الطريقة المثلى التي يجب ان يتعامل بها فني المختبر او طبيب علم الامراض في مختبر علم الامراض الجراحي عند استلام عينة إزالة ثدي

In the surgical pathology laboratory when I received a mastectomy specimen.
On grossing, I have detected a malignant growth .in order to confirm my diagnosis I will select tissue kept in cassettes for fixation and processing after that will do fixation for specimen. This is an important part of the process.
In this to make microscopic slides, the tissue in the cassettes must be enough time in 10% formalin, duration: 6-8 hrs, a fixative that hardens for the tissue and prevents the proteins in the cells from degrading. Fixation helps us to keep the tissue another time. The time needed for fixation depends on size and fat of tissue immunohistochemical stains for prognostic markers .If proteins are not preserved properly in tissue by fixation, they degrade and become impossible to detect like estrogen. After the tissue is hardened by fixation. I will do processing to the tissue. The aim is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut.
Steps processing are:
Put the tissue in water. -
- Transfer tissue to alcohol (70%, 90%, Absolute Alcohol I, Absolute Alcohol II and Absolute Alcohol III) for Dehydration form the tissue by gradual increase concentration of alcohols.
 -transfer tissue from Absolute Alcohol III to xylene I after good time transfer tissue to xylene II then xylene III for Clearing (alcohol replaced by xylene so that it is miscible with paraffin)
- transfer tissue to paraffin wax. Paraffin I then after time paraffin II then paraffin III for Impregnation tissue and embedding tissue by paraffin wax.
After processed tissue I will do embedded in hot wax to form a tissue block. When the block hardens, it is stable at room temperature then Tissue placed in metallic mould in proper orientation and Liquid paraffin poured over tissue into mould then Paraffin solidified by cooling. After the tissue block ready to use. I will put Block mounted on microtome then sections cut 4-5 µm thick with very sharp knife after I prepare sections floated sections on water bath then Tissue ‘fished’ onto plain glass slide .
After prepare slide I will do Hematoxylin and Eosin for staining.
Steps for Hematoxylin and Eosin:
-Immerse sections in xylene I for 2 min.
-then Immerse sections in xylene II for 2 min.
-After that Immerse sections in Absolute Alcohol I, Absolute Alcohol II, 95% Alcohol, 80% Alcohol, 70% Alcohol and 50% Alcohol each for 2 min.
-Then Wash and clean slide thoroughly in running tap water.
-Immerse slide in Harris Hematoxylin for 20 min.
-Rinse slide thoroughly in running tap water.
-Then Differentiate slide in 1% acid alcohol for 1 dip.
-Wash slide in running tap water for 5 min.
-Dip slide in 0.3% ammonia until section appear bright blue for 20 dip.
-Wash slide in running tap water for 5 min.
-Transfer slide and Immerse in Counterstain with eosin for 5 min.
-Then Wash slide thoroughly in running tap water.
-Immerse slide in 80% alcohol for 10 dip.
-Then Immerse slide in 95% alcohol for 10 dips
-Then Immerse slide in absolute alcohol I for 2 min.
-Immerse in absolute alcohol II for 2min.
-Immerse slide in absolute alcohol III for 2 min.
- Immerse slide in xylene I, xylene II and xylene III each for 2 min.
- Put DPX on slide.
The slides are now ready to be examined under the microscope.
Then I will write Pathology Report
Then I will do to confirm my diagnosis. The most common way is by immunohistochemistry to confirm diagnosis
I will then choose a block on which immunohistochemical stains for cancer a mastectomy markers (ER, PR, Ki-67, and her/2neu) will be performed.
In this steps for immunohistochemistry I will cut several (about 5) additional sections from the block that I have made in this laboratory.
-First I will Transfer slides (tissue) section into container with target retrieval solution Tris EDTA (TE) buffer pH 9.0.
-Then Flood section with peroxidase blocking solution.
- After that Add Primary antibody.
- Add 2-3 drops secondary antibody
- Flood DAB substrate on section slide; leave it 5-10 minutes.
- Put in running tap water
- Counter stain with Haematoxylin.
- Results shall be interpreted as negative: if there is no brown precipitate. Results shall be interpreted as positive: when there will be brown precipitate on tumour cell membrane. It will be graded by the pathologist as according to a scale of 0 (negative), 1+ (weak positive) to 2+ (equivocate strongly positive l) and 3+ (strongly positive).
