White blood cell production

Introduction

White blood cells (WBC), also called Leukocytes or Leucocytes’ are cells of the immune system that are involved in protecting the body against disease and foreign invading bacteria, viruses, fungi and parasites. All white blood cells (Leucocytes) are produced and derived from a multi-potent cell in the bone marrow known as a ‘’haematopoietic stem cell’’, white blood cell (Leucocytes)  are found throughout the body, including the blood and lymphatic system.

The Leucocytes are classified according to

1. The presence or absence of specific granules in the cytoplasm, as granulocytes or a granulocytes.
2. The nuclear morphology as poly-morphonucler or monocular cells.
3. The origin as myeloid or lymphoid cells, the granulocytes (Neutrophils, eosinophils and basophil) are by nature, polymorph nuclear cell with granular appearance. In clinical terminology, the cell are referred to as ‘’poly’’ because of the irregular shaped, multi-lobulated nucleus, in the cytoplasm. They are called terminal cells, because the cells are unable to replenish the catabolised proteins after phagocytic function and eventually die.

The agranulocytes, are the lymphocytes and monocytes with regular shaped nuclei, the cytoplasm lack specific granules, the granulocytes and the monocytes protects the body against invading organisms, mainly by digestion i.e. by phagocyteosis (the act of absorbing and digesting fragments or whole organisms as an amoeba does). The lymphocytes and plasma cells function mainly in the immune system.

The life span of granulocytes after release from the bone marrow is normally 4-8 hours in the blood stream and 4-5 days in the tissue. The number of white blood cells (leucocytes) in the blood is often an indicator of disease; the normal white cell count is usually between 4-11 x 109/l which is usually expressed as 4,000-11,000 white blood cells per micro litre of blood; they make up approximately 1% of the total blood volume in a healthy adult. An increase in the number of white blood cells (leucocytes) above the upper limit is called leukocytosis and a decrease in the number of white blood cells below the lower limit is called leukopenia (Abayomi, 2007).

Etymology of white blood cell

The name “white blood cell” derives from the physical appearance of a blood sample after centrifugation. White cells are found in the buff, a thin, typically white layer of nucleated cells between the sedimentated red blood cells and the blood plasma. The scientific term leukocyte directly reflects its description, it is derived from the Greek word “leuko” meaning “white” and kytos meaning “hollow vessel” with-cyte translated as “cell” in the modern use. The Buffy coat may sometimes be green if there are large amount of neutrophils in the sample, due to the Heme-containing Enzymes myeloperoxidase that they produce (Orkin, 2008).

 Different types of white blood cell (leucocytes)

The different types of white blood cells (WBCs) also known as leucocytes are classified according to the presence or absence of specific granules in the cytoplasm as graunlocytes or agranucocyte, the nuclear morphology as polymorphonuclear or mononuclear cells, and the origin as myeloid or lymphoid cells. The granulocytes are (neutrophils, eosinophils and basophils) while the agranulocytes are the (lymphocytes and monocytes).

 Neutrophil

Neutrophil are the most abundant white blood cell constituting 60-70% of the circulating leukocytes; they defend against bacterial or fungal infection, they are usually first responders to microbial infection, their activity and death in large number forms pus. They are commonly referred to as polymorphonuclear (PMN) leukocytes, although, in the technical sense, polymorphonuclear refers to all granulocytes. They have a multilobed nucleus, which consists of 2-5 lobes connected by thin bridges of the nuclear membrane, 10-12mm in diameter. (Bruce, Alexander, Julian, Martin, Keth and Peter, 2002); this gives the neutrophils the appearance of having multiple nuclei, hence the name polymorphonuclear leukocyte. The cytoplasm may look transparent because of fine granules that are pale lilac when stained. Neutrophils are active in phagocytising bacteria and are present in large amount in the pus of wound; these cells are not able to renew their lysosomes (used in digesting microbes) and die after having phagocytised a few pathogens. Neutrophils are the most common cell type seen in the early stages of acute inflammation. The life span of a circulating human neutrophil is about 5.4 days (Pillay et al, 2010).

