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Immunology

Immunology

  • The RCH Immunology Laboratory provides an integrated laboratory and clinical service for the diagnosis of immunodeficiency, autoimmune and allergic disorders.

    Services

    The RCH Immunology Laboratory performs a comprehensive range of tests including:

    • Diagnostic tests for primary immunodeficiency diseases including immunophenotyping and functional testing of lymphocytes and neutrophils
    • Measurement of specific IgG responses to Tetanus and Pneumococcal vaccines
    • Allergy tests, including total IgE and allergen-specific IgE.
    • Diagnostic assays for investigation of autoimmune conditions such as Coeliac Disease, SLE, antiphospholipid syndrome, autoimmune thyroid disease and type 1 diabetes.

     Clinical services are available through the Allergy and Immunology Department at the RCH.

    Guidelines for clinicians

    Antiphospholipid serology

    What is antiphospholipid syndrome?

    Antiphospholipid syndrome (APS) is an autoimmune syndrome characterized by an increased risk of vascular thrombosis or obstetric complications (e.g. foetal loss, premature delivery due to eclampsia, severe pre-eclampsia or placental insufficiency, or recurrent spontaneous abortion), and other clinical manifestations such as livedo reticularis, thrombocytopaenia, stroke, nephropathy and heart valve disease.

    Tests for antiphospholipid syndrome

    According to the International Consensus Statement, APS is present if one of the clinical criteria (vascular thrombosis or obstetric morbidity) and one of the following laboratory criteria are met:

    • Lupus anticoagulant (LA) present on >=2 occasions at least 12 weeks apart
    • Anticardiolipin (ACA) IgG and/or IgM antibodies present in medium or high titer on >=2 occasions, at least 12 weeks apart,
    • Anti-ß2 glycoprotein-I (B2GP1) IgG and/or IgM antibodies present on >=2 occasions, at least 12 weeks apart.

     The relative sensitivities and specificities of these tests are summarised below:

     Sensitivity  Specificity
    LA * ++ +++
    ACA IgG * +++ ++
    ACA IgM ++ +
    B2GP1 IgG * + +++
    B2GP1 IgM + +

    Which tests are performed at RCH?

    If you clinically suspect APS, we recommend testing with:

    • LA (also known as lupus inhibitor): testing is performed weekly in the Haematology Laboratory.
    • ACA and B2GP1 IgG antibodies (also known as antiphospholipid antibodies): testing performed weekly in the Immunology Laboratory.

    This combination of tests (* above) was chosen to achieve high sensitivity and high specificity for APS. At RCH, both ACA IgG and B2GP1 IgG are performed on all requests for “antiphospholipid antibodies”, “cardiolipin antibodies” and “beta-2 glycoprotein antibodies” and both results appear in the same report. If you have a query about a result, please contact the RCH Immunology Laboratory (x55725) and discuss the patient with the senior scientist or the Immunopathology Fellow/Consultant.

    I have a special request for anti-cardiolipin IgM (or B2GP1 IgM). What should I do?

    ACA IgM may give false positive results, especially if rheumatoid factor or cryoglobulins are present. This also occurs with B2GP1 IgM. If APS is strongly suspected and LA, ACA IgG and B2GP1 IgG are all negative, contact the Immunology Lab (x55725) to discuss the patient with the Immunopathology Fellow or Consultant, who will facilitate further antibody testing using alternative methodologies.

    Coeliac serology

    Testing for tTG and DGP antibodies

    Coeliac disease (CD) is an immune-mediated systemic disorder that occurs due to the ingestion of gluten/gliadin in genetically susceptible individuals. Tissue transglutaminase (tTG) catalyses the deamidation of gliadin peptides, enhancing MHC Class II binding and T cell reactivity. tTG IgA, deamidated gliadin peptides (DGP) IgG and DGP IgA antibody assays have high sensitivity and specificity for CD, with higher sensitivities than endomysial IgA, tTG IgG and gliadin antibody assays. To maximise sensitivity, many diagnostic laboratories offer a combination of tTG and DGP antibody assays to screen for CD. As tTG IgA assays have reduced sensitivity in children aged <2 years, both tTG IgA and DGP IgG should be tested when screening young children. Gliadin antibody assays (antibodies directed against native gliadin) are no longer recommended as screening tests for CD.

    Coeliac serology testing at the RCH

    The RCH Immunology Laboratory performs the following tests for CD:

    1. tTG IgA
    2. DGP IgG

    The assays are performed once a week and both tTG IgA and DGP IgG results will appear in the same pathology report.

