Revisited Diagnostic Guidelines for Familial Hemophagocytic Lymphohistiocytosis (FHL) Based on HLH-2004: Updates to Diagnostic Criteria and Development of Multipathway Diagnostic Strategies

Introduction

Familial hemophagocytic lymphohistiocytosis (FHL) is a severe hyperinflammatory disease characterized by abnormal accumulation of macrophages and lymphocytes in tissues, typically accompanied by prolonged fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. The pathogenesis involves autosomal recessive mutations in FHL2-5-related genes, resulting in impaired lymphocyte cytotoxic function. Without timely treatment, patients have a high risk of central nervous system involvement and mortality. Early diagnosis of FHL is thus critically important.

Currently, diagnosis of FHL mainly relies on the HLH-2004 criteria, which were developed for enrollment in a clinical trial and are based on expert opinion. However, in clinical practice, diverse manifestations of hemophagocytic lymphohistiocytosis (HLH) and overlapping symptoms with other inflammatory and infectious diseases pose diagnostic challenges. This study aims to evaluate and optimize the HLH-2004 criteria and propose multipathway diagnostic tools based on clinical, genetic, and cellular pathways to provide accurate and practical diagnostic guidelines for FHL.

Background and Source of the Study

This study was conducted through a multi-institutional collaboration, involving leading researchers and clinicians from renowned institutions worldwide, such as Karolinska Institutet (Sweden), University of Alabama (USA), and Meyer Children’s Hospital (Italy). Published in the November 2024 issue of Blood, this article represents a comprehensive case-control study aiming to validate and enhance existing diagnostic strategies for FHL.

Research Methods and Approach

The study utilized a case-control design to analyze 366 confirmed cases of familial and/or molecular-diagnosed FHL and 1,064 controls (including children with systemic juvenile idiopathic arthritis [sJIA], infectious diseases, and severe infections with similar clinical presentations). Key components include:

1. Comparing and Optimizing Diagnostic Strategies

The study evaluated three diagnostic pathways: 1. Clinical and laboratory-based diagnostic strategy: Assessing the HLH-2004 benchmark (fulfillment of 5 out of 8 criteria) alongside potential refinements. 2. Genetic pathway: Focusing on genetic mutation analysis of FHL-associated genes, emphasizing loss-of-function mutations and their characteristic significance. 3. Cellular functional pathway: Assessing lymphocyte cytotoxic function through NK cell and CD8+ T cell assays to evaluate diagnostic reliability.

2. Data Handling and Statistical Analysis

Data were sourced from the HLH-94 and HLH-2004 databases, supplemented by the Italian HLH registry. Missing data were addressed using multiple imputation, and multivariable logistic regression models, along with receiver operating characteristic (ROC) curve analyses, were employed to evaluate accuracy, sensitivity, and specificity of different diagnostic criteria.

Key Findings

1. Optimizing Clinical Diagnostic Strategies

Statistical analyses revealed that the HLH-2004 criteria yielded high diagnostic accuracy (97.4%). However, optimizing criteria—such as excluding NK cell functionality—raised accuracy further to 99.0%. While modifying thresholds for ferritin and soluble CD25 (sCD25) marginally improved specificity and sensitivity, the study suggested maintaining established cutoffs (ferritin ≥500 µg/L and sCD25 ≥2,400 U/mL) to ensure compatibility with past studies.

Revised clinical diagnostic criteria include nine parameters, with fulfillment of ≥5 sufficient for diagnosis: - Fever (≥38.5°C)
- Splenomegaly (≥2 cm below the costal margin)
- Cytopenias (e.g., hemoglobin <90 g/L)
- Hypertriglyceridemia and/or hypofibrinogenemia
- Hemophagocytosis
- Ferritin elevation (≥500 µg/L)
- Elevated sCD25 (≥5,000 U/mL)

2. Genetic Diagnostic Pathway

The study emphasized genetic pathways with strong diagnostic value, identifying criteria such as biallelic loss-of-function mutations in FHL-associated genes (e.g., PRF1, UNC13D, STXBP2, STX11). For novel or uncertain mutations, functional assays were recommended for diagnostic confirmation.

3. Cellular Functional Diagnostic Pathway

Lymphocyte cytotoxic assays using NK and cytotoxic CD8+ T cells proved effective for diagnosing FHL, with results available within some laboratories in a single day. Observations such as absent perforin expression or defective degranulation strongly indicate FHL. This pathway was especially effective when paired with clinical analyses, helping to confirm diagnoses rapidly.

Significance and Impact

1. Scientific Value

This richly collaborative study validates the efficacy of existing diagnostic strategies for FHL while addressing their limitations. By proposing a multipathway diagnostic framework, it provides clear, flexible, and scientifically supported tools for clinical use.

2. Clinical Utility

The proposed mechanisms are readily translatable into clinical practice. The integration of genetic and cellular analyses enhances diagnostic precision, particularly for complex and early-stage cases. Early identification of high-risk individuals allows expedited curative treatments, such as hematopoietic stem cell transplantation (HSCT), and aids in pre-symptomatic screening within families.

3. Innovation

The combination of three diagnostic pathways marks a significant advance, bridging clinical observations, genetic findings, and cellular assays for FHL diagnosis. The study introduces clear, actionable recommendations, redefining how FHL is detected and managed.

Conclusion and Future Directions

FHL, as a life-threatening inflammatory disease, requires prompt and accurate diagnosis. This study refines the existing HLH-2004 diagnostic criteria and establishes a practical tripartite diagnostic framework. The increasing accessibility of genetic sequencing and improved cellular assay capabilities are expected to further elevate diagnostic accuracy for FHL and related HLH disorders. This comprehensive strategy not only benefits FHL diagnosis but also serves as a model for tackling other hereditary multisystem diseases.