Aplastic anemia presenting as hemophagocytic lymphohistiocytosis/ Hemofagositik lenfohistositoz olarak baslayan aplastik anemi.
Abstract: Two unusual cases of hemophagocytic lymphohistiocytosis (HLH) complicating aplastic anemia (AA) are described. Each patient had a history of preexisting acute hepatitis of unknown cause at the time of HLH diagnosis and infection-associated secondary HLH. They developed high fever and pancytopenia. Hemophagocytes were seen in the bone marrow. With steroid (in combination with etoposide and CyA in 1 patient), high fever disappeared and the patients' liver function gradually recovered. As severe pancytopenia persisted, bone marrow became acellular and AA was diagnosed. Since HLH is known to be able to cause an aplastic bone marrow if untreated for a prolonged time, it is therefore in line that hepatitis-associated AA may also be associated with HLH.

Aplastic anemia-associated HLH has been reported rarely, and problems in the diagnostic procedure are discussed. (Turk J Hematol 2010; 27: 38-42)

Key words: Hemophagocytosis, aplastic anemia, hepatitis

Bu makalede hemofagositik sendrom (HLH) tanisi alip daha sonra aplastik anemi (AA) gelisen 2 hasta sunulmustur. Her 2 hastada da HLH tanisindan once etyolojisi net olarak konulmamis akut hepatit oykusu vardi ve bu enfeksiyon nedeni ile HLH'leri enfeksiyon iliskili HLH olarak degerlendirilmisti. Iki olguda da ates ve pansitopeni vardi. Hemofagositoz iki olguda da kemik iligi aspirasyon materyalinde gosterilmisti. Bir olguda steroid digerinde steroide ek olarak etoposid ve siklosporin (CyA) ile yuksek ates ve karaciger fonksiyonlarinda duzelme saptanmaya baslanmisti. Tedaviye ragmen agir sitopeninin devam etmesi uzerine kemik iliginin tekrar degerlendirilmesi sonucunda hastalara aplastik anemi tanisi konuldu. Hemofagositik sendrom uzun sureli tedavi edilmediginde kemik iliginde aplaziye neden olabilir, bu nedenle hepatite bagli gelisen aplastik anemilerin gelisim surecinde HLH' da olabilir. (Turk J Hematol 2010; 27: 38-42)

Anahtar kelimeler: Hemofagositoz, aplastik anemi, hepatit
Article Type: Clinical report
Subject: Aplastic anemia (Diagnosis)
Aplastic anemia (Drug therapy)
Aplastic anemia (Research)
Immunosuppressive agents (Usage)
Immunosuppressive agents (Health aspects)
Author: Celkan, Tiraje
Pub Date: 03/01/2010
Publication: Name: Turkish Journal of Hematology Publisher: Aves Yayincilik Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Aves Yayincilik ISSN: 1300-7777
Issue: Date: March, 2010
Topic: Event Code: 310 Science & research
Product: SIC Code: 2834 Pharmaceutical preparations
Geographic: Geographic Scope: Turkey Geographic Code: 7TURK Turkey
Accession Number: 220843588
Full Text: Introduction

Differential diagnosis of hemophagocytic lymphohistiocytosis (HLH) and aplastic anemia (AA) is very confusing for a clinician who must initiate life-saving therapy with immunosuppressive/immunomodulatory agents in time [1-3]. Brown et al. [4] reported that post-hepatitis AA typically occurs in young, previously healthy males with self-limited but severe liver inflammation with very high serum aminotransferase and bilirubin levels; profound pancytopenia follows several weeks later.

We present herein our experience with two adolescent boys ages 11 and 12 years who both presented with hyperbilirubinemia and secondary HLH resulting in AA. Both diseases may have some similar immune-mediated conditions involving the activation of T lymphocytes. Moreover, immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporine (CyA) is very effective for AA, while intensive immunosuppressive therapy with ATG and CyA might be a useful strategy for steroid-resistant HLH [5].