    How to request these tests at RCH

    If you request "Coeliac Serology" or “Coeliac Antibodies”, both tTG IgA and DGP IgG tests will be performed. In children aged <2 years, both tTG IgA and DGP IgG will be performed, even if only one of these tests has been requested.

    Interpretation of the results

    If the tTG IgA and/or DGP IgG results are positive and you clinically suspect CD, small bowel biopsy should be performed to confirm the diagnosis. Gluten free diet (GFD) should NOT be commenced based on serology positivity alone. For assistance with result interpretation, contact the Immunology Lab (x55725) to discuss the patient with the senior scientist or Immunopathology Fellow/Consultant.

    What about IgA deficiency? Should I request Total IgA?

    At RCH, you do not need to request Total IgA when you request Coeliac Serology. The tTG IgA assay used at RCH detects IgA deficient samples. If your patient is IgA deficient, you will be informed in the pathology report. As false negative tTG IgA results occur in IgA deficiency, DGP IgG is used to screen for CD in IgA deficient patients.

    Monitoring CD patients on GFD

    Both tTG IgA and DGP IgG assays are used to monitor response to GFD.

    Cellular tests and blood collection

    What are Cellular Tests?

    The RCH Immunology Laboratory performs cellular tests to assess receptors/proteins and function of lymphocytes, monocytes and neutrophils. The tests include: Lymphocyte Subsets, T cells – naïve/memory, T cells – double negative, T cells - HLADR expression, B cell Panel (including transitionals and plasmablasts), High Sensitivity B cells (rituximab monitoring), B cells – HLADR expression, CD40 expression, T cells - regulatory (CD4/CD25/CD127), T cells – follicular helper, Th17 cells, CD46 (MCP) expression, DOCK8 expression, CD40 ligand expression, LRBA expression, SAP and XIAP expression (XLP),  Perforin expression, NK cell degranulation and cytotoxicity, STAT5 phosphorylation, Neutrophil Oxidative Metabolism (DHR), CD62L Shedding, CD18 expression (LAD type 1), CD16 B73.1 epitope, STAT1 phosphorylation, Lymphocyte Proliferation (to PHA, anti-CD3, Tetanus or Candida) and STAT4 phosphorylation.

     How should we collect the blood and in which tubes?

    • Refer to the Specimen Collection Handbook for specimen requirements for individual tests.
    • Collect blood for cellular tests into Lithium Heparin tubes.
    • Try to collect the blood in a sterile manner; Vaccutainer systems are preferred. If you cannot use a Vaccutainer system, do NOT open up all the blood tubes before you start. Open the first blood tube, fill the tube with blood, close the tube, open the second blood tube, fill that tube, close that tube etc. This will reduce the likelihood of contamination of the blood sample and avoid the possibility of tubes being re-capped with the wrong lids.
    • If serum/gel, EDTA and lithium heparin samples need to be collected, fill the serum/gel tube first, followed by the EDTA tube, followed by the lithium heparin tube.
    • Contamination of the blood sample and/or mix-up of blood tubes can cause false test results. It is recommended that you ask a staff member to assist you with the blood tubes.

    When are cellular tests performed at RCH?

    Cellular tests are performed on Tuesdays. However the following tests may be collected Sunday to Thursday, and Friday mornings: Lymphocyte Subsets, B cell Panel (including transitionals and plasmablasts), High Sensitivity B cells (rituximab monitoring), B cells – HLADR expression, T cells – naïve/memory, T cells – double negative, T cells - HLADR expression and T cells - regulatory (CD4/CD25/CD127).

    What time should we collect the blood?

    By 1330 on Tuesdays

    Collect samples for the following tests by 1330, as they must arrive in the Immunology Laboratory by 1400 for immediate set up: Neutrophil Oxidative Burst, CD62L Shedding and Lymphocyte Proliferation to Candida or Tetanus.

    By 1030 on a pre-arranged day

    NK cell functional assays (perforin expression and NK cell degranulation & cytotoxicity) and STAT4 phosphorylation testing need to be booked in advance with the RCH Immunology Laboratory (x55725) and samples collected by 1030 so that they reach the Immunology Lab by 1100.

    Other samples

    The time of collection is not critical for the other cellular tests.

    My patient is on oral corticosteroids -will this affect the results of the cellular tests?

    Immunosuppressants such as oral steroids can affect the results of cellular tests. If you are in the process of weaning the immunosuppression, wean to as low a dose as possible before you request cellular tests. As cell function is most impaired within 4 hours after a dose of oral prednisolone, we recommend performing cellular tests just prior to the prednisolone dose e.g. if your patient takes his/her dose in the morning, we recommend that the blood is collected just before the morning dose. We suggest you discuss the timing of blood collection with the Immunopathology Fellow/Consultant.

