Technical

Question 1

Why do you need forms at embedding

Answer 1

Confirm accession number and check that the number on the block matches the form. Check that the correct number of pieces and size of tissue is in the cassette, check for specific embedding instructions such as on edge or ink down. The form also contains the audit trail, which is signed by the BMS to indicate that they embedded the specimen, this can be used for RCA in the event of an issue arising.

Question 2

What do you do if metal clip in tissue?

Answer 2

If the clip can be removed without destroying the tissue or compromising margins then I would remove it myself. If I was concerned that this might happen then I would consult a Pathologist before removing.

Question 3

Properties of well-embedded block

Answer 3

Orientated according to protocol and embedding instructions. The tissue should also be embedded flat and evenly, allowing for full face trimming without losing much tissue.

Question 4

How do you prevent cross contamination? How would it affect patient

Answer 4

Keep the embedding centre clean, only open one cassette at a time, clean the forceps between each block, open cassette and mesh bags carefully to ensure tissue does not flick and get lost
May cause misdiagnosis of the patient and the patient could potentially receive the wrong treatment if the error is not noticed

Question 5

Describe principles of ICC

Answer 5

A cytoplasmic staining technique in which Antibodies are used to detect antigens in the cells of the tissue section. Specific antigens can be located by using specific antibodies and detecting the antigen-antibody reaction. positivity may be focal

Question 6

Describe the principles of ISH

Answer 6

ISH is a nuclear staining technique in which Labelled Fluorescent or chromogenic probes are used, which hybridize and form complementary base pairs with nucleic acids in the tissue. The labelled probe can then be detected by the immunostainer sensors to detect and localise the DNA or RNA.

Question 7

When would you use positive control

Answer 7

Used to demonstrate that the staining has worked as per the standard set out in the protocol. A positive control is also used to test a new antibody.

Question 8

Main reasons to reject ihc staining?

Answer 8

If known positive control is not stained
If section has lifted or fallen off
If section shows poor or inadequate staining

Question 9

Why do we perform frozens?

Answer 9

Rapid intra-operative preliminary diagnosis. Assist surgeon with patient management. For example it may be able to inform the surgeon whether the tumour is to the margin or not and whether they need to take more tissue

Question 10

What are the hazards associated with cryotomy?

Answer 10

Potential to create aerosols during processing, blade used is a sharps hazard and must be careful when changing blade and cleaning, all tissue should be treated as a biological hazard, surgeon should indicate if there is a known TB or viral infection, wear appropriate ppe

Question 11

Differences between staining frsh tissue and FFPE. Which machine/method used for each?

Answer 11

RFS - Manual staining, no dewxing step, provide rapid diagnosis. FFPE requires dewaxing step and provide a better quality of staining.

Question 12

How do you improve section when cutting fatty tissue on cryostat

Answer 12

Adjust thickness
Adjust temperature of cryostat
Adjust speed of cutting

Question 13

When checking quality of frozen HE what are you looking for

Answer 13

Thickness of the section, complete representation of tissue, check that there are no freezing artefacts, tears, folds or chattering, nuclei should be blue/purple basophils purple/red, cytoplasm red, collagen pale pink, RBC cherry red.

Question 14

What is the decontamination procedure of the cryostat and safety cabinet?

Answer 14

Cryostat - Place container, containing fresh 10% formalin concentrate solution, below drainpipe
6. Once cryostat cabinet has defrosted, fumigate overnight with 30mls 40% formaldehyde in 15mls hot water plus approx. One-teaspoon crystals of potassium permanganate, ensures a vigorous reaction starts. Re-sign “do not open”

Safety cabinet - Empty the cabinet
c) Add 35ml of formaldehyde (40%) to 35ml of HOT water (do this in the cabinet), in an open container.
d) Leave the container open in the cabinet.
e) Press the ‘FORM’ button on the control panel

Question 15

What is internal control in special stains?

