**6. Diagnosis of HLH and AAHS/MAS**

In 1991 the HLH Study Group of the Histiocyte Society published the first diagnostic guidelines for HLH which were later updated in 2004 (Henter et al., 1991c, 2007). According to the current guidelines (HLH-2004), five of the following eight criteria must be fulfilled for the diagnosis of HLH: (1) fever, (2) splenomegaly, (3) cytopenias affecting two or three lineages (Hb <90 g/l; PLT <100 × 109/l; neutrophils <1.0 × 109/l), (4) hypertriglyceridemia (fasting triglycerides >3.0 mmol/l) and/or hypofibrinogenemia (<1.5 g/l), (5) hemophagocytosis in bone marrow, spleen, or lymph nodes, (6) hyperferritinemia (>500 µg/l), (7) low or absent NK-cell activity, (8) elevated level of sIL-2R (sCD25) >2400 U/ml. The last three HLH criteria were introduced in the revised diagnostic guidelines for HLH in 2004 (Henter et al., 2007).

There are no validated diagnostic criteria addressed exclusively for AAHS/MAS, and early diagnosis is often difficult (Filipovich et al., 2010; Fukaya et al., 2008; Grom & Mellins, 2010). In general, in a patient with persistently active underlying rheumatologic disease, a fall in the ESR and platelet count, particularly in combination with persistently high CRP and increasing levels of serum D-dimer and ferritin, should raise a suspicion of impeding MAS (Grom & Mellins, 2010). According to Janka, a C-reactive protein >100 mg/l, increased granulopoiesis with left shift in the bone marrow and peripheral blood, and s-ferritin concentration >10,000 μg/L (if EBV infection has been excluded) are features strongly suggestive of MAS (Janka, 2009). The diagnosis of MAS is usually confirmed by demonstration of hemophagocytosis in the bone marrow, liver, lymph nodes, etc. However, false negative results may occur owing to sampling errors, particularly at the early stages of the syndrome (Grom & Mellins, 2010; Janka 2009). In some patients, subsequent biopsies may reveal hemophagocytic macrophages. In patients with negative bone marrow biopsies, assessment of the levels of sIL-2R and sCD163 in serum may help with the timely diagnosis of MAS (Grom & Mellins, 2010; Komp et al., 1989; Schaer et al., 2005).

In particular, application of the HLH diagnostic criteria to sJIA patients with suspected MAS is problematic. Some of the HLH markers such as lymphadenopathy, splenomegaly, and hyperferritinemia are common features of active sJIA itself and therefore do not distinguish MAS from a conventional systemic JIA flare (Davi et al., 2011; Grom & Mellins, 2010). Other HLH criteria, such as cytopenias and hypofibrinogenemia, become evident only at the late stages. This is related to the fact that sJIA patients often have increased white blood cell and platelet counts and elevated s-fibrinogen as a part of the inflammatory response in sJIA. Therefore, when they develop MAS, they demonstrate cytopenias and hypofibrinogenemia to the extent seen in HLH only at the later stage of MAS, when its management becomes challenging (Davi et al., 2011; Grom & Mellins, 2010). Diagnosis of MAS is even more problematic in SLE patients with autoimmune cytopenias, which are difficult to distinguish from cytopenias caused by MAS (Carvalheiras et al., 2010; Grom & Mellins, 2010; Parodi et al., 2009). In these patients, the presence of extreme hyperferritinemia and elevated LDH should raise suspicion of MAS (Parodi et al., 2009). Attempts to modify the HLH criteria to increase their sensitivity and specificity for the diagnosis of MAS in rheumatic conditions have been initiated and continue today (Ravelli et al., 2005; Davi et al., 2011).

