Total Leukocyte Count

Specimens

• Double oxalated or EDTA blood
• Capillary blood (specimen need not be a fasting sample).

Requirements

Microscope

Improved Neubauer Chamber

WBC pipette

WBC diluting fluid: It is prepared as follows:

• Glacial acetic acid: 2.0 ml
• 1 % (w/v) gentian violet: 1.0 ml
• Distilled water: 97 ml

This solution is stable at room temperature (25°C ± 5°C). A pinch of thymol maybe added as preservative.

Principle

• The glacial acetic acid lyses the red cells while the gentian violet slightly stains the nuclei of the leukocytes. 
• The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid and the cells are counted under low power of the micro scope by using a counting chamber.
• The numbers of cells in undiluted blood are reported per cu mm (μl) of whole blood.

 

Procedure

• Draw blood up to 0.5 mark of a WBC pipette.
• Carefully, wipe excess blood outside the pipette by using cotton. Draw diluting fluid up to 11 marks.
• Mix the contents in the pipette and after five minutes by discarding few drops, fill the counting chamber and allow the cells to settle for two to three minutes.
• Since Bulb pipettes are not recommended following procedure is performed
• Make a 1:20 dilution of blood by adding 20 μl of blood to (1 38 ml of diluting fluid in a glass tube (10 * 75 mm) Cork the tube tightly and mix the suspension by rotating in a cell-suspension mixer for at least 1 minute. Fill the Neubauer counting chamber by means of a Pasteur pipette or glass capillary.
• Focus on one of the ‘W’ marked areas (each having 16 small squares) by turning objective to low power. (10 X).
• Count cells in all four W marked corners.

Calculations

Number of white cells/cu mm (μl) of whole blood = (number of white cells counted * dilution) / (area counted * depth of fluid)

Where: 

• Dilution = 20
• Area counted 4 * 1 sq.mm = 4 sq.mm
• Depth of fluid = 0.1 mm (constant)

Hence number of white cells per cu mm (μl) of whole blood = (No. of cells counted * 20) / (4 * 0.1) = No of cells counted * 50

No. of leukocyte per liter of blood = No. of cells per cu mm (ml) * 106 or

Use the following formula

WBC count / liter = [No. of cells counted (per 1mm2 area) / volume counted (μl)] * dilution * 106

 

Precautions taken are exactly the same as for REC counting technique.

• The sources of error are also same as for RBC counting technique. However; in the case of WBC counting extra care is taken during the preparation and storage of WBC diluting fluid. 
• It should be perfectly free from dust particles and yeast cells, otherwise falsely high counts are obtained due to the presence of yeast cells and dust particles.

Normal values

• Adults: 4,000-10,000/cu mm (μl)
• At birth: 10,000-25,000/cu mm (μl)
• 1 to 3 years: 6,000-18,000/cu mm (μl)
• 4 to 7 years: 6,000-15,000/cu mm (μl)
• 8 to 12 years: 4,500-13,500/cu mm (μl)

Error of the total white cell count

• The inherent distribution error = λ1/2, here λ = total number of cells in each area.
• The error as high as 20% may make difference between 5.0 and 6.0 x 109 cells per liter, which is of little practical significance.
• The error can be reduced by counting more cells. If 400 cells are counted the error is reduced to 5%.
• Error may also be caused due to dirt clumped RBC debris or due to clumping of leukocytes.

Interpretation of Results:

Clinical Significance

Increase in total leukocyte count of more than 10,000/cu mm (μl) is known as

leukocytosis and decrease of less than 4 000 cu mm (μl) as leukopenia.

Causes of leukocytosis

Pathological

• It is common for a transient period in infections. The degree of rise in leukocytes depends on the type and severity of the infection and the response of the body.
• The infection may be 1) bacterial 2) viral 3) protozoal (malaria) or 4) parasitic (filaria, hookworm infection).
• Leukocytosis is also observed in severe hemorrhage and in Leukemia.