Aplastic anemia is an immune-mediated disease that is associated with increased apoptosis of bone marrow stem cells. The increase in apoptosis is due to various cytokines that inhibit hematopoiesis, produced by activated T-cells. As is well known, HLH is characterized by a systemic activation of macrophages/histiocytes, which are induced to undergo phagocytosis of hematopoietic elements. This hyperinflammatory condition is associated with genetic inheritance, infection, malignancy, and immune deficiencies. The cardinal symptoms are prolonged fever, cytopenias, hepatosplenomegaly, and hemo-phagocytosis by activated, morphologically benign macrophages. Biochemical markers include elevated ferritin and triglycerides and low fibrinogen. Impaired function of natural killer (NK) and cytotoxic T-cells is characteristic. Two forms of HLH, primary (genetic) and secondary (acquired), have been reported. Secondary HLH has been reported in association with a variety of conditions [6]. Children with HLH have a higher probability of malignancy, suggesting a possible predisposing role. However, there are only rare reports of an association between AA and HLH.

Generally, the diagnosis of HLH is difficult unless there is suspicion. HLH initially may masquerade as a normal infection since all symptoms may be common.

Case Reports

Written informed consent was obtained from all patients.

Case 1: A 12-year-old boy was followed with hepatitis in a local health center for 40 days. Serologic tests for Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV)-I, HSV-II, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV), and parvovirus B-19 were either negative or consistent with prior exposure. As liver enzymes remained elevated (AST 1975 U/L, ALT 2950 U/L) and total bilirubin increased to 4.73 mg/dl, with international normalized ratio (INR) of 4.67, he was referred to our hospital. Persistent pancytopenia was noticed, and he was consulted to the hematology ward after 25 days. The patient met the diagnostic criteria of HLH described by the Histiocyte Society [2]. Bone marrow aspiration was performed and showed an increased number of histiocytes with hemophagocytosis. As he was suspected to have infection-associated secondary HLH, steroid therapy was started (dexamethasone 10 mg/[m.sup.2] in the beginning and tapered after 2 weeks, planned for a total period of 8 weeks). After steroid, the long-lasting fever disappeared in 2 days and there was an unexpected rapid resolution of hepatosplenomegaly (each 2 cm below the costal margin). Although the clinical manifestations, which were attributed to hypercyto-kinemia, were controlled with steroid, the pancytopenia persisted even after etoposide was added to the HLH regimen after a 4-week period. Bone marrow biopsy performed after 6 weeks of HLH treatment due to no response of cytopenia revealed AA. He was lost 25 days after ATG treatment due to aspergillus in the lung and pseudomonas bacteremia.

Case 2: An 11-year-old boy was followed with persistent hepatitis in a local health center for 2 months until gastrointestinal bleeding was detected. Viral documentation was normal. Liver enzymes remained elevated (AST 2930 U/L, ALT 3460 U/L), INR increased to 6.3, and he was referred to our hospital. Pancytopenia and fever were noticed and he was consulted to the hematology ward after 15 days. The liver biopsy performed before hematology consultation revealed infection. The patient was diagnosed as secondary HLH after an increased number of histiocytes with hemophagocytosis were noticed in the bone marrow aspiration, supported by decreased fibrinogen and increased ferritin, which until the HLH diagnosis were thought to be due to numerous transfusions. He was given dexameth-asone at a dose of 10 mg/[m.sup.2]. Fever and splenomegaly disappeared in the first week, but pancytopenia resulting in near-fatal infection persisted. Granulocyte colony-stimulating factor (G-CSF) was added to steroid treatment due to no hematological relief, and bone marrow biopsy was done. His parents refused any treatment for AA and the patient died due to uncontrolled bleeding and sepsis.

