BRANKO MALENICA, ZORANA GRUBI] and DRAGO BATINI]
Department of Immunology, Clinical Department of Laboratory Diagnosis, Clinical Hospital Center Zagreb, Ki{pati}eva 12, 10000 Zagreb, Croatia E-mail: [email protected]
CHRONOLOGY OF IMPORTANT DATES
1972 Foundation of the Tissue Typing Centre headed by Andrija Ka{telan (1970–2001) and Vesna Brklja~i}-Kerhin (2001–now).
From 1970–2007 the center was part of the Urology Clinic of the Clinical Hospital Center Zagreb and Zagreb Univer- sity School of Medicine.
In 2007 the center became part of the Clinical Institute of Laboratory Diagnosis and changed its original name to De- partment for Tissue Typing.
1977 Foundation of the Laboratory for Cellular Immunology headed by Maja Ka{telan (1977–1981). From 1977–1981 the laboratory was part of the Oncology and Radiotherapy Clinic, Clinical Hospital Center Zagreb.
From 1981–1985 the laboratory was part of the Division of Immunology within the Department for Clinical Labora- tory Diagnostic of the Clinical Hospital Center Zagreb.
1978 Foundation of the Division of Clinical Immunology and Inflammatory Rheumatic Diseases as a part of the Clinic for Internal Medicine, Clinical Hospital Center Zagreb and Zagreb University School of Medicine; the division was headed by Zvonimir Horvat (1978–1990).
Foundation of the Laboratory for Serologic Immunodiagnostics as a part of the Division of Clinical Immunology and Inflammatory Rheumatic Diseases, also headed by Zvonimir Horvat (1978–1981).
1981 Foundation of the Department for Clinical Laboratory Diagnostic (so called »central laboratory«) of the Clinical Hos-
pital Center Zagreb headed by Ana Stavljeni} Rukavina (1981–1985).
Laboratory for Cellular Immunology and Laboratory for Serologic Immunodiagnostics were united and became a new organizational unit – Division of Immunology – within the Department for Clinical Laboratory Diagnostics, Clinical Hospital Center Zagreb. The head of this new division was Maja Ka{telan.
1985 Department for Clinical Laboratory Diagnostics of the Clinical Hospital Center changed its name to the Department for Clinical Laboratory Diagnostics of the Zagreb University School of Medicine (headed by Ana Stavljeni} Rukavina (1985.–1992.)
1986. Matko Maru{i}, a professor of physiology and immunology from the Zagreb University School of Medicine became head of the Division of Immunology of the Department for Clinical Laboratory Diagnostics.
1990 Division of Clinical Immunology and Inflammatory Rheumatic Diseases changed its name to the Department of Clin- ical Immunology and Rheumatology of the Internal Clinic, Zagreb University School of Medicine, headed by Nada ^ike{ (1990–until now).
1990 Department of Clinical Laboratory Diagnostics changed its name to the Clinical Institute of Laboratory Diagnosis of the Clinical Hospital Center Zagreb and the Zagreb University School of Medicine headed by Ana Stavljeni} Rukavina (1992–2005) and Jadranka Serti} (2005–until now).
Division of Immunology changed its name to the Department of Immunology as part of the Clinical Institute of Labo- ratory Diagnosis. In the period 1992–1997 the Department was headed by Matko Maru{i} 1992–1997 and Drago Batini} (1997–until now).
1998 Department of Immunology became the Referral Center of the Croatian Ministry of Health and Welfare for Clinical Laboratory Immunodiagnostics of Hematological and Immunological Diseases.
Department for Tissue Typing obtained the status of the Tissue Typing Referral Center for the Ministry of Health and Welfare of the Republic of Croatia.
2007 Department of Tissue Typing obtained accreditation from the European Federation for Immunogenetics (EFI).
DEPARTMENT OF CLINICAL IMMUNOLOGY AND RHEUMATOLOGY
Staff members (2008): Nada ^ike{, head, Branimir Ani}, Dubravka Bosni}, Jasenka Markeljevi}, Miroslav Mayer and Mirna
Senti}.
