Blood ( Missed information )

Question 1

What is a blood film?

Answer 1

A blood film is a method of blood sample preparation which allows red blood cells, white bloods and platelets to be spread evenly for microscopic inspection.

Question 2

What stain is used most frequently for blood films and why?

Answer 2

H&E (Haematoxylin and Eosin)

Very few cellular components absorb light at visible wavelengths so in order to see cells we stain them with coloured dyes.

Question 3

What does Haematoxylin do?

Answer 3

Haematoxylin is a purple-blue component that binds to acidic components of cells. In particular it binds DNA and shows up the nucleus.

Question 4

What does Eosin do?

Answer 4

Eosin is a pinkish stain that binds protein components, particularly in the cytoplasm.

• ‘eosinophil’ to describe cells with granules which were readily stained by the dye

Question 5

What is the Leishman’s stain used for?

Answer 5

makes the nucleus visible

Question 6

What factors affect a performance of a light microscope?

Answer 6

The wavelength of light

The optical quality

The refractive index of the medium : highest resolution lenses are designed to use oil instead of air

The physical properties of the objective lens : e.g. 10/0.25 means that lens has 10x magnification and a numerical aperture of 0.25.

The geometry of the illuminating cone of light provided by the condenser lens

Question 7

How to set up a microscope?

Answer 7

1 - focus image with main focusing knob

2 - Focus condenser after closing field iris

3 - open field iris

4 - Adjust condenser iris

Question 8

Reference ranges?

Answer 8

Reference ranges are descriptions of data derived from a sample of a reference population

Question 9

Normal range?

Answer 9

generally used to mean a range derived from a healthy reference population

Question 10

Full blood count?

Answer 10

A standard FBC would return the following parameters.

Haemoglobin (Hb) - typically measured in g/L.

White Blood Count (WBC) - typically measured as x 109/L

Platelet Count (Plt) - typically measured as x 109/L

Red Cell Count (RBC) - typically measured x 1012/L

Haematocrit (HCT) - typically measured in L/l.

Mean Cell Volume - Red cell (MCV) - typically measured in fL (x 10-15 L).

Mean Cell Haemoglobin (MCH) - typically measured in pg (x 10-12 g).



Question 11

What is the sickle cell mutation?

Answer 11

Results from a missense mutation at codon 6 of the b globin chain (βS)
Glutamic acid replaced by Valine
Glu			Valine
    Polar		Non polar
    Soluble	Insoluble

Question 12

Autosomal recessive pattern for Sickle cell?

Answer 12

Autosomal recessive inheritance pattern:

- HbSS: sickle cell anaemia 
- HbAS: sickle trait (usually asymptomatic)

Question 13

How are sickle cells made?

Answer 13

HbS polymerises to form tactoids that distort the red blood cell to result in a sickled shape

Question 14

Diseases causing sickling?

Answer 14

HbSC
HbS b thalassaemia
HbSS

Question 15

SCD pathogenesis?

Answer 15

Shortened red cell lifespan: 20 days
Anaemia: baseline Hb concentration in HbSS is ~60-80 g/l (cf 125-160 for HbA)
Gall Stones (increased red cell breakdown products)
Aplastic Crisis (Parvovirus B19 where bone marrow erythropoiesis has been shut down)

Blockage to microvascular circulation (vaso-occlusion)
Tissue damage and necrosis (Infarction)
Pain
Dysfunction

Question 16

Early presentation of SCD?

Answer 16

Onset coincides with switch from fetal to adult Hb synthesis
Early manifestations
Dactylitis (inflammation of digits)
Pooling of rbcs in spleen (splenic sequestration)*
Infection

Question 17

SCD and the spleen?

Answer 17

The spleen is involved in:
Immune defence
Breakdown and removal of old, malformed or damaged red blood cells: ‘quality control’
Repeated splenic vaso-occlusion (usually with no symptoms) in HbSS leads to the spleen no longer working by the age of 5 years: this is known as ‘functional hyposplenism’
Hyposplenism increases susceptibility to encapsulated bacterial infection
Immunisations
Prophylactic (preventative) antibiotics

Question 18

Acute chest syndrome in SCD as an emergency

Answer 18

New pulmonary infiltrate on chest X-ray
 with   Fever
            Cough
            Chest pain
            Tachypnoea (rapid breathing)

Diagnosis often delayed
Mechanical ventilation: 15%
Mortality > 18 yr: 9%

Question 19

Stroke in SCD?