Answer 15

Tissue that has been selected for a particular stain as it has structures which are known to be either demonstrated or not demonstrated with that stain, depending on if it is a positive or negative control

Question 16

Explain trichrome principle (MSB)

Answer 16

Stomach/placenta used as a control. 3 acidic dyes of differing molecular sizes are used to selectively stain basic tissue components, including muscle, collagen fibres fibrin and erythrocytes. First stain nuclei with Wiegerts haem, differentiate in acid alcohol and blue with lithium carbonate. Then use the Small molecular size dye martius yellow, which stains erythrocytes and v. early fibrin yellow. The phosphotungstic acid in the solution limits the staining of other tissue components. intermediate size dye is crystal scarlet and stains muscle, collagen, fibrin red, replaces any trace elements of yellow in these tissues as the small size dye is replaced by the larger dye. Phosphotungstic acid used to differentiate, removes crystal scarlet from collagen and old fibrin, leaving muscle and new fibrin red. Largest dye is aniline blue, stains collagen and old fibrin blue.

If not enough phosphotungstic acid, may under-differentiate collagen. When mixing Wiegerts, should be violet black, discard if brown.

Question 17

How could not using positive control impact diagnosis?

Answer 17

Pos control used to ensure staining procedure works according to the standard. Without pos control could get a false negative from staining errors, resulting in wrong diagnosis.

Question 18

Problems with fatty tissue and how to improve?

Answer 18

Add cold water to water bath, put block in freezer, may need to re-process if problem persists as the tissue may not have been processed properly.

Question 19

Describe use for plain, superfrost

Answer 19

Plain - routine work
Superfrost - IHC, decal, other difficult tissue - prevents tissue from lifting during staining

Question 20

Explain deeper, levels and serials. What slides used?

Answer 20

Deeper - trim until full face
Levels - cut sections with a set interval between each section
Serials - take a continuous series of sections, with no interval
Use plain slides for all unless encounter problems then use supercharged

Question 21

If control placed on same slide as test, where does it go?

Answer 21

Upper area of slide

Question 22

Explain sentinel node protocol

Answer 22

Stage 1 - cut 4 serial sections, stain 2nd with HE, put others on superfrost slides. The 3rd serial should go on a slide with an SMG control, as it will be stained with AE1/3.
Stage 2 - Cut through 125 microns and discard. Cut 4 serial sections, stain 2nd section HE if requested, Continue through block. Label slides level 1 1-6 and so on. Dry spare slides and store.
Stage 3 - Stain 3rd serial section from each level with AE1/3

Question 23

Whiteish hard tissue in block scoring blade, what do you do?

Answer 23

Use surface decal for approx 20-30 mins and attempt to take a section

Question 24

What is full face?

Answer 24

Block trimmed to reveal entire surface of the tissue. Important to ensure that all tissue, including margins is visible to the pathologist after staining.

Question 25

Potential problems with a section and how to rectify

Answer 25

Tissue folded (use superfrost, use 20% alcohol to float section, block may not be cold enough), tissue scored, poorly processed, cut too thick (check micrtotome is set to 4 microns, do not take 1st section), section has bubble (ensure no bubbles in water bath when floating section)

Question 26

Consequences for patient for trimming through small bx, mislabelling slides, cutting a deeper when serials requested

Answer 26

Trimming through - no bx left, unable to report, may need to re-biopsy if possible
Mislabelling - Possible mis-diagnosis
Cutting deeper not serial - May lose part of the tissue that the pathologist needs to see by going deeper.

Question 27

How does a PAS stain work and what would constitute as suitable control material?

Answer 27

The PAS technique stains a varied number of mucopolysaccharides such as glycogen, mucins and glycoproteins. Distinguishing between mucins and glycogen can therefore be difficult.
Periodic acid oxidises the hydroxyl group of carbohydrate into aldehyde. Aldehyde reacts with schiff to form a bright magenta carbohydrate-Schiff complex. Water removes the sulphuric acid and completes the reaction. Schiffs is basic fuschin and sulphuric acid. Should end up with magenta coloured pas positive carbs and blue nuclei.

