# A-level Applied Science/The Role of the Pathology Service/Haematology

Haematology (US English: hematology) is the branch of medicine that is concerned with blood, the blood-forming organs and blood diseases. Haematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases.

Blood diseases affect the production of blood and its components, such as blood cells, haemoglobin, blood proteins, the mechanism of coagulation, etc.

Tests that require examination and measurement of the cells of blood, as well as blood clotting studies, are grouped under haematology. apart from clotting studies, tests on serum or plasma come under biochemistry.

(Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation which packs the more dense blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Plasma is essentially the same as serum, but is obtained by centrifuging the blood without clotting. Plasma therefore contains all of the clotting factors, including fibrinogen.)

Components of blood obtained by centrifuging unclotted whole blood.

# Haematologists

Medical doctors who work in haematology are known as haematologists. Their routine work may range from the management of the haematology laboratory, work at the microscope viewing blood films and bone marrow slides, interpretation of various haematological test results, care of in-patients and care of out-patients.

Haematologists may specialise further or have special interests, for example in:

• treating bleeding disorders such as haemophilia.
• treating haematological malignacies such as lymphoma and leukaemia (onco-haematology).
• treating haemoglobinopathies.
• in the science of blood transfusion and the work of a blood bank.

Haematologists receive whole blood and citrated plasma. They do full blood counts, blood films and coagulation investigations. This would identify conditions such as leukaemia and haemophilia.

# Classification of haematology diseases

• Haemoglobinopathies (congenital abnormality of the haemoglobin molecule or of the rate of haemoglobin synthesis)
• e.g. Sickle-cell disease
• Anaemias (lack of red blood cells or haemoglobin)
• Decreased numbers of cells
• Myeloproliferative disorders (Increased numbers of cells)
• Haematological malignancies
• Lymphomas
• Hodgkin's disease
• Non-Hodgkin's lymphoma
• Myelomas
• Multiple myeloma
• Leukemias
• Coagulopathies (disorders of bleeding and coagulation)
• Disorders of clotting proteins
• Haemophilia
• Disorders of platelets
• Miscellaneous
• HIV/AIDS
• Malaria
• Leishmaniasis

# Tests

Tests used in the investigation of haematological problems include:

• Full blood count
• Erythrocyte sedimentation rate (ESR)
• Blood film
• Bone marrow biopsy
• Coombs test
• Serum ferritin level
• Vitamin B12 and folate levels
• Prothrombin time
• Partial thromboplastin time
• Protein electrophoresis
• Haemoglobin electrophoresis
• D-dimer

# Treatments

Treatments include:

• Oral medication - tablets or liquid medicines.
• Anticoagulation therapy.
• Intramuscular injections (for example, Vitamin B12 injections).
• Blood transfusion (for anaemia).
• Venesection (for iron overload or polycythaemia).
• Bone marrow transplant (for example, for leukaemia).
• Chemotherapy (for example, for leukaemia).
• Radiotherapy (in decline, for example, for leukaemia).

# Type of work

Much work is carried out by technicians using microscopes. Automation is less widespread than in biochemistry because in many tests the cell types need to be identified visually.

## Health and safety

Safe handling of blood products is extremely important. There is a high risk of infection if the blood is infected with pathogens such as HIV or a hepatitis virus.

## Blood culture

blood culture

Blood culture is microbiological culture of blood. It is employed to detect infections that are spreading through the bloodstream (bacteraemia, septicaemia).

### Method

About 30 cm3 of blood are taken through venipuncture and injected into two "blood bottles" with specific media for aerobic and anaerobic organisms.

Care needs to be taken that the bottles are not contaminated with bacteria from staff members or other patients. To that end, the patient's skin and the top of the blood bottles are rubbed or sprayed with denatured alcohol.

To maximise the diagnostic yield of blood cultures, multiple samples are sometimes taken from different veins.

### Uses

Any infection causing fever may prompt hospital physicians to request blood cultures. Identifying the agent may aid in choosing the correct antibiotic and addressing particular risk factors.

