Preparing And Staining A Blood Film Biology Essay

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Along with erythrocytes in the blood, there are leukocytes, also known as White Blood Cells which have different functions in the immune system of the body. There are a number of different cells under the umbrella term of leukocyte

Neutrophils are the most common leukocyte, with an average number of 2.5-7.5x109 cells per Litre. They have a distinct lobed nucleus and have a life span of approximately 5 days. Their primary role is to destroy invading pathogens via phagocytosis and the release of cytokines1. They are generally the first species to react in the inflammatory response.

Lymphocytes are the second most common leukocyte in the blood, and comprises of 3 groups of cell, B-Cells, which produce antibodies which bind to antigens on foreign bodies, T-Cells, which kill cells which are infected by virus and regulate B cells and NK Cells, and Natural Killer Cells (NK Cells) which kill cells which do not express MHC class 1 molecules. They compromise between 20-40% of the leukocytes in the body, with a concentration of around 1.5-3. 5x109 cells per Litre.

Figure 2

http://pathmicro.med.sc.edu/bowers/llympho-darb.jpg

Monocytes are the third largest component of the leukocytes, which compromise between 4-10% of the body's leukocyte, with approximate 0.2-0.8x109 cells per litre. Their main function is the presentation of antigens and phagocytosis of foreign bodies. When Monocytes settle in a tissue, they become Macrophages.

Figure 3. http://pathmicro.med.sc.edu/bowers/monocyte-darb.jpg

Basophils and Eosinophils are the lowest in number compromising around 4% of the total leukocytes. Eosinophils have a role in destroying parasites and Basophils have a large role in the allergic response.

Figure 5 Basophil http://www.cytochemistry.net/microanatomy/blood/more_basophils.htm

Figure 4 http://pathmicro.med.sc.edu/bowers/eosinoph-darb.jpg

Principles

Blood films are prepared to allow investigation into haematological problems and to screen for blood parasites. Blood is fixed and stained onto a slide and examined using a microscope to count the numbers of individual types of white blood cells. This allows us to detect any increases or decreases in the number of blood cells and detect any irregularities.

Aim

To test a blood sample to indentify the levels of different blood components are normal and there are no irregularities.

Method

Spreader

May-Grunwald Stain

Methanol

Wash Buffer

Pipette Tips

Counter

Deionised Water

Fume Cupboard

Coplin Jars

Stop Watch

Giemsa Stain

Light Microscope

Blood Sample

Gloves

BSA treated Slides

Micropipette

Distilled Water

Wax PencilApparatus

Glass slide cleaned and labelled

7µL of blood pipette to the centre of the slide, approximately 1cm from the edge of the slide

Spreader used to spread the blood onto the slide (diagram showing spreading technique used below)

Blood sample allowed to air-dry onto the slide and then fixed using methanol for 5-10 minutes

Slide placed into a Coplin Jar Containing May-Grunwald Stain for 5-10 minutes

Slide washed using wash buffer, ensuring removal of all excess stain

Slide Placed into a Coplin Jar containing Giemsa Stain for 10-15 minutes

Slide rinsed using distilled water

Slide washed using De-Ionised water for 2-5 Minutes

Slide dried and examined under light microscope.

Number of Cells found for each cell type recorded.

blood spread.png

Figure 6. Haematology & transfusion Science Laboratory Manual

Results

Cell Type

Number of Cells

Percentage of Cells (%)

Normal Cell Range (%)

Neutrophils

58

55.24%

55-65%

Basophils

2

1.90%

0-1%

Monocytes

24

22.86%

4-10%

Eosinophils

0

00%

1-3%

Lymphocytes

21

20%

20-40%

Total

105

100%

Conclusion

Neutrophils numbers fell on lower range of normal

Basophils where at normal level

Monocytes where double normal level

No Eosinophils where found

Lymphocyte numbers where on the lower range of normal

Cells appeared well differentiated with smooth nuclear contours and no evidence of rupture or damage. The cells where of normal proportion and there was no evidence of hypertrophy.

Neutrophils and Lymphocytes where low but Monocytes where double normal range. The lack of Eosinophils is of no major concern as the compromise such a lower proportion of the blood that they, along with Basophils, may be difficult to find in a single blood film.

The low levels of Neutrophils and Lymphocytes, coupled with the high number of Monocytes would suggest the recent recovery from an infection, as Neutrophils and Lymphocytes would be used to combat the infection rapidly while the Monocytes migrate to the affected tissue.

If the number of Neutrophils in the blood where much higher, the patient would be suffering from neutrophillia, which is generally caused by a bacterial infection or the effects of drugs or hormones such as cortisol. The Neutrophil level also increases during the inflammatory response or as a result of burns 2

If the Neutrophil level drops, the patient has neutropenia which is caused by anaemia and certain drugs or the lack of intake of essential minerals and vitamins, such as copper and folate, but can also be the result of the increased destruction of cells due to an autoimmune response or if the patient is undergoing chemotherapy3. Mild cases of this can occur during a viral infection.

The high level of Monocytes is known as Monocytosis, and can be caused by a number of issues, such as a chronic inflammatory response and viral disease. But the levels also increase when the body is recovering from an acute infection or during the recovery phase of neutropenia3.

The low number of lymphocytes, called lymphocytopenia, occurs generally after a recent infection which is similar to the cause of neutropenia, but also can be caused through drug use and chemotherapy and as a side effect of cortisol and excercise4. Higher number of lymphocytes, called lymphocytosis, can occur during viral infections and other acute infections and splenomegaly.

When all blood cell types increase, but only a few of the cell types are found to have matured, there is the possibility of leukaemia, which is a cancer of the blood. There are many different forms of leukaemia and there is no single cause. There are many different treatments for leukaemia, including the use of drugs, chemotherapy and bone marrow transplants5. There are many different signs and symptoms of leukaemia, but the diagram below shows the most common signs:

Figure 7 http://commons.wikimedia.org/wiki/File:Symptoms_of_leukemia.png

The main source of error in the experiment was human error. This could be due to things such as not shaking the blood sample before the sample was pipetted onto a slide. There is the possibility that the sample was not spread correctly on to the slide.

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