The role of hemophagocytosis in bone marrow aspirates in the diagnosis of hemophagocytic lymphohistiocytosis

HLH represents the most prevalent and clinically significant macrophage-related histiocytic disorder [1]. Although HLH was initially described as a familial disease [2], it is now clear that even patients without identifiable genetic mutations are at risk for HLH secondary to infections, malignancy, and rheumatological conditions [3,4]. The termmacrophage activation syndrome (MAS) has also been used in the literature. The diagnosis is based on a set of criteria, including genetic mutations, clinical features, laboratory abnormalities, and morphological evidence of hemophagocytosis (HPC) [5]. Genetic mutations in perforin [6], Munc 13-4 [7], and syntaxin 11 [4] can confirm the presence of familial disease, even in the older patients presenting with viral infections [8]. Children suspected ofHLH are often acutely ill and have non-specific clinical findings including fever, hyperferritinemia, hepatosplenomegaly and cytopenias. Thus a multi-disciplinary team approach is often required to confirm the diagnosis with involvement from general pediatrics, rheumatology, infectious disease and haematology/ oncology. Despite the exciting new findings related to the etiopathogenesis of this disease, until the aforementioned specialized genetic and immunological tests become available as routine clinical practice, there persists a lack of confirmatory diagnostic tests available to help the clinician in acute situations. It is often a consensus which navigates both diagnostic conclusions to rule in or out HLH and subsequent therapeutic approaches. Treatment can range from steroids and IVIG in mild cases to full protocol therapy including bone marrow transplantation for familial and severe patients, a distinction which can be anxiety-provoking both for families and the medical team. Even in institutions which perform specialized immunological and genetic testing, the results are usually not available in time to aid in immediate treatment-related decisions. The Munc-13 gene is extraordinarily long and it takes weeks to sequence the gene in its entirety. Furthermore, lowNK cell function and perforin analysis by flow cytometry, can be absent in up to 50% of patients with confirmed genetic abnormalities [9]. Thus, in order to have timely diagnosis, most practitioners continue to rely on the morphological presence of HPC in bone marrow (BM) to distinguish patients with simple infections or rheumatological disease from those with active HLH. Although sites most markedly affected include the splenic red pulp, hepatic portal areas, and lymph node sinuses [3,10], BM represents the most accessible place for morphological examination. BM examinations are relatively easy to organize, results are available the same day, and little risk is associated with the procedure compared to other tissue biopsies, especially in patients who are often acutely ill and coagulopathic. So, the multidisciplinary team often await the results of BM examinations in order to decide whether to diagnose the patient with and treat for HLH. Retrospective cohort analyses show that the majority of the patients have HPC at the time of diagnosis of HLH, suggesting that the diagnosis may be delayed until clinicians prove evidence of HPC [11]. HLH was initially identified and defined by the presence of hemophagocytosis, and some still maintain that it remains a morphological diagnosis [12]. However, it is clear that HPC are neither sensitive nor specific for HLH. The epiphenomenon of HPC is seen even following simple blood transfusions and surgery [13]. Furthermore, hemophagocytosis (HPC) is not always present at the time of the first BM examination in patients suspected of having HLH, and serial examinations are advocated [14]. HPC are found only at autopsy in some patients [15], such as in the original cases of HLH described by Farquhar and Claireaux [2]. There may be a different pattern of involvement of BM HPC in familial and secondary forms of HLH. It is possible that the familial cases are detected earlier in their disease course prior to the development of frank HPC. Others have commented that the degree of involvement is proportional to duration of symptoms and age of the patient. The later in life the disease started and the longer it lasted the ‘more apparent’ erythrophagocytic activity [16]. In young patients identified as having familial HLH due to a history of an affected sibling, HPCmay be absent until the onset of overt clinical symptomatology, and even then, may evolve over time requiring repeated marrow examinations [17]. Thus waiting for HPC to appear may unnecessarily delay appropriate therapy [5]. The number ofHPC required to define a positivemarrow inHLH also remains uncertain. The lack of standardized reporting of BM HPC, introduces variability in the interpretation of a positive finding. The attribution of HPC to the diagnosis of HLH may be more straightforward in situations where there is florid HPC [3], however, the finding of few HPC may not be informative. No quantitative data are available; however, it has been suggested that careful examination of at least three smears should reveal at least two HPC per slide to be significant [18]. Most studies lack information on the relative or absolute amount of HPC seen in tissues. Interpretation of the literature describing the prevalence of marrow involvement in HLH is limited by the variable diagnostic criteria used for study inclusion. A summary of these papers are listed in Table I, reporting variable BM involvement in aspirates or biopsies, ranging from 25 to 100% [3,15,16,19–25]. In combining the results of all the series listed in Table I, HPC inBMappearsmore likely to occur in secondary HLH compared to familial patients, with a Spearman correlation coefficient of 0.64, P1⁄4 0.046. Thus, BM are just as likely to be negative or positive for HPC in patients diagnosed with HLH. In summary, limited by the restriction of genetic and immunological testing to specialized centers and the delay in their reporting, BM HPC are often relied upon by clinicians, as early concrete diagnostic evidence to rule in and rule out HLH. HPC do not appear either sensitive nor specific, and there is marked heterogeneity in BM involvement among both familial and secondary forms of HLH. The problems are further confounded by lack of standardization and quantification of the reporting of HPC. Although there may be a