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Immunofluorescence

 

Direct immunofluorescence is used for the detection of tissue bound proteins such as antibodies and complement. Two types of tissue, skin or renal, are processed, although occasionally others may be submitted (e.g. oral mucosa, conjunctiva). Biopsies are taken from patients; snap-frozen and sectioned prior to incubation with fluorescein isothiocyanate (FITC) conjugated anti-sera. This method allows for the detection of tissue bound antibodies when there may be insufficient levels to detect in serum. Other tissue reactants such as complement and fibrinogen can also be detected. In some disease states patterns of staining can be diagnostic.

 

Instructions for Collection of Samples

 

Do NOT Put Samples For IMF in to Formalin

 

Skin Samples

Skin samples are wrapped in saline soaked gauze and place in a universal container with full patient details label attached.  The skin samples must include the dermal-epidermal junction (DEJ) for tests to be performed.

 

Renal Samples

Renal samples are placed in saline solution in a universal container with full patient details label attached.  Renal samples must include the portion of a kidney containing glomureli.

Any other tissue sample requiring IMF must be placed in saline.  Formalin is NOT an appropriate medium; IMF tests cannot be performed on formalin fixed tissue.

 

 

 

Sending samples to Cellular Pathology department

Samples from patients with known infectious risks ie. Hep B, TB, HIV must be discussed with a pathologist or Immunologist before the sample is taken.

Fill out a Histology form with all the patient and corresponding clinical details. The form must be clearly labelled IMF.

If a sample for routine histology has been taken alongside the IMF sample then one form can be sent detailing the pots and instructions for tests needed.  All samples taken on one patient are to be sent together (thus one uniform report can be generated).

 Samples requiring IMF analysis must be sent as soon as possible, within 24 hours of collection to ensure optimal results.

 

For further information contact

                Mr Gavin Rock – Deputy Head Biomedical Scientist – 0121 424 3188

                Dr Gerald Langman, Renal Specialist – 0121 424 3189

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HER 2 Testing

Author Lisa James

 

Molecular Cytogenetics Service

The Molecular Cytogenetics service within Cellular Pathology based at Heartlands Hospital provides a high quality, fully UKAS accredited FISH service for both internal referrals and external users, both regionally and nationally. Our highly experienced FISH team utilises an integrated approach incorporating morphology, immunohistochemistry (IHC) and fluorescence in-situ hybridisation (FISH) genetics testing to provide a fully comprehensive service for our users.  The section currently offers prognostic IHC and FISH HER2 testing for breast and gastric cancers and diagnostic/prognostic FISH testing of lymphomas.

Breast and Gastric Referrals

IHC testing using the 4B5 antibody clone (Roche) is initially performed to screen patient samples for HER2 protein expression levels to identify tumours which “over-express” this protein. Patients with tumours which are HER2 positive usually benefit from treatment with Trastuzumab (Herceptin). Where expression levels are equivocal (2+ IHC staining, see Fig 1.), FISH is carried out to determine unequivocally whether the tumour is positive or negative for HER2 gene amplification (see Fig 2.). Service users may send referrals for both HER2 IHC and FISH testing when appropriate or FISH analysis only if performing their own HER2 IHC. 

          IHC HER2         

Fig. 1. 2+ IHC staining

        FISH HER2 1           

 Fig2. HER2 amplified FISH result                                                                                                                                                      

The service performs approximately 4,400 HER2 IHC tests and 1,200 HER2 FISH tests per annum. The turnaround times (TaT) for HER2 breast cancer referrals and gastric referrals is 14 days and 7 days respectively including weekends.  However, since the recent introduction of the Abbott Bioview automated digital FISH image capture and analysis system (see Fig 3.) into the department, the TaT for breast and gastric referrals can be quicker than the times stated above. 

                                                                                                      Abbott Bioview1              Abbott Bioview2

Lymphoma referrals

We currently offer an extensive repertoire of FISH testing to identify specific gene rearrangements associated with a range of lymphomas/lymphoproliferative disorders in order to aid diagnosis, tumour classification and disease prognosis (see Table 1.). The variety of FISH testing reflects the 2016 revision of the World Health Organization classification of lymphoid neoplasms 

 

FISH Test Probe Type Disease
MYC Dual colour breakapart Burkitt lymphoma, high grade lymphoma with MYC and BCL2 and or BCL6 translocations, high grade lymphoma NOS, DLBCL
IGH/MYC* Dual colour, dual fusion
MYC/IGK/IGL* Tri-colour, dual fusion
MYC/BCL6* Dual colour, breakapart
BCL2 Dual colour, breakapart High grade lymphoma with MYC and BCL2 and/or BCL6 translocations, folicular Lymphoma
BCL6 Dual colour, breakapart High grade lymphoma with MYC and BCL2 and/or BCL6 translocations, folicular Lymphoma
MALT1 Dual colour, breakapart MALT1 lymphoma
TP53 Dual colour, site specific CLL
ATM Dual colour, site specific CLL
IRF4/DUSP22* Dual colour, breakapart Anaplastic Large Cell Lymphoma (ALCL), large B-cell lymphoma

COND1

Dual colour, breakapart Mantle cell lymphoma
IGH Dual colour, breakapart General FISH marker for all lymphomas

Table 1.  List of lymphoma/LPD FISH probes currently offered by the molecular cytogenetics service.

The FISH testing strategy (see Fig 4.) to exclude a high grade lymphoma with MYC and BCL2 and/or BCL6 translocations involves initial testing using a MYC breakapart probe.

 MYC FISH Chart

 

Fig. 4. Flowchart of current high grade lymphoma FISH testing strategy, DH= double-hit, TH = triple-hit *(Includes non-classical abnormal signal patterns)

 

Additional BCL2 and BCL6 FISH testing will only be performed if the MYC FISH test is abnormal unless specifically requested (see Fig 5.).

