Diagnostic role of blood film

Question 1

Why do we need to stain cells to see them under the microscope?

Answer 1

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

Question 2

What is the most frequently used combinations of stains?

Answer 2

H&E (Haematoxylin and Eosin).

Question 3

What is Haematoxilyn?

Answer 3

• purple-blue component that binds to acidic components of cells.
• In particular, binds to DNA and shows up the nucleus.

Question 4

What is Eosin and what cells got their name because of the dye?

Answer 4

• Pinkish stain that binds protein components, particularly in the cytoplasm.
• Eosin is a bright red synthetic dye
• The German physician Paul Ehrlich published a technique for staining blood films and coined the term ‘eosinophil’ to describe cells with granules which were readily stained by the dye.
• Picture of Eosinophil stained with H&E:

Question 5

What is the alternative to using stains and what advantage does it have?

Answer 5

• To exploit interference properties of light to detect the different refractive index of cells compared with the surrounding fluid.
• This approach (which needs a microscope fitted with special optics) is especially important when examining living cells.

Question 6

What factors affect the performance of a light microscope?

Answer 6

• The wavelength of light (you can’t alter this in this system).
• The optical quality of lenses and all other components in the light path.
• The refractive index of the medium through which the light passes (which is why the highest resolution lenses are designed to use oil instead of air between the objective lens and the slide).
• The physical properties of the objective lens (actually a property called the numerical aperture which is written on the lens next to its magnification, e.g. 10/0.25 means that lens has 10x magnification and a numerical aperture of 0.25. It follows that higher magnification does not necessarily mean higher resolution, though in fact it is normally the case that higher power lenses have better numerical apertures).
• The geometry of the illuminating cone of light provided by the condenser lens (which depends in part on the design properties of the lens, but to a large degree on the way in which the condenser is focused and the iris diaphragms adjusted).
• In practice, the only adjustment you can make to optimise resolution is to set up the condenser properly.

Question 7

Label the Nikon microscope:

Answer 7

Question 8

Label the Leitz microscope:

Answer 8

Question 9

How do you setup a microscope?

Answer 9

1. Focus the image
2. Focus the condenser
3. Adjust the field iris
4. Adjust the condenser iris

Question 10

How do you spread a drop of blood on a slide to make a blood smear?

Answer 10

• Place a drop of blood on a slide (be quick if no anticoagulant used and blood freshly collected from a finger).
• Slowly smear it out in a film, using a second slide as a spreader.
• The direction of spreading is so that the blood drop remains behind the spreader or blood cells might become damaged between the two slides.

Question 11

What is Leishman’s stain?

Answer 11

• Contains a purple-blue dye, which stains nuclei, and a pink one staining components in the cytoplasm.
• Red cells look red
• White cells look blue due to the staining of the nucleus.

Question 12

What is the protocol to stain a blood film with Leishman’s stain?

Answer 12

• Place slide blood-side up in a rack over a draining dish.
• Drop 8 drops of Leishman’s stain using a dropper bottle so that the film is completely covered.
• Leave for a minute to stain.
• Drop 8 drops of a buffer pH 6.8 (again from a dropper bottle) and gently rock so that the buffer and stain are mixed.
• Leave for 7 minutes.
• Pour off stain and rinse slide well with lots of buffer, using a wash bottle.
• Dry slide by tapping the edge of the slide on filter paper and then gently wave it in the air
• Once completely dry, the slide may be examined using the light microscope.

Question 13

How can you identify and differentiate different blood cells under Leishman’s stain?

Answer 13

Question 14

What is a reference population and what are reference ranges?

Answer 14

• A reference population 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).
• A reference range is descriptions of data derived from a sample of a reference population.
• Reference ranges commonly given as 95% ranges, so 2.5% of data are excluded at each end of the range.
• If data have a Gaussian (normal) distribution the mean plus and minus 2 standard deviations gives a 95% range.

Question 15

What other factors need to be considered when deriving reference ranges in haematology?

Answer 15

• Haematological variables are also affected by altitude, cigarette smoking, alcohol intake, whether the person is ambulant or resting, and whether a tourniquet (strap that applies pressure to a limb) has been applied for a long time before taking the blood sample.

Question 16

How is the reference range derived if data does not show normal distribution?

Answer 16

• Mathematical transformation of the data is required before analysis.
• For example, white cell counts have a logarithmic distribution and the mean and standard deviation of the logarithms of the data must be calculated in order to determine the log mean and the 95% range.

Question 17

What is a normal range?

Answer 17

• Less strictly defined term than a ‘reference range’.
• Normal range is generally used to mean a range derived from a healthy reference population.

Question 18

What is a health-related range?

Answer 18

• For some laboratory measurements, a 95% range derived from an apparently healthy population will include data from patients with a high risk of later developing significant disease.
• In the case of cholesterol concentration, If subjects representing the upper 20% of data have a high risk of developing clinically evident coronary artery disease then it is more relevant to interpret data in the light of whether the result is predictive of future good health rather than whether it falls within the 95% limits for apparently healthy people.

Question 19

When interpreting lab data what should you consider?

Answer 19

• A value within the normal range may be abnormal for that individual. For example, a man whose Hb is usually 165 g/l may suffer a gastrointestinal haemorrhage with a fall of Hb to 140 g/l, which is still within the normal range but is abnormal for him. If previous test results are available from a given individual it is always relevant to consider these when deciding if a result is likely to be abnormal for that particular person.
• A value outside the normal range may be normal for that individual. By definition, test results of 5% of healthy subjects are likely to fall outside the ‘normal range’ and if healthy subjects have multiple tests performed there are bound to be one or two which are ‘abnormal’.
• Reference ranges for healthy and sick individuals usually overlap. Calculating reference ranges representing 99% of the population reduces the chance of misclassifying a test result on a healthy subject as abnormal but there will be more abnormal results that are not recognised as such.
• Some haematological variables are dependent on the precise instrument or methodology used. It is, therefore, best to use a reference range derived for a particular instrument/method. Results from various hospitals may differ slightly.

Question 20

When can increased number of basophils be seen?

Answer 20

• In chronic myelogenous leukaemia (CML).
• Certain hypersensitivity reactions.
• If coupled with high eosinophils - parasitic infections

Question 21

he blood film shown above was prepared from a patient with a severe bacterial infection. What can you notice and what does this indicate?

Answer 21

• There is an absence of mature neutrophils and two band form neutrophils can be seen
• This indicates ‘left shift’ - an increase in the proportion of band cells or the presence of neutrophil precursors in the peripheral blood.
• The presence of clear vacuoles within the cytoplasm of the neutrophils can be seen which is a specific indicator of bacterial infection.