There will be 6 rounds of assessment per annum. Each round will consist of two requested specialised staining methods (designated as List A and List B) on issued slides and another two self-provided slides stained with Haematoxylin and Eosin from the participant’s laboratory archive. Such that the participant will be expected to return four slides for assessment of staining technique per round.
Each participant will be issued duplicate slides for each of the two specialised staining methods and will be expected to return one of each (the duplicate is retained by the participant). These will be returned with two slides (that must not be recut or remounted sections) from the participant’s archive that had been stained with H&E on the two requested dates.
Each batch of slides issued is quality control checked to ensure that all slides issued are homogeneous within the batch. The period covering the distribution has been included when the expiry dates of samples were determined.
The main reason for selecting participant’s archived material is to include assessment of all the stages (tissue fixation through to mounting) of routine work used in the preparation of slides for cellular pathology examination.
Participants should submit the first H&E slide from the first case on the given date.
You may substitute for a subsequent slide if the first suitable slide:
Where the tissue has been involved in some other process prior to routine embedding, such as decalcification or cryo-sectioning, slides should still be submitted and an appropriate comment made on the electronic return form.
Generally, the first slide in a multi-block case is requested. This should be submitted unless the quality of the first block is inferior for routine cellular pathology such as if the tissue in the first block is damaged during dissection e.g. the margins of a bowel resection tissue sample may contain suture material or clips. In such cases, a later block from the same case may be substituted.
Where more than one section is cut from a block, the participant is expected to submit the first except where this would render assessment difficult (e.g. deliberately superficial levels). In such case a later section from the same block may be substituted.
The work patterns in some departments may mean that the first specimen is always of a particular, perhaps unrepresentative type. Substitution of a later case is permissible. This should be chosen in accordance with a written departmental operating procedure.
N.B. In all cases of substitution the participants should provide an explanation on the returns form.
It is important to not remove or obscure the original identification system on the self-provided H&E slides. After receipt the participant’s lab identity system will be made anonymous by covering over with removable adhesive labels. Similarly, it is unnecessary for participant’s to label the specialised staining slides.
Details of methods and/or reagents used should be recorded on the result entry sheets. This enables the provision of useful feedback from assessors to participants. This information is not assessed and will not appear on the final report.
The assessment process is to be as fair and consistent as possible. It is subcontracted to both APHA and non APHA personnel who are qualified and well-practised assessors in techniques of Cellular Pathology.
During assessment scores will be allocated to various criteria based on the quality of results in certain cellular pathology techniques.
Reports are issued electronically, these will include:
The participant is responsible for communicating via the reporting form whether a specialised staining method is part of their repertoire.
When a specialised stain is not part of a participant’s repertoire, this will be classified as such and will not be assessed or scored.
Participants who fail to submit a stained slide that is part of their repertoire will be classed as a non submission.
Assessment of Specialised Staining
In total there will be twelve requests for assessment of specialised staining techniques during the year i.e. two requests of specialised staining techniques per distribution.
Each round of assessment will request one stain from either List A and one from List B.
An example (not comprehensive of all staining methods in scheme) for a contract year:
A |
B |
|
| Ziehl Neelson | + | Elastic Van Gieson |
| Martius-scarlet-blue | + | Trichrome |
| Congo Red | + | Alcian Blue/Periodic acid Schiff (PAS) |
| Grocott's | + | Toluidine Blue |
| Giemsa | + | Reticulin |
| Perls' Prussian Blue | + | Masson Fontana |
Where it is the laboratory’s policy to refer specialised stains to another department or laboratory, these should be included in the declared repertoire and the PT material referred to the relevant third party as if it were diagnostic material.
The department’s original submission material will be returned.
Good performance in one category cannot be offset against substandard performance in another.
The scheme organisers will not monitor substandard performance. This should be arranged by the participants themselves and any subsequent investigations dealt with accordingly. However, participants are welcome to contact the scheme organiser or assessor for advice on such matters.
Poor performance is considered to be defined by receiving a score of 2 or lower.
Participants who are dissatisfied with any of their scores can re-submit the slides to be re-assessed. These slides will be reassessed at the next scheduled round of assessment after receipt.
The participant should initially contact the scheme organiser and then return the slides for reassessment.
Any returned slides will be added to the list of the next round of assessment. After reassessment the slides will be returned to the participant.
The appeals procedure will remain confidential.
21 days
Unstained paraffin sections of tissues which may originate from farm, exotic or companion animals.
Transfer may be provided by normal post or Courier. Courier delivery will incur additional charges. Please contact the QA unit (VETQAS) for further details. Participants are liable for the cost of return postage.
It is advisable for participants record receipt and dispatch of all PT materials.
