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2.1 Molecular Immunology > Practical 3: Blood smear and Peripheral Blood Mononuclear Cell Isolation > Flashcards

Practical 3: Blood smear and Peripheral Blood Mononuclear Cell Isolation Flashcards

1
Q

What does whole blood consist of?
(2)

A

Plasma (46-63%)
Formed elements (37-54 %)

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2
Q

What does formed elements consist of?

A

Platelets

White blood cells

Red blood cells (99.9%)

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3
Q

What five wbcs are found in blood?

A

Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes

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4
Q

What percentage of blood leukocytes are neutrophils?

A

40-75

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5
Q

What percentage of blood leukocytes are lymphocytes?

A

20-50%

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6
Q

What percentage of blood leukocytes are monocytes?

A

2-10

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7
Q

What percentage of blood leukocytes are eosinophils?

A

1-6%

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8
Q

What percentage of blood leukocytes are basophils?

A

less than 1%

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9
Q

Write a note on neutrophils
(8)

A

Polymorphonuclear

Granular

Primary, secondary, tertiary granules

Most predominant wbc

Neutral staining

Phagocyte

The older they are the more lobes

Produce neutrophil extracellular nets

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10
Q

Write a note on eosinophils
(5)

A

Polymorphonuclear granulocyte

2-3 lobes

Nucleus dyes purple

Degranulates to degrade parasites

Higher levels seen in allergies

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11
Q

Write a note on basophils
(6)

A

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

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12
Q

Write a note on monocytes
(5)

A

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

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13
Q

Write a note on lymphocytes
(6)

A

Agranular

Much smaller cell

2nd most predominant

Dark purple large nucleus

Thin layer of light blue cytoplasm

B and T lymphocytes

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14
Q

Where would you examine a blood smear?

A

About 1cm in from the feathered edge

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15
Q

Which cells are polymorphonuclear granulocytes

A

Neutrophils
Eosinophils
Basophils

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16
Q

Name two anti-coagulants used on whole blood

A

EDTA
Sodium citrate

17
Q

What type of staining do we carry out?

A

Differential (Diff) quick stianing

18
Q

What type of staining do we carry out?

A

Differential (Diff) quick staining

19
Q

Explain how you carried out a differential stain on a blood smear
(6)

A

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

20
Q

What does eosin dye?

A

The cytoplasm

21
Q

What does methylene blue dye?

A

The nucleus and cytoplasm

22
Q

What does increased central palar of rbcs indicate?

A

Anaemia

23
Q

What does blast dorms of wbcs found in peripheral blood indicate?

A

Leukemia

24
Q

What can be a tell tale sign of leukaemia

A

Hair cells - wbcs with projections

25
Q

What is the most common cause of bacteria found inside cells?

A

Plasmodium falciparum
(malaria)

26
Q

What is a raised lymphocyte count called and what does it indicate?

A

Lymphocytosis

Indicates viral infection

27
Q

What is a raised neutrophil count called and what does it indicate?

A

Neutrophilia

Bacterial infection

28
Q

What is a raised eosinophil count called and what does it indicate?

A

Eosinophilia

Parasitic infection

29
Q

Outline the princ

A
30
Q

Outline the principles of density gradient centrifugation
(3)

A

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

31
Q

What is density gradient centrifugation used for?
(2)

A

Used for enrichment/selection of a dominant cell type

Used to isolate mononuclear cells (monocytes and lymphocytes)

32
Q

Give an example of an iso-osmotic medium suitable for density gradient centrifugation

A

Lymphoprep -> bought in -> contains everything you need

33
Q

What does the centrifuge tube look like before density gradient centrifugation?

A

50% diluted blood on top

50% ficoll on the bottom

34
Q

What does the centrifuge tube look like after density gradient centrifugation?

A

2/8th plasma on top

1/8th mononuclear cells

3/8th Ficoll

2/8th RBCs and PMNs

2.1 Molecular Immunology (19 decks)

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