Former staff member: Zvonimir Horvat (a founder and former head of the department). Patients’ management and treatment
Since its foundation the Department of Clinical Immunology and Rheumatology has been involved in the complex diag- nostic procedures and treatment of patients with systemic connective tissue diseases, systemic vasculitis and primary and sec- ondary immunodeficiency. As will be discussed in more detail, the Department of Clinical Immunology initiated and actually(in fact) founded the first laboratory for specific immunodiagnostic procedures (see below). Concerning the specific treatment of autoimmune patients, clinical investigation protocols included patients with systemic lupus erythematosus, rheumatoid ar- thritis and systemic vasculitis (1–8). Application of intravenous cytotoxic drugs and immunoglobulins as a part of the treat- ment procedures and follow-up of patients is organized in an out-patient fashion (way). The Department is continuously in- troducing new treatment modalities in routine clinical practice. Corticosteroids were first introduced in the treatment of patients with systemic autoimmune diseases in 1951, specific cytototoxic and immunosuppressive drugs in 1960, whereas biologic ther- apy with specific monoclonal antibodies was introduced in 1999. The Department is a training center for subspecialty of clin- ical immunology and allergology as well as subspecialty in rheumatology.
Scientific activity
Four research projects supported by the Croatian Ministry of Science and Technology (project leaders: N. ^ike{, J. Markeljevi})
International collaboration activity:
EULAR: The European League against Rheumatism
1. EULAR Standing Committee for Education in Rheumatology – ESCET (N. ^ike{)
2. EULAR Standing Committee for International Clinical Studies including Therapeutic Trials-ESCISCIT (Branimir
Ani})
3. EULAR Standing Committee for young specialists-EURORITS (Miroslav Mayer);
4. Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA. Project: Unified musculoskeletal undergradu- ate curriculum.
Educational activity
Graduate and Postgraduate courses:
Period biol, Vol 110, Suppl 1, 2008. 101
Specialistic education
Training center for clinical specialties in internal medicine, physical medicine and rehabilitation, infectious diseases, sub- specialty in clinical immunology and allergology as well as in rheumatology. Teaching clinical immunology and rheumatol- ogy for various professional postgraduate medical courses, including dermatovenerology, neurology, ophthalmology, orthorhi- nolaryngology, etc.
DEPARTMENT OF LABORATORY IMMUNOLOGY
Staff members (2008): Drago Batini} (head), Klara Dubrav~i}, Ivana Frani}-[imi}, Ana Kozmar, Branko Malenica, Sanja
Mrsi}, Marija Rudolf
Former staff members: Mila Hr{ak, Antonio Jureti}, Maja Ka{telan, Ljerka Krajina, Matko Maru{i}, Mladen Petrove~ki,
Branka U`arevi}
Immunodiagnostic activity
Immunological tests have been used for the correct diagnosis and management of patients with several categories of dis- eases, such as allergic diseases, autoimmune diseases, neoplastic diseases, immunodeficiency diseases, as well as for histocom- patibility testing and transplantation. During the last three decades, there have been significant changes in methodologies and techniques used for immunodiagnostic purposes: from immunoelectrophoresis, immunodiffusion and radioimmunoassay to nephelometry, turbidimetry, ELISA, microchip and microsphere technology, from UV-microscopy to flow cytometry, from radioactive lymphocytes functional assay to various flow cytometric assays, from standard ELISA and microcytotoxic assays to microsphere technology and molecular biology.