Answer 19

Affects 8% of patients with HbSS

Most common in childhood

Involves major cerebral
vessels

Question 20

Blood film for sickle cell?

Answer 20

Hb low (typically 60 - 80 g/l)
Reticulocytes usually high
Blood film 	
Sickled cells
Boat cells
Target cells
Howell Jolly bodies

Question 21

Sickle solubility test for diagnosis ?

Answer 21

Principle: In the presence of a reducing agent oxyHb converted to deoxy Hb
Solubility decreases
Solution becomes turbid
Does not differentiate AS from SS

• Definitive diagnosis requires Electrophoresis or High Performance Liquid Chromatography (HPLC) to separate proteins according to charge

Question 22

What is Electrophoresis?

Answer 22

is a method of analysing molecules by measuring their migration in an electric field. We often use some sort of support (e.g. agarose for DNA, polyacrylamide for proteins) which acts as a sieve through which the molecules migrate. In this class we will use cellulose acetate as our support in combination with a buffer solution which means that the haemoglobin molecules are negatively charged. You will separate normal haemoglobin (HbA) and sickle cell haemoglobin (HbS) on the basis of their net charge.

Question 23

What is Spectrophotometry?

Answer 23

Spectrophotometry is a way of analysing molecules on the basis of their spectral properties

Question 24

Absorbance?

Answer 24

Absorbance is the fraction of incident light absorbed by a solution, which is typically measured by apparatus known as a spectrophotometer. The absorbance, A (sometimes refereed to as Optical Density or OD) can be defined as follows:

A = log10 (light transmitted through blank solution/light transmitted through test solution)

Thus for a solution with A = 1.0, only 10% of the light is transmitted through the solution and the other 90% is absorbed.

Question 25

If most proteins are transparent to visible light, how is Haemoglobin an exception? ( spectro )

Answer 25

Haemoglobin is an exception to this rule as each of the chains contains an associated haem group. This is an unsaturated organic molecule complexed to the protein which stabilises an Fe2+ ion to form a site to which O2 can bind to reversibly. Binding alters the electron resonance properties of the haem, changing its absorbance spectrum.

Question 26

What is sodium dithionite used for?

Answer 26

The sodium dithionite will strip the oxygen from the haemoglobin and change the binding equilibrium.

Question 27

How to use a spectrophotometer to determine concentration?

Answer 27

A = E × c × ℓ A is the absorbance of the solution c is the concentration of the absorbing substance (typically in mol/L) ℓ is the path length (in cm) E is the extinction coefficient, a constant for the substance being measured at that particular wavelength.

Question 28

What is pulse oximetry?

Answer 28

relies on the difference in absorbance of oxyhaemoglobin and deoxyhaemoglobin, which you have seen for yourself in this practical session. The oximeter is clipped onto a thin appendage (finger, ear lobe) and the device emits light at two wavelengths, 660nm (red light) and 940nm (infrared light) via a couple of LEDs. Oxygenated haemoglobin absorbs more infrared light but less red light than deoxygenated haemoglobin. The transmitted light at each wavelength is corrected for the pulse of arterial blood by subtraction of the minimum absorbance.

Question 29

What is Carboxyhaemoglobin?

Answer 29

generated by the binding of carbon monoxide to ferrous iron (Fe2+) in haemoglobin.

Question 30

What is Methaemoglobin?

Answer 30

generated when the Fe2+ ion is oxidised to the Fe3+ (ferric) state which results in greatly impaired oxygen binding. This gives the blood a bluish/chocolate colour if present at high levels.

Question 31

What does methemoglobin reductase do?

Answer 31

educes​ methaemoglobin back to ​haemoglobin. The disorder methaemaglobinaemia can be hereditary, for example as a deficiency in methemoglobin reductase or production of a mutant form of haemoglobin known as haemoglobin M, which is resistant to reduction.