Services
Guidelines for Clinicans
The Immunology laboratory provides and integrated laboratory and clinical service for the diagnosis of immunodeficiency, autoimmune and allergic disorders.
Services
The Immunology laboratory performs a comprehensive range of
tests including:
- Diagnostic tests for primary immunodeficiency diseases
including lymphocyte/neutrophil function and immunophenotyping
- Measurement of specific IgG responses to vaccines
(Tetanus, Haemophilus influenzae type B and Streptococcus
Pneumoniae)
- Allergy tests, including total IgE and allegen-specific
IgE
- Diagnostic assays for investigation of autoimmune conditions
such as Coeliac Disease, SLE and antiphospholipid syndrome
Clinical services are available through
the Clinical Allergy
Immunology Department at RCH.
Guidelines for Clinicans
Antiphospholipid
Serology
Coeliac Serology
Healthy Controls and Cellular Tests
Collecting blood for Cellular
Testing
Pneumococcal Antibody
Testing
Thiopurine methyltransferase (TPMT) Testing
Antiphospholipid Serology Guidelines
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?
-
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 both high sensitivity and high specificity for
APS.
Which tests should I request?
If you clinically suspect APS, we recommend
testing with:
- LA and
- ACA IgG and B2GP1 IgG (or request "antiphospholipid
antibodies")
To date, ACA IgG and B2GP1 IgG have been
performed as separate tests. From mid March 2011, both ACA IgG
and B2GP1 IgG will be performed on all requests for
"anticardiolipin antibodies", "beta-2-glycoprotein antibodies" and
"antiphospholipid antibodies" and both results will appear in the
same report.
I requested anti-cardiolipin antibodies. Why
did I receive a result for B2GP1 antibodies?
From mid March 2011, the Immunology Laboratory
will change methodology to multiplex technology, which measures
both ACA IgG and B2GP1 IgG antibodies at the same time.
I have a special request for anti-cardiolipin
IgM (or B2GP1 IgM). What should I do?
ACA IgM tends to 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
(x5725) to discuss the patient with the Consultant
Immunopathologist or Immunopathology Registrar, who will facilitate
further serological testing using alternative methodologies.
Interpretation of test results
Reference ranges are provided in the reports.
To assist with interpretation, all equivocal or positive results
will have an interpretative comment added by the Haematology (LA)
or Immunopathology (ACA/B2GP1) Consultant, or Registrar under
Consultant supervision. Results should be interpreted in the
clinical context.
Reference
Miyakis S et al. International consensus
statement on an update of the classification criteria for definite
antiphospholipid syndrome (APS). J Thromb Haemost
2006;4:295-306.
Coeliac
Serology Guidelines
Background
There have been major
developments in serologic testing for Coeliac Disease (CD) over the
last 10 years. Endomysial and gliadin antibody tests are gradually
being replaced by tissue transglutaminase (tTG) and deamidated
gliadin peptide (DGP) antibody assays.
Testing for tTG and DGP
antibodies
tTG catalyses the
deamidation of gliadin peptides, enhancing MHC Class II binding and
T cell reactivity. tTG IgA, 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, an increasing number of diagnostic
laboratories are now offering a combination of tTG and DGP antibody
assays to screen for CD.
Coeliac serology testing
at RCH
The RCH Immunology
Laboratory performs the following tests for CD:
1. tTG IgA
2. DGP IgG
Details about sample
requirements are available in the
Specimen Collection Handbook.The assays are
performed twice a week and both tTG IgA and DGP IgG results
will appear in the same pathology report.
What to write on
the request form
If you request "Coeliac
Serology", both tTG IgA and DGP IgG tests will be
performed.
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. All equivocal and positive serology results are
reported by an immunopathology consultant (or immunopathology
registrar under consultant supervision). If you have a query about
a result, please contact the Immunology Laboratory (x5725) and
discuss with the senior scientist or immunopathology
consultant.
What about IgA deficiency?
Should I request Total IgA?
You do not need to request
Total IgA when you request Coeliac Serology at RCH/RWH. The tTG IgA
assay used in the RCH Immunology Laboratory 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.
Healthy
Controls and Cellular Tests
Why does the RCH
Immunology Laboratory use blood from Healthy Controls for some
tests?
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 DO
NOT require Healthy Control blood?
Lymphocyte subsets,
T cells,
B cells and
Naive T cells do not require Healthy Control blood. You can
request these tests any day of the week except Saturdays and after
3pm on Fridays.
Which cellular
tests DO require Healthy Control blood?
The following cellular
tests require Healthy Control blood:
HLA-DR expression,
CD40 expression,
Memory B cells,
Activated T cells,
Double Negative T cells,
CD46 expression,
CD40L expression,
SAP expression,
Perforin expression,
CD107a granule release,
Neutrophil Oxidative Burst (DHR),
NitroBlue Tetrazolium (NBT) test,
CD62L shedding,
CD11b/CD18 expression,
Chemotaxis, Lymphocyte Proliferation (to
PHA,
anti-CD3,
Tetanus or
Candida) and
IL12/IFNg testing. You should request these tests on Tuesdays
because every Tuesday morning 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?
Please
ring the Immunology Laboratory (93455725) to discuss the patient
and tests required. A few of the above tests are not complex and
can be performed on other days of the week. If the laboratory has
suitable Healthy Control blood available, testing will proceed. If
there is no suitable Healthy Control blood available, the
laboratory will 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, and some can only
be performed on certain days of the week due to overnight, 3-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.