Immunodiagnostics of systemic autoimmune diseases
Classical immunochemical methodologies such as electrophoresis and immunoelectrophoresis were introduced in 1960 in the routine work of the Laboratory for Protein Biochemistry and Immunochemistry at the Internal Clinic (9). More inten- sive immunodiagnostics of systemic autoimmune diseases began approximately three decades ago (1978) in the Laboratory for Serologic Immunodiagnostic of the Division of Clinical Immunology and Inflammatory Rheumatic Diseases (Internal Clinic Rebro). During this early period, the most common tests were LE-cells, anti-nuclear antibodies (ANA) using immu- nofluorescence on tissue imprints and rat liver sections, anti-dsDNA on C. Lucilliae, Waaler-Rose and latex-test for rheuma- toid factor (10–14), complement assays (CH50, C3 and C4) and anti-streptolysin titer (AST). Later , several »in house» ELISA formats for determination of anti-dsDNA and anti-cardiolipin autoantibodies were standardized for routine diagnostics (15). Over time, the number of tests in the panel have increased and today include ANA, anti-phospholipid antibody (aPL), anti- gamma globulins antibody (RF; rheumatoid factors), anti-streptolysin antibody (AST) and anti-cyclic citrullinated peptides antibody (CCP). In addition, CH50, complement components C3 and C4 and C1-inhibitor are performed routinely for mon- itoring of the complement system. All suspected sera for ANA-ENA (extractable nuclear antigens) are screened on Hep-2 cells. The target diagnostic autoantigens in positive are further analyzed by a multiplexed fluorescence microsphere assay (AtheNA-Multi-lyte ANA system – LuminexTM) (16) This system allows simultaneous detection of autoantibodies specific for double stranded DNA, histones, SS-A, SS-B, Sm, RNP, DNA topo I, Scl-70, centromere B (CENP B) and Jo-1. These autoantigens are helpful in differential diagnosis of various forms of systemic autoimmune diseases.
Besides immunodiagnostic work, a number of studies using flow cytometry have been performed to analyze various leu- kocyte subsets in patients with autoimmune diseases at diagnosis and during treatment (1, 2, 4, 17). During the last two years, research interest has been focused on regulatory T-cells enumeration in patients with systemic autoimmune diseases, and the peripheral blood cytokine profile in various conditions, including autoimmune disease and patients on hemodialysis (18). The latter has been done by a novel microchip technology that simultaneously detects a series of cytokines (19).
Immunodiagnostic of systemic vasculitis
Anti–neutrophil cytoplasmic antibodies (ANCA) characterize systemic small vessel vasculitis. All suspected sera are screened using »in house« ethanol fixed cytospin preparation of human granulocytes. The target diagnostic autoantigens (proteinase 3 – PR3, and myeloperoxidase – MPO) in positive sera are further determined by specific ELISA or immunoblotting. These assays are very sensitive, specific and helpful in the diagnosis of different types of small vessel vasculitis such as Wegener’s granulomatosis, microscopic poliangiitis, idiopathic glomerulonephritis and Churg-Strauss syndrome (20, 21). The antibod- ies against glomerular basement membrane (GBM) are also helpful in this respect.
Immunodiagnostics of autoimmune liver diseases
Autoantibodies against mitochondria (AMA), cell nucleus (ANA), smooth muscle (SMA), liver-kidney microsome type-1 antigen (LKM-1), cytosolic liver antigen type 1 (LC-1) and soluble liver antigen/liver pancreas antigen (SLA/LP) are useful diagnostic tools for the management of patients with suspected primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). The analysis of AMA and SMA using indirect immunofluorescence (IIF) was in- troduced in the lab routine three decades ago. During the last decade, the spectrum of autoantigens related to liver diseases has been broadened. All suspected sera are first screened on the appropriate tissue preparations (liver, kidney or stomach) and HEp-2 cells. The target diagnostic autoantigens in positive sera are further determined by antigen-specific ELISA or immu- noblotting. Immunoblotting allows simultaneous detection of autoantibodies specific for AMA-M2 (pyruvate-dehydrogenase complex), Sp100 (nuclear granular protein, nuclear dots), gp210 (integral protein of the nuclear membrane, nuclear pore com- plex), LKM-1 (cytochrome P450 II D6), LC-1 (forminotransferase-cyclodeaminase) and SLA/LP (UGA suppressor tRNA- associated protein). The detection of AMA is of great importance in the diagnosis of PBC. In accordance with the autoanti- body status it is possible to differentiate between the three types of AIH: type I (ANA, SMA), type II (LKM-1, LC-1) and type III (controversial; SLA/LP).
