SAQs Flashcards
Explain H&E principle
Haematoxylin is oxidised to hematein( a basic dye) which is the active dye used. This dye is basic and a mordant(usually Al 3+) is added to improve its ability to attached to anionic compounds ( which are negatively charged) like the cell nuclei which is rich in nucleic acids like DNA and RNA and are stained blue. They are known as basophilic (basic liking) because of the high affinity for hematein. Eosin is an acidic dye ( which is negatively charged) and it’s used to counterstain the plasma or cytoplasm pink this components are known as eosinophilic or acidophilic ( acid liking) because of the high affinity they have for eosin .
Explain the different steps of H&E and why you use them
Differentiate the section in 1% acid alcohol - You will see the red dye streaming (leaching) out of the section which means the mordant bonds are broken.
Differentiation is a de-staining step to remove excess of haematoxylin. In this step non-specific background will be removed and it will improve contrast.
“Blue” in ammonia solution (ammonia water) the process of placing the Hematoxylin stained section in a weak alkaline solution such as ammonia water. At alkaline pH the hematoxylin dye lakes are less soluble and have a strong blue colour. This step converts the haematoxylin to a dark blue colors and forms again the dye lake. The dye lake chemically bind to anionic structures, which are going to be converted to BASOPHILIC structures.
Cover (flood) the slide on the slide rack with Eosin solution for 2-3 minutes Eosin is counterstain which will stain structures in red contrasting to the principal stain (haematoxylin), making the stained structure easily visible using a light microscope. Eosin helps you see structures like cytoplasm, muscle cells, red blood cells and collagen which are positively charged.
Dehydration is the process of removing water by dipping your section in solutions with increasing concentrations of alcohol. Clearing is the process of replacing the dehydrating agent with a reagent (CITROCLEAR) that is miscible in DPX. This will render the tissue completely transparent.
Explain the result you would see with H&E
Nuclei - deep blue Cytoplasm - pink/purple pink Connective tissue - pale pink Muscle fibres - deep pink/red Red blood cells - deep pink/red Eosinophil granules- deep pink/red Calcium - dark blue Mucin - grey/blue
Explain reticulin principle
• Reticulin staining is performed to detect reticulin fibres and assess body cells (liver, kidney and spleen) for conditions such as necrosis (unplanned death of the cells constituting a tissue) and hyperplasia (increase in size of the tissues due to excessive cell division).
• Reticulin stain is made up of ammoniacal silver and formalin and uses silver impregnation to detect reticulin fibers, which are made of type 3 collagen.
the silver binds to the tissue and formalin reacts to produce the coloured precipitate
o The fibers appear black against a gray to light pink background.
o In the liver, such fibers are present as part of the extracellular matrix in the space of Disse.
o By highlighting these fibers, the stain helps in the assessment of the architecture of the hepatic plates,
such as expansion in regenerative and neoplastic conditions,
compression of plates in nodular regenerative hyperplasia,
collapse of the reticulin framework in necrosis
Explain some of the most important steps with reticulin
• Acidified potassium permanganate solution=
Oxidising agent which produces aldehyde groups, it also allows silver to bind to the Reticulin fibers which usually have little natural affinity for silver solutions. On treatment with potassium permangenate it produce sensitised sites on fibers where silver deposition can be initiated.
• Oxalic acid=
Decolourises background stain
• Iron Alum=
A mordant that enhances binding silver salts
• Ammoniacal silver nitrate=
Silver in solution which can easily be reduced
• Formalin=
Reducing agent which causes the deposition of metallic silver
• Sodium thiosulphate=
Excess silver in unprecipitated state is removed
Explain the results you would see with reticulin
Reticular fibres are supposed to be black
Nuclei are supposed to be grey
RBCs stain orange
If there is cirrhosis there are lots of reticulin fibres
Normally reticulin is around single cells but in damages tissue the reticulin is in big clumps
Explain Perl’s principle
The tissue will have granules that contain ferric ion which will react with potassium ferrocyanide to form a blue compound known as Prussian blue. This method of staining is used to stain things like macrophages which contain hemosiderin which is basically iron in ferric state in the spleen and liver. This procedure it is useful to be able to find if a patient is suffering with different diseases associated with hemosiderin like heamochromatosis which is an autosomal recessive disorder and it manifests itself in the liver as cirrhosis, heart as heart myopathy and pancreas as diabetes mellitus.
Explain Perls’ most important steps
Hydrochloric acid : is used because it denatures the binding of the proteins of the haemosiderin molecule and releases ferric ions.
Potassium ferrocyanide : is used because it reacts with the ferric ions and produces ferric ferrocyanide which has the bright blue pigment that can be seen in this reaction also known as Prussian blue.
