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PROTOCOL for viral analysis of intestinal biopsies in autistic children

Sample handling

During ileo-colonoscopy, ileal and colonic biopsies should be taken for routine histopathology, as per standard practice. In addition, fresh frozen biopsies are required.

Viral analysis consists of in-cell and solution phase TaqMan Reverse transcriptase PCR for measles virus N, H and F genes.

Virus has been detected in the terminal ileal lymphoid tissue, and biopsies should be taken from these areas wherever possible. Virus has not, as yet, been detected in the colonic biopsies, and these should only be sent if ileal tissue is not available.

(a) In-cell TaqMan RT-PCR is performed on formalin fixed, paraffin processed tissue sections. The tissue blocks should be sent to the address given below, and these sections will be mounted on to RNAse-free Perkin Elmer in situ PCR slides. The blocks will be returned once the sections have been cut.

(b) Solution-phase TaqMan RT PCR studies are performed on extracted fresh frozen biopsies. The dissected tissue is placed in an Eppendorf tube (or equivalent) and immediately snap frozen in liquid nitrogen. If liquid nitrogen is not available, then submerged the biopsy in approximately 5 volumes of *RNAlaterTM (e.g. a 0.5g sample requires 2.5ml of RNAlater). Store at 4oC overnight and then transfer to -20oC.

(c) Collect at least 8.5mls of whole blood into a plastic EDTA tube, mix well and freeze. (If a plastic EDTA tube is not available, transfer blood into plastic conical after collection in EDTA tube, and freeze at -20oC.)

Where possible, all three types of samples should be provided: formalin fixed paraffin processed tissue, fresh frozen biopsy and frozen whole blood sample. Complete the sample checklist form, which is included in this package and send with samples.

 


 

Sample shipping

Paraffin embedded tissues should be shipped at room temperature.

Frozen blood samples and fresh frozen biopsies should be sent on dry ice in a sealed, insulated container in accordance with shipping regulations.

Ship samples Monday and Tuesday only, to guarantee arrival before Friday. Use sufficient dry ice to keep samples frozen for at least 48 hours.

The sender will be responsible for paying all shipping costs.

Fax notification form at time of dispatch so samples can be handled appropriately in the receiving laboratory.

Samples should be sent to: Andrea Doolan

Unigenetics Ltd,

Research Laboratory,

Coombe Women's Hospital,

Dublin 8,

Ireland

Ph. 353-1-4737142

Fax 353-1-4737144

 

 

 

* RNAlaterTM is a non-toxic tissue storage reagent that rapidly permeates tissue to stabilize and protect cellular RNA in situ in specimens. It can be purchased from Ambion, Inc. @ 1-800-888-8804 (USA) or , catalogue number 7020 (100mls) or 7021 (500mls).

 

 

 


 

 

SAMPLE CHECKLIST:

 

Patients' Name: __________________ Date of Birth: __________________

Patient Identification: ______________ Sex: Male / Female

Date Sample Collected: ____________ Diagnosis: _______________

 

 

Please check which samples are being sent:

PE In situ slides: ___ Biopsy site: ________________ How many: ___

Paraffin Block: _____ Biopsy site: ________________ How many: ___

Fresh frozen biopsies: ___ Biopsy site: ____________ How many: ___

Whole Blood/EDTA — Frozen: _________________ Volume: ________

Other type of sample: ___________________________________

 

 

Please note that there will be a cost of IR£1000 to do the analysis. Please complete the following information to for result reporting and invoicing purposes.

Parent's Name: _________________________

Address: ___________________________

___________________________

___________________________

___________________________

 

Name of Physician: ______________________

Address: ___________________________

___________________________

___________________________

___________________________

 


Fax Notification Form

 

Attention: ______Andrea Doolan _______

Fax number: ______353-1-4737144 _______

Phone Number: ____ 353-1-4737142_______

 

 

From: __________________________________

Phone number: ___________________________

 

Air bill number: _________________________

(For tracking purposes)

 

Please check:

Paraffin embedded tissue (ambient) _____

Clotted blood sample (dry ice) _____ Fresh frozen biopsies (dry ice) _____

Please complete & fax this form when sending samples

If you have any questions or need further information please contact Unigenetics Ltd.