Diagnostic role of the blood film Flashcards
What is a blood film?
a method of blood sample preparation which allows red blood cells, white bloods and platelets to be spread evenly for microscopic inspection
What is microscopy?
a technique used to examine samples (tissue, fluid, cells) that are too small for the human eye to see unaided
Why do we stain cells?
Very few cellular components absorb light at visible wavelengths so we cannot see them
What is the most frequent combination of stains?
H&E (Haematoxylin and Eosin)
What is haematoxylin?
a purple-blue component that binds to acidic components of cells
in particular it binds to DNA and shows up the nucleus
What is Leishman’s stain?
Contains a purple-blue dye, which stains nuclei, and a pink one staining components in the cytoplasm
What is eosin?
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.
What is a good general purpose stain?
H&E
What is the alternative to using stain?
exploit interference properties of light to detect the different refractive index of cells compared with the surrounding fluid
important when examining living cells
What is the resolution of a microscope?
the closest limit at which you can distinguish two adjacent small objects
What factors affect the performance of a light microscope?
- the wavelength of light ( cant alter this)
- the optical quality of lenses
- the refractive index of the medium through which the light passes
- The physical properties of the objective lens
- The geometry of the illuminating cone of light provided by the condenser lens
How do you set up a microscope correctly?
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
How is the microscope is adjusted?
Step 1: Focus the image
Step 2: Focus the condenser
Step 3: Adjust the field iris
Step 4: Adjust the condenser iris
How do we prepare a blood film?
- blood sample is mixed thoroughly
- Blood dispenser is pushed through the lid of the sample
- single drop is taken from the sample and placed onto the slide
- Spread the drop with another slide
- Label the slide
How can a drop of blood be taken from a finger
using a sterile lancet to puncture the fleshy pad alongside the base of a fingernail
How do you spread the blood on the slide?
- spreader slide placed at an angle in front of the drop
- spreader slide drawn back to touch the drop
- the drop spreads between the 2 slides
- The drop is spread by surface tension “dragging” it along the slide as the spreader is pushed forward
How to stain a blood film using Leishmans stain?
- The slide is first placed blood-side up in a rack over a draining dish.
- 8 drops of Leishman’s stain are dropped on the using a dropper bottle so that the film is completely covered.
- The slide is left for a minute to stain.
- 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.
- The stain is poured off and the slide rinsed well with copious amounts of buffer, using a wash bottle.
- The slide is dried by first tapping the edge of the slide on filter paper and then gently waving it in the air
- Once completely dry, the slide may be examined using the light microscope.
What are reference ranges?
descriptions of data derived from a sample of a reference population
What is 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).
What else are haematological variables affected by?
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
What is a normal range?
used to mean a range derived from a healthy reference population
When interpreting laboratory data you should consider the following points:
- A value within the normal range may be abnormal for that individual
- A value outside the normal range may be normal for that individual
- Reference ranges for healthy and sick individuals usually overlap
- Some haematological variables are dependent on the precise instrument or methodology used –> reference range derived for a particular instrument/method
What do elevated numbers of eosinophils show?
allergic reactions e.g. asthma and also parasitic infections.
What do increased numbers of basophils show?
chronic myelogenous leukaemia (CML) and also in certain hypersensitivity reactions
What does “left shift” mean?
may indicate either an increase in the proportion of band cells or the presence of neutrophil precursors in the peripheral blood
What are the indicators of bacterial infections?
an absence of mature neutrophils and two band form neutrophils
presence of clear vacuoles within the cytoplasm of the neutrophils
How do we distinguish acute lymphoblastic leukaemia (ALL)?
very irregular shape of the nuclei
How do we distinguish chronic granulocytic leukaemia?
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
How do we distinguish Megaloblastic anaemia (maturation of the nucleus is retarded in relation to that of the cytoplasm) ?
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.
What are blood films used together with?
Full blood counts (FBC)
Which following parameters are returned by a standard FBC?
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).
