Hemophagocytic Lymphohistiocytosis
Hemophagocytic Lymphohistiocytosis
Based on these common clinical and laboratory findings, diagnostic criteria for HLH were proposed in 1991 and updated in 2004 to include NK-cell activity measured by the 51-Cr release assay, sCD25, and elevated ferritin (Table 3). These criteria, generated based on studies of FHL, are the only guidelines available for the diagnosis of acquired HLH.
The diagnostic criteria include fever; splenomegaly; cytopenias affecting at least 2 of 3 lineages in the peripheral blood; hyperferritinemia greater than 10,000 μg/L; hypertriglyceridemia and/or hypofibrinogenemia; hemophagocytosis in the bone marrow, spleen, or lymph nodes (see Image 1); low or absent NK-cell activity determined by the 51-Cr release assay; and high levels of sCD25. Five of these 8 criteria are required for diagnosis, although in patients with an established genetic abnormality (eg, FHL mutations), the diagnosis can be established without meeting the 5 criteria. The diagnostic criteria are listed in Table 3.
The 2004 diagnostic criteria for HLH do not apply to MAS because of the overlap of clinical and laboratory findings between HLH and autoimmune diseases. Modified diagnostic criteria for MAS have been suggested by Ravelli et al, who proposed a change in baseline laboratory findings as an indication of MAS in conjunction with the appropriate clinical symptoms. In the absence of arthritis, a very high C-reactive protein, only moderate cytopenias, reduced erythropoiesis, increased granulopoiesis with a left shift, and a high level of interleukin 1β might suggest MAS. Ongoing projects are being conducted to validate these criteria.
The perforin gene mutation was the first genetic defect to be described in association with HLH in 1999. The perforin protein is one of the major cytolytic proteins in cytotoxic cells, and mutations involving perforin gene 1 (PRF1) account for 20% to 50% of familial cases of HLH (FHL2) (see Table 1). Mutations in other genes involved in the perforin pathway account for the other types of FHL—namely, UNC13D (FHL3),STX11 (FHL4), and STXBP2 or UNC18B (FHL5). A potential gene locus on chromosome 9q21 is associated with FHL1. The types of FHL are summarized in Table 1.
Mutation analysis should be requested for all cases of confirmed or suspected HLH, even when an associated infectious disease has already been identified. The demonstration of a characteristic genetic defect alone can be used to make the diagnosis of HLH in the appropriate clinical setting, without the need to fulfill 5 of the 8 diagnostic criteria. It should include the analysis of the known FHL mutations (PRF1, UNC13D, STX11, and UNC18B) at a minimum. When XLP is suspected (eg, upon documentation of EBV infection), mutation analysis for SH2D1A/SAP and BIRC4 also must be included. Specific testing for the other types of immunodeficiency syndromes, such as Chédiak-Higashi syndrome, Griscelli syndrome type 2, or Hermansky-Pudlak syndrome type 2, may be warranted in the appropriate clinical setting.
Heterozygous patients for the perforin gene mutation may manifest the disease despite having normal perforin expression levels. Children with a strong family history of HLH also may be offered the test prophylactically. Prenatal and preimplantation diagnosis is possible by genetic analysis once the gene defect within a family is known. Prenatal diagnosis was first performed in 2 unrelated Turkish families harboring a perforin mutation.
Importantly, HLH cannot be ruled out solely on a negative mutation study, since at least half of childhood and most adult cases cannot be attributed to any known mutation.
Flow cytometry recently has been added to the arsenal of screening tools for HLH, although it is not currently cited in any formal diagnostic algorithms. It is based on the detection of perforin expression in all cytotoxic cell types by intracellular staining. Thus, the absence of perforin staining reflects the presence of homozygous perforin gene mutations, and even heterozygous carriers also demonstrate abnormal perforin staining patterns. This method has enabled a 2-hour screen for FHL2 in the medical centers that use this technique.
However, the fact that some mutations do not result in significant reduction of the protein levels and that disease can sometimes occur in heterozygous patients limits flow cytometry's sensitivity to perforin defects. In addition, alternative defects in gene regulation unrelated to HLH may affect protein expression, which affects the specificity of the study. Despite these limitations, the fast turnaround time of flow cytometry makes it advantageous over mutation analysis.
Mutations in other genes related to granule trafficking and exocytosis also can be determined by quantifying the expression of surface CD107a (LAMP-1) on peripheral blood mononuclear cells following stimulation with phytohemagglutinin or anti-CD3. CD107a is normally released to the surface of cytotoxic T cells and NK cells upon exocytosis; thus, the absence of CD107a expression on the cell surface may be indicative of defects throughout the pathway involving secretory granule migration, docking, priming, or fusion. A similar technique is used for the detection of SAP/SH2D1A expression.