Control for pas is mucosa

Precipitation may occur if haem not filtered before use

Question 28

pasd

Answer 28

Use of amylase (diastase) to digest glycogen and demonstrate the pas positive carbohydrates that remain in the tissue. Glycogen removed, other mucopolysaccharides magenta, nuclei blue.

If amylase not used long enough then glycogen may still be present.

Control is mucosa

Question 29

Gram

Answer 29

Differentiate between gram pos and gram neg bacteria. Section is treated with crystal violet. Gram positive retains the purple colour. Section then treated with iodine, followed by acetone. Other bacteria and structures are subsequently decolourised by the acetone and stain with neutral red counterstain. So gram pos is purple/black, gram neg is colourless/pale pink
Control is bacterial infection

Filter neutral red before use to avoid precipitate. Be careful not to over-differentiate with acetone.

Question 30

Grocott

Answer 30

Fungal walls contain carbohydrates which are oxidized to aldehyde with chromic acid. Aldehyde reduces hexamine silver solution to form a black/silver deposit. Gold chloride used to remove excess silver deposit and bring retic fibres into sharper contrast. Sodium thiosulphate then used to make the stain permanent. Light green is then used to stain background and give contrast to the Fungi which are black/grey.
Control is fungal infection

Do not over incubate as intense fungal staining may obscure fine internal detail of hyphal septa (basic building blocks of fungi)

Question 31

HE

Answer 31

has the ability to clearly demonstrate an enormous number of different tissue structures.
Haematoxylin is a basic dye which stains acidic tissue components such as cell nuclei. The stain only becomes permanent in moderately high pH conditions, such as provided by hard tap water. To control the degree of staining more precisely, a differentiation step with acid alcohol is added to remove any excess stain, a process known as regressive staining.
Eosin is an acid dye that stains basic tissue components such as cytoplasm, collagen and muscle. Eosin is differentiated with water and further differentiated with alcohol during the dehydration step which follows.

The key difference between progressive and regressive staining is that in progressive staining, the tissue is left in the staining solution just long enough to reach the desired endpoint while in regressive staining, the tissue is deliberately left for over staining until the dye saturates all tissue elements and then de-stained.

If over-stained in haem or under differentiated, section will be too purple. If under-stained in haem, not blued, or over differentiated then section will appear too red.

Question 32

HE control

Answer 32

A locally selected H&E quality control composite block is made containing representative tissue most frequently processed in the department (thyroid, lymph node, salivary gland)

Question 33

Why do we use 10% NBF and how does it work?

Answer 33

The fixative 10% buffered formalin is commonly used to preserve tissues for routine histology in many labs. The formaldehyde has a greater chance for oxidation in this concentration of tissue fixative and eventually the solution will start to drop in pH, in spite of the buffer. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5? Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine.

Question 34

Difference between pas and abpas

Answer 34

abpas used to differentiate acidic and neutral mucins, as it stains acidic mucins a deep blue while it does not stain neutral mucins. Acidic mucins are now chemically blocked and will not be stained during the following pas method, will then be able to differentiate acidic mucins stained with ab from pas positive neutral mucins. Not c/s with haem in head and neck.

Question 35

How do bascilli stain?

Answer 35

gram negative

Question 36

What are oxidation and reduction reactions

Answer 36

Oxidation is loss of electrons, gain of oxygen or loss of hydrogen. Reduction is gain of electrons, loss of oxygen or gain or hydrogen.

