Diagnostic role of the blood film

Question 1

What is a blood film?

Answer 1

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 is microscopy?

Answer 2

a technique used to examine samples (tissue, fluid, cells) that are too small for the human eye to see unaided

Question 3

Why do we stain cells?

Answer 3

Very few cellular components absorb light at visible wavelengths so we cannot see them

Question 4

What is the most frequent combination of stains?

Answer 4

H&E (Haematoxylin and Eosin)

Question 5

What is haematoxylin?

Answer 5

a purple-blue component that binds to acidic components of cells

in particular it binds to DNA and shows up the nucleus

Question 6

What is Leishman’s stain?

Answer 6

Contains a purple-blue dye, which stains nuclei, and a pink one staining components in the cytoplasm

Question 7

What is eosin?

Answer 7

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

Red cells look red while white cells actually look blue due to the staining of the nucleus.

Question 8

What is a good general purpose stain?

Answer 8

H&E

Question 9

What is the alternative to using stain?

Answer 9

exploit interference properties of light to detect the different refractive index of cells compared with the surrounding fluid

important when examining living cells

Question 10

What is the resolution of a microscope?

Answer 10

the closest limit at which you can distinguish two adjacent small objects

Question 11

What factors affect the performance of a light microscope?

Answer 11

1. the wavelength of light ( cant alter this)
2. the optical quality of lenses
3. the refractive index of the medium through which the light passes
4. The physical properties of the objective lens
5. The geometry of the illuminating cone of light provided by the condenser lens

Question 12

How do you set up a microscope correctly?

Answer 12

start with the low power (4x) objective lens, turned into position on the lens turret (A).
A microscope slide is inserted into the clips on the stage and the lamp is turned (B).
The distance between the eyepieces (C) should be adjusted to suit your own eyes

Question 13

How is the microscope is adjusted?

Answer 13

Step 1: Focus the image
Step 2: Focus the condenser
Step 3: Adjust the field iris
Step 4: Adjust the condenser iris

Question 14

How do we prepare a blood film?

Answer 14

1. blood sample is mixed thoroughly
2. Blood dispenser is pushed through the lid of the sample
3. single drop is taken from the sample and placed onto the slide
4. Spread the drop with another slide
5. Label the slide

Question 15

How can a drop of blood be taken from a finger

Answer 15

using a sterile lancet to puncture the fleshy pad alongside the base of a fingernail

Question 16

How do you spread the blood on the slide?

Answer 16

1. spreader slide placed at an angle in front of the drop
2. spreader slide drawn back to touch the drop
3. the drop spreads between the 2 slides
4. The drop is spread by surface tension “dragging” it along the slide as the spreader is pushed forward

Question 17

How to stain a blood film using Leishmans stain?

Answer 17

1. The slide is first placed blood-side up in a rack over a draining dish.
2. 8 drops of Leishman’s stain are dropped on the using a dropper bottle so that the film is completely covered.
3. The slide is left for a minute to stain.
4. 8 drops of a buffer pH 6.8 (again from from a dropper bottle) should be dropped onto the slide and gentle rocking performed so that the buffer and stain are mixed. This is then left for 7 minutes.
5. The stain is poured off and the slide rinsed well with copious amounts of buffer, using a wash bottle.
6. The slide is dried by first tapping the edge of the slide on filter paper and then gently waving it in the air
7. Once completely dry, the slide may be examined using the light microscope.

Question 18

What are reference ranges?

Answer 18

descriptions of data derived from a sample of a reference population

Question 19

What is a reference population?

Answer 19

has characteristics that have been carefully defined with regard to age and gender and, when relevant, other variables such as state of health, ethnic origin and physiological status (pregnant or not).

Question 20

What else are haematological variables affected by?

Answer 20

altitude, cigarette smoking, alcohol intake, whether the person is ambulant or resting, and whether a tourniquet has been applied for a long time before taking the blood sample

Question 21

What is a normal range?

Answer 21

used to mean a range derived from a healthy reference population

Question 22

When interpreting laboratory data you should consider the following points:

Answer 22

1. A value within the normal range may be abnormal for that individual
2. A value outside the normal range may be normal for that individual
3. Reference ranges for healthy and sick individuals usually overlap
4. Some haematological variables are dependent on the precise instrument or methodology used –> reference range derived for a particular instrument/method

Question 23

What do elevated numbers of eosinophils show?

Answer 23

allergic reactions e.g. asthma and also parasitic infections.

Question 24

What do increased numbers of basophils show?

Answer 24

chronic myelogenous leukaemia (CML) and also in certain hypersensitivity reactions

Question 25

What does “left shift” mean?

Answer 25

may indicate either an increase in the proportion of band cells or the presence of neutrophil precursors in the peripheral blood

Question 26

What are the indicators of bacterial infections?

Answer 26

an absence of mature neutrophils and two band form neutrophils

presence of clear vacuoles within the cytoplasm of the neutrophils

Question 27

How do we distinguish acute lymphoblastic leukaemia (ALL)?

Answer 27

very irregular shape of the nuclei

Question 28

How do we distinguish chronic granulocytic leukaemia?

Answer 28

A spectrum of granulocytic cells from myeloblasts to mature neutrophils

An essential feature of CML is the presence of a BCR-ABL1 fusion gene, often as a result of a t(9;22)(q34;q11.2) translocation

Question 29

How do we distinguish Megaloblastic anaemia (maturation of the nucleus is retarded in relation to that of the cytoplasm) ?

Answer 29

hypersegmented neutrophils and macrocytes, particularly oval macrocytes

In early deficiency, before anaemia has yet developed, there may be macrocytosis without poikilocytosis or hypersegmented neutrophils but in severe deficiency the blood film is distinctive.

Question 30

What are blood films used together with?

Answer 30

Full blood counts (FBC)

Question 31

Which following parameters are returned by a standard FBC?

Answer 31

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).