Molecular Diagnostics

Introduction

The Molecular Laboratory offers tests for the detection of a wide range of infectious agents by molecular methods. These methods include in-house assays based on conventional and real-time polymerase chain reaction (PCR), as well as commercial kit-based assays on platforms such as real-time PCR, hybrid capture and line probe assays. It also offers molecular tests for monitoring of viral loads, detection of mutations and genotyping of viruses.

In addition, the Molecular Laboratory offers tests for diagnosis of molecular markers of non-infectious diseases. In particular, it is currently expanding its menu to include molecular tests for mutation analysis of tumours and oncogenes.

Sample Labeling & Completion of Request Forms

Please see requirements for sample labeling and completion of test requests under the section “Sample Collection & Handling (General)". The date and time of sample collection must be indicated. In addition, the requesting doctor’s full name must be indicated. For urgent requests, the hand-phone number should also be included.

A verbal add-on test order must be followed by a written request from an authorized person, e.g. by fax, within 3 days.

Molecular Microbiology Tests

A. GENERAL GUIDELINES

For most viral infections, the causative agents can only be detected during the first 5 days of the acute illness.
Generally, samples should reach the laboratory within 6 hours of collection. If this is not possible, the sample must be refrigerated at 4°C (for a maximum period of 24 hours) until transport. As far as possible, samples should be collected during opening hours of the laboratory. For samples collected after office hours, refrigerate at 4°C until transport to the laboratory. DO NOT FREEZE.
Blood samples for molecular tests should be sent in EDTA tubes rather than plain blood tubes. Blood that is collected in plain blood tubes for PCR tests on RNA viruses (e.g. influenza, dengue and hepatitis C viruses) and which arrive at the laboratory after 24 hours of collection will be rejected.
Swabs used for sampling should be made of dacron or rayon, not of calcium alginate or cotton, and the shaft of the swab stick should preferably be made of plastic. Materials other than dacron or rayon are inhibitory to the PCR process. Laryngeal swabs are rejected for this reason.
Aqueous or vitreous fluid must collected in a sterile leak-proof container or in a syringe with the needle removed. Cap the syringe with a rubber bung or plastic cap to prevent leaking. Do not collect samples using syringes with undetachable needles. Samples received in syringes with needles attached will be rejected.
Test requests for urgent samples must be communicated to the Molecular Laboratory (ext. 6920) before sending. These samples must be hand-delivered to the Molecular Laboratory at Level 12, Diagnostics Tower, Academia, if sent within SGH.
Transport the sample with an ice pack or in a bag of ice cubes where a delay in transportation is expected. First seal the sample in a plastic bag, then place this bag inside an outer plastic bag containing the ice pack or ice cubes. The ice cubes must not come into direct contact with the sample bottle as contamination will occur. Place the accompanying request form into another plastic bag to keep the form dry. Do not place blood tubes adjacent to ice packs as this will cause blood to haemolyse and lead to rejection of blood samples
Molecular tests detect the presence and absence of DNA or RNA regardless whether the organism is alive or dead. Qualitative molecular tests should not be used for monitoring the patient’s response to therapy.

Collection Methods For Molecular Microbiology Tests by Sample Type

Blood
Collect at least 5 mL of blood in EDTA and mix gently. EDTA-treated blood is preferred to plain blood as EDTA stabilizes DNA and RNA and prevents haemolysis which can inhibit the PCR process.

Body fluid (CSF, Vitreous Fluid, Amniotic Fluid, Pleural Fluid etc)
Send at least 0.5 mL in a sterile container without any additives. Transport on ice immediately to the laboratory. For eye fluid, send as much sample as possible. Collect sample in a sterile leak-proof container or in a syringe with the needle removed. Cap the syringe with a rubber bung or plastic cap to prevent leaking.

Bronchoalveolar Lavage and Endotracheal Aspirate
Send at least 1 mL in a sterile container without any additives.

Cervical Swab
Use a speculum during collection. Use a cotton-tipped swab to clean the exocervix of mucus and discard. (Mucus present in the sample may inhibit the PCR assay). Insert a Dacron-tipped swab about 1cm into the cervical canal. Rotate and leave for a few seconds to absorb the secretions. Send swab in swab sheath without any media.

