EUROIMMUN Medizinische Labordiagnostika AG EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail [email protected]MV_5110_I_UK_A02, 04/2009 HLA-B27 determination using EUROArray A molecular genetic microarray test system for the diagnosis of ankylosing spondylitis Easy test performance due to prefabricated PCR components and known incuba- tion technique Simple protocol – expert knowledge in molecular biology is not required Fully automated unbiased evaluation and diagnosis using EUROArrayScan Highest sensitivity: all known HLA-B27 alleles are detected Improved specificity: non-disease-associated HLA-B27 alleles are detected Many integrated control mechanisms document correct test performance Test system, scanner and software are validated in accordance with IVD guide- lines In contrast to cytotoxicity tests, blood samples can be collected and processed together, e.g. once per week; the test performance can be interrupted at different steps and resumed the next working day • • • • • • • • Order entry and automatic protocol creation (EUROArrayScan software) DNA extraction from EDTA blood PCR amplification using HLA-B27 specific primers Microarray hybridisation Scanning of reactions using Microarray scanner Data analysis and creation of result report (EUROArrayScan software) HLA-B27 Microarray test system EUROIMMUN Microarray scanner Evaluation using EUROArrayScan Protocol for each individual report Test performance 1 mm
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EUROIMMUNM e d i z i n i s c h eL a b o r d i a g n o s t i k aA G
HLA-B27 determination using EUROArrayA molecular genetic microarray test system for the diagnosis
of ankylosing spondylitis
Easy test performance due to prefabricated PCR components and known incuba-tion technique
Simple protocol – expert knowledge in molecular biology is not required
Fully automated unbiased evaluation and diagnosis using EUROArrayScan
Highest sensitivity: all known HLA-B27 alleles are detected
Improved specifi city: non-disease-associated HLA-B27 alleles are detected
Many integrated control mechanisms document correct test performance
Test system, scanner and software are validated in accordance with IVD guide-lines
In contrast to cytotoxicity tests, blood samples can be collected and processed together, e.g. once per week; the test performance can be interrupted at different steps and resumed the next working day
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Order entry and
automatic protocol creation
(EUROArrayScan software)
DNA extraction from EDTA blood
PCR amplifi cation using HLA-B27
specifi c primers
Microarray hybridisation
Scanning of reactions using
Microarray scanner
Data analysis and creation of
result report (EUROArrayScan
software)
HLA-B27 Microarray test system EUROIMMUN Microarray scanner
Evaluation using EUROArrayScan Protocol for each individual reportTest performance
1 mm
EUROIMMUNM e d i z i n i s c h eL a b o r d i a g n o s t i k aA G
Other mosaics also available Special substrate combinations on request
* Currently not available as IVD in the EU.
Made in Germany
Indication: Test system for the determination of disease-associated HLA-B*27 alleles in human, genomic DNA for the diagnosis of the following diseases: Bechterew’s disease (spondylitis anky-losans, ankylosing spondylitis, AS), Reiter’s disease (combination of symptoms from urethritis, conjunctivitis/uveitis, arthritis), reactive arthritis (para-/postinfectious arthritis), acute uveitis an-terior or acute iridocyclitis, periarthritis (periarthropathia) humeroscapularis, arthritis psoriatica (psoriasis arthritis), juvenile idiopathic arthritis, enteropathies (chronic-inflammatory bowel dis-eases).
Clinical significance: Human leukocyte antigens (HLA) are tissue antigens (membrane-associated glycoproteins) of the human major histocompatibility complex (MHC). HLA-B belongs to the HLA antigens of class I (also called MHC I antigens) which are present in all nucleus-containing body cells. Their function is the control of the T-cell-mediated immune response. Due to an extreme genetic polymorphism there are a large number of HLA phenotypes. For HLA-B over 1000 differ-ent alleles have been described. The HLA-B*27 allele alone can be divided into 51 subtypes, which differ only in a few bases (B*2701 to B*2743).
The membrane protein HLA-B27 is associated with the occurrence of several autoimmune dis-eases. In Western Europe the prevalence of HLA-B27 is around 6 to 9%. When HLA-B27 is present, the relative risk of Bechterew’s disease (ankylosing spondylitis) is 87.4, for Reiter’s disease (symp-tom combination urethritis, conjuncativitiy(uveitis, arthritis) 37, for reactive arthritis (para-/post-infection arthritis) 14 to 21 (depending on the infectious agent), for acute uveitis anterior or acute iridocyclitis 10.4, for periarthritis (periarthropathy) humeroscapularis 6.3, for arthritis psoriatica (psoriasis arthritis) 4 and for juvenile idiopathic arthritis 3.2. Enteropathies (chronic-inflammatory bowel diseases) are also associated with HLA-B27.
