Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

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Malenica B.S01-1: Immunodiagnosis of paraneoplastic syndrome. Biochemia Medica 2009;19(Suppl 1):S18.
Department of Immunology and Referral Center for Clinical-laboratory Immunodiagnosis of Immunological and Hematological Diseases, Ministry of Health, Republic of Croatia, and Clinical Institute of Laboratory Diagnosis, Zagreb Clinical Hospital Center, Zagreb, Croatia
Corresponding author:b_malenica [at] yahoo [dot] com
Paraneoplastic neurological syndromes (PNS) occur very rarely in patients with cancer provided that they are not caused by invasion of the tumor or its metastases, by infection, ischemic or metabolic disorders, or neurotoxicity of the standard cancer treatments such as a radiotherapy and chemotherapy. Immunologic factors appear important in the pathogenesis of PNS because anti-neuronal autoantibodies and T-cell responses against nervous system antigens have been defined for many of these disorders. Immunologic response is elicited by ectopic expression of neuronal antigens by the tumor. The clinical presentation of neurological symptoms is caused by autoimmune reactions directed against antigens common to both the cancer and the nervous system, designated as “onconeuronal“ antigens. Paraneoplastic neurological syndrome can affect any part of the central and peripheral nervous system. In recent years, a few specific antibodies directed against different „onconeuronal“ antigens have been identified and molecularly characterized by serologic screening of complementary DNA (cDNA) expression libraries of patients with PNS. In routine laboratory immunodiagnosis, these autoantibodies are detected by indirect immunofluorescence (IIF) on primate tissue preparations of cerebellum, peripheral nerve and fetal intestine (screening test) and by Western blot analysis with specific recombinant antigens (confirmation test). Diagnostic paraneoplastic autoantibodies directed against “onconeuronal“ antigens recognize neuronal nuclear proteins (anti-Hu-ANNA-1; anti-Ri-NOVA1, NOVA2-ANNA-2; anti-MA2) and neuronal cytoplasmic proteins (anti-Yo- CDR2-PCA-1; anti-Tr-PCA-Tr; anti-CV2-CRMP5; anti-amphiphysin). All these paraneoplastic autoantibodies are included in diagnostic criteria in reaching decision between “definite“ and only “possible“ paraneoplastic neurological syndromes. One or more positive neuronal nuclear or neuronal cytoplasmic autoantibodies observed in patients with paraneoplastic disorders are connected with specific neurological syndrome and with patient’s neoplasm.
Kulić A1, Vrbanec D2. S01-2: The role of autoantibodies in early detection of cancer. Biochemia Medica 2009;19(Suppl 1):S19.
1Department of Pathophysiology, Zagreb University Hospital Center, Zagreb, Croatia
2Department of Medical Oncology, Zagreb University Hospital Center, Zagreb, Croatia
Corresponding author: ana [dot] kulic1 [at] zg [dot] t-com [dot] hr
An ideal tumor marker would be assessable by simple blood test, would have potential to detect cancer and its levels would correlate with the stage and progression of malignant disease. During past thirty years many proteins, hormones and enzymes have been used as tumor markers. However, due to the lack of sensitivity and specificity no one of these markers could fulfill the criteria of ideal tumor marker. Detection of various autoantibodies in serum of cancer patients has opened a new gateway for cancer diagnosis. These antibodies have been detected in serum of patients with cancer of various cell types. The factors that may have impact in pathogenesis of these autoantibodies include: host immune response to tumor-associated antigens, antigen release from destroyed cells and immune dysregulation of the host induced by neoplastic process. The regulation and localization of various proteins may be different in tumor cells in comparison with their normal counterparts. For example two types of cAMP-dependent protein kinase (PKA) are localized strictly intracellularly in normal cells, while PKA is secreted in the medium by various cancer cells. That secreted form is called extracellular PKA (ECPKA) and it is accompanied with host immune response. Autoantibodies to ECPKA could be detected in serum using immunoassay method (EIA). Autoantibodies against ECPKA have been detected in various tumors including carcinoma of the lung, breast and prostate. EIA method measures the anti-IgG autoantibodies to ECPKA. This method has shown 90% sensitivity and 87% specificity compared to the 83% sensitivity and 80% specificity of the method that detects directly PKA (PKA enzymatic essay). Higher sensitivity and specificity has been observed in testing various other autoantibodies to tumor markers like VEGF, CA125 and AFP, in comparison with conventional methods. Detection of autoantibodies may be helpful in distinguishing the elevated levels of tumor markers caused by cancer from elevated levels due to inflammation. In conclusion, detection of autoantibodies in serum may be valuable new approach in early diagnosis of malignant disease. The advantages of this approach are its increased sensitivity and specificity.
