Haematology - WBC

Question 1

What is qualitative blood analysis?

Answer 1

This analyses the red and white blood cell and platelet morphology (shape and structure) and is an essential part of a blood smear examination.

Morphological changes are important markers of underlying disease or can give clues to the mechanisms behind cytopenias (reduction in the number of circulating blood cells) in peripheral blood.

Question 2

Manual blood film assessment can be…

Answer 2

far more accurate than automatic analysers, as eyes can detect very subtle changes that machines are unable to.

Question 3

Why is a manual blood smear examination used?

Answer 3

to complement a complete blood count haematology panel, especially when the CBC has been performed on an automated analyser.

A smear is used to identify, count and visualise abnormal cell morphology allowing monitoring or diagnosis of a range of disease processes.

A smear is prepared with peripheral blood and stained, to allow visualisation of individual cells, with a haematological stain e.g. Wright’s stain or a rapid stain like Diff Quick.

Question 4

Define a Differential leukocyte count

Answer 4

Determines the percentage of each type of white blood cell within the blood sample.

Question 5

Define a RBC morphology assessment

Answer 5

Identifies changes within the shape, sizes, structure, presence of inclusions and formation of RBC’s

Question 6

Define a WBC morphology assessment and WBC count

Answer 6

identifies immature or abnormal changes is WBC cell structure, presence of intracellular inclusions- Total WBC count can be performed using a good blood smear.

Question 7

Define Platelets morphology and count

Answer 7

Changes in appearance, formation e.g. clumping. Estimated counts can also be performed to verify figures generated by automatic analysers.

Question 8

Why is the push technique often used for a blood smear?

Answer 8

As this is considered the most effective technique for obtaining a diagnostic smear.

Question 9

How and why do you pre-clean microscope slides?

Answer 9

Slides should be pre-cleaned, premium quality with a frosted edge for easy labelling.

Non pre-cleaned slide and spreader slides will require wiping thoroughly with lint free tissue prior to use. This removes grease and gritty particles that may interfere with the smearing process.

Question 10

Describe the smearing technique

Answer 10

Ensure that the EDTA sample is sufficiently mixed by gently rolling or inverting the sample.

Collect the blood from the EDTA tube using a haematocrit tube (this fills by capillary action).

DO NOT be tempted the tip the blood onto the slid from the blood tube or lid, or to use a 1ml syringe. These methods have limited control and often result in thick, undiagnostic smears.

Place a dot of blood at approximately 4mm in diameter on one end of the slide. If the blood does not flow sufficiently from the haematocrit tube DO NOT repeatedly tap the tube on the slide. Go back to the EDTA tube and collect some more blood.

Hold the spreader slide at a 30-45° angle and draw it back into the blood spot.

Capillary action will cause the blood spot the travel along the length of the spreader slide.

Push the spreader slide away from the blood spot smoothly and moderately quickly to spread the blood evenly over the surface of the slide.

Question 11

What are the qualities of a good blood smear?

Answer 11

It should have a dense body that takes up 2/3rd of the entire smear.

A well developed edge that is fine and feathery in appearance. If there is a thick line of blood where the slides stops its and indication of a poorly made smear.

Often only ½ cm in length, the mono layer should be just behind the feathered edge, this region should be noticeably thinner than the body of the smear. Prior to staining, if the slide is held up to the light a rainbow effect should be seen through this section of the smear.

t should not cover the entire surface of the slide, hence the use of a spreader slide.

It should have a smooth and even appearance.

The blood film should be free from waves, lines and holes.

It should not have an irregular tail.

Question 12

What can dirty or faulty equipment cause?

Answer 12

Poor quality smear

Question 13

What can using a full sized slide to spread the sample cause?

Answer 13

will result on a overly wide smear with loss of cells at the edges. A bevelled edge slide can be used in place of a spreader slide if unavailable.

Question 14

What can excessive downward pressure cause?

Answer 14

Producing short smears with hesitation lines and poorly developed monolayer and feathered edge.

Question 15

What can a slow spreading motion cause?

Answer 15

Causing long, thin smears, that lack a dense body, thin monolayer and developed feathered edge. Often a straight edge of blood is seen where the spreader slide has been lifted too quickly and hesitation lines can be seen throughout the smear.

Question 16

What can wobbling the spreader slide on the surface of the smear cause?

Answer 16

This is commonly due to inexperience and trying to exert pressure instead of letting the spreader glide along the surface of the smear.

Question 17

What can lifting the spreader slide too soon cause?

Answer 17

The spreader should remain in contact with the slide right to the end. Lifting the spreader too soon will result in a line, straight end of blood at the end of the smear instead of a thumb shaped, feathered edge.

Question 18

What can pulling instead of pushing cause?

Answer 18

Holding the spreader around the wrong way, therefore pushing the spreader into the blood spot and then pulling it back across the side. Instead of pulling the spreader backwards into the blood spot and then pushing it across the slide. This results in a very short and thick smear.

Question 19

Name some biological causes of a poor quality smear

Answer 19

Sample is too cold – cold agglutination. Warm the sample at 37° for 5 mins then re make the smear.

Lipaemia – holes will appear on the blood film, there is nothing that can be done to rectify this.

Rouleux – Stacking of RBC’s, nothing can be done to rectify this.

Question 20

What does blood smear staining do?