                                                                          MYC PIC Edit

Fig 5. Abnormal 1F1R1G split signal pattern using the Vysis MYC dual colour, breakapart probe specific for the MYC gene region at 8q24. BCL2 and BCL6 FISH studies will be carried out (see Fig 6).

                                                                                                                        BL2andBLC6

 

Fig. 6 Vysis dual colour, breakapart BCL2 and BCL6 FISH results. The BCL2 probe shows an abnormal 1F1R1G signal pattern consistent with a BCL2 gene rearrangement at 18q21.  The BCL6 probe shows a normal 2F signal pattern. As the MYC gene is also rearranged this result is consistent with a High grade lymphoma with MYC and BCL2 translocations.

Turnaround Times (TaT’s) for FISH testing

  • The TaT for HER2 IHC is 1-2 days from receipt of the sample.
  • The TaT for breast HER2 FISH testing is 14 days from receipt of the sample.
  • The TaT for gastric HER2 and MYC FISH testing is 7 days from receipt of the sample

Sample Requirements

All samples submitted for FISH testing must be accompanied by a request form detailing the patient name, DOB, NHS number and laboratory number of the requesting laboratory. A copy of the histology report should also be included.

A representative paraffin wax block should be submitted for testing, which should contain sufficient tumour material for FISH testing. Alternatively, we do accept tissue sections on charged, Menzel Glazer slides.

If slides are to be sent please provide 5 sequential sections, cut at 3-4μm, on to charged slides, maintaining placement and orientation on the slide. The slides must be labelled with the laboratory number, including any suffix/part number, patient name and the date that the sections were cut. Sections must be dried overnight at 37OC. Do not heat on.

If slides are being sent for HER2 FISH analysis only, please ensure that the original HER2 immunohistochemistry slide is also included.

Please ensure that spare sections being sent are representative of the IHC slide.

Limitations of test

Samples should preferably be fixed in 10% neutral buffered formalin for 6 – 48 hrs. Suboptimal fixation, either insufficient or extended, can cause issues with interpretation of FISH results.

The use of marker dyes, such as eosin and carbol fuchsin, on breast core biopsies and resection specimens should be avoided as this causes auto-fluorescence on FISH samples which are difficult to interpret.

FISH studies involving bone marrow trephines where tissue decalcification has occurred may fail due to destruction of cellular DNA.

For lymphoma FFPE testing, FISH results are not diagnostic and should be considered in the context of the morphology and immunophenotype. FISH false positive signal patterns may be seen in up to 5% of cells for breakapart probes consequently abnormal clones below these levels may not be excluded.

Contact Details For Molecular Cytogentetics (FISH) Service

 

Hours of Service: The HER2 service is staffed Monday – Friday 08:30 – 17:00

Please contact our team via our generic email address, This email address is being protected from spambots. You need JavaScript enabled to view it. or telephone the Cellular Pathology secretaries on 0121 4243487 who will transfer you to the FISH service.   Alternatively, contact our senior scientist team individually via the following email addresses;

  • Dr. Jane Starczynski – Consultant Clinical Scientist This email address is being protected from spambots. You need JavaScript enabled to view it.
  • James Dowds - Senior BMS This email address is being protected from spambots. You need JavaScript enabled to view it.
  • Julie Pritchard – Senior BMS This email address is being protected from spambots. You need JavaScript enabled to view it.

 

For Clinical advice and interpretation please contact one of the pathologists listed below.

Additional Information

- Referred blocks are returned to the referring laboratory by recorded delivery once the case has been reported.

- HER2 IHC and FISH tests are fully FDA approved and validated assays (Pathway 4B5 antibody from Roche and PathVysion dual colour, site specific HER2 FISH probe from Abbott Molecular (both CE marked)).

- The Bioview automated system has been fully validated and is in scope of ISO 15189 UKAS accreditation.

- All FISH and IHC testing is performed in accordance with National Guidelines. We are a national reference centre laboratory for HER2 testing.

- We actively participate in national UKNEQAS/GENQA schemes for HER2 and lymphoma/LPD FISH testing.

- We are continually monitoring and auditing all aspects of our service to provide high quality results for our users.

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Specimen Labelling And Completion Of The Request Form

All Histology specimens must be labelled appropriately and accompanied by a fully completed request from.

Specimen Labelling Requirements

  • Forename and surname
  • Date of birth
  • NHS number and or Patient Identification number
  • Address
  • Nature of specimen

Request Form Labelling Requirements

  • Forename and surname
  • Date of birth
  • NHS number and or Patient Identification number
  • Address
  • Nature of specimen
  • Clinical features
  • Any known concomitant diseases
  • Requesting doctor
  • Location
  • Date and time specimen taken

The DATE AND TIME that the specimen is taken is particularly important for breast core biopsies and bone marrow trephine biopsies to ensure optimal results of subsequent testing.

Items in bold type are essential. Failure to provide the essential information may result in a delay in reporting as confirmation of patient demographics will be required prior to specimen processing Where there are inadequate patient or specimen identification details the clinician will be informed and requested to attend the department, identify the case and provide the necessary details. If the clinician cannot be contacted the request form and specimen will be returned to the sender with a "discrepancy" proforma to be completed by the clinician. The completed and signed proforma must then be returned with the corrected form/specimen pot to the department. A Datix incident report will be completed if a sepcimen has to be returned for relabelling.

If a report is required urgently the request form must be labelled URGENT/TWO WEEK WAIT/ETC and the specimen must be sent to the laboratory as soon as possible. If a report is required for an MDT it is helpful if the date of the MDT is stated on the form.

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