Also I have to do another technique May the cancer has made by the gene.
I will do Chromogenic in situ hybridization which includes:
The first one I will also to take the tissue from the same block. This technique will take 2 day to give the result.
First day I will do:
A- Pretreatment (Dewax/Proteolysis) (day 1)
- Incubate slides at 70°C (e.g. on a hot plate) and 100% ethanol then wash
- Heat Pretreatment Solution in a staining jar standing in boiling water
bath to at least 95°C .Place slides in the Pretreatment Solution and incubate
- Transfer slides immediately to distilled water and wash
- Apply (dropwise) Pepsin Solution to the air-dried tissue/cell section and Incubate at RT in a humidity chamber and Wash in distilled water
- Dehydration: in 70%, 85%, 95% and 2x 100% ethanol then air dry sections
B- Denaturation and Hybridization (day 1)
- Vortex the ZytoDot SPEC EGFR Probe and pipette 15 μl onto individual
Samples
- Avoiding trapped bubbles, cover the samples with a coverslip (22 x 22 mm) Seal the coverslip, e.g., with a layer of hot glue from an adhesive
- Pistol or rubber cement
- Denature the slides at 94-95°C e.g., on a hot plate
- Transfer the slides to a humidity chamber and hybridize overnight at 37°C (E.g. in a heating oven).
The second day will do:
C- Post-Hybridization and Detection (day2)
- Carefully remove the rubber cement or glue
- Remove the coverslip by submerging in Wash Buffer SSC at RT and wash in Wash Buffer SSC at 75-80°C and Wash in deionized or distilled water
- Incubate slides in 3% H2O2 and Wash in PBS/Tween
- Apply Blocking Solution dropwise (3-4 drops/slide) to the slides and Incubate
- Blot off Blocking Solution, but don't rinse
- Apply Mouse-Anti-DIG dropwise (3-4 drops/slide) to the slides and
Incubate at RT and Wash in PBS/Tween.
- Apply Anti-Mouse-HRP-Polymer dropwise (3-4 drops/slide) to the slides and incubate at RT and Wash in PBS/Tween.
- During the wash steps, prepare DAB Solution by adding DAB Solution B
- Dropwise in a graduated cup up to 1 ml and add one drop DAB Solution A
- Apply DAB Solution dropwise (3-4 drops/slide) to the slides and incubate
- Transfer slides into a staining jar and wash in running tap water
- Counter stain the tissue or cell samples with Mayer’s Hematoxylin Solution
- Transfer the slides into a staining jar and wash in running tap water
- Dehydration: in 70%, 85%, 95% and 2x 100% ethanol and Incubate in xylene and then air dry sections.
- Avoiding trapped bubbles, cover the samples with a coverslip (22 x 22 mm; 24 - 32 mm) by using Mounting Solution and air dry slides.
- Evaluation of the sample material is carried out by light microscopy
I will Interpretation of Results like:
Amplification >10 copies, or large clusters, of the gene present per nucleus in >50% cancer cells.
Low Amplification 6-10 copies of the gene, or a small gene cluster, present per nucleus in >50% cancer Cells.
No Amplification 1-5 copies of the gene present per nucleus in >50% of cancer cells.
I have to do another technique to conform diagnosis
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a cytogenetic technique done in specialized laboratories which can be used to detect and localize DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of Sequence similarity. Fluorescence microscopy can be used to find outwhere the Fluorescent probe bound to the chromosome
-The same block will be subjected to FISH.
-The tissue section on a microscope slide is treated with a specific fluorescence labeled DNA probe assay kit. The procedure will be followed according to the manufacturers’ instructions.
-The Probe mix contains fluorescence – labeled PNA probe targeted at Centromere of chromosome 17 and Texas – red labeled DNA probe to gene (on chromosome 17).
-Fluorescence labeled DNA probes bound to the interphase nuclei of cells mark the candidate cells.
-The labeled sample is placed under the microscope and examined - 20 nuclei per tissue specimen will be counted.
-Results of ratio gene will be recorded.
-Specimens with a gene ratio above or equal to 2 is considered 2 genes amplified.
Also I can do other techniques to confirm diagnosis like:
PCR (polymer chain reaction)
DNA/RNA/protein by use PCR. Detection of mutations in oncogenes & tumour suppresor genes, gene amplifications, MSI, microorganism etc
Preparation of tissue from fresh tissue, FFPE tissue
Microdissection of tissue using light microscope
Laser capture microdissection

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