Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using HEp-20-10 substrate.

Interpretation: Positive IF (nucleus) at titre 1/80 or more. IF patterns suggest ANA specificity.

Clinical significance: ANA is a screening test for suspected connective tissue disease. Positive result, especially high titres indicate a possible connective tissue disease. Negative result in 95% to 99% of cases rule out systemic lupus erythematosus (SLE). ANA is also detected (low titre) in other autoimmune diseases such as chronic active hepatitis (CAH), Sjögren’s syndrome, primary biliary cirrhosis (PBC) and rheumatoid arthritis (RA).

Result: Within 8 working days.

Note: If ANA is positive, confirm with test for anti-dsDNA, anti-Sm and anti-RNP for SLE [see Anti- Extractable Nuclear Antigens (ENA)].

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Flourescene Enzyme ImmunoAssay (FEIA) 

Clinical significance: High titre is suggestive of active SLE, however it is negative in 25% of SLE patients.

Result: Within 8 working days.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using tissue section as substrate.

Interpretation: Positive IF at titre of 1/20 or more.

Clinical significance: High titre of ASMA is detected in patients with autoimmune chronic active hepatitis (CAH) (60-70%), lupoid hepatitis (80%) and other liver diseases including Epstein Barr viral infection.

Result: Within 10 working days.

Note: Tests are done together with AMA, LKM and APCA

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using tissue section as substrate.

Interpretation: Positive IF at titre of 1/20 or more.

Clinical significance: High titre of AMA is detected in 90% patients with primary biliary cirrhosis (PBC) but also found in other liver diseases (low titre), syphilis, connective tissue disorders and myocarditis.

Result: Within 10 working days.

Note: Tests are done together with ASMA, LKM and APCA.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using tissue section as substrate.

Interpretation: A specific staining is seen in the cytoplasm of proximal renal tubules but not in distal. The liver shows homogenous staining of the hepatocytes and there is no staining seen in the stomach.

Clinical significance: Antibodies that bind to cytochrome p450 and are common associated to type 2 autoimmune hepatitis that predominantly occurs in a subgroup of girls and young women (80% of prevalence). They can be also associated with hepatitis C.

Result: Within 10 working days.

Note: Tests are done together with ASMA, AMA and APC.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using tissue section as substrate.

Interpretation: Finely granular fluorescence of the parietal cells in the gastric mucous membrane indicates APCA. Since AMA also reacts with parietal cells, anti-mitochondrial antibodies (renal tubules) should be excluded in the APCA assessment.

Clinical significance: Circulating antibodies against the sctructures of the parietal cell of the gastric mucosa are generally due to pernicious anemia. They may, however, also be detected with other deseases of the stomach (chronic atrophic gastritis, gastric ulser), diseases of the thyroid (Hashimoto’s thyroiditis, myxedema) and more rarely with hypoferric anemia, diabetes mellitus and in older patients.

Result: Within 10 working days.

Note: Tests are done together with ASMA, AMA and LKM.

Extractable Nuclear Antigen Antibodies (dsDNA, Nucleosomes, Histones, SmD1, PCNA, RPP/PO, SS-A/Ro 60kD, SS-A/Ro 52 kD, SS-B/La, CENP-B, Scl-70, U1-snRNP, AMA M2, Jo-1, PM-Scl, Mi-2 & Ku)

Type of specimen: Minimum 3 ml of blood in plain tube

Method/Principle: The test is based on the principle of an enzyme immunoassay. The test strip is composed of a membrane fixed on a plastic support. The intensity of the coloration is directly proportional to the amount of antibody present in the sample.
Interpretation: A sample is positive for a specific antibody if the colour intensity of the corresponding Antigen Dot is higher than the intensity of the Negative Control Dot. A sample is negative for a specific antibody if the colour intensity of the corresponding Antigen Dot is lower or equal than the intensity of the Negative Control Dot.