Physiological

• Age: At birth the total leukocyte count is about 18,000/cu mm (μl). It drops gradually to adult level.
• Pregnancy: At ‘full term’ the total count tends to be about 12,000 to 15,000/cu mm (μl). It rises soon after delivery and then gradually returns to normal.
• High temperature
• Severe pain
• Muscular exercise

Causes of Leukopenia

Certain viral and bacterial infections (typhoid) lead to leukopenia rather than leukocytosis.

Infections

• Bacterial (typhoid. paratyphoid, tuberculosis, etc)
• Viral (hepatitis, influenza, measles, etc)
• Protozoal (malaria)

Functions of WBCs:

Neutrophil

• Neutrophils defend against bacterial or fungal infection and other very small inflammatory processes that are usually first responders to microbial infection; their activity and death in large numbers forms pus.
• Neutrophils are very active in phagocytosing bacteria and are present in large amount in the pus of wounds.
• These cells are not able to renew their lysosomes used in digesting microbes and die after having phagocytosed a few pathogens. Most common cell seen in acute inflammation comes in and kills foreign substance.
• They stick to walls of the blood vessels of the infected or injured area and prevent the entry of any foreign particles into the blood stream. They can be found in the pus of wounds.

Eosinophil

• Eosinophils primarily deal with parasitic infections and an increase in them may indicate such.
• Eosinophils are also the predominant inflammatory cells in allergic reactions.
• The most important causes of eosinophilia include allergies such as asthma, hay fever, and hives; and also parasitic infections.
• Disorder in the eosinophils may lead to over active response to a disease in the Immune system in an attempt to destroy foreign organisms

Basophil

• Basophils are chiefly responsible for allergic and antigen response by releasing the chemical histamine causing inflammation.
• They secrete anti coagulant and anti bodies which mediate hypersensitivity reaction within the blood. They are the reason for immediate immune reaction against external germs and diseases.

Lymphocyte

They produce antibodies against toxins secreted by bacteria and infecting germs. These antibodies will be excreted into the plasma to kill bacteria in the blood and also act as anti toxins.

The blood has three types of lymphocytes:

B cells:

B cells make antibodies that bind to pathogens to enable their destruction. (B cells not only make antibodies that bind to pathogens, but after an attack, some B cells will retain the ability to produce an antibody to serve as a 'memory' system.)

T cells:

CD4+ (helper) T cells co-ordinate the immune response and are important in the defense against intracellular bacteria. In acute HIV infection, these T cells are the main index to identify the individual's immune system activity.

CD8+ cytotoxic T cells are able to kill virus-infected and tumor cells.

Natural killer cells: Natural killer cells are able to kill cells of the body which are displaying a signal to kill them, as they have been infected by a virus or have become cancerous.

Monocyte

• Monocytes share the "vacuum cleaner" (phagocytosis) function of neutrophils, but are much longer lived as they have an additional role: they present pieces of pathogens to T cells so that the pathogens may be recognized again and killed, or so that an antibody response may be mounted.
• Monocytes eventually leave the bloodstream to become tissue macrophages which remove dead cell debris as well as attacking microorganisms.
• Neither of these can be dealt with effectively by the neutrophils. Unlike neutrophils, monocytes are able to replace their lysosomal contents and are thought to have a much longer active life. 

Leukogram

A tabulation of the leukocytes present in a blood sample.

Leukergy

The clumping of white blood cells that accompanies some inflammations and infections

Lupus Erythematosus

• Lupus erythematosus is a category for a collection of diseases with similar underlying problems with immunity. This is an autoimmune disease in which any part of the body may be attacked by the immune system resulting in inflammations and tissue damage.
• Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blood cells, heart, and lungs.
• Four main types of lupus exist — systemic lupus erythematosus, discoid lupus erythematosus, drug-induced lupus erythematosus and neonatal lupus erythematosus. Of these, systemic lupus erythematosus is the most common and serious form of lupus.