Tešija Kuna A.S01-3: News in serologic diagnosis of celiac disease. Biochemia Medica 2009;19(Suppl 1):S20.
University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
Corresponding author:andrea [dot] kuna [at] gmail [dot] com
Celiac disease (CD) is a chronic inflammatory autoimmune disease induced in genetically susceptible individuals by dietary gluten (a mixture of proteins present exclusively in cereals) with interaction of other environmental cofactors. Disease is characterized by flattened villi on the small bowel mucosa, increased number of intraepithelial lymphocytes and crypt hyperplasia. Due to the diverse clinical heterogenity ranging from asymptomatic to severely malnourished patients, CD has been seriously underdiagnosed worldwide.
Serology represents an essential part in the diagnostic evaluation of CD but still, histology of small bowel biopsies is regarded as the gold standard for diagnosis. The development of new tests and technologies has improved the testing options for CD. Nowadays, the measurement of antibodies against native gliadin (AGA) in diagnosing CD becomes obsolete owing to its lack of sensitivity and specificity. The new recommended screening panel for CD comprise the measurement of total IgA (in order to exclude the IgA deficiency) followed with measurement of IgA class anti-tissue transglutaminase (anti-tTG) autoantibodies, utilizing recombinant or purified human tTG as antigen in test. Although historical anti-endomysial autoantibodies (EMA) are slightly more specific than anti-tTG, the interpretation of the test result is affected by subjectivity and therefore mostly replaced by tTG test. Both tests have very high negative predictive value, while positive test results identify patients that should undergo small-bowel biopsy in order to confirm CD. In the case of IgA deficiency, measurement of anti-tTG-IgG is recommended. Recently, specific antibodies against synthetic peptides derived from partially deamidated gliadin peptides (DGP) through the action of tTG, were described. According to present data, anti-DGP test has comparable sensitivity and specificity as that of anti-tTG for CD and should be used in the event of anti-tTG negative or equivocal result in patients with strong clinical suspicion of CD. On the other hand, anti-DGP test perform better than anti-tTG in the assessment of dietary compliance and mucosal recovery.
Salamunić I.S01-4:Laboratory diagnosis of autoimmune diseases – new technologies, old dilemmas. Biochemia Medica 2009;19(Suppl 1):S21-S22.
Department of Medical Laboratory Diagnosis, Split University Hospital Center, Split, Croatia
Corresponding author:ilza [dot] salamunic [at] gmail [dot] com
Autoimmune disease occurs when the body’s immune system begins to attack its own antigens. A hallmark is the production of high-affinity autoantibodies. The diagnosis of autoimmune diseases depends on the identification of disease-associated clinical symptoms and is associated with the detection of autoantibodies.