    I am a rural/interstate clinician or laboratory wanting to arrange cellular tests for a patient. What should I do?

    Please ring the RCH Immunology Laboratory (03-93455725) to book in the tests. You will be advised about sample and transport requirements. The samples should be couriered to RCH at room temperature. Transport time is crucial for some assays e.g. neutrophil tests should be performed within 6 hours of blood collection. For cellular tests that require a Healthy Control, please send the Patient sample with a Healthy Control sample (see “Healthy Controls and Cellular Tests” section). This will control for transport problems, should they occur. 

    Healthy controls and cellular tests

    Why does the RCH Immunology Laboratory use blood from Healthy Controls?

    Quality Control (QC) is an essential component of a diagnostic laboratory service. Although QC materials are available for common cellular tests such as lymphocyte subsets, they are not available for the majority of cellular tests offered by the RCH Immunology Laboratory. For such tests the laboratory uses blood from healthy adults (“Healthy Controls”) for the purposes of QC.

    Which cellular tests require healthy control blood?

    The following cellular tests require Healthy Control blood: CD40 expression, T cells – follicular helper, Th17 cells, CD46 (MCP) expression, DOCK8 expression, CD40 ligand expression, LRBA expression, SAP & XIAP expression (XLP), Perforin expression*, NK cell degranulation and cytotoxicity*, STAT5 phosphorylation, Neutrophil Oxidative Metabolism (DHR), CD62L Shedding, CD18 expression (LAD type 1), CD16 B73.1 epitope, STAT1 phosphorylation, Lymphocyte Proliferation (to PHA, anti-CD3, Tetanus or Candida and STAT4 phosphorylation*). Excluding tests marked *, which are only available by appointment, the above tests should be requested on Tuesdays because every Tuesday morning (excluding public holidays) the RCH Immunology Laboratory arranges for blood to be collected from a Healthy Control.

    It is not a Tuesday but I want to request one of the above tests. What should I do?

    Contact the RCH Immunology Laboratory (x55725) to discuss the patient and tests with the senior scientist or the Immunopathology Fellow/Consultant. A few of the above tests are less complex and can be performed on other days of the week, if the laboratory has Healthy Control blood available. If there is no/insufficient Healthy Control blood available, the laboratory may ask you to arrange for blood to be collected from a Healthy Control to enable testing to proceed. On the other hand, some of the above tests are very time-consuming to perform, and some can only be performed on certain days of the week due to overnight, 3-day, 4-day or 6-day incubation periods. In these situations, if the test is not urgent, the laboratory will ask you to postpone testing until the following Tuesday. If you need urgent testing for a patient after hours, ask the RCH Switchboard to page the Allergy-Immunology Consultant on call, who will liaise with the Immunology Laboratory.

    Who can be a healthy control?

    The Healthy Control needs to be:

    • A healthy adult aged >=18 years

    • Not the sibling or parent of a patient being tested

    • Immunocompetent (e.g. not an oncology, transplant or HIV patient) and not on immunosuppressive medications (e.g. chemotherapy, oral corticosteroids, cyclosporin, azathioprine, mycophenolate, anti-TNF, anti-CD20)

    • Not pregnant or <6 weeks postpartum

    It is recommended that the Healthy Control reads an information statement and signs a consent form. These forms are kept in RCH Pathology Collection. For external clinicians/laboratories requesting functional tests, these forms may be used for the external Healthy Control and the completed consent form should accompany the Healthy Control specimen - refer to links below to download the forms.

    Pneumococcal antibody testing

    Pneumococcal vaccines

    Three pneumococcal vaccines are available in Australia: Pneumovax 23, Prevenar 13 (which replaced Prevenar) and Synflorix. Each of these vaccines contains a different range of serotypes.

    Measuring antibodies to pneumococcal vaccines

    Measurement of antibody responses to vaccines is recommended in patients with suspected antibody deficiency or combined (T and B cell) immunodeficiency. In children aged ≥2 years and in adults, this evaluation involves the measurement of antibodies to "T-cell independent" vaccines (e.g. polysaccharide based: Pneumovax 23) and "T-cell dependent" vaccines (e.g. protein-based: Tetanus, or conjugated: Hiberix, Prevenar, Synflorix, Prevenar 13. The pneumococcal antibody test should not be used to assist in the diagnosis of pneumococcal infection and should not be used to assess if an individual is "protected" from pneumococcal disease.

    Which pneumococcal antibodies are measured and why?