Diagnostic Criteria
Based on these common clinical and laboratory findings, diagnostic criteria for HLH were proposed in 1991 and updated in 2004 to include NK-cell activity measured by the 51-Cr release assay, sCD25, and elevated ferritin (Table 3). These criteria, generated based on studies of FHL, are the only guidelines available for the diagnosis of acquired HLH.
The diagnostic criteria include fever; splenomegaly; cytopenias affecting at least 2 of 3 lineages in the peripheral blood; hyperferritinemia greater than 10,000 μg/L; hypertriglyceridemia and/or hypofibrinogenemia; hemophagocytosis in the bone marrow, spleen, or lymph nodes (see Image 1); low or absent NK-cell activity determined by the 51-Cr release assay; and high levels of sCD25. Five of these 8 criteria are required for diagnosis, although in patients with an established genetic abnormality (eg, FHL mutations), the diagnosis can be established without meeting the 5 criteria. The diagnostic criteria are listed in Table 3.
The 2004 diagnostic criteria for HLH do not apply to MAS because of the overlap of clinical and laboratory findings between HLH and autoimmune diseases. Modified diagnostic criteria for MAS have been suggested by Ravelli et al, who proposed a change in baseline laboratory findings as an indication of MAS in conjunction with the appropriate clinical symptoms. In the absence of arthritis, a very high C-reactive protein, only moderate cytopenias, reduced erythropoiesis, increased granulopoiesis with a left shift, and a high level of interleukin 1β might suggest MAS. Ongoing projects are being conducted to validate these criteria.
Mutation Analysis
The perforin gene mutation was the first genetic defect to be described in association with HLH in 1999. The perforin protein is one of the major cytolytic proteins in cytotoxic cells, and mutations involving perforin gene 1 (PRF1) account for 20% to 50% of familial cases of HLH (FHL2) (see Table 1). Mutations in other genes involved in the perforin pathway account for the other types of FHL—namely, UNC13D (FHL3),STX11 (FHL4), and STXBP2 or UNC18B (FHL5). A potential gene locus on chromosome 9q21 is associated with FHL1. The types of FHL are summarized in Table 1.
Mutation analysis should be requested for all cases of confirmed or suspected HLH, even when an associated infectious disease has already been identified. The demonstration of a characteristic genetic defect alone can be used to make the diagnosis of HLH in the appropriate clinical setting, without the need to fulfill 5 of the 8 diagnostic criteria. It should include the analysis of the known FHL mutations (PRF1, UNC13D, STX11, and UNC18B) at a minimum. When XLP is suspected (eg, upon documentation of EBV infection), mutation analysis for SH2D1A/SAP and BIRC4 also must be included. Specific testing for the other types of immunodeficiency syndromes, such as Chédiak-Higashi syndrome, Griscelli syndrome type 2, or Hermansky-Pudlak syndrome type 2, may be warranted in the appropriate clinical setting.
Heterozygous patients for the perforin gene mutation may manifest the disease despite having normal perforin expression levels. Children with a strong family history of HLH also may be offered the test prophylactically. Prenatal and preimplantation diagnosis is possible by genetic analysis once the gene defect within a family is known. Prenatal diagnosis was first performed in 2 unrelated Turkish families harboring a perforin mutation.
Importantly, HLH cannot be ruled out solely on a negative mutation study, since at least half of childhood and most adult cases cannot be attributed to any known mutation.
Flow Cytometry
Flow cytometry recently has been added to the arsenal of screening tools for HLH, although it is not currently cited in any formal diagnostic algorithms. It is based on the detection of perforin expression in all cytotoxic cell types by intracellular staining. Thus, the absence of perforin staining reflects the presence of homozygous perforin gene mutations, and even heterozygous carriers also demonstrate abnormal perforin staining patterns. This method has enabled a 2-hour screen for FHL2 in the medical centers that use this technique.
However, the fact that some mutations do not result in significant reduction of the protein levels and that disease can sometimes occur in heterozygous patients limits flow cytometry's sensitivity to perforin defects. In addition, alternative defects in gene regulation unrelated to HLH may affect protein expression, which affects the specificity of the study. Despite these limitations, the fast turnaround time of flow cytometry makes it advantageous over mutation analysis.
Mutations in other genes related to granule trafficking and exocytosis also can be determined by quantifying the expression of surface CD107a (LAMP-1) on peripheral blood mononuclear cells following stimulation with phytohemagglutinin or anti-CD3. CD107a is normally released to the surface of cytotoxic T cells and NK cells upon exocytosis; thus, the absence of CD107a expression on the cell surface may be indicative of defects throughout the pathway involving secretory granule migration, docking, priming, or fusion. A similar technique is used for the detection of SAP/SH2D1A expression.
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