Question 37

Name a silver stain

Answer 37

grocott and masson fontana

Question 38

What stain do you use for spirokits

Answer 38

warthin stariy

Question 39

Fungal stains

Answer 39

grocott and pas. Pas is easier to perform but may need to do grocott to confirm the presence of fungi as it can be harder to see in a pas, pas stains a number of cellular structures whereas grocott is more specific, it stains fungi black on a green background

Question 40

bacterial stains

Answer 40

zn is used to diagnose tb

Question 41

preparation of solutions

Answer 41

to make a 1% solution dissolve 1g of reagent in 100ml of solution

Question 42

Congo red

Answer 42

To demonstrate amyloid. Cut at 5-6 microns. Differentiate with potassium hydroxide, c/s with haem, blue, differentiate with acid alcohol. Red staining is not specific to amyloid, other tissues are also stained with Congo red. To ensure the red stain is amyloid, it must display an apple green birefringence using a polarized microscope

Question 43

Perls

Answer 43

Demonstrates iron. Haemosiderin used as a control. Perl’s is a mix of potassium ferrocyanide and hydrochloric acid. The hydrochloric acid releases ferric iron from protein, which then reacts with pottasium ferrocyanide producing a blue staining of the iron. Nuclei C/s with neutral red to provide contrast.

C/S could mask the pigment, not providing contrast.

Question 44

Abpas

Answer 44

To differentiate between acid mucin and neutral mucins

Question 45

Zn

Answer 45

Section treated with carbol fuchsin. Zn positive organism takes the red carbol fuchsin, other structures are decolourised with acid alcohol and take the counter stain methylene blue. Acid fast bascilli stain red.

Question 46

What staining would be used for a liver panel?

Answer 46

While I haven’t done a liver panel myself as I completed my training in Head and Neck, I believe a liver panel would use a masson trichrome, which is used for staging of chronic liver diseases, reticulin , which stains reticulin fibers and highlights the architecture of the hepatic plates, Perl’s which demonstrates the distribution of iron in the hepatocytes and can diagnose conditions such as hemochromotosis, and pas and pas diastase for various glycogen storage diseases

Question 47

p16 and HR HPV ISH

Answer 47

p16 is a tumour suppressor protein which is activated by the HR-HPV oncoprotein E7. Overexpression of p16 is an accepted surrogate marker for HR-HPV in cervical and oropharyngeal cancers. HPV DNA ISH detects HPV nucleic acids, not only allowing the virus to be detected, but also showing the location of the virus and how it is affecting the morphology of the tissue. HPV subtype-specific probes are used depending on which subtypes are trying to be detected, with different probes allowing for the detection of different LR and HR subtypes.

Question 48

Positive control/negative control

Answer 48

Positive control shows that the staining technique has been carried out correctly and there are no false negatives, whereas a negative control shows that there has been no contamination during testing and there are no false positives.

Question 49

Antigen retrieval

Answer 49

When fixed, tissue undergoes chemical modifications which can make it more difficult to detect proteins in the tissue, antigen retrieval reduces or eliminates these modifications. Main methods are heat-induced epitope retrieval and protease induced epitope retrieval. PIER cleaves peptides which mask the epitope, whereas HIER reverses some crosslinks and restores secondary and tertiary structure of the epitope.

Question 50

if you need to make 20mls of a 5% gold chloride solution but only have 2g of gold chloride how much could you make?

Answer 50

so need 5g to make 100ml, so do 5/2 = 2.5, then 100/2.5 =40. So could make up 40ml

Question 51

two types of negative control and why required

Answer 51

1. known internal neg control
2. neg control slide run with the test slide to ensure accurate staining

Question 52

Pre-treatments on ultra

Answer 52

ultra cc2 - pre-diluted solution used as pre-treatment step for ihc and ish
ultra cc1 - tris-based buffer pre-treatment
protease

Question 53

How to evaluate new antibody for use on benchmark

Answer 53

using MDS to setup a protocol and run a known positive control section. If doesnt show adequate staining then adjust antibody dilution or pre-treatment time until optimal staining. Complete validation with consultant

Question 54

Describe waste composition from benchmark and how to dispose

Answer 54

2 layers. collected in carboys beneath instrument. Upper foamy layer is liquid coverslip, it is an oil that protects against drying. Disposed of with flammable waste. Bottom layer made up of bulk reagents and waste reagent from kits, dispose down sink with running water

Question 55

Which markers useful for identifying epithelial neoplasms, which staining pattern occurs?