    The RCH Immunology Laboratory measures antibodies to 15 pneumococcal serotypes (see table below). 7 of the 15 serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) are contained in all 4 vaccines, and the other 8 serotypes (2, 8, 10A, 11A, 15B, 17F, 20 and 33F) are contained in Pneumovax but are not included in Prevenar, Synflorix or Prevenar 13 vaccines (see table below).* These serotypes are "T-cell independent" in adults who have not previously received Prevenar, Synflorix or Prevenar-13.

     

     Pneumococcal Serotypes

     

    "T-cell dependent" serotypes

    "T-cell independent" serotypes

    Vaccines

    4

    6B

    9V

    14

    18C

    19F

    23F

    2

    8

    10A

    11A

    15B

    17F

    20

    33F

    Pneumovax 23

    Y*

    Y*

    Y*

    Y*

    Y*

    Y*

    Y*

    Y

    Y

    Y

    Y

    Y

    Y

    Y

    Y

    Prevenar

    Y

    Y

    Y

    Y

    Y

    Y

    Y

    -

    -

    -

    -

    -

    -

    -

    -

    Synflorix

    Y

    Y

    Y

    Y

    Y

    Y

    Y

    -

    -

    -

    -

    -

    -

    -

    -

    Prevenar 13

    Y

    Y

    Y

    Y

    Y

    Y

    Y

    -

    -

    -

    -

    -

    -

    -

    -

    If a patient has received Prevenar, Synflorix or Prevenar 13 in infancy, followed by Pneumovax at >=2 years of age, it is important to consider the "T-cell dependent" and "T-cell independent" serotypes separately when evaluating the antibody response to Pneumovax, because the patient may have T-cell memory to the "T-cell dependent" serotypes.

    Blood collection

    A "pre-immunisation" blood sample should be taken just prior to the pneumococcal vaccine, and a "post-immunisation" sample taken 4-6 weeks after the vaccine.

    Result interpretation

    This test is used to evaluate the immune response to a Pneumococcal vaccine in patients with suspected immune deficiency. Pneumococcal antibody levels in the pre- and post-immunisation samples are compared. All results are reported by a Consultant Immunopathologist, or Immunopathology Fellow under consultant supervision, and an interpretative comment provided.

    Information required on the request form

    Request "Pneumococcal antibodies" and specify (1) the name of the pneumococcal vaccine the patient will be receiving, (2) the date of pneumococcal immunisation and (3) whether the sample is "pre" or "post" immunisation. With 3 pneumococca l vaccines currently available, this information is essential for result interpretation.

    Cost of pneumococcal antibody testing

    This test is currently performed using a complex “in-house” ELISA method consisting of 16 ELISA plates, performed over a period of two days. RCH bears the cost of this test only for RCH patients. External laboratories and external/private patients should contact the RCH Immunology Laboratory (03-93455725) for information relating to test costs.

    Thiopurine methyltransferase (TPMT) testing

    What is TPMT?

    Thiopurine drugs (including 6-mercaptopurine and azathioprine) are widely used in the treatment of autoimmune and allergic conditions, malignancies and to prevent transplant rejection. The metabolism of these drugs is reliant on three main enzymes: TPMT, xanthine oxidase and hypoxanthine-guanine phosphoribosyl transferase enzymes. 

    The main therapeutic effects of these drugs are through the incorporation of 6-thioguanine nucleotides (6-TGN) into DNA. If there is a reduced amount of TPMT available, there is increased production of cytotoxic 6-TGN, resulting in a significantly increased risk of myelosuppression. Approximately 1 in 300 people have undetectable TPMT activity, about 11% demonstrating low or intermediate activity and the remainder having normal activity.

    Tests for TPMT

             1. TPMT Genotyping

    Genotypic assessment of TPMT is now performed as the first-line assessment and can identify the most common polymorphisms of the TPMT gene: the normal or wild-type allele (TPMT*1), and those associated with reduced function (TPMT*2, *3A or *3C).   Heterozygosity of *2, *3A or *3C with *1 correlates with intermediate or reduced TPMT activity, and homozygosity of any of these latter alleles correlates with undetectable TPMT activity and high risk of myelosuppression.  

             2. TMPT Phenotyping

    Phenotypic assessment of TPMT involves measurement of TPMT function in red blood cells, with reduced activity predicting myelosuppression. As this test is sensitive to specimen storage/transport, and is unreliable in individuals who have recently received a blood transfusion, TPMT genotyping is preferred. If the genotype is difficult to interpret (see below), TPMT phenotyping may be useful as a second-line test.

    How to request TPMT tests at RCH

    In the first instance, all requests for TPMT testing will be sent for genotyping. TPMT phenotyping is a second-line test and requires approval from the RCH Consultant Immunopathologist.

