Table 3 Summary of actions and recommendations for the analytical phase

Sample reception at the pathology laboratory

1.Responsible personnel: administrative or technical employee

2.Verify the list of dates/times registered for the steps/procedures previously performed

3.Register date and time of sample receipt

4.Confirm the type of tissue (fresh or fixed) and the type of fixative, and register the date of entry at the laboratory

5.The criteria for sample acceptance and rejection and the recommendations for exams to be performed in samples with restriction must be clearly specified in written instructions

6.Reasons for samples rejection:

• Samples lacking patient identification or with doubtful or incorrect data

• Inconsistency between the type of sample mentioned in the exam request form and the type of material received

• Samples without a medical request form

7.Factors that limit sample condition (notified at the registry of exam entry)

• Fixative is inadequate or absent

• Broken or cracked containers/slides with possible partial leakage of material

• Information about the dates/times of the previous steps is unavailable

• Inadequate proportion of fixative to specimen

• Large specimen not previously sectioned

• Inadequate containers

• Exam request form incomplete

8.Specimen registration and transfer to macroscopy

Specimen registration in the laboratory

• Verify if specimens retrieved from the container used for transportation match the information provided in the labels and in the request form

• If specimen and identification data match, a unique identification number is attributed for the sample to allow tracking during the process

• When possible, use barcode labels to improve traceability of all materials of a single case (sample fragments, paraffin blocks, histological slides, routine and special staining, etc.)

Macroscopic examination

• Manually performed by pathologist or laboratory technician

• Verify the correspondence between the specimen/sample identification on the label and the request form, confirming the laterality and tumor location in breast quadrants

• Follow the test and sampling protocols recommended by scientific societies of pathology and international institutions

• Verify if fixation was properly performed

• Measure the size and weight of the tissue surgical piece

• Ink the surgical margins with different ink colors

• Cut the specimen into thin, parallel, and cross-sectional slices, avoiding damaging or clamping the tissue

• Describe the observed alterations in relation to the color, texture, consistency, delimitation of the adjacent tissue

• Measure the lesions found in the macroscopic examination

• Use clean cut surfaces and instruments to avoid cross-contamination with other samples

• Special care is required for fine-needle biopsies to assure the inclusion of all fragments

• Choose appropriate and labeled cassettes for each type of material, avoiding placing excess material

• Describe and measure the lesions visualized in the macroscopic examination, registering information regarding the topography in relation to the anatomic position and distance from the nipple (when present) and surgical margins

Histological processing

• Performed by laboratory technicians using tissue processors

• Use of adequate time of tissue processing for each type of specimen

• Needle biopsies require shorter time in each reagent during processing than specimens from surgical resections

Paraffin embedding technique

• Performed by laboratory technician

• Manually (handling-processing) or with the use of a paraffin embedding machine

• Avoid excessive heating of paraffin

• Check the paraffin temperature regularly

• Avoid overfilling of each mold/block

• Samples should be carefully oriented, handled and positioned in the inclusion blocks

Microtomy

• Performed by a laboratory technician

• Use high quality blades

• Optimize the knife angle of inclination in the microtome

• Slice the paraffin embedded tissue blocks carefully

• Avoid freezing damages

• Slice blocks in thin Sects. (3 to 5 µm), gently and slowly

Tissue floatation in water bath and placement of the paraffin embedded tissue sections on slides

• Use clean water

• Certify that blades/knives are clean to avoid cross-contamination

• Avoid simultaneous floating of various cuts in the water bath chamber

• Check water bath temperature

• Avoid excessive expansion and damage of tissue sections

• Carefully choose tissue section with no folding or extensive distension

• Avoid the formation of bubbles under the tissue sections that could lead to the detachment of the sections during histological staining

Dehydration of histological sections

• Dry the histological section before placing it in the histological incubator to dehydrate

• Incubator temperature and dehydration time should be monitored

Routine staining

• Staining with hematoxylin and eosin are routinely performed manually by the histotechnician or using specific equipment (autostainer)

• Histological sections must be completely deparaffinized before staining

• Reagent should be regularly renewed

• Use standardized conditions and protocols for staining, adopting precise times and quality constant monitoring

Coverage of tissue sections with coverslip

• Histological sections should completely dehydrate before mounting

• Place the mounting medium and cover with cover slip

• Avoid excessive drying, formation of crystals or bubbles