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Review
. 2019 Jul 23;10:1740.
doi: 10.3389/fimmu.2019.01740. eCollection 2019.

Current Flow Cytometric Assays for the Screening and Diagnosis of Primary HLH

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Free PMC article
Review

Current Flow Cytometric Assays for the Screening and Diagnosis of Primary HLH

Samuel Cern Cher Chiang et al. Front Immunol. .
Free PMC article

Abstract

Advances in flow cytometry have led to greatly improved primary immunodeficiency (PID) diagnostics. This is due to the fact that patient blood cells in suspension do not require further processing for analysis by flow cytometry, and many PIDs lead to alterations in leukocyte numbers, phenotype, and function. A large portion of current PID assays can be classified as "phenotyping" assays, where absolute numbers, frequencies, and markers are investigated using specific antibodies. Inherent drawbacks of antibody technology are the main limitation to this type of testing. On the other hand, "functional" assays measure cellular responses to certain stimuli. While these latter assays are powerful tools that can be used to detect defects in entire pathways and distinguish variants of significance, it requires samples with robust viability and also skilled processing. In this review, we concentrate on hemophagocytic lymphohistiocytosis (HLH), describing the principles and accuracies of flow cytometric assays that have been proven to assist in the screening diagnosis of primary HLH.

Keywords: HLH; XLP; clinical diagnostics; clinical laboratory tests; diagnostic accuracy; flow cytometry; hemophagocytic lymphohistiocytosis; primary immunodeficiencies.

Figures

Figure 1
Figure 1
Intracellular staining of perforin and granzyme B in different individuals. Histograms represent gated NK cells showing varying levels of perforin expression as a consequence of the different PRF1 variants as shown.
Figure 2
Figure 2
Cytotoxic lymphocyte evaluation of an STXBP2 patient. We performed NK cytotoxicity as well as NK and T cell degranulation using fresh PBMC from a case with homozygous c.1430C>T (p.Pro477Leu) mutations. While (A) control NK cells and CD8+CD57+ T cells degranulated as expected when stimulated, respectively with K562 or anti-CD3 antibody, (B) the patient's cytotoxic lymphocytes did not. (C) NK cytotoxicity was also evaluated via 51Cr release and found deficient. In addition, we included cytotoxicity data from a sibling carrying the same homozygous mutation.
Figure 3
Figure 3
Indirect diagnosis of XLP1. (A) While most SH2D1A mutations result in absent or lowly expressing SAP protein levels, we found (B) a clinically suspicious patient with c.125G > A (p.Cys42Tyr) missense mutation with only a slight reduction in SAP protein expression by flow cytometry. The patient was thus further evaluated for (C) iNKT numbers on bulk CD3+ cells and (D) restimulation-induced cell death (RICD) via anti-CD3 antibody repeated on two occasions. The low iNKT counts and reduced cell death upon TCR restimulation provided evidence that the missense SH2D1A variant found was indeed pathological.
Figure 4
Figure 4
Phenotyping and functional evaluation for XLP2. (A) While a majority of BIRC4 mutations present with absent or lowly expressing XIAP protein levels, we found (B) a patient with c.632A>G (p.Glu211Gly) variant of uncertain significance (VUCS) with only a slight reduction in XIAP protein expression by flow cytometry. Functional evaluation of XIAP can be done through stimulation of NOD2 with L18-MDP. This signaling pathway requires XIAP for TNF transcription through NF-κB. (C,D) When examined, both these cases show equally defective TNF production regardless of XIAP expression revealing the VUCS is in fact a damaging mutation. LPS acts as a positive control that signals through TLR4 demonstrating preserved cellular function in patient cells.

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