    TPMT Genotype

    • A patient with a *1/*1 genotype is a normal metabolizer of TPMT. These patients have normal TPMT activity.

    • Heterozygosity of *1 and a mutant allele (*2, *3A, *3B or *3C) indicates intermediate TPMT activity and increased risk of myelosuppression at standard doses. The recommendation is to commence the individual on approximately 50% of the standard dose of thiopurine drug.

    • Due to technical limitations of the genotyping method, differentiation between heterozygosity of *1/*3A and compound heterozygosity of *3B/*3C (extremely rare) is not possible. TPMT phenotyping may be useful in such cases as compound heterozygotes will have undetectable TPMT activity and heterozygous individuals will have intermediate activity. Alternatively, the clinician may choose to assume that the patient is heterozygous and commence the patient on approximately 50% of the standard dose of the thiopurine drug, followed by a FBC one week later.

    • If a patient has compound heterozygosity or homozygosity of a mutant allele, the patient has undetectable TPMT activity. Thiopurine drugs should be avoided, or commenced at approximately 10% of the standard dose, given three days per week instead of weekly, with close FBC monitoring.

    Thiopurine Metabolites

    • The level of 6-TGNs can be measured as a marker of efficacy/response to thiopurine drugs in patients already established on thiopurine treatment.  This test may be used to distinguish noncompliance from treatment failure. Low levels of 6-TGN in a patient who is not responding to thiopurine treatment suggests poor compliance or use of a subtherapeutic dose of the thiopurine drug. High levels of 6-TGN may be associated with myelotoxicity.

    • This test is not covered by Medicare. RCH bears the cost of the test only for RCH patients. External and private patients should contact the Immunology Laboratory (93455725) for information relating to test cost.

    Haemophagocytic Lymphohistiocytosis (HLH) Testing

    Investigation of suspected HLH

    When a patient is first admitted with fever and cytopenias, it is important to consider the possibility of HLH so that appropriate blood samples can be collected BEFORE treatment with blood products and/or immunosuppression (e.g. corticosteroids). In addition to the many investigations required for the diagnostic workup of patients with suspected HLH, laboratory tests for HLH should include:

    • FBC
    • LFT
    • Ferritin
    • Fibrinogen (Coags)
    • Soluble CD25: note serum needs to be frozen at minus 200C within 24h of collection
    • Serum storage: 7-8ml of blood in a serum tube to store at minus 200C, for consideration of
      • Immunoglobulins IgG, IgA and IgM
      • Tetanus IgG
      • EBV PCR
    • Cellular tests for HLH (see below)
    • Bone marrow examination
    • CSF examination: protein/glucose, automated white cell and red cell counts, cytospin

    How to arrange cellular tests for HLH

    For all patients (RCH and external), cellular tests for HLH must be BOOKED AHEAD with the RCH Immunology Laboratory. To arrange testing, ring the laboratory on x55725, or page or email the RCH Immunopathology Consultant (pager 6744 or sharon.choo@rch.org.au) or Fellow (pager 5554). If you need urgent testing on a patient after hours, please ring the RCH Switchboard (03-93455522) and page the on-call Allergy-Immunology Consultant, who will liaise with the Immunology Laboratory.

    Recommended cellular tests

    1. Perforin expression
    2. NK cell degranulation (CD107a expression)

    3. SAP and XIAP expression (XLP)
    4. Activated T cells
    5. [NK cell cytotoxicity – second line test – performed if perforin or NK degranulation abnormal]

    Additional tests to consider

    • Lymphocyte subsets
    • T cells - naïve/memory
    • B cell panel
    • Molecular testing
      • PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, SH2D1A, BIRC4

    If the patient is a RCH patient

    Contact the Immunology Laboratory (x55725) to discuss the timing of blood collection.

    If the patient is not a RCH patient

    Guidelines for external patients are available from the RCH Immunology Laboratory. If you are an external clinician or an external laboratory wanting to arrange HLH cellular tests for a non-RCH patient, ring the RCH Immunology Laboratory on 03-93455725, or page or email the RCH Immunopathology Consultant (pager 6744 or sharon.choo@rch.org.au) or Fellow (pager 5554).

    Documentation required for external samples

    Refer to the “Healthy Controls and Cellular Tests” section above for Healthy Control information.

    Food specific IgE testing 

    What is food specific IgE?

    Serum food specific IgE (food sIgE) tests are used in the diagnosis of IgE mediated (immediate) food allergy. The results must be interpreted in the context of the clinical history, as food sIgE can be detected in individuals without any clinical evidence of allergy.

    Which food sIgE tests should I request?