#### **7. Case presentations**

84 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

suppressive acitivity of TNF-α, INF-γ, and the heavy unit of ferritin on hematopoiesis;

supressive action of increased levels of

high levels of plasminogen activator secreted by macrophages stimulate plasmine and in consequence lead to

hemophagocytosis

hiperfibrynolisis

TNF-α on lipoprotein lipase

histiocytes/macrophages

organ infiltrations with activated

lymphocytes and histiocytes/macrophages

**HLH symptom/sign Causative factors** 

Fever IL-1; IL-6, TNF-α

High concentration of ferritin in serum released by activated

sIL-2R in blood secreted by activated T lymphocytes

HLH – hemophagocytic lymphohistiocytosis; IL – interleukin; TNF – tumor necrosis factor; sIL-2R –

autoimmune/autoinflamatory diseases and MAS fulfill at the beginning diagnostic criteria for HLH (Janka, 2009). In patients, who already have signs of inflammation such as high leukocytosis, elevated platelet count, and elevated levels of fibrinogen, a decline in these parameters, without reaching pathological values, may herald MAS (Ravelli et al., 2005). MAS as a first symptom of sJIA may be indistinguishable from other cases of HLH when arthritis is missing. A high interleukin-1β concentration in blood may also suggest MAS rather than classic HLH (Janka, 2009; Henter et al., 1996). Although mild elevation of sIL-2R has been reported in many rheumatic diseases including JIA and SLE, a several-fold increase in the levels of sIL-2R in these diseases is highly suggestive of MAS (Grom & Mellins, 2010). Importantly, other clinical entities associated with high levels of sIL-2R include malignancies and some viral infections, such as viral hepatitis, and so these conditions should be considered in the differential diagnosis. Nevertheless, sIL-2R receptor and sCD163 are now increasingly recognized as important biomarkers of AAHS/MAS

In 1991 the HLH Study Group of the Histiocyte Society published the first diagnostic guidelines for HLH which were later updated in 2004 (Henter et al., 1991c, 2007). According to the current guidelines (HLH-2004), five of the following eight criteria must be fulfilled for the diagnosis of HLH: (1) fever, (2) splenomegaly, (3) cytopenias affecting two or three

Cytopenia in peripheral blood

blood

High concentration of triglycerides in

Low concentration of fibrinogen in blood

High concentration of the α chain of the

Increased liver transaminases and

soluble IL-2 receptor (also named sCD25).

(Filipovich, 2009; Grom & Mellins, 2010).

**6. Diagnosis of HLH and AAHS/MAS** 

Table 2. Signs and symptoms of HLH and their causes

Neurological abnormalities

Hepatosplenomegaly

bilirubin in blood

Here we present three illustrative cases of patients with different autoimmune diseases developing severe AAHS/MAS in the course of their autoimmune disorder.

#### **7.1 MAS complicating juvenile arthritis and ankylosing spondylitis**

A 31-year-old male was referred from community hospital to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum, Jagiellonian University, Krakow,

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 87

Hemoglobin g/l 120–170 99 White blood cells × 109/l 4.0–10 1.0 Lymphocytes % 20–40 64 Neutrophils % 58–66 29 Platelets × 109/l 150–400 90 Reticulocytes ‰ 3–15 2

AspAT U/l 17–59 263 AlAT U/l 21–72 116 LDH U/l 313–618 7,700 CRP mg/l <5 56.1

INR 0.85–1.15 1.30 aPTT sec 25–31.5 55.60 Fibrinogen g/l 1.8–3.5 undetectable Fibrinogen (nephelometry) g/l 1.8–3.5 0.7 Trombin time sec 14–21 60.1 D-dimer ng/ml <500 >10,000 Factor II % 70–120 45.9 Factor V % 70–120 106.5 Factor VII % 70–120 109.7 Factor VIII % 50–120 39.8 Factor IX % 70–120 94.6 Factor X % 70–120 76.2 Factor XI % 70–120 110.9 Factor XII % 70–120 81.5

Because of a rapidly progressive thrombocytopenia and neutropenia, bone marrow biopsy was reinterpreted towards a diagnosis of hemophagocytic syndrome. Multiple macrophages (10–15% of nucleated bone marrow cells) together with several hemophagocytes were found

A diagnosis of macrophage activation syndrome complicating recent adalimumab treatment was established. Six days after the patient's admission, treatment with etoposide, dexamethasone, and cyclosporine A was instituted as recommended by the modified HLH-2004 protocol (Fig. 3). Body temperature normalized at day 3 of therapy. Etoposide-related

Table 3. The patient's laboratory results on admission

**Biochemistry** 

**Coagulation tests** 

(Fig. 2).