Autoimmunity laboratories analyse and measure an increasing number of autoantibodies employing broad spectrum of techniques and methods. Conventional immunoassay methods were first developed in 1957. Since then, several technologies have been developed and used in clinical practice. Immunochemical methods like immunofluorescence (IIF), passive agglutination (PA), immunodiffusion (ID), immunoprecipitation (IPA), immunoblot (IB), have allowed qualitative research defining the presence or absence of autoantibodies in the serum of patients. Immunometric methods like radioimmunoassay (RIA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), have allowed quantitative measurement of antibody concentrations. A variety of techniques have been used to developed specific tests for antinuclear antibody (ANA). IIF method used of HEp-2 cells as substrate has remained the gold standard for ANA detection. This highly sensitive and specific method has some limitations. The HEp-2 antinuclear antibody EIA (HEp-2 ANA EIA) is automated method with high reproducibility and with internal calibration as a basis for standardisation. But, for example, evaluation of clinically well-defined samples in case of scleroderma patients, HEp-2 ANA EIA yielded a lower rate of positive results compared to ANA IIF. The accurate determination of single antibody specificities requires a perfect understanding of their clinical significance, as well as the biochemical characteristics of the target antigens and selection the most appropriate test systems. Many studies conducted under standardized conditions showed the analytical variability of different test systems. Results of these studies underline the need for a drastic standardization of the procedures used and the importance of independent calibrators or international standards.
Multiplex technologies for the study of autoantibody profiles are the new technologies. The possibility of simultaneous measuring a number of correlated analytes (multiplexing) overcomes some limitations of conventional methods. However, the data do not correlate well with results obtained from IIF testing or EIA suggesting high rates of both false positive and false negative results.
Results obtained in laboratories or in different clinical studies are not interchangeable always, which impairs evidence-based medicine (EBM).
The application of proteomic technologies to the diagnosis of autoimmune disease has opened up new diagnostics possibility which may revolutionize diagnostics procedures in future.
Standardisation of autoantibody assay „old“ or „new“ is critical to their use in the clinic to predict diagnose and treat a very diverse group of autoimmune disorders.
The Autoantibody Standardisation Committe (ASC) was established in the early 1980s based on the recognised needs for reference human autoimmune sera in standardisation. European Autoimmunity Standardisation Initiative (EASI) was formed to discuss how interaction between laboratories and clinics could be improved in practice, how algorithms in autoantibody testing could be harmonized, and what an international concept of standardisation of diagnostic strategies in this area could be look like.
Matišić D.S01-5: New strategy in diagnosis of monoclonal gammopathies. Biochemia Medica 2009;19(Suppl 1):S22-S23.
Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb, Croatia
Corresponding author:dmatisic [at] kbc-zagreb [dot] hr
Monoclonal gammopathies (MG) belong to the group of immunoproliferative diseases characterized by the presence of monoclonal components in serum and/or urine that are produced by B-cell clone subjected to genetic transformation. Such condition can be the consequence of benign or malignant disease that manifests in clinical symptoms from asymptomatic nonprogressive stage to malignant aggressive multiple myeloma or lymphoma. Laboratory diagnosis of MG comprises determination of serum total proteins, serum protein electrophoresis (SPE), immunoglobulins G, A and M (IgG, IgA, IgM), definition of the class and type of possibly present monoclonal immunoglobulin (M Ig), beta-2-microglobulin, creatinine and serum calcium, and confirmation of monoclonal free light chains (BJP) by immunofixation (IF) in serum and 24-h urine.
The development of the Free Light Chain reagent enabled routine quantitative determination of kappa and lambda type BJP, and increased the clinical significance of BJP.
Serum BJP concentration correlates only with bone marrow activity, indicating that BJP finding can be an independent marker of MG. A particularly important aspect of BJP is their short half-life: 2-3 hours for kappa and 5-6 hours for lambda, which is a 150-fold shorter period than 21 days that is, e.g., an IgG half-life. Thus, BJP concentration allows very rapid assessment of chemotherapy effects: 92-128 days faster than M Ig concentration.
BJP concentration depends on the production of plasma cells, but also on renal clearance. Polyclonal immunoglobulin synthesis and/or kidney lesion may result in 10-20-fold increased synthesis of serum BJP. Renal BJP excretion increases with BJP serum concentration but significantly drops when the high concentration is combined with kidney lesion. In two separate studies, Katzmann JA. and Hill PG. showed on a high number of patients that false positive results of serum BJP, and false negative in urine, can be found due to kidney lesion (in approximately 25% of MG patients).