In collaboration with the Department of Gastroenterology (head Boris Vuceli}) and Referral Center for Chronic Hepatic Diseases (head Rajko Ostoji}) of the Clinical Hospital Center Zagreb, there has been a fruitful study on the diagnostic accu- racy of an atypical perinuclear anti-neutrophil cytoplasmic antibody (a/P-ANCA) in patients with AIH. The results show that a/P-ANCAs have importance in the diagnosis in AIH type I (22) and may also be helpful in the diagnosis of PSC.
Immunodiagnostics of inflammatory bowel diseases
The test for antibodies against parietal cells (PCA), neutrophil cytoplasmic antigens (ANCA), Saccharomyces cerevisiae (ASCA) and endomysium (EMA) have been used as diagnostic tools for the proper (correct) management of patients with suspected inflammatory bowel diseases, chronic atrophic gastritis, pernicious anemia and celiac disease. All suspected sera are first screened by immunofluorescence on the appropriate tissue preparations (stomach and esophagus) and smear of the Sac-
charomyces cerevisiae. The diagnostic target autoantigens in positive sera are determined with antigen-specific ELISAs. The
determination of the tissue transglutaminase (tTg) in EMA positive sera is of great importance in the diagnosis of celiac dis- ease. Autoantigens H+/K+ ATPase and intrinsic factor are helpful in the diagnosis of autoimmune gastritis and pernicious anemia.
During the last decade research interest has been focused on the diagnostic accuracy of the simultaneous determination of perinuclear anti-neutrophil cytoplasmic antibodies (a/P-ANCA) and ASCA in patients with inflammatory bowel diseases. The results of this study show that the simultaneous determination of these antibodies may be important in differential diag- nosis of ulcerative colitis (UC) and Crohn’s disease (CD) (23, 24).
Immunodiagnostic of autoimmune neurological diseases
Autoantibodies against various gangliosides, striated skeletal muscle, myelin and neurons have been used as laboratory tools in the proper (correct) management of patients with suspected autoimmune neurological diseases and especially for di- recting the search for underlying tumors. The laboratory determination of antibodies against myelin oligodendrocyte glyco- protein (MOG) and against myelin basic protein (MBP) may be helpful in the diagnosis of multiple sclerosis (MS). On the other hand, antibodies against gangliosides asialo-GM1, GM1, GM2, GD1a, GD1b and GQ1b are helpful in distinguishing acute neuropathies such as Guillan-Barré syndrome and its subtypes. Antibodies to myelin–associated glycoprotein (MAG) are associated with chronic inflammatory demyelinating polyradiculoneuropathy. The determination of the autoantibodies against acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) is a sensitive and specific laboratory test in the di- agnosis of myastenia gravis. The presence of these autoantibodies is routinely checked in the Laboratory for neurochemistry (head Milica Trbojevi}-^epe) as a part of the Department of Special and Molecular Biochemistry (head Dubravka ^vori{}ec). Antineuronal antibodies characterize paraneoplastic neurological diseases (PND). All suspected sera are first screened on the appropriate tissue preparations (primate cerebellum, intestinal tissue and peripheral nerve). The diagnostic target autoanti- gens in positive sera (cut off dilution 1:20) are detected by immunoblotting (IB). IB allows for simultaneous determination of autoantibodies specific for Hu (ANNA-1), Yo (PCA-1), Ri (ANNA-2), amphiphysin, Tr, CV2 and Ta antigens. Hu antigens are associated with small cell lung carcinoma (SCLC) and paraneoplastic encephalomyelitis/paraneoplastic sensory neurop- athy (PEM/PSN), Hu and Ta with SCLC and testicular tumors and paraneoplastic encephalitis (PLE), Yo with ovarian, breast and uterus tumors and paraneoplastic cerebellar degeneration (PCD), Ri with breast and SCLC and opsoclonus/my- oclonus syndrome (OMS) and amphiphysin with breast and paraneoplastic Stiff-Person syndrome (SPS).