**Hematological parameters Reference range Patient's result** 

Poland) because of persistent fever and progressive haemostatic abnormalities (skin bruises, undetectable fibrinogen level, prolonged aPTT and INR, markedly elevated FDP and Ddimer levels). The patient was admitted to a community hospital 3 weeks earlier with signs of a possible upper respiratory tract infection (fever, dry cough, sore throat) accompanied by herpes labialis. Broad spectrum antibiotic and acyclovir treatment were both ineffective. Because of a progressing bi-cytopenia (thrombocytopenia with neutropenia; Fig. 1) a fine needle bone marrow biopsy was performed showing no signs of significant primary bone marrow suppression.

Past medical history revealed that the patient suffered from a polyarticular seropositive juvenile arthritis since the age of 8 years. In the following years the patient was hospitalized several times because of disease exacerbations. Non-steroidal inflammatory drugs, gold salts, azathioprine, methotrexate and systemic corticosteroids together with physiotherapy were administered at various time periods; synoviectomy was performed twice. At the age of 29 a diagnosis of ankylosing spondylitis was established (sacroilitis, presence of HLA-B27). Approximately 5 months before admission to our Center the patient's immunosuppressive treatment was modified because of poor disease control, and he received a TNF-α inhibitor (adalimumab) with good clinical response.

On admission the patient complained of pain localized to the right subcostal region and persistent fever. On physical examination body temperature was 39.2°C, and slight hepatomegaly and skin ecchymoses with mucosal bleeding were present. Selected laboratory results and hematologic parameters at admission are shown in Table 3.

Differential diagnosis included DIC with sepsis (blood cultures later showed negative results), neutropenic fever (neutrophils 0.29 × 109/l), and opportunistic infections (HBV, HCV, EBV, CMV, and HIV infection were excluded). Adalimumab side effects were also taken into consideration. Filgrastim, ganciclovir and intravenous gammaglobulins were instituted with no improvement. Concomitant medications included ceftazidime, vancomycin, amikacin, sulfamethoxazole/trimethoprim, linezolide, tranexamic acid, fresh frozen plasma and cryoprecipitate.

Fig. 1. Changes in neutrophils and lymphocyte counts before the admission

Poland) because of persistent fever and progressive haemostatic abnormalities (skin bruises, undetectable fibrinogen level, prolonged aPTT and INR, markedly elevated FDP and Ddimer levels). The patient was admitted to a community hospital 3 weeks earlier with signs of a possible upper respiratory tract infection (fever, dry cough, sore throat) accompanied by herpes labialis. Broad spectrum antibiotic and acyclovir treatment were both ineffective. Because of a progressing bi-cytopenia (thrombocytopenia with neutropenia; Fig. 1) a fine needle bone marrow biopsy was performed showing no signs of significant primary bone

Past medical history revealed that the patient suffered from a polyarticular seropositive juvenile arthritis since the age of 8 years. In the following years the patient was hospitalized several times because of disease exacerbations. Non-steroidal inflammatory drugs, gold salts, azathioprine, methotrexate and systemic corticosteroids together with physiotherapy were administered at various time periods; synoviectomy was performed twice. At the age of 29 a diagnosis of ankylosing spondylitis was established (sacroilitis, presence of HLA-B27). Approximately 5 months before admission to our Center the patient's immunosuppressive treatment was modified because of poor disease control, and he