Clinical significance: Anti-Sm is specific for SLE (30-40%), but negative result does not rule out SLE. Anti-RNP is detected in mixed connective tissue diaseses (MCTD) (95-100%) but also in SLE and scleroderma. Anti-SS-A occurs in SLE (25%), Sjogren syndrome (40-50%), congenital complete heart block, cutaneous lupus and is a serologic marker for neonatal lupus. Patients with anti-SS-B most commonly have Sjogren syndrome (23%) and SLE (5-10%) and Sjogren syndrome sicca complex (60%) and neonatal lupus. Anti-Scl-70 occurs in 70% of patients with progressive systemic sclerosis (PSS) and is a specific marker for PSS. Anti-Jo-1 is detected in patients with polymyositis/dermatosytis. Anti-PM-Scl is found in patients with polymyositis and scleroderma. Anti- CENP-A/B is detected in patients with CREST (60%). Anti-PCNA is another specific marker for SLE but is only detected in 5-10% of the patients. Anti-Ku and anti-Mi-2 antibodies are detected in patients with myositis.

Result: Within 5 working days.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using a monolayer of cells as substrate.

Interpretation: Positive IF at titer of 1/20 or more.

Clinical significance: cytoplasmic (c-ANCA) is detected in patients with Wegener’s granulomatosis, microscopic polyarteritis. Perinuclear (p-ANCA) may occur in polyarteritis nodosa, SLE, severe vasculitis and rheumatoid vasculitis.

Result: Within 10 working days.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: ELISA

Interpretation: the absorbance values of the samples are multiplied by the Conversion Factor to obtain the anti-cardiolipin antibody concentration in GPL or MPL units.

Clinical significance: Positive anti-cardiolipin Ab is found in patients with repeated abortions associated with SLE, various venous and arterial thrombotic disorders including cerebral infarction, deep venous thrombosis, thrombocytopenia and pulmonary embolism.

Result: Within 10 working days.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Latex agglutination

Interpretation: Agglutination of latex particles compared to positive control.

Clinical significance: RF is detected in >80% patients with rheumatoid arthritis (RA). It is also detected in other diseases such as SLE, Sjogren’s syndrome, chronic infections (infective endocarditis & tuberculosis).

Result: Within 8 working hours.

Type of specimen: 20 ml of urine in urine container (preferable early morning urine).

Method/Principle: Immunoblot (strip).

Interpretation: Band formation is compared with the control. 2 bands formation indicate positive result.

Clinical significance: Positive result indicates pregnancy (in pregnancy hCG is increased exponentially for the first 8 weeks, peaks at 10 weeks and start to decline after that until 1/5 of peak level). But very high hCG up to 200,000IU/L suggestive of chrio-epithelioma. hCG can also be detected in other conditions such as persistent trophoblastic diseases, hydatidiform mole, chorioepithelioma of uterus or testis and testicular tumours (monitoring some ovarian and testicular malignancies). False positive UPT can be seen in UTI (urine full of bacteria), proteinuria, hematuria and patient on methadone treatment. False negative UPT maybe found in ectopic pregnancy, toxaemia, foetal death or threatened abortion.

Result: Within 8 working hours.

Type of specimen: Minimum 5 ml of blood in plain container.

Method/Principle: FEIA (Fluorescent enzyme immunoassay).

Interpretation: Test is considered positive if the antibody titre was greater than 12 IU/ml.

Clinical significance: Highly specific marker with comparable sensitivity for rheumatoid arthritis compared to RF.

Results: 10 working days.

Type of specimen: minimum 3 ml blood in plain bottle.

Method/principle: Patients samples, calibrators and controls are incubated with the allergen. After washing, all these solid phase are incubated with a conjugate. After another series of washing, a flurogenic substrate is incubated with these solid phase and then stop solution is added. The end points are read by fluorometry. The standard curve is generated. Calibration data are fit and control values are interpolated on the standard curve.

Clinical Significance: Assessing the level of allergen specific IgE in a Patients’ serum in conjunction with a clinical evaluation based on patient history. This will help the clinician to confirm a diagnosis of allergy and assist in the treatment of the patient.Result: within 5 working days.