BJP accumulation is the direct cause of pathological processes in tubulointerstitial lesion. After filtration, BJP are bound to heteromeric receptors that consist of cubilin and megalin and thus initiate reabsorption into proximal tubules. Following endocytosis and hydrolysis, BJP return as amino acids into circulation. BJP are toxic due to possible interactions with all nephron segments. Nephrotoxic potential of BJP is dependent on their physico-chemical content and on the impact of their environment.
The collection of 24-h urine is cumbersome for patients and mostly incorrect. The laboratory procedure of monoclonal protein evaluation is considerably prolonged due to 24-h urine analysis. This is why many clinicians suggest that 24-h urine should be eliminated from protein M screening. Urine is to be analyzed discontinuously, i.e. in case of various disproteinemias and for monitoring diseases after a monoclonal protein had already been identified.
The application of the program of serum total protein determination, a quantitative determination of kappa and lambda type BJP in serum and of their ratio eliminates the need to collect 24-h urine, which considerably reduces the process of defining monoclonal components.
Dodig S.S01-6: Interferences special to immunoassays. Biochemia Medica 2009;19(Suppl 1):S24-S25.
Srebrnjak Children’s Hospital, Reference Center for Clinical Allergology in Children, Zagreb, Croatia
Corresponding author:slavica-dodig [at] zg [dot] t-com [dot] hr
The main feature of immunoassays - from immunoprecipitation to micro-arrays - is that an antibody used as a reagent, detects an analyte (antigen) of interest. Although the noncovalent bound between analyte and antibody is specific, false-positive and false-negative interferences are possible. Interferences in immunoassays could be detected: A. during development/evaluation of an assay, and B. during routine use. The later are: 1. cross-reactivity with endogenous and exogenous non antibody-structured substances, 2. cross-reactivity with endogenous and exogenous antibody-structuree substances 3. the hook effect, and 4. the matrix effect.
1. Cross-reactivity with endogenous and exogenous non antibody-structured substances: Cross-reactivity (the most common interference in competitive immunoassays) imples that non-specific influence of substances occures in a sample that structurally resembles analyte and competes for binding site on antibody. The interference grade depends on three factors: antibody specificity, test design and sample preparation. Interference most often occurs: during determination of hormone concentration, drugs and allergene-specific IgE, respectively.
2. Cross-reactivity with endogenous and exogenous antibody-structured substances: Immunoreaction can be affected by antibodies present in biological samples of a patient or antibodies from the reagent. Human samples can contain exogenous (i.e. biological drugs) and endogenous antibodies (heterophilic antibodies, i.e. natural antibodies and autoantibodies and anti-animal antibodies, i.e. human anti-animal antibodies-HAAAs). Endogenous antibodies could be found in about 40% of patients, especially in those who were subjected to immunotherapy with monoclonal antibodies.
3. Prozone effect: The prozone effect (hook effect) is based on the saturation curve of antibody with antigen. Primarily prozone effect depends on analyte concentration. It implies the presence of huge excess of analyte which saturates all binding sites on antibody, and it occurs mostly in assays where antigen, antibody and marker, respectively, incubate simultaneously (single step assay). The prozone effect does not occur in competitive immunoassays.
4. The matrix effect: Each component of immunoassays (biological sample, buffer, antibody, additive) has its own matrix. All of them, whether coming from biological sample (lipids, proteins, carbohydrates, salt, water), or from exogenous sources, can affect the target analyte to be measured. Endogenous elements cause inter- and intra-individual variability of results. Matrix components have low affinity of binding to analyte or antibody - usually disguise the analyte or the antibody causing the absence of the binding reaction of analyte to antibody.

In the future, in some areas (esspecially associated with implementation of biological drugs) interferences will increase, and in other areas (implementation of high specificity immunoassays) interferences will recede. However, problems of interferences in immunoassays will persist. Therefore, the laboratory medicine experts, the physicians and the experts who develop the immunoassays must be aware of these problems, and to minimize them to a lesser extent.