On admission the patient complained of pain localized to the right subcostal region and persistent fever. On physical examination body temperature was 39.2°C, and slight hepatomegaly and skin ecchymoses with mucosal bleeding were present. Selected

Differential diagnosis included DIC with sepsis (blood cultures later showed negative results), neutropenic fever (neutrophils 0.29 × 109/l), and opportunistic infections (HBV, HCV, EBV, CMV, and HIV infection were excluded). Adalimumab side effects were also taken into consideration. Filgrastim, ganciclovir and intravenous gammaglobulins were instituted with no improvement. Concomitant medications included ceftazidime, vancomycin, amikacin, sulfamethoxazole/trimethoprim, linezolide, tranexamic acid, fresh


Days to admission

laboratory results and hematologic parameters at admission are shown in Table 3.

 Lymphocytes Neutrophils

Fig. 1. Changes in neutrophils and lymphocyte counts before the admission

received a TNF-α inhibitor (adalimumab) with good clinical response.

marrow suppression.

frozen plasma and cryoprecipitate.

%


Table 3. The patient's laboratory results on admission

Because of a rapidly progressive thrombocytopenia and neutropenia, bone marrow biopsy was reinterpreted towards a diagnosis of hemophagocytic syndrome. Multiple macrophages (10–15% of nucleated bone marrow cells) together with several hemophagocytes were found (Fig. 2).

A diagnosis of macrophage activation syndrome complicating recent adalimumab treatment was established. Six days after the patient's admission, treatment with etoposide, dexamethasone, and cyclosporine A was instituted as recommended by the modified HLH-2004 protocol (Fig. 3). Body temperature normalized at day 3 of therapy. Etoposide-related

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 89

Fever (°C) 39.2 35.8 Splenomegaly slight - Hepatomegaly + - Hemoglobin (g/l) 85 137 Platelets (× 109/l) 14 260 Neutrophils (× 109/l) 0.29 5.3 Triglycerides (mmol/l) 6.5 2.84 Fibrinogen (g/l) undetectable 4.8 Ferritin (µg/l) >20,000 233.2 AlAT (U/l) 116 25 Bilirubin (mol/l) 19 15 LDH (U/l) 7,687 624 Hemophagocytosis + -

Table 4. Changes in selected laboratory and clinical parameters characteristic for MAS

A 58-years-old patient, suffering from rheumatoid arthritis for 12 years, about 13 months after a course of TNF-α inhibitor (etanercept) treatment was admitted to hospital in another country due to progressive weakness, weight loss, intensive ankle joint pain and fever reaching 40°C. RA flare and infection were excluded and etanercept-related bone marrow dysfunction was suspected due to agranulocytosis, leukopenia, and thrombocytopenia. The patient had decided to leave the hospital and sought consultation in our University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland). On admission his general condition was satisfactory; physical examination having revealed *livedo reticularis* on the lower limbs, swelling and redness of the left ankle joint, slight splenomegaly and caries of teeth 11 and 24. The most important laboratory abnormalities are shown in Table 5. Rheumatoid arthritis with Felty syndrome was diagnosed 12 years ago based on the presence of 5 ACR criteria, positive rheumatoid factor (RF) and elevated anti-cyclic citrullinated peptide antibodies (anti-CCP). About a year ago etanercept was started due to the ineffectiveness

After admission the patient received three injections of filgrastim (48 mln. units each) and antimicrobial treatment was implemented (ceftazidim, amikacin, fluconazole). Teeth 11 and 24 were extracted. The patient was seen by a hematologist and a fine needle bone marrow

A macrophage activation syndrome associated with rheumatic disease (RA) was diagnosed. On day 8 of hospitalization, dexamethasone (20 mg qd) and cyclosporine A (dose adjusted to trough blood levels) were initiated. Treatment led to a quick decrease in body

temperature and little later to normalization of the peripheral blood picture (Table 6).

biopsy was performed showing numerous, typical hemophagocytes (Figure 4).

during the applied therapy (HLH-2004)

of corticosteroids and methotrexate.