Result: Within 8 working days

Detection of Antibodies in AutoImmune Vasculities 

Type of specimen: Minimum 3 ml of blood in plain tube

Method/Principle: The test is based on the principle of line immunoassay (LIA). The antigens ara applied as line on a nitrocellulise membrane: PR3, MPO and GBM. The nitrocellulose membrane is blocked to prevent unspecific reactions. During Incubation of a strip with diluted patient samples autoantibodies present in the sample will bid to the antigens on the strip. For the detection of the bound antibodies a secondary horseradish peroxidase labelled anti human IgG antibody is used. After addition of the substrate and stop solution the appearance of brown lines indicate the existance of (auto) antobodies against the respectivge antigen.  

Clinical significance: The assay is intended for in-vitro diagnostic use of only as an aid in the diagnosis of systemic vasculitis. Protenase 3(PR3) is the main target of cytoplasmic anti- neutrophils cytoplasmic antibody (cANCA). In contast to that, perinuclear ANCA (pANCA) aminly react with myeloperoxidase (MPO). Anti GBM antibodies (anti glomerular basement membrane antibodies) can be detectedin abaout 90% of patient with GOODposture's syndrome. 

Result: Within 5 working days.

Detection of Autoantibodies in AutoImmune Liver Disease (IgG; AMA M2, Sp100, LKM1, gp210, LC1, SLA) 

Type of specimen: Minimum 3 ml of blood in plain tube

Method/Principle: The test is based on the principle of line immunoassay (LIA). The antigens ara applied as line on a nitrocellulise membrane: PDH(AMA M2), Sp100, LKM 1, gp210, LC 1, & SLA. The nitrocellulose membrane is blocked to prevent unspecific reactions. During Incubation of a strip with diluted patient samples autoantibodies present in the sample will bid to the antigens on the strip. For the present in the sample will bind to the antigens on the strip. For the detection of the boi=und antibodies a secondary horseradish peroxidase labelled anti human IgG antibody is used. after addition of the substrate and stop solution the appearance of brown lines indicate the existance of (auto) antobodies against the respectivge antigen.  

Clinical significance: This assay is intended for in vitro diagnostic use only as an aid in the diagnosis of autoimmune live disease. 

Result: Within 5 working days.

Type of specimen: Minimum 3 ml of blood in plain tube.

Method/Principle: Immunofluorescence (IF) using Dermatology Mosaics 7.

Interpretation: Fluorescence pattern (positive reaction). Antibodies against spine cell desmosomes react with the surface antigens of keratinocytes.

Tissue sections of oesophagus and tongue show a characteristic, honeycomb-like, smooth fine granular fluorescence of the intercellular substance, which is mainly spread over the entire stratum spinosum.

Autoantibodies against epidermal basement membrane show a fine-linear staining between the stratum basale and the connective tissue.

Antibodies against plakins react with the transitional epithelium (basement membrane and desmosomes) of rat urinary bladder. Antibodies against epidermal basement membrane cause a fine linear staining between the stratum basale and the connective tissue. Additionally, a reaction of the intercellular substance of the transitional epithelium can be visible (antibodies against desmosomes).

Antibodies against basement membrane structures react with salt-split skin. Anti-BP180, anti-BP230 and anti-LAD97 cause a staining of the epidermal side, while antibodies against laminin 332, collagen VII and other antigens stain the dermal side of salt-split skin.

Antibodies against laminin 332 (LAM332, formerly laminin 5) react specifically with the transfected cells of the test substrate. They cause a smooth to finely granular cytoplasmic fluorescence, in part with slightly pronounced cell membranes. The cell nuclei are usually not or only slightly stained.

Antibodies against BP230gC and collagen VII show a smooth to finely granular cytoplasmic fluorescence in the transfected cells of the respective test substrate, partly with fluorescence of the cell membrane. The cell nuclei are usually not or only slightly stained.

Antibodies against desmoglein 1 and desmoglein 3 show a smooth to finely granular cytoplasmic fluorescence in the cells of respective test substrate, partly with slightly pronounced cell membranes. The area of the cell nucleus is usually not or only slightly stained.

