Journal Highlights: article review.

Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry.  Zhao M, Kanegane H, Kobayashi C et al., Cytometry, Part B, Clinical Cytometry, ePub

Flow cytometry has become an important tool for the diagnosis of many primary immune deficiencies.  Most importantly, flow cytometry is rapid.  Flow cytometric screening tests can lead to patient diagnosis in as little as 1 day, as opposed to the many weeks that are often required for genetic sequencing. 

Dr. Toshio Miyawaki and colleagues have recently described their experience using flow cytometry for the diagnosis of X-linked lymphoproliferative disease (XLP) due to deficiency of SLAM-Associated Protein (SAP).  SAP deficiency is caused by mutations in the SH2D1A gene found on chromosome Xq24-25,^1-3 and is the most common cause of XLP.  The ability to rapidly diagnose XLP is extremely important because the majority of patients present with hemophagocytic lymphohistiocytosis (HLH) due to Epstein-Barr virus, which is rapidly fatal in the absence of recognition and treatment.

Dr. Miyawaki’s group previously developed a rat monoclonal antibody against SAP, KST-3, that can be used for flow cytometric detection of intracellular lymphocyte SAP.⁴  The authors originally described this technique to work well in T-lymphoblasts, as stimulation of T cells results in increased SAP expression, which enabled good detection of intracellular SAP using FITC-conjugated secondary antibodies.⁴  The presence of SAP was also observed in peripheral blood T cells and NK cells, but with considerable overlap in fluorescence intensity with the isotype control antibody.⁴ 

Now, Dr. Miyawaki’s group reports their observation of an increase in the sensitivity of their flow-cytometric technique to detect SAP within peripheral blood T cells and NK cells using Alexa-Fluor 488-labeled secondary antibodies (Molecular Probes, Eugene OR) in place of the FITC-labeled secondary antibodies.  This allowed the authors to evaluate patients for SAP deficiency using only peripheral blood samples.  Deficiency of SAP was observed in T cells and NK cells from 6 patients with suspected XLP, all of whom possessed mutations in SH2D1A.  Additional patients who were suspected of having XLP who possessed normal or near-normal SAP expression were not found to possess SH2D1A mutations.  The work presented by the authors lends further evidence that flow cytometric techniques are able to reliably diagnose this primary immune deficiency.  Similar assays have been employed by other laboratories, and have also yielded good results.⁵ 

In addition to screening male patients with HLH for SAP deficiency, flow cytometric techniques can also be used to screen for related conditions.  Deficiency of X-linked inhibitor of apoptosis (XIAP), sometimes referred to as XLP-2 or X-linked familial HLH, and deficiency of perforin, the cause of familial HLH type 2, are also observed in male patients presenting with HLH (as are the other genetic causes of familial HLH).^6-7  Flow cytometry can be used to detect XIAP and perforin in peripheral blood lymphocytes,^8-9 and flow cytometric screening tests can be used to screen patients for all 3 disorders simultaneously.  A swift definitive diagnosis of patients with these devastating disorders can hasten initiation of HLH therapy and speed preparations for allogeneic hematopoietic cell transplantation.  Thus, flow cytometry is a powerful tool to both hasten diagnosis and improve patient outcome.



Rebecca Marsh, MD
University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

References

1.         Coffey AJ, Brooksbank RA, Brandau O, et al. Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene. Nat Genet. Oct 1998;20(2):129-135.

2.         Nichols KE, Harkin DP, Levitz S, et al. Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proc Natl Acad Sci U S A. Nov 10 1998;95(23):13765-13770.

3.         Sayos J, Wu C, Morra M, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature. Oct 1 1998;395(6701):462-469.

4.         Shinozaki K, Kanegane H, Matsukura H, et al. Activation-dependent T cell expression of the X-linked lymphoproliferative disease gene product SLAM-associated protein and its assessment for patient detection. Int Immunol. Oct 2002;14(10):1215-1223.

5.         Tabata Y, Villanueva J, Lee SM, et al. Rapid detection of intracellular SH2D1A protein in cytotoxic lymphocytes from patients with X-linked lymphoproliferative disease and their family members. Blood. Apr 15 2005;105(8):3066-3071.

6.         Rigaud S, Fondaneche MC, Lambert N, et al. XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome. Nature. Nov 2 2006;444(7115):110-114.

7.         Stepp SE, Dufourcq-Lagelouse R, Le Deist F, et al. Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science. Dec 3 1999;286(5446):1957-1959.

8.         Marsh RA, Villanueva J, Zhang K, et al. A rapid flow cytometric screening test for X-linked lymphoproliferative disease due to XIAP deficiency. Cytometry B Clin Cytom. Sep 2009;76(5):334-344.

9.         Kogawa K, Lee SM, Villanueva J, Marmer D, Sumegi J, Filipovich AH. Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members. Blood. Jan 1 2002;99(1):61-66.