**7.2 MAS complicating rheumatoid arthritis** 

**(nadir/zenith) After treatment** 

**Parameter (units) Baseline** 

nadir occurred at day 20 (platelets 14 × 109/l; leukocytes 0.42 × 109/l) (Table 4). One dose of etoposide was omitted and filgrastim (G-CSF) was administered (48 mln units b.i.d) with a beneficial effect. Etoposide was then continued for the next 9 months.

Fig. 2. Bone marrow aspirate smears showing the centrally placed macrophage laden with erythroblasts. Normally developed myeloid cells are present nearby. Wright's stain, lower (× 400) and higher (× 1000) magnification

Fig. 3. The patient's treatment scheme with a HLH-2004 protocol

Control examination of bone marrow with a fine needle biopsy performed at week 40 of treatment confirmed the disappearance of activated macrophages. The patient felt well and continues treatment with cyclosporine and low-dose corticosteroids.


Table 4. Changes in selected laboratory and clinical parameters characteristic for MAS during the applied therapy (HLH-2004)

#### **7.2 MAS complicating rheumatoid arthritis**

88 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

nadir occurred at day 20 (platelets 14 × 109/l; leukocytes 0.42 × 109/l) (Table 4). One dose of etoposide was omitted and filgrastim (G-CSF) was administered (48 mln units b.i.d) with a

Fig. 2. Bone marrow aspirate smears showing the centrally placed macrophage laden with erythroblasts. Normally developed myeloid cells are present nearby. Wright's stain, lower

beneficial effect. Etoposide was then continued for the next 9 months.

(× 400) and higher (× 1000) magnification

Fig. 3. The patient's treatment scheme with a HLH-2004 protocol

continues treatment with cyclosporine and low-dose corticosteroids.

Control examination of bone marrow with a fine needle biopsy performed at week 40 of treatment confirmed the disappearance of activated macrophages. The patient felt well and A 58-years-old patient, suffering from rheumatoid arthritis for 12 years, about 13 months after a course of TNF-α inhibitor (etanercept) treatment was admitted to hospital in another country due to progressive weakness, weight loss, intensive ankle joint pain and fever reaching 40°C. RA flare and infection were excluded and etanercept-related bone marrow dysfunction was suspected due to agranulocytosis, leukopenia, and thrombocytopenia. The patient had decided to leave the hospital and sought consultation in our University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland). On admission his general condition was satisfactory; physical examination having revealed *livedo reticularis* on the lower limbs, swelling and redness of the left ankle joint, slight splenomegaly and caries of teeth 11 and 24. The most important laboratory abnormalities are shown in Table 5. Rheumatoid arthritis with Felty syndrome was diagnosed 12 years ago based on the presence of 5 ACR criteria, positive rheumatoid factor (RF) and elevated anti-cyclic citrullinated peptide antibodies (anti-CCP). About a year ago etanercept was started due to the ineffectiveness of corticosteroids and methotrexate.

After admission the patient received three injections of filgrastim (48 mln. units each) and antimicrobial treatment was implemented (ceftazidim, amikacin, fluconazole). Teeth 11 and 24 were extracted. The patient was seen by a hematologist and a fine needle bone marrow biopsy was performed showing numerous, typical hemophagocytes (Figure 4).

A macrophage activation syndrome associated with rheumatic disease (RA) was diagnosed. On day 8 of hospitalization, dexamethasone (20 mg qd) and cyclosporine A (dose adjusted to trough blood levels) were initiated. Treatment led to a quick decrease in body temperature and little later to normalization of the peripheral blood picture (Table 6).


Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 91

Fig. 4. Bone marrow aspirate smears. The centrally located macrophage shows phagocytosis of erythrocytes, erythroblasts and myeloid cells. Wright's stain, lower (× 400) and higher (×

A 46-year-old man was admitted to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland) in 2010 because of persistent fever and pulmonary nodules and consolidations. He has been treated for the last 10 years for systemic lupus erythematosus (SLE) with renal involvement. His SLE was diagnosed in 1991 based on the presence of several ARA criteria (fever, arthralgias, pleuritis and pericarditis, proteinuria, anemia, and the presence of ANA in high titer as well as ds-DNA antibodies) and typical results of renal biopsy. His medical history revealed arterial hypertension, peripherial artery occlusive disease and deep vein thrombosis of both lower limbs. Initially SLE was treated with corticosteroids and oral cyclophosphamide, then treatment was frequently modified according to the disease activity (azathioprine,

**Hematological parameters Reference range Patient's result** 

Hemoglobin g/l 120–150 134 White blood cells × 109/l 4.0–10 9.0 Lymphocytes % 20–40 12.2 Neutrophils % 58–66 83.0 Platelets × 109/l 150–400 57 Reticulocytes ‰ 3–15 10

AspAT U/l 21–72 38 AlAT U/L 21–72 50 Bilirubin mol/l 3–22 11 LDH U/l 313–618 1,114 CRP mg/l <5 75.7

INR 0.85–1.15 0.94 aPTT sec 25–31.5 25.9 Fibrinogen g/l 1.8–3.5 5.0

Table 7. The patient's laboratory results on admission to the University Hospital

**7.3 MAS complicating systemic lupus erythematosus** 

cyclosporine A, mycophenolate mofetil and plasmapheresis).

**Biochemistry**

1000) magnification

**Coagulation tests** 

Table 5. The patient's laboratory results on admission to the University Hospital


Table 6. Changes of selected laboratory and clinical parameters of MAS during the treatment with modified HLH-2004 protocol

Fig. 4. Bone marrow aspirate smears. The centrally located macrophage shows phagocytosis of erythrocytes, erythroblasts and myeloid cells. Wright's stain, lower (× 400) and higher (× 1000) magnification

#### **7.3 MAS complicating systemic lupus erythematosus**

90 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

Hemoglobin g/l 120–170 108 White blood cells × 109/l 4.0–10 0.62 Lymphocytes % 20–40 80 Neutrophils % 58–66 13 Platelets × 109/l 150–400 56 Reticulocytes ‰ 3–15 43

AspAT U/l 21–72 16 AlAT U/l 21–72 11 Bilirubin mol/l 3–22 12 LDH U/l 313–618 420 CRP mg/l <5 54.3

INR 0.85–1.15 1.08 aPTT sec 25–31.5 31 Fibrinogen g/l 1.8–3.5 1.5

**(nadir/zenith) After treatment** 

Table 5. The patient's laboratory results on admission to the University Hospital

Fever (°C) 37.2 36.6 Splenomegaly + - Hepatomegaly - - Hemoglobin (g/l) 108 133 Platelets (× 109/l) 25.2 173 Neutrophils (× 109/l) 0.6 3.43 Triglycerides (mmol/l) 2.83 2.7 Fibrinogen (g/l) 1.40 5.2 Ferritin (µg/l) 1,855 717.5 AlAT (U/l) 16 52

Bilirubin (mol/l) 12 not determined LDH (U/l) 420 not determined Hemophagocytosis + not determined

Table 6. Changes of selected laboratory and clinical parameters of MAS during the treatment

**Parameter (units) Baseline** 

**Biochemistry** 

**Coagulation tests** 

with modified HLH-2004 protocol

**Hematological parameters Reference range Patient's** 

**result** 

A 46-year-old man was admitted to the University Hospital (The Second Chair of Internal Medicine, Collegium Medicum of the Jagiellonian University, Krakow, Poland) in 2010 because of persistent fever and pulmonary nodules and consolidations. He has been treated for the last 10 years for systemic lupus erythematosus (SLE) with renal involvement. His SLE was diagnosed in 1991 based on the presence of several ARA criteria (fever, arthralgias, pleuritis and pericarditis, proteinuria, anemia, and the presence of ANA in high titer as well as ds-DNA antibodies) and typical results of renal biopsy. His medical history revealed arterial hypertension, peripherial artery occlusive disease and deep vein thrombosis of both lower limbs. Initially SLE was treated with corticosteroids and oral cyclophosphamide, then treatment was frequently modified according to the disease activity (azathioprine, cyclosporine A, mycophenolate mofetil and plasmapheresis).