Bechterew’s disease is a chronic inflammatory rheumatic disease of the axial skeleton (spinal column, iliosacral joints, pubic symphysis and facet joints), the extremity joints and tendon inser-tions. It affects mainly men between the ages of 15 and 30. There is a clear relationship between the disease and the presence of HLA-B27. Around 90% of Bechterew’s disease patients are car-riers of this tissue antigen, in particular subtypes B*2702, B*2704 and B*2705. Subtypes B*2706 and B*2709 are, on the other hand, not associated with this disease. HLA-B*27 determination is therefore important for differential diagnostics.
Application of the EUROArray HLA-B27: HLA-B27 can be determined accurately and precisely with molecular biological methods via the detection of the corresponding allele (HLA-B*27) in the genomic DNA of the patient. The method competes with the lymphocytotoxicitiy tests used up until now. In particular, PCR (polymerase chain reaction) using allele-specific primers has the potential to deliver reliable results, especially for the various HLA-B*27 subtypes. Because of cross reactions with antibodies (with e.g. HLA-B7) and potential false-negative results in immu-nophenotyping when HLA-B*27 expression is low, molecular genetic determination of HLA-B*27 is more specific and sensitive than serological methods. In this EUROIMMUN test system the HLA-B*27 primers are chosen and optimised so that all currently known HLA-B*27 subtypes are detected. Furthermore, when a positive result is obtained, it is indicated whether subtypes HLA-B*2706 or HLA-B*2709 could be involved. These two subtypes are not associated with the occur-rence of ankylosing spondylitis.
EUROArray HLA-B27 Molecular genetic microarray test system
Evaluation using EUROArrayScan Protocol for each individual report
EUROIMMUNM e d i z i n i s c h eL a b o r d i a g n o s t i k aA G
Test principle: The EUROArray is an in vitro test for molec-ular genetic determination of disease-associated HLA-B*27 alleles in human, genomic DNA. In the first reaction step, two sections (exon 2 and exon 3) of the HLA-B gene and a ß-globin gene fragment as positive control are amplified from a genomic patient DNA sample using the polymerase chain reaction (PCR). The HLA-B gene sections are only amplified if the sample contains an HLA-B*27 allele. All PCR products are labelled with a fluorescence dye as they are produced. In the second reaction step, the products are analysed using the microarray, which contains immobi-lised probes that are complementary to the amplified DNA. The specific binding (hybridisation) of the fluorescing PCR product to the corresponding microarray spot is detected using a EUROIMMUN microarray scanner. A fluorescence signal on the HLA-B*27-specific spots indicates the pres-ence of an HLA-B*27 allele in the patient DNA sample.
Test procedure: The PCRs are first incubated in the thermo-cycler and then, using the TITERPLANE™ technique, on EUROArray slides containing microar-ray BIOCHIPs. Scanning and evaluation are performed using the EUROIMMUN Microarray scan-ner and the EUROArrayScan programme. The programme enables fully automated evaluation of EUROArray analyses and detailed documentation of results.
Sensitivity and specificity: The microarray test system detects all HLA-B*27 subtypes and indicates the possible presence of the non-disease associated subtypes HLA-B*2706 and HLA-B*2709.
Robustness: A total of 106 DNA samples from blood donors were investigated simultaneously in one test run. All determinations were successful.
Prevalence: In the investigation of 106 randomly selected samples 9.4% of cases were positive. This value corresponds to the known prevalence of the marker in Central European popula-tion.
Technical data:
Substrate Single-stranded DNA probes, length: 20 to 50 nucleotides.
Test procedure 60 min (PCR) / 60 min (hybridisation) / 5 min (automated evaluation).
Reagents Ready for use.
Controls Integrated HLA-B27 positive control (no additional positive control re-quired). Integrated DNA positive control.
CE-IVD certified Complete process incl. DNA extraction is validated.
Test kit format 20 slides, each containing 5 test fields.
Order number MN 5110-2005
Test characteristics Molecular genetic microarray test system
Reference samples nReference method
Sensitivity Specificity
EDTA blood samples from patients and blood donors, Germany(15 precharacterised as HLA-B*27 positive, 48 as HLA-B*27 negative)
63molecular
genetic100 % 100 %
DNA samples from patients and blood donors, Germany(30 precharacterised as HLA-B*27 positive, 30 as HLA-B*27 negative)
60molecular
genetic100 % 100 %
DNA samples from the “International Histocompatibility Working Group” (IHWG): “Sequence Polymorphism Reference Panel (SP Reference Panel)” and “HLA (Anthropology) Reference Panel” (6 precharacterised as HLA-B*27 positive, 49 as HLA-B*27 negative)