Discussion

Both of the presented patients had no family history suggestive of familial HLH. The patients died before any mutational analysis or NK cell function studies could be performed. The diagnosis of secondary HLH, therefore, is based on fulfillment of a minimum of five clinical criteria [6]. Gupta et al. [7] mentioned in their commentary that despite the availability of genetic and immunological tests in the diagnosis of HLH, there is a lack of a confirmatory diagnostic test for acute situations. There are also no clear-cut definitions for HLH bone marrow infiltration. Three smears with at least 2 histiocytes demonstrating hemophagocytosis on each are suggested for HLH diagnosis, which were detected in both of our patients [7]. No fatty infiltration was detected during HLH diagnosis. Bone marrow examination showed normal maturation of the three series without dysplastic features or any blast. The most prominent feature was an increase in hemophagocytosing histiocytes. Based on these findings, the patients were diagnosed as HLH and not AA. Then, after 15-21 days of HLH treatment, the bone marrow became severely hypoplastic. However, the hemophagocytic features that were apparent before the HLH regimen had disappeared in both patients.

Both patients were diagnosed as having severe bone marrow failure after HLH that was similar to severe AA. Severe pancytopenia with fatty infiltration of the bone marrow is characteristic of AA, but high fever or liver dysfunction is not common if there is no coexisting infection.

Hemophagocytic lymphohistiocytosis and AA represent distinct disease entities, but they have some pathologically similar aspects with activation of T lymphocytes. As mentioned in the literature, HLH can be diagnosed before or after AA [8-10]. In our patients, post-hepatitis AA, or HLH associated with an undiagnosed viral infection in a patient with AA, or a coexistence of HLH and AA could be considered in the differ- ential diagnosis. However, no fatty change of the marrow was found in the beginning, and its cellularity was not severely depressed at the time of admission, while hemophagocytic histiocytes were prominent.

These findings were characteristic of HLH rather than AA. The diagnosis of AA is often difficult because of the presence of local hemapoietically active spots that can lead to an erroneous assessment before fatty bone marrow. Thus, marrow examinations need to be repeated if pancyto-penia persists. The histiocyte count is sometimes increased in AA just like plasma cells, but this is a relative increase and is not associated with hemophagocytosis. Ost et al. [11] reported that the cellularity of the marrow was decreased in some patients and this tissue was severely hypoplastic, even in some children who had not been treated with cyto-static drugs. They also reported that the histological pattern in HLH resembles that of chronic persistent hepatitis. In other words, there is a potential link between AA, HLH and hepatitis-associated AA [11].

Hyperbilirubinemia usually coexists with pancytopenia in HLH, while bone marrow failure secondary to acute hepatitis generally occurs after the hepatitis has resolved if the diagnosis is AA. Immunosuppressive therapy with ATG and CyA, which is a common treatment strategy for severe AA, is sometimes used successfully for HLH, especially for steroid-refractory HLH [5,12].

Impaired function of NK cells and cytotoxic T-cells is characteristic for both genetic and acquired forms of HLH. Frequent triggers are infectious agents, mostly viruses of the herpes group [13,14]. HLH has been identified to be related with genes encoding perforin (PRF1/FHL2), Munc 13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In order to show the relation of AA and HLH, Solomou et al. [15] studied perforin levels in patients with AA. They found that in AA disease with hematopoietic stem cells, which were destructed by activated T-cells and Th1 cytokines, perforin protein levels were very low or absent, and perforin granules were completely diminished [15]. NK cells are the predominant perforin-containing cell type. As a result, NK cell cytotoxicity in these patients was significantly decreased [13]. In one of the HLH subgroups with normal perforin levels, syntaxin-deficient patients, it was shown very recently that their NK cells failed to degranulate when they encountered susceptible target cells [16,17].

Patients with HLH, however, cannot control the hyperin-flammatory response which, if untreated, is fatal. Awareness of the clinical symptoms and of the diagnostic criteria of HLH is important to start life-saving therapy with immunosuppressive/ immunomodulatory agents in time. Therefore, we suggest that patients with hepatitis who develop pancytopenia and high liver enzymes that remain elevated for longer than expected should be examined with bone marrow aspiration and biopsy for demonstration of hemophagocytosis or for bone marrow failure resulting in AA.

