Mohamed Abdelhaleem

The role of the initial bone marrow aspirate in the diagnosis of hemophagocytic lymphohistiocytosis.

The identification of hemophagocytosis (HPC) in tissue or bone marrow (BM) represents only one of 5/8 criteria needed for the diagnosis of hemophagocytic lymphohistiocytosis (HLH). Yet, confirmation of HPC in bone marrow aspirates (BMA) is often relied upon to make therapeutic decisions. There is no standardized reporting criteria for the definition of “positive” BMA, and likely differs between institutions. The purpose of this study was to quantify the number of HPC in the initial BMA in patients diagnosed with HLH at our institution.

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

Complex Biological Profile of Hematologic Markers across Pediatric, Adult, and Geriatric Ages: Establishment of Robust Pediatric and Adult Reference Intervals on the Basis of the Canadian Health Measures Survey

BACKGROUND In a collaboration between the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) and the Canadian Health Measures Survey (CHMS), we determined reference value distributions using an a priori approach and created a comprehensive database of age- and sex-stratified reference intervals for clinically relevant hematologic parameters in a large household population of children and adults. METHODS The CHMS collected data and blood samples from 11 999 respondents aged 3-79 years. Hematology markers were measured with either the Beckman Coulter HmX or Siemens Sysmex CA-500 Series analyzers. After applying exclusion criteria and removing outliers, we determined statistically relevant age and sex partitions and calculated reference intervals, including 90% CIs, according to CSLI C28-A3 guidelines. RESULTS Hematology marker values showed dynamic changes from childhood into adulthood as well as between sexes, necessitating distinct partitions throughout life. Most age partitions were necessary during childhood, reflecting the hematologic changes that occur during growth and development. Hemoglobin, red blood cell count, hematocrit, and indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) increased with age, but females had lower hemoglobin and hematocrit starting at puberty. Platelet count gradually decreased with age and required multiple sex partitions during adolescence and adulthood. White blood cell count remained relatively constant over life, whereas fibrinogen increased slightly, requiring distinct age and sex partitions. CONCLUSIONS The robust dataset generated in this study has allowed observation of dynamic biological profiles of several hematology markers and the establishment of comprehensive age- and sex-specific reference intervals that may contribute to accurate monitoring of pediatric, adult, and geriatric patients.

Experience with hemophagocytic lymphohistiocytosis/macrophage activation syndrome at a single institution.

Background Hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS) is a serious and potentially life threatening histiocytic disorder in children and adults. The most commonly used protocol-based therapy includes corticosteroids, cyclosporine-A, and etoposide. However, patients are often started on corticosteroid alone with or without the addition of intravenous γ-globulin. The role of the various therapies in HLH/MAS remains undefined. Objective To identify patient-related factors that led to the use of full protocol therapy (HLH 1994/2004) and to determine treatment-related factors that were associated with adverse outcome including relapse and death. Design/Methods Patients who were diagnosed with HLH/MAS between January 1998 and December 2005 were included in this study. Results Thirty-eight patients had a median age of 9.1 years at diagnosis. Underlying diagnoses were: viral/other 42%; rheumatologic 37%; and malignancy 21%. Initial treatment included corticosteroids 29%; intravenous immunoglobulin (IVIG) 18%; steroids+IVIG 8%; cyclosporine 5%; etoposide 5%; HLH protocol 32%. Etoposide was eventually used in 21% (3/14) of rheumatology and 75% (18/25) viral/other patients. In all, 5/14 (36%) rheumatology and 12/16 (75%) viral/other patients required intensive care unit admission, and 1/14 (7.1%) rheumatology, and 6/16 (38%) viral/other patients died. Three children received a bone marrow transplant. Eleven of 38 (29%) patients died, despite 8 having received etoposide therapy. Three deaths were secondary to underlying malignancy and one from transplant-related complication for malignancy. Conclusions Patients with HLH are at high risk for death early in their disease course. However, corticosteroids and/or IVIG may be sufficient as first-line therapy for patients with underlying rheumatologic disease who present with HLH/MAS. Further prospective studies are required to more precisely define early risk factors for poor outcomes in this often fatal disease.

Helicases: An Overview

Helicases are essential enzymes involved in all aspects of nucleic acid metabolism including DNA replication, repair, recombination, transcription, ribosome biogenesis and RNA processing, translation, and decay. They occur in vivo as part of molecular complexes that include the components required for each specific step of nucleic acid metabolism. The role of the helicases is to utilize the energy derived from nucleoside triphosphate hydrolysis to translocate along nucleic acid strands, unwind/separate the helical structure of double-stranded nucleic acid, and, in some cases, disrupt protein-nucleic acid interactions. Because of their essential function, helicases are ubiquitous and evolutionary conserved proteins. This chapter briefly highlights helicase structure and activities and provides examples of the helicases involved in nucleic acid metabolism.