Period biol, Vol 110, Suppl 1, 2008. 103
Immunodiagnostics of autoimmune skin diseases and hypersensitivity
Immunodiagnostics of autoimmune skin diseases (by direct and indirect immunofluorescence) and skin testing with var- ious allergens are performed by specialists in the Dermatovenerology Clinic (head Jasna Lipozen~i}) of the Clinical Hospital Center Zagreb. The laboratory testing for skin allergy includes total and specific IgE to »classical« allergens. Hypersensitive reactions to various drugs are determined by the lymphocyte transformation test (LTT) and cellular antigen stimulation test (CAST) in vitro (25–26).
The immune status and diagnostics of primary and secondary immunodeficiencies
Diagnostic activities of the Department of Immunology have been intensively focused on primary and secondary immu- nodeficiencies. The pioneering work in that sense have been studies on the immune status in various pathologic conditions, using at the time standard methods for lymphocyte enumeration (E-rosettes and immunofluorescent surface Ig) and func- tional lymphocyte proliferation assay, respectively (27–29).
The immune status in various pathologic conditions and the functioning of the immune cells was a continuous subject of the research of staff members (1, 2, 4, 30, 31).
During the 1990s , activities were focused on primary immune deficiency (PID) diseases which sometimes represent a real diagnostic challenge. Thanks to fruitful collaboration with pediatricians within the Clinical Hospital Center Zagreb and other pediatric units in Croatia, a number of cases with various forms of immunodeficiencies have been determined and success- fully treated by substitution therapy or bone marrow transplantation (BMT). Among them are children with various forms of severe combined immunodeficiency (SCID), Brutton’s agammaglobulinemia, hyper-IgM syndrome and chronic granuloma- tous disease (CGD). In addition to standard flow cytometric (FC) lymphocyte enumeration, a number of complex and time- consuming cellular assays based on FC have been developed and introduced, including lymphocyte proliferation assay, CD40- ligand assay, phagocytosis and respiratory burst.
As previously mentioned, the early 1990s witnessed significant work on the analysis of specific lymphocyte subsets in pa- tients with systemic autoimmune diseases (1, 2, 4). Recently, the laboratory introduced several new FC assays, including those for enumeration of regulatory T-cells (Tregs) and allergen-activated peripheral blood.
Immunophenotyping of leukemia, lymphoma and other hematological diseases
Immunological classification of leukemia and lymphoma began during the late 1970s at the Clinical Hospital Center Za- greb (32, 33) and soon after at the Ru|er Bo{kovi} Institute (34, 35).
The older methods (such as E-rosettes) were soon replaced by monoclonal antibodies (33–36) and flow cytometry (37–40). Namely, the Division of Immunology of the Department of Clinical Laboratory Diagnostics was the first institution in this part of Europe (Middle and South-East Europe) to purchase and install fluorescence activated cell sorter (flow cytometer, FC). The Coulter’s cell sorter EPICS was installed in 1986 and since then this technology has been engaged (used) in rou- tine diagnostics of immunological and hematological diseases, as well as in scientific and educational activities within Repub- lic of Croatia and at international level.
From the very beginning, the machine (apparatus?) was used for lymphocyte enumeration in various clinical conditions
(1, 2, 4, 17, 31, 41), but was especially valuable for the immunophenotyping of leukemia and lymphoma (39, 42) and DNA-
analysis of tumors (43). At that time, novel approaches to the immunodiagnosis of leukemia/lymphoma were tested, such as computer assisted method for recognition of leukemia/lymphoma phenotype pattern (44).
Although for many years it served as an unofficial referral center for immunophenotyping of leukemia and lymphoma, in 1998 the Department of Immunology finally became the official National Referral Center for Immunodiagnostics of Hema- tological and Immunological Diseases for the Croatian Ministry of Health and Welfare.