Table 7. The patient's laboratory results on admission to the University Hospital

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 93

Macrophage activation syndrome was diagnosed and one month after the patient's admission treatment with dexamethasone, cyclosporine A, and etoposide was started resulting in the normalization of body temperature and peripherial blood morphology. Etoposide-related nadir occurred at day 14 (platelets 18 × 109/l, leukocytes 1.0 × 109/l). Two etoposide doses were omitted and filgrastim was administered twice (48 mln units qd). Intravenous pulses of etoposide were given for the next 2 months, followed by oral administration. A control bone marrow examination performed on week 40 of treatment showed disappearance of activated macrophages. The patient has continued treatment with cyclosporine A in combination with low-dose corticosteroids with continuous improvement

Although significant progress in understanding the genetics and pathophysiology of primary HLH has been achieved during recent years, the pathogenesis of acquired forms of HLH is still not fully understood. An exaggerated immune response is the final common pathway of HLH, however, there are multiple roads leading to it (Arceci, 2008; Janka, 2009). The immune response is often triggered by different stimulants (e.g., infection) and the underlying inherited or acquired immune defect. It has been proposed that the clinical presentation of HLH is due to uncontrolled activation of immune cells, macrophages and CD8+ T lymphocytes (cytotoxic), leading to a massive release of various mediators of inflammation such as TNF-α (tumor necrosis factor α), interleukin(IL)-6, IL-8, IL-10, IL-12, IL-18, interferon γ, macrophage inflammatory protein (MIP 1-α), and hematopoietic growth factors (e.g., GM-CSF) (Filipovich, 2009; Henter et al., 1991a, 1996, 2007; Janka & Schneider, 2004; Osugi et al., 1997). IL-10 with its anti-inflammatory properties plays many important roles in the regulation of autoimmune inflammatory responses, particularly of systemic autoimmune disorders such as HLH/MAS. The role of IL-10 as part of an important regulatory mechanism involved in HLH has long been proposed (Behrens et al., 2011; Benveniste et al., 2000; Osugi et al., 1997). Recently, the roles of T regulatory cells in HLH have also been discussed (Verbsky & Grossman, 2006). Low or absent NK-cell function is present in many HLH patients and results in difficulties in termination of the exaggerated

There are two major subtypes of genetic causes of HLH. First are those genetic defects, grouped under the term FHL, that present with HLH as the primary and only manifestation of disease (Gupta & Weitzman, 2010; Henter et al., 2007). A second group of genetic disorders include HLH as only one, although often fatal, manifestation of the disease (Gupta & Weitzman, 2010; Janka, 2009). All known genetic abnormalities causing FHL involve genes that regulate proteins important in the secretory cytolytic pathway of NK-cells and CD8+ T lymphocytes. In 1999, the first FHL-inked locus was discovered on chromosome 9q21.3–22 in several Pakistani families and was later defined as the FHL1 subtype (Ohadi et al., 1999). Shortly thereafter, mutations in the perforin gene *PRF1* were discovered on chromosome 10q21 in a group of patients with FHL (FHL2 subtype) (Stepp et al., 1999). Furthermore, mutations in genes *UNC13D* (located on chromosome 17q25; FHL3 subtype)*, STX11* (located on chromosome 6q24; FHL4 subtype), and most recently *STXBP2* (located on chromosome 19p13; FHL5 subtype) were described (Feldmann 2003; zur Stadt et al., 2005, 2009). In view of the remarkable progress since the discovery of the first genetic defect in

of his clinical and laboratory parameters (Table 8).

immune response (Filipovich, 2009; Henter et al., 2007).