 Eosinophil

Eosinophils compose about 2-4% of the total WBC, 12mm in diameter, this count fluctuates throughout the day, seasonally, and during menstruation eosinophil count rises in response to allergies, parasitic infections, collagen diseases, and disease of the spleen and central nervous system. They are rare in blood, but numerous in the mucous membranes of the respiratory, digestive, and lower urinary tracts. They primarily deal with parasitic infection. 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 (swollen red areas of the skin) and also parasitic infections. They secrete chemical that destroy these large parasites, such as hookworms and tape worms that are too big for anyone WBC to phagocytise. In general their nucleus is Bi-lobed. The lobes are connected by a thin strand. The cytoplasm is full of granules that assume a characteristic pink-orange colour with eosin staining (Saladin and Kenneth, 2012).

 Basophil

Basophils are chiefly responsible for allergic and antigen response by releasing the chemical “histamine” causing the dilation of blood vessels. They are the rarest of the white blood cells (less than 0.5% of the total leucocytes count), 9mm in diameter, and share physiochemical properties with other blood cells, they are difficult to study. They can be recognized by several coarse, dark violet granules, giving them a blue hue. The nucleus is bi or trilobed, but it is hard to see because of the number of number of coarse granules that hide it. They excrete two chemicals that aid in the body defences; histamine and heparin. Histamine is responsible for widening blood vessels and increasing the flow of blood to injured tissue. It also makes blood vessels more permeable so neutrophils and clotting protein can get into connective tissue more easily. Heparin is an anticoagulant that inhibits blood clotting and promotes the movement of white blood cells into an area. Basophil can also release chemical signals that attract eosinophils and neutrophils to an infection site.

 Lymphocyte

Lymphocytes are of two morphological forms, the large and small lymphocytes. The small lymphocyte is about 7-10 um in diameter and has a round purple nucleus which occupies most of the cell having a thin rim of pall blue cytoplasm. They are the predominant form found in normal blood.  The large lymphocyte has a diameter between 12-20μm and the nucleus strain a little paler than the small lymphocyte. The cytoplasm is abundant, straining a pale blue colour and contain few reddish granules. The normal lymphocyte count for an adult is between 1.5-4.0×10⁹ per litter of blood, and a differential count of 20-45%. From experimental work, it has been found that there are many subtraction of lymphocyte population, each with specific complimentary roles; two major population of cell, the B-lymphocyte and T-lymphocyte.

The B-lymphocyte is derived from the bone marrow and is concerned with the production of antibodies in response to the stimulation from antigens foreign to the body. The B-lymphocyte having recognized an antigen, transform into a plasma cell which matures and secretes a specific antibody from the lymph nodes; the antibody is carried into the plasma and lymph, fluid it coats the antigen and thus make it susceptible to phagocytisis. The T-lymphocyte is derived from the thymus gland and concern with cell mediated immunity, a process which involves the cells surrounding the antigenic materials causing its destruction by direct cellular involvement; the T-lymphocyte has a very long span possible 20 years and are thought to hold a memory of previously encountered antigens such that on subsequent exposure, the body can rapidly respond. Lymphocytes are immunologically component cells which assist the phagocytes in the defence of the body against infection and other foreign invasion and added specificity to attack. They also play major role in the rejection of transplanted tissue which is antigenically different to the recipient.

 Monocyte

Monocytes are larger than other white cells, measuring between 16-22mm in diameter, having one large nucleus, which is centrally placed within the cell and often have kidney shaped nucleus with surrendered appearance like closely coiled bundle of wool and pale violet colour when strained, the cytoplasm strain a pale greyish-blue and contains reddish-bleu dust like granules and a few clear vacuoles. The normal monocyte count in an adult is between 0.2-0.8 x 10⁹ per litre of blood and differential count of 2-10%. Monocytosis often occur in certain bacterial infection e.g. tuberculosis and in some protozoa infection e.g. malaria and monocytes are motile and phagocytic, capable of ingesting bacteria and particulate matter, acting as scavenger cells at site of infections, the blood monocyte is an intermediate stage of development, they migrate to tissue and develop into fixed tissue, macrophages of the reticulo-endothelial system.