Conflict of interest

No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

Received: August 14, 2008 Accepted: April 8, 2009

Gelis tarihi: 14 Agustos 2008 Kabul tarihi: 8 Nisan 2009

References

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[2.] Matsunawa M, Kawakami K, Hisatake J, Suzuki J, Nakamaki T, Hino K, Tomoyasu S. Hepatitis-associated aplastic anemia preceded by a hemophagocytic syndrome-like state. Rinsho Ketsueki 2003;44:1010-4.

[3.] Omagari K, Ashida R, Oh-I H, Minamino Y, Sasaki O, Ozono Y. Successful treatment with cyclosporine in a case of hemophagocytic syndrome manifesting as severe liver dysfunction. Am J Med Sci 1997;314:403-7.

[4.] Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia. N Engl J Med 1997;336: 1059-64.

[5.] Kaito K, Otsubo H, Takei Y, Usui N, Kobayashi M. Immunosuppressive therapy with antithymocyte globulin and cyclosporine for prolonged marrow failure after hemophagocytic syndrome. Ann Hematol 2003;82:699-701.

[6.] Henter JI, Horne A, Arico M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-31.

[7.] Gupta A, Weitzman S, Abdelhaleem M. Commentary: the role of hemophagocytosis in bone marrow aspirates in the diagnosis of HLH. Pediatr Blood Cancer 2008;50:192-4.

[8.] de la Serna FJ, Lopez JI, Garcia-Marcilla A, Ortiz-Conde MC, Mestre MT. Hemophagocytic syndrome causing complete bone marrow failure. Report of an extreme case of a reactive histiocytic disorder. Acta Haematol 1989;82:197-200.

[9.] Stephan JL, Galambrun C, Pozzetto B, Grattard F, Bordigoni P. Aplastic anemia after Mycoplasma pneumoniae infection: a report of two cases. J Pediatr Hematol Oncol 1999;21:299-302.

[10.] Yashima A, Narigasawa Y, Ishida Y, Uchiyama T, Oyake T, Enomoto S, Kaneko J, Ono Y, Sugawara T, Numaoka H, Shimosegawa K, Murai K, Itoh S, Ito T, Kuriya S. Hemophagocytic syndrome due to miliary tuberculosis in the course of aplastic anemia. Rinsho Ketsueki 1998;39:392-7.

[11.] Ost A, Nilsson-Ardnor S, Henter JI. Autopsy findings in 27 children with haemophagocytic lymphohistiocytosis. Histopathology 1998;32:310-6.

[12.] Erduran E, Gedik Y, Sen Y, Yildiran A. Successful treatment of reactive hemophagocytic syndrome with cyclosporin A and intravenous immunoglobulin. Turk J Pediatr 2000;42:168-70.

[13.] Janka GE. Hemophagocytic syndromes. Blood Rev 2007; 21:245-53.

[14.] Gurgey A, Secmeer G, Tavil B, Ceyhan M, Kuskonmaz B, Cengiz B, Ozen H, Kara A, Cetin M, Gumruk F. Secondary hemophagocytic lymphohistiocytosis in Turkish children. Pediatr Infect Dis J 2005;24:1116-7.

[15.] Solomou EE, Gibellini F, Stewart B, Malide D, Berg M, Visconte V, Green S, Childs R, Chanock SJ, Young NS. Perforin gene mutations in patients with acquired aplastic anemia. Blood 2007;109:5234-7.

[16.] Bryceson YT, Rudd E, Zheng C, Edner J, Ma D, Wood SM, Bechensteen AG, Boelens JJ, Celkan T, Farah RA, Hultenby K, Winiarski J, Roche PA, Nordenskjold M, Henter JI, Long EO, Ljunggren HG. Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. Blood 2007;110:1906-15.

[17.] Horne A, Ramme KG, Rudd E, Zheng C, Wali Y, al-Lamki Z, Gurgey A, Yalman N, Nordenskjold M, Henter JI. Characterization of PRF1, STX11 and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis. Br J Haematol 2008;143:75-83.