Stain nuclei with 1% neutral red : this is a counterstain which shows tissue components like nuclei and cytoplasm.
Explain the results you would see with Perls’
Iron ( hemosiderin) - blue
Nuclei - red
Background - pink
We use Perls’ to demonstrate ferric iron in tissue sections.
Small amounts of iron are found normally in spleen and bone marrow.
Excessive amount are present in hemochromatosis- with deposits found in the liver and pancreas and hemosiderosis- with deposits in the liver, spleen, and lymph nodes
Martius Scarlet Blue
Allows Fibrin to be seen
Made up of 3 acidic dyes, martius yellow, brilliant crystal scarlet (red) and aniline blue
Chemical attraction between the acid dye and basophillic tissue.
Principle:
- Martius yellow- binds to RBCs, washes out of collagen/muscle/cytoplasm
- Brilliant crystal scarlet (red) stains cytoplasm/muscle/collagen. NOT RBC as stained by martius yellow
- Phosphotungstic acid acts as a dye excluder/differentiator, and competes with the red dye and removes it from tissue (Cytoplasm and muscle are still red)
- Aniline blue is added and stains collagen blue
Explain how PAS works with Diastese PAS and what would you see
We use the PAS and Diastase PAS methods to locate where the glycogen is within the tissues. A sample of the same tissue is put onto two separate microscope lsides and one is washed with only PAS and this stains the tissue pink. the second slide is stained with Diastase PAS and the tissue is stained purple. Glycogen is diastase sensitive, hence section containing glycogen when pretreated with diastase, the enzyme will break down the glycogen into glucose and maltose and will give negative PAS reaction. The purpose of using PAS with diastase staining procedure is to differentiate glycogen from other PAS positive elements such as mucin that may be present in the tissue sample
Explain the ABO group principle- the idea behind it
ABO grouping follows Landsteiner’s law which states that whenever an antigen (A and /or B) is not present on the red cells, the corresponding antibody is found in the serum. Hence a person naturally possesses antibodies against the AB antigens which they lack. An antigen is a substance capable of stimulating the formation of an antibody whereas an antibody is a protein produced in the blood in response to a foreign antigen. These interactions are very specific. When red blood cells carrying one or both the antigens are exposed to the corresponding antibodies they interact with each other to form visible agglutination or clumping.
Explain the principle of Antibody identification ( why does the test work, the idea behind it , what does each of the component parts do and how do they work, why do we get a positive result , why do we get a negative result , limitations of this procedure)
The principle of antibody identification involves the indirect antiglobulin technique. It allows for the detection of 37 degrees active antibodies, most commonly IgG and also detects complement fixing antibodies such as anti-K, Fya, Fyb, Jka etc. The patient’s plasma which contains the antibody (ies) is incubated with the panel cells containing various antigens. Any antibody (ies) in the sample will bind to the antigens on the red cell surface/within the gel forming an antibody-antigen complex. This is known as agglutination. RBCs carry a net negative charge and often repel one another. The repulsion pushes them too far apart for the cells to clump together properly so a solution contained in the gel of the cards (the anti human globulin) acts as a bridging agent and closes the gap between the red cells causing visible agglutination. The patient’s plasma which possibly contains an antibody (ies) is incubated with panel cells containing various antigens. These antigens are found within the wells of the card and the detectable, corresponding antibody will bind.
Explain the principle of coagulation ( why does the test work, the idea behind it , what does each of the component parts do and how do they work, why do we get a positive result , why do we get a negative result , limitations of this procedure)
How are the pathways monitored in the lab?
o Extrinsic is monitored by the PT
o Intrinsic is monitored by the APTT.
The principle of coagulation is to see if there are any abnormalities in the extrinsic or intrinsic pathways. If PT and APTT are normal then the secondary haemostasis is okay.
Secondary Haemostasis
· End of the coagulation cascade is a fibrin clot.
o Makes it more stable
o Once healing starts, the fibrin clot needs to be broken down
· Extrinsic. Intrinsic. Common pathway
· Fibrinolysis occurs generating plasmin which dissolves the clot.
o By breaking down the fibrin clot, it produces fibrinogen split products
o The fibrinogen split products generated prevent further clotting by inhibiting fibrin polymerisation and platelet aggregation and stops the clot from becoming too big.
Talk about the coagulation pathway
Right side- INTRINSIC FACTORS o XI, XII, VIII, IX Left side- EXTRINSIC FACTORS o VII, II, X, V, I Middle- COMMON PATHWAY (X, V, II) o If you have a deficiency in one of these factors it will effect both PT and APTT
If APTT is high then its abnormal Intrinsic factors (FVIII, FIX,FXI,FXII)
If PT was high then its abnormal Extrinsic factors
(FII, FV, FVII, FX) (FVII is the most important)