When using plastic slide mailers, please take particular care when packing to protect the slides from movement such as filling any upper vacant space with padding. Also, seal the lid onto the container with adhesive tape to avoid spillage and breakage.
6
Various aspects of the Proficiency testing schemes may at times be subcontracted. When this occurs it is placed with a competent subcontractor and the Quality Assurance Unit is responsible to the scheme participants for the subcontractors work.
2011
ISO/IEC 17043: 2010 Conformity assessment – General requirements for proficiency testing.
Alcian Blue/PAS
Results:
Acid mucopolysaccharides - Bright Blue
Neutral mucopolysaccharides - Bright Magenta
Mixed mucins - Purple
Counterstain (optional) - Very Light Blue
Comments:
There should be clear distinction between acid, neutral and mixed mucins.
Background staining should be negligible. A (typically haematoxylin) counterstain is considered optional. If present, it should be light, even and not mask or alter the primary stains. It should assist location. Section quality and presentation must not impair the result.
Congo Red
Results: Amyloid Bright Pink/Orange
Counterstain (optional) Very Light Blue
Comments: Red blood cells, elastin, eosinophils and keratin may also stain.
Background staining should be negligible.
The (typically haematoxylin) counterstain, if present, should be light, even and not mask or alter the primary stains. It should assist location.
Section quality and presentation must not impair the result.
N.B. Assessment will not involve polarised light microscopy.
Diastase/PAS
Results: Intestinal mucopolysaccharide. Bright Magenta
Counterstain (optional) Very Light Blue
Comments: The scheme will distribute sections from a composite block containing glycogen-rich liver and intestine. Only one diastase-digested section should be returned by participants.
The Schiff reaction is a histochemical method for aldehyde groups. Periodic acid oxidation creates these groups on a variety of tissue structures notably glycogen, mucins, basement membrane and fungi. This modification uses the enzyme diastase (or amylase) to digest out any glycogen and is normally used in conjunction with a straight PAS (not required for EQA unless requested).
The requirement of this stain is the complete removal of glycogen and precise, complete demonstration of intestinal mucopolysaccharide. Membrane staining should not be confused with background, which should be minimal.
Counterstain (typically haematoxylin) should provide good colour contrast and assist location.
Section quality and presentation must not impair the result.
Elastic Van Gieson (Elastin)
Results:
Elastic Fibres - Very Dark Blue/Black
Muscle Fibres - Yellow
Collagen - Red
Comments:
Elastin stains fall into two main groups; haematoxylin-based (e.g. Verhoeff) and hydrophobic dye mixes (e.g. Miller's and Weigert's).
The requirement of this stain is the precise staining of all coarse and fine elastin fibres.
The counterstain is a simple trichrome and there should be good colour separation between muscle, red blood cells (yellow) and collagen (red). Picro-Sirius red is a suitable alternative, popular in the US. The counterstain should provide good colour contrast, assist location and not mask or hinder elastic fibre location.
Giemsa
Results:
Microorganisms/Nuclei - Blue or Purple
Mast Cell Granules/Basophils - Purple-Violet
Rbc’s/Eosinophils - Pink Rose
Collagen - Pink Rose
Comments:
The Giemsa technique is used for the demonstration of various substances including mast cell granules and Rickettsia (in this case cryptosporidia organisms attached to the mucosal surface). It is a mixture of toluidine blue and eosin and for the purposes of these criteria only, the eosin is regarded as a counterstain.
The requirement of this stain is that organisms must be easily distinguishable from the other well differentiated tissue structures.
Gram
Results: Gram Positive Bacteria: Blue
Gram Negative Red
Comments: The requirement for this stain is that the crystal violet staining should render all Gram positive organisms blue/black without over- or under-differentiation. There should be no violet background.
There are many variations of the gram technique and most are based on the counterstain used. The counterstain serves two purposes: to demonstrate all Gram negative organisms in a contrasting colour and to assist location. There is no restriction on the use of variations.
Grocott's
Results:
Fungal Spores and Hyphae - Black
Other tissue components - Green
Comments:
The requirement for this stain is there should be dark grey/black staining of all fungal spores and/or hyphae or pneumocysts. Where appropriate, internal structures of the organisms should be visible. Impregnation of other structures, particularly collagen, should be negligible. The counterstain should provide good colour contrast and assist location.
Haematoxylin & Eosin
The H&E part of the scheme also includes assessment on pre-staining elements of the discipline.