Malignant hematological disorders in children with Wolf-Hirschhorn syndrome

Wolf–Hirschhorn syndrome (WHS) is a rare chromosomal disorder attributable to a deletion at the short arm of chromosome 4. This syndrome is associated with characteristic facial appearance, multiple congenital abnormalities, mental retardation, feeding difficulties and failure to thrive. We report two girls with WHS who developed myelodysplastic syndrome (MDS). According to the “Category, Cytology, Cytogenetic (CCC)”classification of childhood MDS, patient 1 had refractory cytopenia with ring sideroblasts at the age of 6 years, while patient 2 had refractory cytopenia with dysplasia at the age of 5½ years. Patient 1 progressed to refractory cytopenia with excess blasts within a year, while patient 2 progressed to acute lymphoblastic leukemia within 1 month of presentation. It is possible that allelic loss of a tumor suppressor gene such as WHSC1 and/or FGFR3 from the deleted segment 4p16.3 plays a critical role in the process of malignant transformation. To our knowledge, this is the first report of severe hematological complications like MDS and leukemia in children with WHS and may be an important genetic model for understanding malignant hematological transformation. This report also underscores the importance of evaluating children with WHS for hematopoietic dysfunction.

The novel helicase homologue DDX32 is down-regulated in acute lymphoblastic leukemia

We identified DDX32 as gene predicted to encode a 743 amino acid protein containing a helicase-like domain based on its homology to the conserved helicase domain of the DEAH family of helicases. The helicase domain of DDX32 has a novel sequence including a signature sequence of DDIH in motif II and several substitutions in other motifs. DDX32 has a murine orthologue and shares the presence of conserved sequences, other than the eight canonical helicase motifs, with DEAH helicases across species. Expression studies show that DDX32 is widespread but specifically down-regulated in acute lymphoblastic leukemias. DDX32 gene has 11 exons and is located on 10q26 chromosomal band. An alternatively spliced message lacking exon 4 in the coding region is present in several tissues. Our results suggest that DDX32 is a novel gene, which might play a role in normal and/or abnormal lymphopoiesis.

Thrombocytopenia and megakaryocyte dysplasia: an adverse effect of valproic acid treatment.

We describe a 10-year-old girl with thrombocytopenia for 5 months. She has severe cerebral palsy, quadriplegia, developmental delay, and a seizure disorder. She had been treated with valproic acid (Depakene) 53 mg/kg/day t.i.d. for 2 years before presentation. Her platelet count decreased to 28 × 10/L a short time before presentation. Five months ago she was admitted for severe pneumonia; her laboratory values were as follows: platelet count 38 × 10/L, fibrinogen 6.37 (normal 1.60–4.0) g/L, DDI >7,000 (normal 0–449) ng/mL, and International Normalized Ratio 1.6 (normal 0.9–1.1). Other hematologic findings included hemoglobin 66 (normal 120–160) g/L, absolute reticulocyte count 517 (normal 10–100) × 10/L, mean corpuscular volume 98 (normal 80–94) fL, erythrocyte sedimentation rate 130 mm/h, and negative direct antiglobulin test. Vitamin B12 and folate levels were normal. She was successfully treated for pneumonia and did not require platelet transfusions. With the improvement of her clinical condition, her complete blood count and coagulation screen returned to normal except for prolonged thrombocytopenia with limited response to intravenous immunoglobulin. Therefore, a bone marrow aspiration was performed (Fig. 1). This case illustrates one of the hematologic adverse effects of valproic acid, namely thrombocytopenia with megakaryocytic dysplasia. The long-standing thrombocytopenia with valproic acid levels up to 1,115 (normal 350–700) mmol/L before bone marrow aspiration and the improvement after dose reduction of valproic acid, resulting in levels of 517 mmol/l, strongly suggest that they are related to

Novel mutation of the perforin gene and maternal uniparental disomy 10 in a patient with familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis is a rare disorder characterized by lethal primary immunodeficiency associated with hypercytokinemia and a concomitant defect in natural killer cell cytotoxicity. We report a fatal case of familial hemophagocytic lymphohistiocytosis homozygous caused by a novel nonsense mutation of the perforin gene. Homozygosity was established to be the result of uniparental disomy of the maternal chromosome 10. Uniparental disomy increases the risk of autosomal recessive disease.

Unusual functional manifestations of a novel STX11 frameshift mutation in two infants with familial hemophagocytic lymphohistiocytosis type 4 (FHL4).

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an autosomal recessive, early‐onset, life‐threatening immune disorder. Loss‐of‐function mutations in STX11 have been found to impair NK cell degranulation and cytotoxicity. Here, we describe two unrelated infants of Punjabi descent presenting with FHL and carrying a novel, homozygous STX11 frameshift mutation [c.867dupG]. Western blot analysis indicated absence of syntaxin‐11. Unexpectedly, degranulation by NK cells from one of the patients was not impaired, although patient NK cells showed mildly and significantly decreased cytotoxicity, respectively. Importantly, these observations imply that STX11 should be sequenced in HLH patients even when impaired NK cell degranulation is not found. Pediatr Blood Cancer 2011;56:654–657.