Blood culture is essential in the diagnosis of infective endocarditis. In this elusive disease, blood cultures may have to be repeatedly taken during febrile episodes, when bacteria are shed from the heart valves into the bloodstream (bacteraemia).

## Haemocytometry

A haemocytometer. The two semi-reflective rectangles are the counting chambers.
The parts of the haemocytometer (as viewed from the side) are identified.

The haemocytometer is a device originally used to count blood cells (as the name suggests). It is now used to count other cells and many types of microscopic particles. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a grid of perpendicular lines.

The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. Therefore it is possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

### Principles

Diagram of the microscopic grid.

The ruled area of the haemocytometer consists of one large square known as a type A square. Within the type A square there are 16 smaller squares known as type B squares. These are further divided into 16 smaller squares known as type C. Each type C square has an area of 1/256 mm2 and each type B square has an area of 1/16 mm2. There are three lines around each side of the type B squares. The cell-sized structures to be counted are those which lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square.

Some designs have 25 type B squares in each type a square. In this design, each type C square has an area of 1/400 mm2 and each type B square has an area of 1/25 mm2.

### Usage

When a liquid sample containing immobilised cells is placed on the chamber, it is covered with a cover glass, and capillary action completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture from which the sample was taken: it is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions and counting shortcuts:

$\mbox{concentration of cells in original mixture} =$

$\left (\frac{\mbox{number of cells counted}}{(\mbox{proportion of chamber counted})(\mbox{volume of chamber})} \right ) \left (\frac{\mbox{volume of sample dilution}}{\mbox{volume of original mixture in sample}} \right )$

Haemocytometers are often used to count blood corpuscles, organelles within cells, blood cells in cerebrospinal fluid after performing a lumbar puncture, or other cell types in suspension. Using a haemocytometer to count bacteria results in a 'total count' as it is difficult to distinguish between living and dead organisms.

#### Usage tricks

Empty haemocytometer grid at 100x power.
• Mix your original mixture thoroughly before taking a sample. This ensures that your sample is representative, and not just an artefact of the particular region of your original mixture from which you drew it.
• Use an appropriate dilution of your mixture with regard to the number of cells you hope to count. If your sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, your sample size will not be enough to make strong inferences about the concentration in the original mixture. Naturally, you must have a rough idea of the concentration before you begin in order to guess an appropriate dilution. If your mixture is coloured, it may be helpful to memorise a particular intensity of that colour at which the mixture tends to be easy to analyse.
• Analyse multiple chambers. By performing a redundant test on a second chamber, you can compare the results. If they differ greatly, your method of taking the sample may be unreliable (e.g. the original mixture is not mixed thoroughly). You can use the average of your results for a more accurate calculation.
• Make sure to put enough liquid on the instrument that some leaks out of the cover glass when it is placed over the chamber. Otherwise, it is uncertain whether the space under the cover glass is completely filled with liquid. This volume should be the same every time you use the instrument.
• Do not use a paper wipe to dry the excess liquid. The same capillary action that filled the chamber will then dry it out.
• Watch out for the objective lens. Remember that the haemocytometer is thicker than a normal microscope slide. If you focus too closely, your objective lens may contact the instrument. This may affect your choice of which objective lens you use - carefully figure out what will fit, before you start.
• Count across the rows or down the columns. Use the gridlines to help you remember which areas' cells have already been counted.
• You don't have to count the whole chamber. If there are lot of cells, you can just perform your count in a section of the chamber and use the grid to determine what proportion of the chamber that is. You can then extrapolate to estimate how many cells are in the chamber, and use that figure in your final calculation. This gives you speed at the expense of potential accuracy; if possible, using a more appropriate dilution is better.
• Are the lines in or out? Some cells inevitably fall smack on top of the outside gridlines that mark the edges of the chamber. The usual practice is to include cells overlapping the top and left lines, but not those overlapping the bottom or right lines - this has the advantage of eliminating redundant counting if you count adjacent regions.