Practical 3: Blood smear and Peripheral Blood Mononuclear Cell Isolation Flashcards
What does whole blood consist of?
(2)
Plasma (46-63%)
Formed elements (37-54 %)
What does formed elements consist of?
Platelets
White blood cells
Red blood cells (99.9%)
What five wbcs are found in blood?
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
What percentage of blood leukocytes are neutrophils?
40-75
What percentage of blood leukocytes are lymphocytes?
20-50%
What percentage of blood leukocytes are monocytes?
2-10
What percentage of blood leukocytes are eosinophils?
1-6%
What percentage of blood leukocytes are basophils?
less than 1%
Write a note on neutrophils
(8)
Polymorphonuclear
Granular
Primary, secondary, tertiary granules
Most predominant wbc
Neutral staining
Phagocyte
The older they are the more lobes
Produce neutrophil extracellular nets
Write a note on eosinophils
(5)
Polymorphonuclear granulocyte
2-3 lobes
Nucleus dyes purple
Degranulates to degrade parasites
Higher levels seen in allergies
Write a note on basophils
(6)
Polymorphonuclear granulocyte
Dark blue granules which obscure the nucleus
Bi-lobed
Less than 1 per 100 wbcs
Produce heparin, histamine and cytokines
Higher levels seen in allergic reactions
Write a note on monocytes
(5)
Large, agranulocyte
Horseshoe shaped nucleus which is lighter than other wbcs
Cytoplasm is pale blue and has vacuoles
Differentiate into macrophages and dendritic cells
Phagocytic and antigen presenting
Write a note on lymphocytes
(6)
Agranular
Much smaller cell
2nd most predominant
Dark purple large nucleus
Thin layer of light blue cytoplasm
B and T lymphocytes
Where would you examine a blood smear?
About 1cm in from the feathered edge
Which cells are polymorphonuclear granulocytes
Neutrophils
Eosinophils
Basophils
Name two anti-coagulants used on whole blood
EDTA
Sodium citrate
What type of staining do we carry out?
Differential (Diff) quick stianing
What type of staining do we carry out?
Differential (Diff) quick staining
Explain how you carried out a differential stain on a blood smear
(6)
Let blood smear air dry at room temperature
Dip in methanol for 5 seconds - fixative solution
Dip in eosin for 12 seconds
Dip in water for 2 seconds
Dip in methylene blue for 5 seconds
Rinse with water - 5 seconds
Let air dry
What does eosin dye?
The cytoplasm
What does methylene blue dye?
The nucleus and cytoplasm
What does increased central palar of rbcs indicate?
Anaemia
What does blast dorms of wbcs found in peripheral blood indicate?
Leukemia
What can be a tell tale sign of leukaemia
Hair cells - wbcs with projections
What is the most common cause of bacteria found inside cells?
Plasmodium falciparum
(malaria)
What is a raised lymphocyte count called and what does it indicate?
Lymphocytosis
Indicates viral infection
What is a raised neutrophil count called and what does it indicate?
Neutrophilia
Bacterial infection
What is a raised eosinophil count called and what does it indicate?
Eosinophilia
Parasitic infection
Outline the princ
Outline the principles of density gradient centrifugation
(3)
Mononuclear cells have a lower buoyant density than erythrocytes and polymorphonuclear granulocytes
Agranulocytes have a density below 1.077g/ml therefore they can be isolated from whole blood by centrifugation using an iso-osmotic medium with a density of 1.077g/ml
The erythrocytes and PMNs sediment through to the bottom
What is density gradient centrifugation used for?
(2)
Used for enrichment/selection of a dominant cell type
Used to isolate mononuclear cells (monocytes and lymphocytes)
Give an example of an iso-osmotic medium suitable for density gradient centrifugation
Lymphoprep -> bought in -> contains everything you need
What does the centrifuge tube look like before density gradient centrifugation?
50% diluted blood on top
50% ficoll on the bottom
What does the centrifuge tube look like after density gradient centrifugation?
2/8th plasma on top
1/8th mononuclear cells
3/8th Ficoll
2/8th RBCs and PMNs