 Fixed Leukocyte

Some white blood cell (Leukocytes) migrate into the tissues of the body to make up a permanent residence at that location rather than remaining in the blood; often these cell have specific names depending upon which tissue they settle in, such as fixed macrophages in the liver which become known as kupfer cell. These cells serve a role in the immune system, histiocytes, dendritic cells, mast cells and, microglia.

 White blood cell disorder

White blood cell disorders are two major categories; Proliferative and Leucopoenia, in the proliferative disorder there is an increase in the number of white blood cell, this increase is commonly reactive (due to infection), but may also be cancerous. In leucopoenia disorder there is decrease in the number of white blood cells; both proliferative disease and leukopenias are quantitative disorders of white blood cell. Qualitative disorders of white blood cells are another category. These are disorder in which the number of white blood cell is normal but the cell do not function normally Kenneth Kaushanky et al (2010).

 Method of estimating white blood cell (leukocyte)

White blood cells (leukocytes) can be estimated in the laboratory by visual white cell count and differential cell count; visual white blood cell count estimates the total number of white blood cells in a cubic millimetre of blood, the range of visual white cell count in a normal individual is 4.0-11.0 x 10⁹ cells per litre. The differential white cell count estimate the different white cell in a patient blood, the normal percentages for differential white cell count of an adult blood is neutrophils 60-70%, lymphocyte 25-40%, monocytes 2-6%, Eosinophils 1-4% and basophils 0-1%.

Visual white cell count

Principle:

Blood is diluted 1 in 20 with a fluid containing 2% glacial acetic acid, tinged with gential violet; the acid lyses the red cells but not the leukocytes or nucleated red blood cells leaving the white blood cells to be counted microscopicically using an improved neubauer counting chamber (Haemocytometer) and the WBC per litre of blood calculated the gential violet stains the nuclei.

Equipment

1. Haemocytometer (new improved neubauer counting chamber) and cover.
2. 75 x 12mm test tubes.
3. Capillary tube.
4. White cell pipette or 0.02ml pipette.
5. 1ml graduated pipette.

Reagent: Turk’s Fluid

• 500ml Turk’s WBC diluting fluid.
• Glacial acetic acid –           –           –           –           –           –           –           –           10ml
• Gentian Violet, 1% aqueous –           –           –           –           –           –           1ml
• Distilled Water- –           –           –           –           –           –           –       –      500ml

1. Fill a 1 litre cylinder with distilled water to the 490 mark.
2. Add 10ml of concentrated glacial acetic acid and mix.
3. Dissolved 0.1g gentian violet in 10ml distilled water and filter.
4. Add 1ml of the gentian violet solution and mix.
5. Transfer to a storage bottle and label.

Turk’s fluid specimen: EDTA Antiloagulanted Blood of Free Flowing Capillary Blood

Procedure:

1. Pipette 0.38ml of Turk’s fluid into a clean test.
2. Using the 0.02ml pipette, pipette 0.02ml of well mixed EDTA anticoagulanted blood or free flowing capillary blood into the test-tube and mix properly by gentile inversion.
3. Clean the chamber and the special haemocytometer cover glass.
4. Slide the cover glass into position over the grid area and press down on each side until a rainbow effect called Newton’s rings are seen.
5. Re-mix the diluted blood sample; withdraw little quantity of the diluted blood with Pasteur pipette or capillary tube.
6. Fill the chamber by holding the capillary tube at angel of 45^oC, allow the tip to touch the edge of the cover glass, taking care not to over fill the area.
7. Leave the chamber undisturbed for 2 minutes for the white cells to be settled.
8. Use x 10 objective to count the white cells present in the 4 corner square (1mm²) areas.

 Calculation

The total white cell counted is calculated using the formula;

White cell count = N x DF x 10⁶

A x D

Where:

N         = Total number of cell counted.

DF       = Diluting factors.

10⁶        = Factors to convert count /μ/ to count 1L

A         = Total area counted in mm²

D         = the depth of the chamber (0.1mm)

(Abayomi, 2007).