Clinical significance: Autoimmune bullous dermatoses belong to the organ-specific autoimmune diseases. They are characterised by the formation of autoantibodies against structural proteins of the skin. These structural proteins establish the cell-to-cell contact in keratinocytes within the epidermis and the adhesion of the epidermis to dermis. Autoimmune bullous dermatoses are divided into 4 groups based on their target antigens and the localisation of the blisters: pemphigoid diseases, including epidermolysis bullosa acquisita, dermatitis herpetiformis, pemphigus diseases and paraneoplastic pemphigus.  

Result: Within 4 months.

Type of specimen: minimum 3 ml x 3 tubes in EDTA bottle

Method/Principle: Extraction of genomic DNA from the mononuclear cells followed by  low-resolution  sequence-specific primer amplification (PCR- SSP).Primer pairs are designed to have perfect matches only with a single allele or group of alleles.  The perfectly  matched primer pairs result in the amplification of target sequences while mismatched primer pairs do not result in amplification.

Interpretation: The presence or absence of appropriately sized bands will be assessed and analyzed using worksheet.  The results of molecular typing were converted to the

serologically   stablished  HLA  phenotypic equivalents.

Clinical Significance: Pre-requisite for organ especially bone marraw transplantation in order to find matched donor.   Tissue typing is done for HLA class I (HLA-A, HLA-B, HLA-C) and class II  (HLA-DR, HLA-DQ).

Result:  3 months

Note: An appointment with the Laboratory is essential for this test. Blood sample of donors and recipient need to be send together.   For donors HLA class II wil be typed first, if DR locus is matched, then  proceed for HLA class I

Type of specimen: paediatric - 1 ml of fresh anti-coagulated blood in EDTA container; adult – 3 ml fresh anti-coagulated blood in EDTA container.

Method/Principle: Flow cytometry using monoclonal antibody against various lymphocyte subpopulations.

Interpretation: Percentage of cells with positive staining.

Clinical significance: Lymphocyte immunophenotyping is mainly done to support diagnosis of AIDS/HIV infection (low CD4 population (<200 cells/ul of blood is characteristic of AIDS). Lymphocyte counts are also useful for B & T cells deficiencies.

Result: Within 8 working hours. 

Note: An appointment with the Laboratory is essential for this test (at least one day earlier). All  specimen must arrive at Immunology Laboratory before 12pm and within 4 hours of collection. 

Type of specimen: Suspected patient: At least 2 ml of fresh blood in sodium heparin tube (green-top) AND healthy control (at least 2 ml of fresh blood in sodium heparin tube)

Method/Principle: Flow cytometry (CFSE)

Interpretation: Percentage of T cells that proliferate in the suspected patient compared with healthy control samples. 

Clinical significance: Severe combined immunodeficiency (SCID) and other primary immunodeficiency disorders. 

Results: Within 7 working days.

Note: An appointment with the Laboratory is essential for this test (at least one day earlier). All specimen must arrive at Immunology Laboratory before 10am and within 24 hours of collection.

Type of specimen: minimum 3 ml x 3 tube of blood in EDTA bottle.

Method/Principle: Extraction of genomic DNA from the mononuclear cells followed by  low-resolution  sequence-specific primer amplification (PCR- SSP).Primer pairs are designed to have perfect matches only with a single allele or group of alleles.  The perfectly  matched primer pairs result in the amplification of target sequences while mismatched primer pairs do not result in amplification.

Interpretation: The presence or absence of appropriately sized bands will be assessed and analyzed using worksheet.  The results of molecular typing were converted to the

serologically   stablished  HLA  phenotypic equivalents.

Clinical Significance: The human leukocyte antigen HLA-B27 is strongly associated with development of a group of inflammatory arthritides collectively known as the spondyloarthritides, a group of related diseases including ankylosing spondylitis and reactive arthritis.  It can precede the onset of ankylosing spondylitis by approximately three years

 Acute anterior uveitis, are also highly correlated with possession of HLA-B27. With lower frequency, the presence of the HLA-B27 allele has correlated with inflammatory bowel disease, psoriatic arthritis, and reactive arthritis.

Result:  3 months

Note: An appointment with the Laboratory is essential for this test. Blood sample of donors and recipient need to be send together.