Week 13 Flashcards
Was is Cytochemistry?
In vitro staining of cells
What are some elements that may be stained in cytochemistry?
Leucocytes, erythrocytes
In Cytochemisty it is difficult to determine blast cell origin from morphology what is it helpful with?
Acute leukaemias
Even though cytochemisty is now largely replaced by Immunophenotyping, high resolution cytogenetic studies, molecular genetics. It is still used to some extent in differentiating what?
Differentiate M0 from M1 leukaemia (Myeloperoxidase, Sudan Black B)
List some specimen types that can be used – smears and imprints:
- Bone marrow (most common)
- Lymph node
- Spleen
- Peripheral blood
List 2 things that can affect enzymatic techniques.
- Some fixatives
* Age of specimen
What is the main use for the Kleihauer – Betke test?
• Evaluate foeto-maternal haemorrhage
Guide dosage for anti-D administration
• Rh(D) negative mother with Rh(D) Positive baby
Do maternal cells stain more strongly or do foetal cells?
Foetal stain more strongly
Maternal stain like ghosts
List the steps of the Kleihauer – Betke test for preparing maternal blood film
- Fix in ethanol
- Immerse in acid-citrate buffer (pH 3.3)
- Stain
- Determine % foetal cells
- Calculate volume of bleed
What cells is myeloperoxidase enzyme is found in?
Neutrophil and eosinophil primary granules
Monocytes - to some extent
What cells is myeloperoxidase enzyme not found in?
Lymphocytes
Erythrocytes
Myeloperoxidase is useful for differentiating blasts from which leukaemias?
AML blasts from ALL blasts
Sudan Black B is useful for differentiating which leukaemias?
AML from ALL
Sudan Black B has similar results to myeloperoxidase however it’s a little more sensitive for what?
early myeloid cells
What does sudan black B stain? give examples
lipids eg: • Sterol fats, neutral fats, phospholipids
Note: SBB is lipid soluble
Where are the lipids found that Sudan Black B stain?
- Primary and secondary granules of neutrophils
* Lysosomal granules of monocytes
How would the following cell types stain?
Granulocytes
Monocytic cells
Lymphoid cells
Granulocytes positive from myeloblast onwards
Monocytic cells negative to weakly positive
Lymphoid cells Negative - note burkitts cell vacuoles may stain positive
What would the myeloperoxidase and sudan black b results be for M0 Acute myeloid leukaemia minimally differentiated?
- Myeloperoxidase negative
* Sudan Black B negative
What would the myeloperoxidase and sudan black b results be for M1 Acute myeloid leukaemia without maturation?
- Myeloperoxidase positive
* Sudan Black B positive
What are esterases used for?
Used to differentiate granulocyte vs monocyte cell lines.
How many esterase isoenzymes are present in leucocytes?
Nine
Esters used as substrates may be “Specific”, meaning
Only granulocyte series stain positive
• Napthol AS-D chloroacetate (specific esterase)
Esters used as substrates may be “Non-specific”, meaning
- Monocyte series and some other cell lines stain positive
- a-napthyl acetate (non-specific esterase)
- a-napthyl butyrate (non-specific esterase)
Leucocyte alkaline phosphatase is used to differentiate what?
• Chronic myeloid leukaemia and Leukaemoid reaction in severe infection
Would an untreated CML have an elevated or decreased LAP?
Decreased LAP
Would a Leukaemoid reaction have an elevated or decreased LAP?
High normal to increased LAP
Would the following conditions have a High, Normal. or low LAP score?
• 3rd trimester of pregnancy
• Polycythaemia vera
High scores
Would Secondary polycythaemia have a High, Normal. or low LAP score?
Normal score
Would the following conditions have a High, Normal. or low LAP score?
• Paroxysmal nocturnal haemoglobinuria
• Sideroblastic anaemia
• Myelodysplastic disorders
Low score
Acid phosphatase enzyme present in what cell lines?
Myeloid, monocytic, lymphoid, plasma cell, platelets
Acid phosphatase is tartrate __________
resistant
Hairy cells produce isoenzyme ____
5
tartaric acid inhibits all isoenzymes except
5
In ACP the addition of L-(L+)-tartaric acid make
• Haemopoietic cells positive or negative
• Hairy cells remain positive or negative
- Haemopoietic cells negative
* Hairy cells remain positive
Masson’s trichrome stain Used to study
connective tissues, muscle, collagen fibres
Masson’s trichrome stain is used to demonstrate increased collagen in ________
myelofibrosis
Silver stain is used to stain _______ ______ that replace normal marrow stroma in _________
reticulin fibres, myelofibrosis
Reticulin fibres comprised of Type ___ collagen
III
What 3 things would you check if control cells do not exhibit expected staining pattern ie:
• No colour when they should be positive
• Incorrect colour
Reagents - correct or expired
Specimen - age, storage
Method - correct steps
Smears for which of the following could be stable for several months at room temperature
a) Myeloperoxidase
b) Sudan Black B
c) Leucocyte Alkaline Phosphatase
d) a-Napthyl butyrate esterase
b) Sudan Black B
Which cytochemical stain can be used to differentiate AML from ALL?
a) TRAP
b) Leucocyte Alkaline Phosphatase
c) Myeloperoxidase
d) Alpha-napthyl acetate
c) Myeloperoxidase
Sudan Black B stains which cellular components?
a) Glycogen
b) Lipids
c) Structural proteins
d) Enzymes
b) Lipids
Hairy cells produce an abundance of which isoenzyme of acid phosphatase? How can you demonstrate this isoenzyme? a) 1 b) 3b c) 4 d) 5
d) 5
• Demonstrate tartrate resistance
An LAP score of 250 would be consistent with a diagnosis of:
a) Normal cells
b) CML
c) Hairy cell leukaemia
d) Leukaemoid reaction
d) Leukaemoid reaction
The cytochemical stain a-napthyl butyrate is a non-specific esterase stain that reacts with cells of which lineage?
a) Erythroid
b) Monocytic
c) Granulocytic
d) Lymphoid
b) Monocytic
Which of the following stains is used to evaluate mature neutrophils and bands when discriminating reactive changes from CML?
a) Acid phosphatase
b) Leucocyte Alkaline Phosphatase
c) Romanowsky
d) Sudan Black B
b) Leucocyte Alkaline Phosphatase
Cytochemical staining on a spleen imprint stained positive for acid phosphatase. The stain was repeated after treating with tartrate, and remained positive. This suggests which condition?
a) Erythrocytic leukaemia
b) AML
c) Hairy cell leukaemia
d) Acute megakaryocytic leukaemia
c) Hairy cell leukaemia
A normal blood film can act as a suitable positive control for testing all of the following staining methods, except:
a) Myeloperoxidase
b) Alpha-napthyl acetate esterase
c) Acid phosphatase with tartrate pre-treatment
d) Leucocyte alkaline phosphatase
c) Acid phosphatase with tartrate pre-treatment
Which of the following is not a suitable fixative for slides that are to be cytochemically stained?
a) Phosphate buffered saline
b) Methanol
c) Acetone
d) Formaldehyde
a) Phosphate buffered saline