Differential white cell count

Principle:

A drop of blood is spread on a slide, stained and examine under the microscope to establish the morphology of red blood cells, leukocytes and platelets and the relative frequently frequency of different leukocytes.

Material

1. Clean glass free slide/cover-slip.
2. EDTA anticoagulanted blood or free flowing capillary blood.
3. IEISHMAN stain.

 Procedure

1. Place a drop of properly mixed EDTA anticoagulanted blood or free flowing capillary blood in the centre line of a clean grease free slide about 1cm from one end.
2. Place a spreader in front of the blood at an angle of 45⁰C, push backward to touch the drop of blood.
3. Allow the blood to spread through the edge of the spreader and push forward to form a thin firm.
4. Label the slide with the patient name and date and allow to air-dry.
5. Fix the thin film with methanol for two minutes.

Staining procedure

6. Arrange the air-dried blood firm on a horizontal staining rack with the film facing upward.
7. Flood the slide with Leishman stain and allow to stain for 2 minutes.
8. Dilute the stain twice, its volume with buffered distilled water and allow the diluted stain to stand for 8 minutes.
9. Rinse thoroughly with stream of buffer water until it has acquired a tinge or salmon pick colour.
10. Wipe the back of the side with a piece of cotton wool to remove all trace of excess strain.
11. Set the slide upright to air-dry at room temperature.
12. Mount on a microscope and examine with x 100 oil immersion objective. (Abajomi, 2007).

Some signs and symptoms observed in high or low WBCS

A high white blood cell count called leukocytosis, may result from a number of conditions, some includes;

1. Infections, most commonly caused by bacterial and some viruses, less commonly by fungi or parasite.
2. Inflammation or Inflammatory conditions such as Rhematoid arthritis, Vasculitis or inflammatory bow/disease.
3. Allergic response e.g. allergic asthma.

A low white blood cell count, called leucopoenia can result from condition such as;

1. Bone marrow damage (e.g. Toxin, Chemotherapy, Radiation therapy, Drugs).
2. Bone marrow disorder: when the bone marrow does not produce sufficient WBCS (e.g. myelodysplastic Syndrome, Vitamin B₁₂ or folate deficiency.
3. Autoimmune Disorder: when the body attacks and destroy its own WBCs (e.g. Lupus).
4. Disease of the immune system such as HIV, which destroys T-lymphocyte.
5. Overwhelming infection e.g. sepsis.

Conclusion

Since white blood cell plays important role in the immune system by protecting the body against infection and foreign invader and its disorder leads to proliferative disorder and leukopenias; such as increase in the number of white blood cells (due to infection). And decrease in the number of white blood cells (due to bone marrow disorder), therefore it is concluded that each individual should check and know there visual white cell count and differential white cell count from time to time, in order to keep it within the normal range.

Recommendations

Since white blood cell plays important role in the immune system by protecting the body against infection and foreign invaders and various complications, it is therefore, recommended that all individuals should check their visual white cell count and differential cell count from time to time, in other to keep it within the normal range.

References

Abayomi, A. (2007). Method of white blood cell analysis in: A test book for medical laboratory practice (2^nd ed). Lagos; Edoson Book Centre pp 192-199.

Bruce, A., Julian, M., Keith, R. and Peter, W. (2002). ‘’Leukocyte functions and percentage   breakdown’’, Molecular Biology of the         cell (4^th ed). New York: Garland Science

Kenneth, K (2010). Eosinophil Count Williams haematology (8^th ed) New York: McGraw-Hill Medical.

Pillay, J., Den Braber. I., Vrisekoop. N., Kwase, L.M., De Boer, R.J, Borghans, J.A.M., Tesselaar, K. and Koenderman, L. (2010). ‘’In invo labelling with 2H₂O, reveals a human neutrophil life span of 5.4 days: Journal of Clinical Haematology 116(4): 625-7.

Saladin, K. (2012). Anatomy and physiology: the unit of forms and functions (6^th ed). New York: McGraw Hill.