Tiraje Celkan

Department of Pediatric Hematology-Oncology, Istanbul University, Cerrahpasa Faculty of Medicine, Istanbul, Turkey

Address for Correspondence: Assoc. Prof. Tiraje Celkan, Konaklar mah. Sebboy sok. Armakent sitesi B2/8 4. Levent/ 80620 Istanbul, Turkiye Phone: +90 212 283 69 22 Office: +90 212 414 30 00 / 21956 E-mail: tirajecelkan@yahoo.com
Table 1. Findings of patients (ND: not done)

                                                   Case 1

Clinical findings
Fever                                                +
Hepatomegaly                                        2 cm
Splenomegaly                                        2 cm
Infection                                            +
Jaundice                                             +
Bleeding

Laboratory findings

Hemoglobin gr/dl
  At the beginning of complainment                  9.6
  At the admission our hospital                     5.6
  At the diagnosis of HLH                           3.6
  At the diagnosis of AA                            3.7

WBC [mm.sup.3]
  At the beginning of complainment                 12.600
  At the admission our hospital                     5400
  At the diagnosis of HLH                           3000
  At the diagnosis of AA                            2200

Platelet [mm.sup.3]
  At the beginning of complainment                178.000
  At the admission our hospital                    98.000
  At the diagnosis of HLH                           6000
  At the diagnosis of AA                            9000

Reticulocyte                                        <0.1

AST iu/L
  At the beginning of complainment                  1108
  At the admission our hospital                     1975
  At the diagnosis of HLH                            46
  At the diagnosis of AA                             44

ALT iu/L
  At the beginning of complainment                  1963
  At the admission our hospital                     2950
  At the diagnosis of HLH                            27
  At the diagnosis of AA                            660

bilirubin
  At the beginning of complainment                  4.8
  At the admission our hospital                     1.35

Triglyceride mg/dL
  At the diagnosis of HLH                           456
  At the diagnosis of AA                            115

Fibrinogen g/dL
  At the admission our hospital                      ND
  At the diagnosis of HLH                            80
  At the diagnosis of AA                            170

Bone marrow aspiration
  At the diagnosis of HLH                 hemophagocytic features
  At the diagnosis of AA              Bone marrow failure, hypoplasia

Bone marow biopsy
  At the diagnosis of AA                  devoid of hematopoietic
                                        elements, fat and reticulum
                                                   cells

                                                   Case 2

Clinical findings
Fever                                                +
Hepatomegaly                                       1.5 cm
Splenomegaly                                        2 cm
Infection                                            +
Jaundice                                             +
Bleeding

Laboratory findings

Hemoglobin gr/dl
  At the beginning of complainment                  10.8
  At the admission our hospital                     6.8
  At the diagnosis of HLH                 8.7( With transfusions)
  At the diagnosis of AA                            7.5

WBC [mm.sup.3]
  At the beginning of complainment                 17.000
  At the admission our hospital                     6700
  At the diagnosis of HLH                           1400
  At the diagnosis of AA                            1100

Platelet [mm.sup.3]
  At the beginning of complainment                186.000
  At the admission our hospital                    79.000
  At the diagnosis of HLH                           8000
  At the diagnosis of AA                            4000

Reticulocyte

AST iu/L
  At the beginning of complainment                  2089
  At the admission our hospital                     1790
  At the diagnosis of HLH                            47
  At the diagnosis of AA

ALT iu/L
  At the beginning of complainment                  2298
  At the admission our hospital                     1810
  At the diagnosis of HLH                            45
  At the diagnosis of AA                             43

bilirubin
  At the beginning of complainment               Not known
  At the admission our hospital                     1.77

Triglyceride mg/dL
  At the diagnosis of HLH                           780
  At the diagnosis of AA                             ND

Fibrinogen g/dL
  At the admission our hospital                     180
  At the diagnosis of HLH                            56
  At the diagnosis of AA                             ND

Bone marrow aspiration
  At the diagnosis of HLH                 hemophagocytic features
  At the diagnosis of AA                         Hypoplasia

Bone marow biopsy
  At the diagnosis of AA                  Diminished hematopoietic
                                        elements, increased showing
                                          largely fatty tissue and
                                              reticulum cells
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