Transplantation &

Immunolab Urmi Chouhan

Biomedicinska analytikerprogrammet

BMA053, Tillämpad laboratorievetenskap special diagnostik

VT-2014.

Sahlgrenska Universitetssjukhuset

Patologi, Transplantation och immunolab.

Sahlgrenska Akademin

Rules

If I talk too fast wiggle your hand like a beauty queen.

The bold green text are abbreviations.

The Organization

Sahlgrenska University Hospital (SU) is organized in a linear structure.

Dictated by Barbro Fridén who together with a board of directors decides about health care issues in SU.

Lars Lundgren. Director of department 4.

Department 4 = Clinical Pathology and Genetics (KPG).

Mattias Åbom. Branch head for Special Lab Transplantation & Immunolab (T&I). NOTE! It’s not called

Clinical Pathology and

Cytology but Clinical

Pathology and

Genetics. This change

took place 01/2014

The Organization

Who works here?

Biomedical laboratory scientists(BLS) Laboratory assistents Doctors Cleaners Janitors IT technicians Admin and specialists Autopsy technicians and more!

Approx. 40 employees at special lab.

NOTE! It’s not called

Clinical Pathology and

Cytology but Clinical

Pathology and

Genetics. This change

took place 01/2014

The Machines

PT Link

pH High/Low

(Deparaffinization)

Autostainer

EnVision™

Flex / Flex+

Artisan

Special staining

Tissue-Tek Film

Coverslipper

The Laboratory

The samples obtained at this lab is after the full pathology circle has gone; Now the doctor wishes to specify the tissue to a certain antibody staining ex.

antibody KI-67!

Patient groups:

All transplanted patients.

Investigations on kidney, liver, hearts.

Usually a pointed question e.g. Reaction question, CMV, Routine and/or Medication control.

Immunostaining can be helpful when it comes to identifying the primary origins of a tumour (if it’s metastatic)

BLS looks at the completed staining to assess if it’s good/bad then delivers the slides to the doctor.

Transplantation & Immunolab » Basically one unit «

Lab-assistent brings

blocks to freezer. Prints work-lists fr.

SymPathy.

BLS cuts the tissue. Burns slides in an incubator for 1hr

in 60°C.

Pretreatment for target retrieval

ag.

Autostainer visualizes the

slides

Dehydration approx 15min

Clearing

Mounting (glass/film)

Controlling slides & colouring

Unticked from Sympathy

Delivered to doctors.

Pretreatment of slides

Several different ways to pretreat the tissue for staining.

1. PT Link: pH 6 and 9 (TRS High/Low)

2. Tris/EDTA pH 9

3. Citrate ph 6 and/or 2

4. Protease (”digration”) in microwave.

5. Or none at all!

Why pretreat the slides? Because of the antigen-antibody interaction. (The antibodies bind to the epitopes in the tissue)

Autostrainer:

EnVision™, Flex & Flex+ EnVision™

Two-step polymer method.

The 1° ab is targeted by a 2° ab with a Dextran backbone with enzymes on it.

Flex

Smaller polysaccharide chain than in EnVision.

Ag activated by the polymer.

DAB binds around the complex.

Results? Stronger staining.

Dilute the ab more than in EnVision. $$$ profit shorter lastability

Flex+

Only difference is the linker which

multiplies the signal.

Results?

More staining

Shorter incubation

Visualization Process: EnVision™ FLEX

T&I runs 3 times a day:

Morning, Mid-day, Over night = Huge production.

Workload varies.

Some statistics

Month Total slides

Jan 2013 4145

Jan 2014 4560

These slides are all in total. The first slides, immunostained, Hercep & Artisan.

*Light grey = EnVision™ FLEX

**Dark grey = Other reagents

”HP Block”-

bottle in

Autostainer.

Possible outcomes (Kidney – Glomeruli, Breast - Proliferation)

Summary of

Workflow & Data management at T&I

1. Tissue sample acquired

2. Tissue preparation

•Patient ID

•Slicing tissue

•Tissue onto slides

•Labelling + Control specimens

•Specific Pretreatments

3. Immunohistochemistry

•Target retrieval (PT Link)

•Deparaffinization

•Antibody staining

•Autostainer and/or Artisan

•Coverslipper

•Image analysis

4. Data management: There is an integration with

Sympathy and DakoLink to process information

technically.

5. Doctor receives slides. Produces results.

6. Patient medical records.

7. Treatment adjusted accordingly

Quality Assessments Why? To assure the staining has above adequat quality.

T&I follow guidelines and requirements from SWEDAC.

*Internal: Checklists; Weekly/monthly routine controlling utensiles, reagents & ab. POS/NEG or Internal sample controls.

A multiblock control for ab (pancreas, liver & appendix)

Tonsil-, Tumour-, Normal tissues

Multi-multi (not here though!)

*External: NordiQC, NEQAS & EQUALIS

Mostly used in

research. Worth the money.

Quality Assessments

How to gain a Certificate of Eligibility. Coaching until independence. This certificate must annually be updated.

Traceability and documentation = ♥? YEP! SymPathy

If a divergence is made or noted it must be register in the datasystem RMT+. Depending on the nature of the problem it may also need a report in MedControl Pro.

Biobanking All the tissues is saved until an answer has been delivered to the customer (within 4-5 weeks.)

Patients can decide in the referral if they don’t want their tissue to be archieved.

The glass are saved for approx. 10 years.

Wax-blocks are saved forever.

In conclusion Accredited special lab with a lot of cooperation.

Pretreatment is important to open up epitopes on the antigen for the antibodies to bind in to.

Autostainer. EnVision™, Flex & Flex+. Varied use to obtain the best & most efficient staining

Flow:

Biobanking: Glass saved for 10 years. Blocks saved forever

Pathology has a huge production and an increasing workload so employees are wanted!

New order from Doc

BLS cut/slice the tissue

1hr incubation

Pretreatment+PT-Link

Autostainer Mounting Drying

& Sorting

Controlling Unchecking Glass to doctor.

Thank you for listening!

Created by Urmi Chouhan, 2014