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Chapter 11.16 : Neutrophil Assays

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Neutrophil Assays, Page 1 of 2

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Citation: Leber A. 2016. Neutrophil Assays, p 11.16.1.1-11.16.3.6. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.16
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Figures

Image of Figure 11.16.2–1
Figure 11.16.2–1

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 axis and right angle light scatter (side scatter) on the 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 as described in the text. Note that the level of expression of CD11b on these -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.

Citation: Leber A. 2016. Neutrophil Assays, p 11.16.1.1-11.16.3.6. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.16
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Image of Figure 11.16.3–1
Figure 11.16.3–1

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 axis and right-angle light scatter (SSC-Height) on the 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 gene (NOI = 21.6) and the oxidative burst generated by those neutrophils expressing the normal X chromosome (NOI = 165.7).

Citation: Leber A. 2016. Neutrophil Assays, p 11.16.1.1-11.16.3.6. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.16
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References

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1. Prince HE. Lapé-Nixon M. 1995. Influence of specimen age and anticoagulant on flow cytometric evaluation of granulocyte oxidative burst generation. J Immunol Methods 188:129138.
2. Mauch L, Lun A, O’Gorman MR, Harris JS, Schulze I, Zychlinsky A, Fuchs T, Oelschlagel U, Brenner S, Kutter D, Rosen-Wolff A, Roesler J. 2007. Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD. Clin Chem 53:890896.
3. Alvarez-Larrán A, Toll T, Rives S, Estella J. 2005. Assessment of neutrophil activation in whole blood by flow cytometry. Clin Lab Haematol 27:4146.
4. Bass D, Parce W, Dechatelet L, Szejda P, Seeds M, Thomas M. 1983. Flow cytometric studies of oxidative product formation by neutrophils: a grade response to membrane stimulation. J Immunol 136:19101917.
5. Centers for Disease Control and Prevention. 1997. Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb Mortal Wkly Rep 46(RR-2):129.
6. Emmendorffer A, Hecht M, Lohmann-Matthes ML, Roesler J. 1990. A fast and easy method to determine the production of reactive oxygen intermediates by human phagocytes using dihydrorhodamine 123. J Immunol Methods 131:269275.
7. Hassan NF, Campbell DE, Douglas SD. 1988. Phorbol myristate acetate oxidation of 2′7′ dichlorofluorescein by neutrophils from patients with chronic granulatomatous disease. J Leukoc Biol 43:317322.
8. Malech HL, Gallin JI. 1987. Neutrophils in human diseases. N Engl J Med 317:687694.
9. O’Gorman MRG Mcnally AC. 1993. A rapid whole blood lysis technique for diagnosis of moderate or severe leukocyte adhesion deficiency. Ann N Y Acad Sci 677:427430.
10. O’Gorman MRG Corrochano V. 1995. Rapid whole blood flow cytometry assay for the diagnosis of chronic granulomatous disease. Clin Diagn Lab Immunol 2:227232.
11. Todd RF Freyer DR. 1988. The CD11/CD18 leukocyte glycoprotein deficiency. Hematol Oncol Clin N Am 2:1331.

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