Answer 20

Staining facilitates visualisation of normally colourless leucocytes, by enhancing contrast in the microscopic image. This allows identification of particular cells, cell contents and parasites.

Question 21

What are the two types of stains?

Answer 21

An acid dye eosin, that is attracted to the alkaline cytoplasm, producing a red colouration.

Basic azure dyes (methylene blue) that bind acid nuclei and result in a blue to purple colour.

Question 22

Describe different types of romanowsky stains

Answer 22

Wrights stain

Modified Wrights (Diff quick / Rapi-Diff)

Giemsa stain

Question 23

Describe giemsa

Answer 23

Classic blood peripheral blood film stain.

More complex technique than the rapid stains.

Slides must be immediately ‘wet’ fixed in methanol for 5 mins prior to commencing the staining process.

Commercially prepared powder containing methylene blue, eosin and Azure B. Reconstitute with pH7.2 buffered water shortly before use as has limited shelf life.

Staining technique is slow (10-15mins), can be up to 30mins to produce a stained sample.

Question 24

Describe Diff Quik

Answer 24

Rapid blood staining technique with stains contained within coplin jars.

Solvent (methanol) - light blue Air dried blood smears are fix prior to staining, to avoid the smear being washed off by the following staining processes.

Solution I (Eosin Y) -orange / red The stain is attracted to the alkaline cytoplasm, producing a red colouration (eosinophil dye).

Solution II (Thiazine dyes, methylene blue and Azure A) – Purple / blue These stains bind to the acid nuclei and result in a blue to purple colour (Basophil dye).

Question 25

Describe how you would examine a blood smear

Answer 25

Using the light microscope scan the smear at low x 10 magnification.

Look for platelet clumps, large cells or infectious agents.

Identify the monolayer, where cells are uniformly distributed and well spread out.

Check that leukocytes are well distributed throughout the slide. Concentration of WBC’s at the feathered edge can be a sign of poor smear technique. If this is highlighted, repeat the smear using a faster action.

Once the optimal area has been located (monofilament layer) move to a higher x100 oil immersion magnification and begin your differential white cell count of 100 WBC’s.

Move back and forth across the slide to avoid going over the same area twice. This technique is called a ‘battlement’ pattern

Question 26

Describe a neutrophil

Answer 26

Produced in the bone marrow and release into the blood stream once mature.

Segmented, full mature neutrophils are the dominant leukocyte in dogs and cats (approx. 65%)

Segmented purple nucleus and pale pink granules.

Neutrophils are phagocytes.

Question 27

How would you interpret neurophilia?

Answer 27

An increase in the number of circulating ‘band’ immature neutrophils (horse shoe shaped nucleus)

Acute inflammation or infection, that results in the release of immature cells.

Question 28

How would you interpret neutropaenia?

Answer 28

An abnormally low concentration of circulating neutrophils.

Chemotherapy, congenital conditions affecting the bone marrow, overwhelming viral infection, autoimmune conditions.

Question 29

Describe basophils

Answer 29

Produced in the bone marrow, very low numbers within the blood of healthy animals (approx. 0.1%)

Purple nucleus with blue granules.

Contain heparin to reduce blood clotting and manufacture histamine, promoting blood flow to tissues through vasodilation.

Question 30

How would you interpret basophilia?

Answer 30

An abnormal abundance of basophils – Allergic reactions and myeloproliferative conditions.

Question 31

Describe eosinophils

Answer 31

Produced in the bone marrow.

Relatively low numbers within the blood stream (approx. 4%)

Purple bi-lobed nucleus with red / orange granules.

Involved in allergic responses and immunity against parasites.

Question 32

How would you interpret eosinophilia?

Answer 32

An increase in the number of circulating eosinophils in the blood, can be caused by an a response to allergens, drugs and parasites and in some times of leukaemia.

Question 33

How would you interpret eosinopaenia?

Answer 33

A reduction in the normal number of circulating eosinophils. This can be as result of Stress reactions, steroid therapy, Cushing’s syndrome

Question 34

Define lymphocytes

Answer 34

Produced in the lymphoid tissue NOT bone marrow.

Moderate numbers within the blood stream (Approx 25%)

Large oval or kidney shaped nucleus, that is stained purple.

3 different types of lymphocyte:

B cells - produce immunoglobulin,

T cells – are involved in cell mediated immunity.

Killer cells (slightly larger in size)- control certain cancer cells and microbial infection by limiting their spread.

B and T cells are indistinguishable

Question 35

How would you interpret Lymphocytosis?

Answer 35

A increase in the number of circulating lymphocytes as a result of chronic infection, stress and some types of neoplasia e.g. leukaemia

Question 36

How would you interpret Lymphopaenia?

Answer 36

A reduction in the number of lymphocytes within circulating blood cells. This may be as a result of stress, viral infection, bone marrow suppression,

Question 37

Describe a monocyte

Answer 37

Large immune cell produced in the bone marrow.

Relatively low numbers found within the blood stream (approx. 5%)

Largest of the white blood cells with a large purple, bean shaped nucleus.

Chronic phagocyte

Question 38

How would you interpret monocytosis?

Answer 38

A increase in the number of circulating monocytes. This may be caused by stress, chronic infection or hepatic inflammation.

Question 39

How would you interpret Monocytopaenia?

Answer 39

A decrease in the number of circulating monocytes. Difficult to assess due to the low reference ranges for this leukocyte.