HTLV load testing and genotyping
If you are using the service for the first time please contact Professor Graham Taylor to discuss your requirements and to answer any questions that you may have.
+44 (0)20 7594 3910
FAO Dr Claire L. Greiller
Molecular Diagnostic Unit
Imperial College London
4th Floor, Medical School Building
St. Mary’s Hospital
London W2 1PG
Imperial College Healthcare NHS Trust hosts the National Centre for Human Retrovirology, the national referral service for HTLV infection, at which details of this service can be found. MDU provides diagnostic tests in support of this service.
HTLV DNA qPCR
Scope of the test
HTLV-I DNA viral load measurement for diagnosis and monitoring of disease in individual patients infected with human T-lymphotropic virus types 1 and 2.
An in-house method to measure HTLV-1/2 viral load in patient peripheral blood mononuclear cells (PBMC) is used. Primers are specific for HTLV-1 and HTLV-2 Tax gene. HTLV-1 or 2 DNA copy number and β-globin gene copy number of each sample of extracted PBMC DNA are quantified using real-time quantitative PCR monitored by SYBR Green I dye incorporation. HTLV-1/2 copy number is estimated by interpolation from standard curves and is expressed as HTLV-1/2 DNA copies per 100 PBMCs. When extracted DNA has appropriate β-globin gene copy number but no quantifiable HTLV-1/2 DNA is amplified using the RealTime PCR, a PCR is carried out using nested primers specific for HTLV-1 or for HTLV-2 (see typing section below). PCR products are detected on an agarose gel.
HTLV typing by PCR
Scope of the test
To discriminate between HTLV-1 and HTLV-2 infection.
PCR amplification of the tax gene sequence of HTLV DNA extracted from PBMCs is performed. PCR is carried out using generic outer primers in the first round and discriminatory inner primers in the second round. PCR products are detected on an agarose gel. HTLV-1 , HTLV-2 and negative controls are run in parallel.
HTLV clonal analysis
Scope of the test
To facilitate rapid detection of Adult T-cell Leukaemia/Lymphoma. The method can also be used to monitor therapy response and provide early detection of relapse/remission.
The test is performed to determine whether the dominant integration sites of HTLV-1 are monoclonal, oligoclonal or polyclonal, using a vectorette linker and specific proviral primers BIO2 and BIO3. Patients’ DNA is extracted and digested with four different restriction enzymes that do not cut HTLV-1. The vectorette units are ligated to the restriction digested DNA fragments and then used as a template in a two-step touchdown PCR to allow amplification of DNA between the known sequence of HTLV-1 and the vectorette units. After PCR amplification the clonal pattern is analysed by electrophoresis of the amplicons on a 1.5% agarose gel.
Reports are routinely made as a hard copy but can be supplied as an email attachment if required. MDU do not provide clinical interpretation of results but clinical advice can be obtained from Prof. Graham Taylor of the National Centre for Human Retrovirology (+44(0)20 3312 1521).
We aim to issue reports within two weeks of receiving a sample for the proviral load and within four weeks for genotyping and clonal analysis. However, turnaround times may be extended if repeat testing is required.
- Whole EDTA blood and CSF to arrive (at ambient temperature) at MDU within 24 hours of venesection/lumbar puncture. Do not refrigerate or freeze. CSF HTLV-1 DNA viral load to be accompanied by EDTA whole blood obtained at the same time point
- Tissues: for tissues, including lymph nodes, send fresh tissue or shavings from the paraffin-embedded blocks, do not send blocks of tissues or slides.
- Extracted DNA
- Do not send plasma (HTLVs are not detected in plasma)
New: Dried Blood Spots (DBS)
Please see Dried Blood Spot DBS procedure (PDF)
Assay failure rate
The most common cause of assay failure is old age of sample.
If you are unhappy with the service provided by MDU or if you wish to make suggestions on how our service can be improved, please contact Unit Manager, Simon Dustan (firstname.lastname@example.org).
For any further information regarding HTLV samples shipment or suitability, please contact Dr Claire L Greiller (email@example.com), +44 (0)20 7594 9118
If requesting CSF HTLV-1 DNA viral load, send sample EDTA whole blood obtained at the same time point.
Each sample must be accompanied by the HTLV request form document available for download at the button above. If using your own form, it must contain the following details:
- patient clinic number (not the patient’s name)
- date of birth
- sample date
- name and telephone number of the requesting physician
- reason for the test
- if the HTLV type is already known from the serology please specify
Other details such as ethnicity are useful but not essential.