    Food sIgE tests should only be requested where the history suggests an IgE-mediated allergy e.g. onset of urticaria, angioedema, vomiting, abdominal pain or anaphylaxis within 1 hour of ingestion of a food. Only the suspected food should be tested. If a child has been eating a food without reaction, food allergy is unlikely and testing should not be performed. In addition, ‘screening’ for food allergy (testing for foods that the patient has not eaten) is not recommended. Food sIgE testing for fruits and vegetables is not recommended because these tests have reduced sensitivity.

    Result interpretation?

    In general, food sIgE tests have good negative predictive value i.e. if the result is negative ( <0.35 kUA/L), and the history does not suggest an IgE-mediated allergy to the food, the patient is very unlikely to be allergic to that food. However, if the result is positive (≥0.35 kUA/L), the patient may or may not have clinical allergy. A history of immediate allergic reaction, together with a positive test result, suggests IgE mediated food allergy. If the history is unclear, published 95% specificity values, above which there is a 95% chance of clinical allergy (see table below), should be used to assist with interpretation of the food sIgE results. For further assistance with result interpretation, contact the Immunology Laboratory (x55725) to discuss the patient with the Immunopathology Fellow/Consultant. For peanut allergy, an additional test (Ara h2 sIgE) may assist with diagnosis (see next section).

    95% specificities at different food-sIgE levels (Sampson H and Ho D, 1997; Sampson HA, 2001)

      Egg Milk Peanut Fish Soy* Wheat*
    Food sIgE level (kUA/L) 7 15-23 15 20 65 79
    Specificity (%) 95 94 92-100 99-100 99-100 99

    * Soy sIgE and Wheat sIgE tests have poor positive predictive value.

    How do food component sIgE tests differ from standard food sIgE tests?

    Most food sIgE tests detect IgE antibodies against extracts of the whole food, and therefore the result can be positive if an individual has antibodies against any component of that food, even if some of those antibodies don’t cause an allergic reaction. Food component sIgE testing allows measurement of antibodies to the clinically relevant allergen component/s of the food. In peanut allergy, individuals with antibodies to specific allergen components are more likely to have clinical allergy to peanut. In particular, sIgE against Ara h 2 (the major allergen of peanut) has higher sensitivity and higher specificity for the diagnosis of peanut allergy than peanut sIgE (sIgE against whole peanut) alone.


    When should I request Ara h 2 sIgE?

    If peanut allergy is suspected clinically, peanut sIgE (or peanut skin prick test (SPT)) is the most appropriate initial investigation. If the peanut sIgE (or peanut SPT) result is positive and there is a history of clinical reaction consistent with immediate IgE mediated peanut allergy, the diagnosis is confirmed. If the history is uncertain and the peanut sIgE result is positive but below the 95% specificity threshold for diagnosing clinical allergy (peanut sIgE 0.35-15.0 kUA/L; peanut SPT 3-8 mm), Ara h 2 sIgE testing may be helpful in differentiating those individuals who have a peanut allergy from those who are tolerant to peanut. An Ara h2 sIgE test is not required if the peanut sIgE or SPT is above the 95% specificity threshold for diagnosis of peanut allergy (>15 kUA/L or >8mm respectively).

    How do I interpret the Ara h2 sIgE results?

    For Ara h 2 sIgE testing, levels of >=0.1 kUA/L are positive. Based on local data (Dang et al, 2012), a value of 0.1 kUA/L Ara h 2 sIgE offers a sensitivity of 95% i.e. peanut allergy is unlikely if Ara h 2 sIgE is <0.1 kUA/L. The study also identified a 95% specificity threshold for diagnosis of peanut allergy of 0.46 kUA/L, ie results above this level indicate a 95% probability of peanut allergy.

    Does a high level of food sIgE mean my patient is anaphylactic?

    There is no correlation between the level of food sIgE and the severity of the allergic reaction. High Ara h2 sIgE levels do not predict anaphylaxis.

    Which sIgE tests will be performed by the RCH Immunology Laboratory?

    Currently, due to Medicare billing restrictions, our laboratory performs a maximum of four sIgE tests per patient sample for RCH patients, not more than four times per year. Ara h 2 sIgE will ONLY be performed if the peanut sIgE result, on the same or previous sample, is 0.35-15.0 kUA/L (or the peanut SPT result is 3-8mm). External laboratories and external/private patients should contact our laboratory (9345 5725) for information relating to test costs.

    Should I request total IgE?

    As high levels of total IgE (>1000 kUA/L) may reduce the specificity of sIgE tests, we recommend measurement of total IgE when sIgE is requested in individuals who may have an elevated total IgE (e.g. eczema patients).

    Complement studies

    What is the complement system?

