysis gate of tube 3. These events correspond to the level of CD11b-PE expressed on the surface of neutrophils stimulated in vitro as described in the text. Note that the level of expression of CD11b on these in vitro-activated neutrophils is at least 10-fold higher than the level of CD11b-PE on resting neutrophils and that all of the neutrophils have upregulated the expression of CD11b. The results of this assay would be interpreted as consistent with normal.
Whole-blood flow cytometry assay for the screening diagnosis of chronic granulomatous disease. The plots represent the results of three whole-blood assays performed on a healthy control individual, a patient with CGD, and the X-linked carrier mother of the patient with CGD. The top diagram is representative of the light scatter pattern generated by lysed whole blood. An analysis region is drawn around the cluster of events representative of neutrophils in a dot plot of forward-angle light scatter (FSC-Height) on the x axis and right-angle light scatter (SSC-Height) on the y axis. Each of the three histograms displayed below the light scatter dot plot represents overlays of the gated events in each of the three tubes from each patient. The first histogram represents the results of a healthy individual, with an NOI result of 133.4, with a normal result being greater than an NOI of 30. The second histogram represents the results generated from a patient with a confirmed mutation in the gene encoding gp91phox located on the X chromosome. Note that there is a low level of fluorescence generated by the neutrophils of this patient with a corresponding NOI of 27.6. The third histogram represents the results generated in the test performed on the mother of this patient. Note that there are 2 peaks of fluorescence generated by the stimulated neutrophils of the mother. This corresponds to the oxidative burst generated by the neutrophils expressing abnormal X chromosome, i.e., carrying the mutated cybb gene (NOI = 21.6) and the oxidative burst generated by those neutrophils expressing the normal X chromosome (NOI = 165.7).