Conjunctival Swab
Using a Dacron-tipped swab moistened with tiny amount of sterile saline, pull down the lower lid and swab the conjunctiva firmly, then evert the upper lid and swab similarly. Send swab in swab sheath without any media.

Nasal Swab
Use a Dacron-tipped swab or flocked swab. Advance the swab tip past the anterior nares to the nasal mucosa (approximately 2–3 cm from the nostrils in adults) and gently rotate to collect nasal secretions from the anterior portions of the turbinate and septal mucosa. Send swab in swab sheath or sterile empty container without any media.

Nasopharyngeal Swab
A flexible shafted flocked swab is essential. Insert the swab into the nostril and back to the nasopharynx. The swab should be slid straight into the nostril with the patient’s head held slightly back. The swab is inserted following the base of the nostril towards the auditory pit and will need to be inserted at least 5–6 cm in adults to ensure that it reaches the posterior pharynx. Leave the swab in place for a few seconds. Withdraw slowly with a rotating motion. Send swab in swab sheath or sterile empty container without any media.

Nasopharyngeal Aspirate

Attach a disposable polythene catheter (French catheter 8 for infants) to a mucus trap and a suction pump.
Insert the catheter through the nose for a distance equal to that from the tip of the nose to the angle of the jaw.
Using the suction pump, aspirate mucus into the trap. If secretions are thick and trapped in the catheter, dislodge the mucus by sucking up 1 - 3 mL of phosphate buffered saline (PBS).
Disconnect the trap when collection is complete and empty contents into an empty bottle.
If a suction pump is not available, attach the catheter to a 20 mL syringe, aspirate secretions, then expel secretions into an empty bottle.

Rectal Swab
Collect only when stools are difficult to collect, as rectal swabs are inferior to stools. Insert a Dacron-tipped swab into the anal orifice, (at least 3cm deep in an adult) and rotate to ensure collection of faeces. Send swab in swab sheath without any media.

Semen
Collect semen in an empty sterile container.

Sputum
Instruct patient to rinse mouth with water prior to sputum collection. Collect sample resulting from deep cough into a sterile screw-capped container. Instruct patient not to expectorate saliva into the container.

Stool
Collect at least 1ml of liquid stool or a pea-sized amount of stool in an empty sterile container.

Throat Swab
Swab the pharynx and both tonsils vigorously with a Dacron-tipped or flocked swab. Send swab in empty container or swab sheath without any media.

Tissue Sample
Fresh samples are preferred for molecular tests. Fresh tissue samples, at least 5 X 5 X 5 mm in size, should be sent immediately to the laboratory on ice in a sterile empty container with no media. Formalinised or fixed tissues can be used as well, but are less ideal with reduced detection sensitivity.

Urethral Swab
Use aluminium Dacron-tipped swab. Insert swab 2-4 cm through the urethra and rotate 3-5 seconds to ensure adequate sampling. Send swab in swab sheath.

Urine
First morning void urine is best as microorganism is most concentrated. Alternatively, patient must not urinate within two hours prior to urine collection. Collect the first 15-20 mL for Chlamydia trachomatis and 2-10 mL for BK virus into a sterile container without any media.

Vesicular Swab
Use Dacron-tipped swabs. Sample fresh skin vesicles during the first three days following the appearance of the eruption. (Crusted lesions have a lower chance of yielding viable virus.)

Gently clean the surface of the vesicle with sterile saline. Do not use alcohol.
If the vesicle is intact, use a sterile needle to lift off the roof of the vesicle.
Using a sterile swab, soak up all the fluid from the vesicle, then swab the base of the lesion vigorously to dislodge cells on the base (which contain the virus) onto the swab.
Using the same swab, repeat the procedure with a number of vesicles, if present, in order to increase the yield of virus.

Molecular Oncology Tests

Sample requirements

Fresh/frozen tissue or paraffin embedded tissue block is preferred but slides are also accepted. For slides, at least 8 sections of 8 mm thickness, unstained with no cover slips, are required. For smaller biopsies, at least 12 sections of 5 mm thickness are required. Please also include 1 H&E slide, 4 mm thick, with a coverslip as a reference slide for the unstained slides. The tumour content must be documented on the request form. The tissue sample should be predominantly tumour cells (optimally at least 50%). The Molecular Laboratory will reject all samples where the tumour content is less than 30%, with the exception of EGFR PCR where a tumour content of less than 10% will be rejected.

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