    The complement system consists of soluble and cell surface proteins which, when activated, cascade to form the membrane attack complex (C5b-9) and enhance the ability of antibodies and phagocytic cells to kill bacteria. The complement system consists of 3 pathways: classical (THC/CH50/CH100), alternative (AH50/AP50/AP100) and lectin (MBL/MBP) pathways.

    Functional assessment of the complement system

    The following tests are performed at RCH.

    • Complement C3 and Complement C4 (gel tube): Measurement of C3 and C4 may be useful in the diagnostic workup of the following conditions: glomerulonephritis, thrombotic microangiopathy (TMA), primary immunodeficiency disorders (PID), systemic lupus erythematous (SLE), vasculitis and cryoglobulinaemia.
    • Complement function (neutral tube): This is a functional ELISA method used to assess the integrity of the classical, alternative and lectin complement pathways. At RCH all 3 pathways are tested simultaneously on all samples (Note that “old” CH50 hemolytic assays only measure function of the classical pathway). Functional testing of the complement system is indicated in patients with suspected atypical haemolytic uremic syndrome (atypical HUS), C3 glomerulopathy (C3G) and complement deficiency. C3 and C4 should always be requested alongside complement function to aid in the interpretation of complement function results.

    How to request complement function testing at RCH

    If you request “complement function”, THC/CH50, AH50/AP50 or MBL at RCH, all 3 complement pathways (classical, alternative and MBL) will be tested. For assistance with diagnostic workup of the complement system or complement result interpretation, contact the Immunology Laboratory (x55725) to discuss the patient with the senior scientist or Immunopathology Fellow/Consultant. As there are no published paediatric reference ranges, all results are reported by a Consultant Immunopathologist, or Immunopathology Fellow under consultant supervision, and an interpretative comment provided.

    Investigation of suspected thrombotic microangiopathy or C3 glomerulopathy

    When a patient is first admitted with suspected TMA, it is important to consider the possibility of atypical HUS, so that appropriate blood samples can be collected for complement studies BEFORE treatment with blood products and/or plasma exchange. In addition to the many investigations required for the diagnostic workup of patients with suspected HUS (see Haemolytic Uraemic Syndrome (HUS): Management Guideline), laboratory tests for TMA (including atypical HUS) and C3G should include:

    • Complement C3 & Complement C4
    • Complement function
    • Serum storage: 7-8ml of blood in a serum tube to store at minus 200C for consideration of
      • Complement Factor H antibodies (performed at RCH)
      • Factor H, factor I and factor B (sent to an external laboratory)

    Additional tests recommended for the investigation of atypical HUS include:

    • ADAMTS13 activity – arrange with the Haematology Laboratory
    • CD46 (MCP) expression - performed on Tuesdays or by prior arrangement with the Immunology Laboratory (x55725).

    Additional tests recommended for the investigation of C3G include:

    • C3 nephritic factor (sent to an external laboratory)

    For sample requirements, refer to Specimen Collection Handbook ( http://ww2.rch.org.au/specimen).

    Molecular testing for mutations in several complement genes and complement regulatory genes is performed in external laboratories. To discuss these tests and sample requirements, and to ensure that correct documentation is provided to the external labs, contact the Immunology Lab on x55725.

    Eculizumab therapy

    Eculizumab is a monoclonal anti-C5 antibody, which prevents formation of C5a and C5b-9, leading to terminal complement blockade. It is used to treat aHUS and other TMAs, including post haematopoietic stem cell transplant (HSCT). Effective treatment with eculizumab is determined by inhibition of terminal complement activation, which can be monitored by hemolytic CH50 assays, sC5b-9 ELISAs or the complement function ELISA (classical and alternative pathways). As both the sC5b-9 and complement function ELISAs measure C9, the RCH Immunology Laboratory uses the complement function assay to monitor patients receiving eculizumab. Current recommendations suggest that complement function should be <10% of the lower limit of normal with effective therapy.

    Prior to commencement of eculizumab, consider collecting samples for

    • Complement C3 and Complement C4
    • Complement function - post-HSCT TMA patients should be discussed with the Immunology Lab (x55725) prior to starting eculizumab as these patients may require more frequent monitoring and more frequent dosing (eg a second dose within 72 hours).
    • Request for serum storage

    Investigation of suspected Hereditary Angioedema

    Hereditary angioedema (HAE) is a rare inherited condition associated with recurrent episodes of angioedema in the absence of urticaria, due to uninhibited activation of the bradykinin pathway. Three forms of HAE have been described. Type 1 and type 2 HAE are due to a deficiency in the function of C1 esterase inhibitor (C1-INH), either from low absolute levels or the production of a dysfunctional protein. Type 3 HAE is not due to C1-INH deficiency. In a subset of patients (with Type 3 HAE), mutations in the Factor XII gene have been identified. Acquired C1-INH deficiency occurs more commonly in adults, and results from increased metabolism or destruction of C1-INH.

