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Chapter 31 : Clinical Evaluation of Myeloid and Monocytic Cell Functions

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Abstract:

With the implicit understanding that monocytes and granulocytes represent distinct cell lineages, this chapter reviews laboratory procedures which can be utilized to assess their functions and phenotypes. In the chapter, assays which have proven to be adaptable for routine clinical use or have potential for increased utility in a clinical context are reviewed. Eosinophils and basophils complete the peripheral myeloid cell subsets. Basophil function is briefly described in the context of allergy in the chapter. Physical isolation procedures may also lead to the selective loss of specific monocyte subsets. Phagocytosis is a complex physiological process involving the engulfment and internalization of material bearing appropriate surface molecules. The measurement of phagocytosis is confounded by the difficulty of differentiating bound from internalized material, regardless of whether this process is evaluated by microscopy or flow cytometry. The chapter talks about a procedure that assesses phagocytosis by flow cytometry and differentiates between surface-bound and internalized particles. It describes a method that is commercially available as a kit (Orpegen) and includes an optional procedure to exclude from analysis cells with bound bacteria that have not been internalized (i.e., phagocytosed).

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31

Key Concept Ranking

Innate Immune System
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White Blood Cells
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Immune Response
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Cell-Mediated Immune Response
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Figures

Image of FIGURE 1
FIGURE 1

Two-dimensional dot plots illustrating the separation of monocytes and granulocytes (and lymphocytes) by their inherent light-scattering properties (forward-angle light scatter versus right-angle light scatter) (left) or the combination of a light-scattering property (right-angle light scatter) and a specific lineage-defining surface antigen (CD14-PE)(right).

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31
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Image of FIGURE 2
FIGURE 2

Two-dimensional dot plots illustrating the inherent light-scattering properties of monocytes and granulocytes (left) as in Fig. 1 . (Right) For the same tube, the same populations were visualized on the basis of right-angle light scatter ( axis) versus the level of cell surface CD33-PE expression ( axis). Note the high level of expression of CD33 on monocytes, the less densely expressed levels of CD33 on granulocytes, and the negative expression on lymphocytes.

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31
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Image of FIGURE 3
FIGURE 3

Summary of the results of a flow cytometric screening assay for the dignosis of LAD-1. The large dot plot represents the light-scattering characteristics of lysed whole blood and is used to electronically gate the granulocyte population (R1). The events in this gate are then evaluated for the surface expression of CD11b. The top histogram on the right shows the expression level of the isotype control antibody (negative control), which is used as a positive versus negative discriminatory set (so that the granulocyte population is contained within the first decade on a 4-decade log scale). The middle histogram on the right illustrates the level of expression of CD11b-PE on resting unstimulated granulocytes. The bottom histogram on the right indicates the level of expression of CD11b-PE on granulocytes which have been stimulated in vitro with a phorbol ester (PMA; for details, see the text). Results are expressed in MFC. Note the level of expression of CD11b on resting PMN (MFC = 60.4) relative to that of the isotype control (MFC = 3.8) and to the level achieved after in vitro stimulation (MFC = 982.1). These results would be consistent with normal expression and not with a diagnosis of LAD-1 ( ).

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31
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Image of FIGURE 4
FIGURE 4

Summary of the flow cytometry procedure used to screen patients suspected of having CGD. The results illustrated are for a healthy control (D), an X-linked CGD carrier (E), and a CGD patient (F). The large two-dimensional dot plot on the left (A) illustrates the innate light scatter properties of lysed whole blood, with an electronic gate (R1) drawn around the granulocyte cluster. The histogram on the top right (B) represents the innate fluorescence emitted by granulocytes which have not been incubated with the oxidation-sensitive dye DHR-123; this tube is used to establish the baseline fluorescence settings for the flow cytometer. Histogram C represents gated granulocytes which have been incubated with the dye (DHR-123) but which have not been stimulated (baseline oxidative state). Histogram D represents granulocytes which have been incubated with the dye and which have been stimulated in vitro with PMA (see the text for details). Note that the NOI, which is the geometric MFC of the granulocyte population in histogram D (MFC = 990) divided by the MFC of the granulocytes in histogram C (MFC = 11), is 90, which is consistent with normal expression (i.e., an NOI of >30). Note that in histogram E, there are two populations of cells expressing different levels of fluorescence, one with an NOI of 2 (abnormal) and the other with an NOI of 168. This result is consistent with an X-linked CGD carrier status. The last histogram (F) illustrates an example of the results obtained for a patient with a diagnosis of CGD (note that the NOI [ ] is significantly less than 30).

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31
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References

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Tables

Generic image for table
TABLE 1

Sensitivity and specificity of flow cytometric analysis of in vitro allergen-activated basophils

Citation: O’Gorman M. 2006. Clinical Evaluation of Myeloid and Monocytic Cell Functions, p 272-280. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch31

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