Chapter 20 : Functional Flow Cytometry-Based Assays of Myeloid and Lymphoid Functions for the Diagnostic Screening of Primary Immunodeficiency Diseases

Flow cytometry-based LAD screening assay on a nondiseased healthy donor. Granulocytes are gated based on their innate forward and right angle light scatter properties. The level of CD11b-PE fluorescence on resting (purple) versus PMA-stimulated granulocytes is displayed. Note that in LAD-1 both the resting and activated levels of CD11b are considerably lower than normal. Normal ranges for the level of CD11b, expressed as the geometric mean fluorescence, were determined on 30 healthy nondiseased control patients. Normal CD11b was expressed as greater than the 5th percentile of normal.

Gating algorithm and histogram display of CD40-ligand upregulation on in vitro activated CD4 positive T cells. (Upper left) Dot plot of right angle light scatter versus CD3 (T cells). A gate is drawn around the CD3 positive cluster (R1). The events within R1 are then displayed on a dot plot of CD3 (x-axis) vs. CD8 (y-axis). A gate is drawn around the CD3 positive events and the CD8 negative events which contain primarily CD4 positive T cells. The expression of CD40 ligand is then assessed on the CD3+CD8− T cells. Each dot plot illustrates the level of CD40-ligand (purple) overlaid on an isotype control (green). Note the middle histogram displays the results from the CD40-ligand deficient patient's mother, who is an X-linked carrier. Note the two populations of T cells, one with a normal level and one with a level essentially overlapping the CD40 ligand level observed on the activated T cells of her son. These results are essentially diagnostic of the X-linked hyper IgM syndrome.

The flow cytometry oxidative burst assay for normal healthy control father (top), X-linked CGD carrier mother, and CGD patient. Granulocytes are gated on their innate forward and right angle light scatter properties (left column). The right-hand column presents histograms of the DHR (FL1/FITC) fluorescence levels of unstimulated whole blood granulocytes incubated with the dye (purple) overlaid on PMA-stimulated granulocytes that had been preincubated with DHR (green). Note the middle histogram results of the X-linked carrier mother with two populations of granulocytes, ne with a normal NOI (NOI = 198) and one with an abnormal NOI (NOI = 12). Normal NOI is >30 (developed in our laboratory by testing 35 normal healthy controls).