Besides routine immunodiagnostic and scientific activities, laboratory members have been engaged in several international studies, especially those dealing with treatment of adult acute leukemia (EORTC Leukemia Group) and pediatric acute leuke- mia (BFM; ALL-IC group) (46). Recently, a significant amount of work has been devoted to the problem of minimal residual disease (MRD) in acute leukemia, again in the context of international collaborative studies, especially BFM-ALL-IC for pediatric leukemia (46).
In addition to hematological neoplasms, the laboratory has gradually developed and introduced many valuable flow cyto- metric assays, including CD34+ enumeration, assay for paroxysmal nocturnal hemoglobinuria (PNH), analysis of platelet glycoproteins, leukocyte functional assays such as respiratory burst, etc. (47).
Hematopoietic stem cell transplantation
From the very beginning, the Department of Immunology was actively involved in the field of hematopoietic stem cell transplantation (HSCT). (In that sense) Consequently, flow cytometry (41) and short term bone marrow culture for the de- tection of colony forming units (CFU) (48) were important for the development of a clinical stem cell program and corre- sponding scientific activities carried out in collaboration with the Department of Hematology. Besides routine diagnostics in patients undergoing HSCT, the laboratory was actively engaged in the immunological follow-up of patients after HSCT and research work dealing with cellular composition of bone marrow transplant (41).
The Immunology Laboratory started to play an even more critical role during the early 1990s , when autologous HSCT came to light: enumeration of hematopoietic CD34+ stem/progenitor cells in peripheral blood and leukapheresis product (i.e. transplant) by FC, as well as short term bone marrow culture, became essential laboratory tools for planning and per- forming auto-HSCT (49). Although modified over time, these two methods have preserved their importance for auto-HSCT until today. In parallel with diagnostic CD34+ enumeration, the Department of Immunology was actively involved in the first clinical program of CD34+ selection for auto-HSCT. Namely, in 1994 the laboratory staff performed the first CD34+ positive selection from leukapheresis product, using an affinity cell-chromatography-based device (CellProTM) (49).
During next two years, a total of 40 CD34+ selections for auto-HSCT and two combined procedures (CD34+ selection followed by T-cell depletion) for haploidentical allo-HSCT were performed. By the end of the 1990s , the chromatography- based CD34+ selection was replaced by an immunomagnetic one routinely performed by specialist transfusiologists.
It should be mentioned that the Clinical Hospital Center (quickly) recognized the value of umbilical cord blood as a po- tential source of hematopoietic stem/progenitor cells for transplantation (50). Soon after, a study of cord blood was performed including lymphocyte subsets enumeration, CD34+ content and CFU-content (V. [neler, unpublished). Fifteen years after those pioneering steps, the Clinical Hospital Center established the Cord Blood Bank »Ana Rukavina« to serve public and private needs for hematopoietic cells (51).
Immunocytogenetic analysis of hematological and solid malignant tumors
The Laboratory of Immunocytognetics was formed (established) during the late 1990s to serve increasing demands for accurate cytogentic and molecular diagnosis of various hematological neoplastic diseases as well as solid tumors. In addition to conventional cytogenetics, the laboratory soon introduced sophisticated molecular methodologies, including M-FISH and I-FISH (45, 52). FISH technology has been used in several collaborative research sudies, especially a study investigating te- lomere dynamics in human tumor cell line (53).
Scientific activity
Six research projects supported by the Croatian Ministry of Science and Technology (project leaders: M. Maru{i}, D. Batini}, B. Malenica)
Five collaborative projects supported by the Croatian Ministry of Science and Technology (project leaders: B. Labar, H. Banfi}, B. Vuceli})
International collaboration activity
1. UEMS, Union European of Medical Specialty, Section of Medical Biopathology-Immunology Division (B. Malenica) 2. EORTC, European Organization for Research and Treatment of Cancer (D. Batini})
3. BFM, International cooperative study on diagnosis and treatment of childhood acute lymphoblastic leukemia (ALL-IC) (D. Batini}, Klara Dubrav~i})(46).
Educational activity
Graduate and Postgraduate courses:
1. Zagreb University School of Medicine (D. Batini}, B. Malenica) 2. Mostar University School of Medicine, BIH (D. Batini})