Differentiation of white blood cells by light microscopy
Most mammals have blood consisting of three types of blood cells, Red blood cells (Erythrocytes), White blood cells (Leukocytes) and Platelets (Thrombocyte). These are collectively termed as hematocyte, which simply means blood cell.
These cells constitute to around 40% to 45% of the blood volume. The remaining consist of Plasma, which is referred to the liquid part of blood. The total volume of blood can be determined by centrifugation or flow cytometry. The 45% of blood component (blood cells) is termed as hematocrit. Another important component of blood is hemoglobin, which gives blood its red color. It is the carrier of oxygen in blood and any defects in it can lead to many health problems. It is an iron containing protein and also helps in transporting different gases and components to tissues and organs of the body.
White blood cells (Leukocytes) are often regarded as immune cells. They are involved in fighting infections and other foreign components which enter the body. They have vital role in diagnosis of a disease, since their number increases in case a person is diseased or ill. Abnormal or high number of Leukocytes (WBC) is also seen in blood cancer (Leukemia). There are 5 different types of Leukocytes found in humans: Neutrophil, Eosinophil, Basophil, Lymphocyte and Monocyte. They are all generated from the Bone marrow. They are found in most organs of the body and their survival rate is around 3 to 4 days in the body. They are also found in the lymphatic system.
Clinical Relevance of White Blood Cells
White blood cells have great importance in Immune system. As mentioned earlier, Leukocytes are derived from hematopoietic stem cell, which is present in the Bone marrow. On an average in a healthy individual, there are around 6500 to 7000 WBC’s per ml of blood. An increase in Leukocyte is an indication of a disease and is referred to as Leukocytosis. They are called white, since they appear as a white cloudy coat on centrifugation and the word Leukocyte is derived from the German word Leuko (white) and kytos (Hollow vessel).
Leukocytes can be broadly classified on the basis of granular structures, present within the cells. They are distinguished as Granulocytes and Agranulocytes respectively.
Granulocytes are characterized by the presence of granules within the cytoplasm of the cell and mainly observed under Light microscope using differential staining, mainly Giemsa blue stain. The granules found are mostly enzymes (Lysozymes) and help in digestion of foreign particles. The leukocytes belonging to Granulocytes are Neutrophil, Eosinophil and Basophil. These cells can be stained using different kinds of stains like, Giemsa blue stain, H&E staining etc.
Neutrophil Granulocyte
Neutrophils are widely found in blood and consist of nearly 45% to 65% of the total white blood cells. They are normally 10-12 µm in diameter. They have the shortest life span of near 6 hours a day and show motility. The name is derived from the staining technique hematoxylin and eosin (H&E) by which they are stained light pink in color. Their nucleus have found to be multi-lobed and mainly consisting of 2-5 lobes. Neutrophils usually have phagocytic features and found in the bloodstream, Neutrophils play important role in immune system and are the first cells to induce any kind of response. They have good influence in inflammatory response and migration to tissue through signal transmission. They are spherical when inactive but change appearance once activated in presence of antigens. Their main roles are phagocytosis and degranulation.
Neutrophil Granulocyte
Image Source: Astarte Biologics (Human Neutophils) [Image Online] Available through Astarte Biologics website.
< http://www.astartebio.com/files/images/Segmented_neutrophils__2_.jpg>
[Accessed 18 December 2013]
Eosinophil Granulocytes
These are second type of white blood cells and usually associated with asthma and allergic reactions. They are acid loving, as they appear to have affinity to coal dyes and being easily stained by eosin red dye. The name eosinophil granulocytes is derived because of its stained properties with eosin. They are normally transparent, but appear red with eosin dye. They have smaller granules and contain chemicals like lipases, RNA’s and plasminogen. They constitute to around to around 2% to 4% of white blood cells. They are similar in size to neutrophils, around 1o-12µm in diameter. Their nucleus is however bi-lobed. They are usually found in lymph nodes, ovary and spleen. The average life cycle of eosinophil is around 8 to 12 days. Their main targets are multicellular parasites and to mediate allergic and inflammatory responses.
Eosinophil Granulocyte
Image Source: Karen Kendall. Eosinophil. [Electronic Print] Available at:
< http://libweb.anglia.ac.uk/referencing/harvard.htm> [Accessed 18 December 2013]
Basophil Granulocytes
Basophils constitute to around 0.55 to 1% of total white blood cells in the body. The name basophil is derived with the fact that these cells have affinity to basic dyes. They are usually larger and are around 12-15µm in diameter. They maybe bi-lobed or tri0lobed which can be seen upon staining with a basic dye and visualizing under light microscope. They have significant importance in blood clotting. These cells consist of Heparin (Anticoagulant). They play an important role in proper blood flow and contain vasodilator histamine for this purpose. They are present mainly in body tissues and contribute to allergic and inflammatory reactions. Studies also reveal the role of basophils in T cell regulation.