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,
azathiaprine, anti-CD20, anti-TNF)
- Not pregnant or <6 weeks
postpartum
At RCH, the Healthy Control
is required to read and sign a consent form. This form is kept
in Pathology Collection.
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, please wean to as
low a dose as possible before you perform cellular tests. As cell
function is most impaired 4 hours after a dose of oral steroid, we
recommend performing cellular tests just prior to the steroid dose
e.g. if your patient takes his/her dose in the morning, we
recommend that blood is taken for the cellular test before
the morning dose.
I am a rural or interstate
clinician wanting to arrange testing for a patient. What should I
do?
Please
ring the Immunology Laboratory (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
must 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. This will control for
transport problems, should they occur.
Collecting Blood for Cellular
Testing
What are Cellular
Tests ("Tuesday Tests")?
The RCH Immunology Laboratory performs
cellular tests to assess receptors/proteins and function of
lymphocytes and neutrophils. The tests include:
Lymphocyte Subsets,
T cells,
B cells,
Naive T cells,
HLA-DR expression,
CD40 expression,
Memory B cells,
Activated T cells,
Double Negative T cells,
CD46 expression,
CD40 Ligand expression,
SAP Expression,
Perforin Expression,
Granule Release (CD107a),
Neutrophil Oxidative Burst (DHR),
NBT test,
CD62L Shedding,
CD11b/CD18 expression,
Chemotaxis, Lymphocyte Proliferation (to
PHA,
anti-CD3,
Tetanus or
Candida) and
IFNg-IL12 pathway evaluation.How should we collect the blood
and in which tubes?
When are Cellular Tests
performed?
Cellular tests are performed on Tuesdays.
However the following tests: Lymphocyte Subsets, T cells, B cells
and Naive T cells, may be collected Sunday to Thursday, and Friday
morning. For information about Healthy Control requirements, please
refer to Healthy Controls and
Cellular Tests.
On Tuesday, what time should we
collect the blood?
- Collect samples for CD40L expression,
Granule Release, Neutrophil Oxidative Burst, NBT, CD62L Shedding
and CD11b/CD18 by 1200 as they must arrive in the
Immunology Laboratory by 1330.
- Chemotaxis and IFNg-IL12 tests need to be
booked in advance with the Immunology Laboratory (9345 5725) and
the specimens collected by 0930 so that the
samples reach the Immunology Laboratory by
1100.
- Time of collection is not critical for the
other cellular tests.
We may not have enough time on
Tuesday to collect the blood. What should we do?
If Pathology Collection is required to
collect the blood, please ring ext 5821.
Pneumococcal Antibody
Testing
Pneumococcal vaccines
Four pneumococcal vaccines are now available
in Australia: Pneumovax 23, Prevenar, Synflorix and Prevenar 13.
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: Hib, Prevenar, Synflorix,
Prevenar 13). The pneumococcal antibody test should
not be used to diagnose a pneumococcal
infection and should not be used to
assess if an individual is "protected" from pneumococcal
disease.
Which pneumococcal antibodies are measured
and why?
From 2 May 2011, the RCH Immunology Laboratory
will measure 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).
|
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
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
* These serotypes are "T-cell independent" in
adults who have not previously received Prevenar, Synflorix or
Prevenar-13.
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.
Specimen collection details are located in the
Specimen Collection Manual.
Result interpretation
Pneumococcal antibody levels in the post- and
pre- immunisation samples are compared. All results are reported by
a consultant immunopathologist, or immunopathology registrar under
consultant supervision, and an interpretative comment provided.
Information required on the request form
Request "Pneumococcal antibodies" and specify
(1) name of the pneumococcal vaccine the patient is receiving and
(2) whether the sample is "pre" or "post" immunisation. With four
pneumococcal vaccines available, this
information is essential for result interpretation. If there is
insufficient information on the request form, an "Immunology
Specimen Comment" report will be sent to the clinician requesting
the information and the sample will not
be analysed until the clinician contacts the laboratory with the
details required. If the clinician does not provide this
information within two weeks, the sample will be discarded.
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 (see
Figure).
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 the
measurement of TPMT enzymatic function in red blood cells, with
reduced activity predicting myelosuppression. As this test is
sensitive to specimen storage/handling, 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.
3. 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. 6-methylmercaptopurine (6-MMP)
may also be measured as a response
measure.
Which tests should
I request?
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 Immunopathologist.
How do I interpret
the results?
All TPMT genotyping, TPMT phenotyping and
thiopurine metabolite results will be reported by the external
laboratory and reviewed and validated by the RCH Immunopathology
Consultant or Registrar.
TPMT
Genotyping
- 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 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. In this instance, TPMT
phenotyping may be useful 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 (*2, *3A
or *3C), 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 monitoring of the FBC.
Thiopurine
Metabolites
This test may be used to distinguish
noncompliance from treatment failure. Low or undetectable 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.
References
Ford and Berg. Thiopurine S-methyltransferase
(TPMT) assessment prior to starting thiopurine drug treatment; a
pharmacogenomic test whose time has come. J Clin Path
2010;63:288-95.
TPMT testing before azathioprine therapy? DTB 2009;47:9-12.
Relling MV et al. Clinical Pharmacogenetics
Implications consortium guidelines for Thiopurine Methyltransferase
Genotype and Thiopurine dosing. Clin Pharm Ther
2011;89:387-91.
Sheffield LJ et al. Thiopurine
methyltransferase and thiopurine metabolite testing in patients
with inflammatory bowel disease who are taking thiopurine
drugs.Pharmacogenomcs.2009;10:1091.