**8. Pathophysiologic and molecular mechanism of HLH** 

The patient's general condition on admission was satisfactory. His body temperature was 38.4°C, physical examination revealed neither hepatosplenomegaly nor lymphadenopathy. Selected laboratory results and hematologic parameters on admission are shown in Table 7. Microbial analysis of bronchoalveolar lavage obtained at bronchoscopy revealed a group I Mycobacteria-other-than-tuberculosis (MOTT). Targeted therapy was instituted (rifampicin 450 mg qd, isoniazide 250 mg qd, ethambutol 750 mg qd and clarithromycin 1000 mg bid) with no effect on the fever. Because of the persistent fever, bronchoscopy was repeated one month later, but MOTT's were no longer detectable. Due to the progressive anemia and thrombocytopenia a fine needle bone marrow biopsy was performed showing typical hemophagocytes, with some of these forming cell conglomerates (Fig. 5).

Fig. 5. Wright's stain of bone marrow aspirate smears. The centrally placed large cell conglomerate consists of activated macrophages presenting hemophagocytosis of erythroblasts and myeloid cells. Lower (× 200) and higher (× 400) magnification


Table 8. Changes of selected laboratory and clinical parameters typical for MAS during treatment (HLH-2004)

The patient's general condition on admission was satisfactory. His body temperature was 38.4°C, physical examination revealed neither hepatosplenomegaly nor lymphadenopathy. Selected laboratory results and hematologic parameters on admission are shown in Table 7. Microbial analysis of bronchoalveolar lavage obtained at bronchoscopy revealed a group I Mycobacteria-other-than-tuberculosis (MOTT). Targeted therapy was instituted (rifampicin 450 mg qd, isoniazide 250 mg qd, ethambutol 750 mg qd and clarithromycin 1000 mg bid) with no effect on the fever. Because of the persistent fever, bronchoscopy was repeated one month later, but MOTT's were no longer detectable. Due to the progressive anemia and thrombocytopenia a fine needle bone marrow biopsy was performed showing typical

hemophagocytes, with some of these forming cell conglomerates (Fig. 5).

Fig. 5. Wright's stain of bone marrow aspirate smears. The centrally placed large cell conglomerate consists of activated macrophages presenting hemophagocytosis of erythroblasts and myeloid cells. Lower (× 200) and higher (× 400) magnification

Fever (°C) 40.0 36.5 Splenomegaly - - Hepatomegaly - - Hemoglobin (g/l) 120–170 85 153 Platelets (× 109/l) 150–400 42.2 118.2 Neutrophils (× 109/l ) 6,100 3,500 Triglycerides (mmol/l) 0.3–2.26 2.63 1.07 Fibrinogen (g/l) 1.8–3.5 3.60 3.1 Ferritin (µg/l) 13–400 1,387 105 AlAT (U/L) 21–72 142 57 Bilirubin (mol/l) 3–22 12 11 LDH (U/L) 313–618 804 452 Hemophagocytosis ++ single cells Table 8. Changes of selected laboratory and clinical parameters typical for MAS during

**Baseline** 

**(nadir/zenith) After treatment** 

**range** 

**Parameter (units) Reference** 

treatment (HLH-2004)

Macrophage activation syndrome was diagnosed and one month after the patient's admission treatment with dexamethasone, cyclosporine A, and etoposide was started resulting in the normalization of body temperature and peripherial blood morphology. Etoposide-related nadir occurred at day 14 (platelets 18 × 109/l, leukocytes 1.0 × 109/l). Two etoposide doses were omitted and filgrastim was administered twice (48 mln units qd). Intravenous pulses of etoposide were given for the next 2 months, followed by oral administration. A control bone marrow examination performed on week 40 of treatment showed disappearance of activated macrophages. The patient has continued treatment with cyclosporine A in combination with low-dose corticosteroids with continuous improvement of his clinical and laboratory parameters (Table 8).