Haematoxylin staining
Haematoxylin choice varies across the scheme with some haematoxylins being darker/bluer than others and some such as Ehrlich’s tend to leave some tissue components looking a bit grey. Some use haematoxylin progressively and some regressively. However, regardless of the haematoxylin used the nuclei should be stained to provide good contrast against the eosin and provide sharp strong detail of the nuclear chromatin where present. For those using regressive haematoxylins, decolourisation should be carried out to the point where the background cytoplasm is clear but nuclear detail remains strong and sharp. Marks are generally lost due to under differentiation resulting in a blue/purple cast in the cytoplasm which alters the subsequent eosin staining colour.
Eosin staining
Eosin staining should be carried out to provide good selectivity (obvious shade differences between cytoplasm, muscle and red blood cells). The staining should not be overdone to the point of producing a very dark dense stain which can overpower and mask the haematoxylin stain. Most marks are lost due to poor selectivity or over staining. The eosin should provide a good contrast to the haematoxylin.Martius Scarlet and Blue (MSB)
Martius, Scarlet and Blue (MSB) is a specialised trichrome method designed to demonstrate fibrin.
Comments:
Accuracy: all fibrin should be brightly coloured red and distinguishable from other structures. As with other trichromes, there should be good colour separation between the red, blue and yellow dyes, best seen in RBCs (yellow), muscle (red) and collagen/background (blue). Nuclear staining should be dark blue or black. Section quality and presentation must not impair the result.
Masson Fontana
Comments:
All melanin should be coloured black after toning and easily identified from unreacted melanin. Impregnation should be such that the melanin is stained black but without non-specific background deposit. Counterstaining should compliment the melanin staining and provide good contrast without masking fine melanin deposits.
PAS
Results: Basement membranes. Bright Magenta
Counterstain (optional) Very Light Blue
Comments: The scheme will distribute sections from kidney tissue.
The Schiff reaction is a histochemical method for aldehyde groups. Periodic acid oxidation creates these groups on a variety of tissue structures notably glycogen, mucins, basement membrane and fungi. Membrane staining should not be confused with background, which should be minimal.
Counterstain (typically haematoxylin) should provide good colour contrast and assist location.
Section quality and presentation must not impair the result.
Perls' Prussian Blue
Results:
Haemosiderin - Blue
Nuclei - Red
Comments:
A histochemical reaction for ferric iron ions adapted for the demonstration of
haemosiderin. The requirement of this stain is the Prussian blue coloration of haemosiderin, this should not be modified by the counterstain or any underlying, unreacted pigment. The counterstain should provide good colour contrast and assist location.
Section quality and presentation must not impair the result.
Reticulin
Results:
Reticulin - Black
Comments:
This method is primarily a low power scanning method so contrast is all-important. There are many methods, the most popular in the UK being Gordon & Sweet, while Gomori is more popular in Europe and the US. The latter impregnates nuclei which, when done well, renders counterstaining unnecessary.
The main variation in method is toning. Without it, collagen should remain golden brown and cell cytoplasm a pale yellow. Again, counterstaining is unnecessary if the rest of the method is well done.
The requirement of this stain is that all reticulin should be black. Under-impregnation will result in incomplete staining, while over-impregnation yields knobbly fibres. Fine reticulin fibres should be clearly seen at high power. There should be no non-specific silver deposits and staining of cytoplasmic structures is undesirable. Counterstaining, if used, should provide good contrast and assist location. Gomori and its variants may render toned collagen rose-pink.
Section quality and presentation must not impair the result.
Toluidine blue
This is a metachromatic dye and metachromasia in certain tissue elements produces staining of a different colour from the dye solution. Mast cells are found in the connective tissue (in this case a mast cell tumour) and their cytoplasm contains granules composed of heparin and histamine. The requirement of this stain is that the mast cells stain purple (metachromatic) providing good contrast against the background which remains blue (orthochromatic).
Trichrome
Results:
Muscle - Red
Collagen - Blue or Green
Nuclei - Dark Blue or Black
Comments:
Trichrome methods are designed to give colour separation between smooth muscle and collagen. There are a variety of methods; Masson's being the most popular in the UK. The requirement of this stain is that all muscle should be stained red, collagen blue or green, nuclei blue/black. The collagen (milling) dye may be green or blue.
Section quality and presentation must not impair the result.
N.B. MSB is a fibrin stain and should not be submitted as a "standard" trichrome stain.
Ziehl Neelsen
Results:
Mycobacteria - Red/Magenta
Nuclei- Light Blue
Comments:
A stain for Mycobacterium tubercolosis and closely related organisms.
The requirement for this stain is that all mycobacteria, including isolated bacilli, should be sharply stained magenta/red by carbol fuchsin with minimal background staining. The counterstain should be light, even and predominantly nuclear. It should provide good colour contrast and assist location. There should be no dye precipitation and the tissue should not be damaged by heating.
Section quality and presentation must not impair the result.