Answer 55

CK7 - diffuse cytoplasmic.
CK20, AE1/3, VIMENTIN, EMA - cytoplasmic staining

Question 56

What kind of cells identified by cd20 and cd3

Answer 56

cd3 express in t cell and their neoplasms such as t/nk cell lymphoma. Used for detection of t cell malignancies, both mature and immature. cd20 expressed on b cells. Used to identify b cell lymphoma and large b cell lymphoma.

Question 57

how to set up new prep kit and rtu dispenser

Answer 57

prep kit - scan and use a prep kit number and assign to the desired antibody. Make up antibody using antibody diluent up to 5ml and fill dispenser kit. Edit protocol to include new kit. Run control to validate.
RTU - scan dispenser and edit protocol to state no manual titration. Run control to validate.

Question 58

Explain forward and reverse pipetting

Answer 58

forward - used for most reagents. Use blowout function of pipette to ensure complete delivery of liquid.
Reverse - Use for viscous liquids, biological foaming liquids or very small volumes of liquid. Delivery performed without blowout, so excess remains in tip.

Question 59

When setting volume on pipette, what need to be careful of?

Answer 59

Always use calibrated pipette. Thumbwheel must be turned slowly when adjusting volume. Do not turn thumbwheel outside pipette volume range.

Question 60

Things to remember for safe handling pipettes

Answer 60

Store in upright position using pipette holders. Do not drag or bang the pipette when handling. When drawing up solutions always ensure the pipette is held vertically and insert it approx 2-3mm into the liquid.

Question 61

How often pipettes calibrated and why

Answer 61

Done annually by a UKAS accredited calibration lab. Is needed to maintain accuracy of measurement.

Question 62

How could incorrect volume of antibody pipetted impact patient results

Answer 62

Could result in false positive or false negative results, this will then end up in either a delayed result, if the issue is spotted and corrected, or a wrong diagnosis if not spotted.

Question 63

Masson fontana

Answer 63

Melonoma control used. The section is placed in silver solution in the dark, either overnight or for 1hr at 60 degrees. The endocrine cell granules and melanin reduce the silver solution causing a black deposit at reaction site. Section then toned with gold chloride to provide cleaner background. Unreduced silver then removed with sodium thiosulphate. Also prevents any precipitation of silver by reduction by light. Light neutral red c/s then used. The stain is useful in distinguishing melanin from other pigments, such as haemosiderin, and identifying malignant melanoma. The melanin and endocrine cell granules stain black, nuclei red.

Inadequate impregnation cause lack of reduction of pigment. Prolonged impregnation may cause undesirable background staining. Too much ammonia in silver solution causes weak or no impregnation.

Question 64

Miller’s elastin

Answer 64

Either vein, vessel or lung control used. Miller’s is a combination of victoria blue, new fuchsin and crystal violet. It binds elastin fibres via hydrogen bonds. First oxidise using potassium permanganate, then decolourise with oxalic acid. Rinse in IMS, stain in millers for 90 mins. Rinse in IMS until blue is removed and elastic fibres are clear. c/s with van gieson. Should show elastic fibres and mast cell granules black, collagen bright red, cytoplasm and muscle a yellowish green and rbcs grey or black

If you wash in ims too long may over-differetiate and lose staining of finer elastic fibres.

Question 65

HVG

Answer 65

Differentiate between collagen and muscle. Skin used as control. Celestine blue used to limit differentiation by van gieson solution. Stain nuclei with haem, differentiate with acid alcohol and blue with tap water. Apply van gieson stain which works using two component dyes of differing sizes. Smaller is picric acid and penetrates all tissue but only remains in least permeable structures, which are muscle and rbcs. Larger dye is ponceau s, which displaces picric acid from collagen which are more porous, staining collagen red. End up with muscle yellow, collagen red, nuclei brown.

Differentiation may cause sections to be too pale