    Tests recommended for investigation of patients with suspected Type 1 or 2 HAE include:

    • Complement C3 and Complement C4

    • Complement – C1 esterase inhibitor studies (includes C1-INH level and function)

    Additional investigations for possible Type 3 HAE or acquired C1-INH deficiency include:

    • Complement C1q

    • Factor XII gene mutation analysis - contact the Immunopathology Fellow/Consultant (x55725) prior to collecting the sample.

    Diabetic autoantibodies

    What is diabetes mellitus?

    Diabetes mellitus is a chronic disease characterised by abnormally high levels of blood glucose over a prolonged period of time. There are two main types of diabetes. “Type 1” diabetes (T1DM) is an autoimmune condition that results in destruction of the insulin secreting cells of the pancreas. This type more commonly occurs in young children, and is associated with autoantibodies directed against components of the insulin secreting cells. “Type 2” diabetes is a progressive condition where the body becomes resistant to the normal effects of insulin - it is not associated with autoantibodies.

    What are diabetic autoantibodies?

    There are several different diabetic autoantibodies which target different autoantigens that are present on the surface of or within pancreatic islets cells, or are the product of the islet cells. The relative sensitivities and specificities of currently available diabetic autoantibody tests are summarised below:

    Autoantibody Test
    Sensitivity Specificity
    Islet Cell antibodies +++ +++
    GAD antibodies +++ +++
    Insulin antibodies + +++
    IA2 antibodies ++ +++
    ZnT8 antibodies ++ +++

    Not every patient with T1DM will have each autoantibody present, therefore the sensitivity of testing is increased if the autoantibodies are tested in combination.

    Which of these tests are performed at RCH?

    The diabetic autoantibodies tested at RCH are:

    • GAD: antibody directed against glutamate decarboxylase. This test has replaced islet cell antibodies.
    • IA2: antibody directed against protein tyrosine phosphatase IA2. At RCH this test is performed instead of insulin antibodies.
    • ZNT8: antibody directed against zinc transporter ZnT8.

    Combining the above 3 tests increases sensitivity to >95%. GAD and IA2 antibody testing is performed every 2-3 weeks, and ZnT8 antibody testing is performed every 4-6 weeks.

    How to request these tests at RCH

    If you request “diabetic antibodies”, all 3 antibodies (GAD, IA2 and ZnT8) will be tested.

    I have a special request for Insulin or Islet Cell autoantibodies. What should I do?

    If T1DM is strongly suspected and GAD, IA2 and ZnT8 antibody results are all negative, contact the Immunology Laboratory (x55725) to discuss the patient with the senior scientist or Immunopathology Fellow/Consultant, who will facilitate additional testing.

    Faecal calprotectin

    What is Calprotectin?

    Calprotectin is a neutrophil cytoplasmic protein. During active intestinal inflammation, neutrophils migrate from the circulation to the intestinal mucosa. Calprotectin is released from the neutrophils and excreted into the faeces. Faecal calprotectin may be increased in inflammatory bowel disease, gastrointestinal infections (in particular bacterial infections) and gastrointestinal malignancies.

    Why do you need a separate sample for Calprotectin?

    Faeces samples for Calprotectin testing must be stored at 2-8 degrees Celsius for a maximum of 6 days after collection. The Microbiology and Molecular Microbiology laboratories require faecal samples for 4-6 days to complete their tests (e.g. M/C/S and rotavirus/adenovirus/enterovirus), by which time the samples are no longer suitable for Calprotectin testing. Therefore Calprotectin and Microbiology tests should be performed on separate faeces samples. However faecal biochemistry tests (such as sugars/pH, electrolytes and occult blood) can be performed on the same sample as Calprotectin (Calprotectin testing is performed first, then faecal chemistry).

    Result interpretation

    In general, faecal calprotectin has a good negative predictive value i.e. if the result is negative, the patient is very unlikely to have active intestinal inflammation. As there are no published paediatric reference ranges, and young children are known to have higher calprotectin levels than adults, all Calprotectin results are reported by a Consultant Immunopathologist, or Immunopathology Fellow under consultant supervision, and an interpretative comment is provided.

    Cost of Calprotectin testing

    The Calprotectin test not covered by Medicare. RCH bears the cost of the test only for RCH patients. External laboratories and external/private patients should contact the RCH Immunology Laboratory (03-93455725) for information relating to test costs.