Basophil Granulocyte
Image Source: Basophils: stains very dark, large histamine granules. [Electronic Print] Available at:
< http://www.cbv.ns.ca/young/bio12a/blood/Basophil.html> [Accessed 18 December 2013]
The next type of Leukocytes are Agranulocytes. These cells lack any kind of granules within the cytoplasm and can be differentiated by alternative staining methods and can be also visualized under Light microscope. The two agranulocytes are Lymphocytes and Monocytes.
Lymphocytes
Lymphocytes mainly constitute the adaptive immune system. Under microscopic visualization these can be differentiated into Small and Large Lymphocytes. The small lymphocytes are mainly T cells and B cells, while large consist of Natural killer cells (NK Cells) Each of these have significance in immune response. B cells mediate in T cell activation and release certain antibodies. Cell mediated immunity is mostly related to T cells, whereas humoral immunity is mainly of B cells. T cells produce toxins which help in destruction of foreign proteins while some cells produce cytokine which paly important role in immune response.
Natural Killer cells on the other hand are part of innate immunity. They help in necrosis of tumor or any malignant cells within the body. They recognize MHC cells, which helps in distinguishing between tumor cells and healthy cells. However these cells do not require any mediator and thus are termed as Natural and kill any tumor cell in its way. On the whole lymphocytes have a long life cycle, from weeks to many years and act as memory cells of the human body.
Lymphocytes:
Image Source: Cytohematology Proficiency Test Event - Slide 012. August 1998. Normal lymphocytes. [Electronic Print] Wadsworth.org website. Available at:
< http://www.wadsworth.org/chemheme/heme/glass/slide_012_lymphs.htm> [Accessed 18 December 2013]
Monocytes
Monocytes are part of the innate immune system and the largest of leukocytes. They are usually 15-20µm in diameter. These cells constitute of around 5% to 8% of total leukocytes. They have a distinguishing feature, of being kidney shaped and extensively large compared to all other leukocytes. These cells normally migrate from bloodstream to target organs. Their average life span is around 6 hours a day in the human body. The main function of monocytes is phagocytosis, antigen presentation and cytokine production.
Monocytes
Image Source: Blood Cells. [Electronic Print] Available at:
< http://nhscience.lonestar.edu/biol/cardio/blood.htm> [Accessed 18 December 2013]
Use of Light Microscope to Visualize White Blood Cells
The most common method to differentiate different white blood cells is use of differential staining techniques. Giemsa stain is widely used to distinguish and identify agranulocytes. Lymphocyte cytoplasm usually takes up a sky blue color while monocytes are stained light blue in color.
Alternatively Lieshman stain is used alone for granulocytes.
The basic steps employed for microscopic analysis of leukocytes (White Blood cells) are as follows:
- Preparing a blood film on a clean grease free slide
- Staining of the film using differential stains (Giemsa, Lieshman or Eosin red dye)
- Observing under light microscope (Under 10x and then under 45x)
- Counting the number of cell types
- Interpretation of observed findings.
Observation under light microscope is quite simple and it enables one to differentiate between different leukocytes. Under 45x power of the microscope, it is possible to visualize the different components of each cell. Cytoplasmic studies can also be revealed.
Slide number: 100
Observations:
A total of 136 cells were observed. Of the total number of Leukocytes, Neutrophil finding was the highest. Nearly 51% of the total cells were neutrophils. Followed by this nearly, 41% of Basophilic granulocytes were observed. Only a single eosinophil was observed. However agranulocytes like lymphocytes and monocytes were not observed.
Conclusion
Use of differential staining method enabled to distinguish between different Leukocytes. Observation under light microscope revealed the presence of Neutophils in the given sample to be the highest. A substantial amount of Basophilic granulocytes were also observed. However the amount of Basophil count was higher than expected, which implies that the individual may have mediated inflammatory responses and hence the number of Basophilic granulocytes was high. Eosinophilic granulocytes were quite less, in fact only a single cell was observed, which is quite normal. The absence of Lymphocytes may have been due to poor staining technique or disruption of lymphocytes during the process. The same satnds for the absence of monocytes in the observation.
References:
Astarte Biologics (Human Neutophils) [Image Online] Available through Astarte Biologics website.
< http://www.astartebio.com/files/images/Segmented_neutrophils__2_.jpg>
[Accessed 18 December 2013]
Karen Kendall. Eosinophil. [Electronic Print] Available at:
< http://libweb.anglia.ac.uk/referencing/harvard.htm> [Accessed 18 December 2013]
Basophils: stains very dark, large histamine granules. [Electronic Print] Available at:
< http://www.cbv.ns.ca/young/bio12a/blood/Basophil.html> [Accessed 18 December 2013]
Cytohematology Proficiency Test Event - Slide 012. August 1998. Normal lymphocytes. [Electronic Print] Wadsworth.org website. Available at:
< http://www.wadsworth.org/chemheme/heme/glass/slide_012_lymphs.htm> [Accessed 18 December 2013]
Blood Cells. [Electronic Print] Available at:
< http://nhscience.lonestar.edu/biol/cardio/blood.htm> [Accessed 18 December 2013]
