Cell Identification; in order to familiarise yourself with leukocyte morphology you have also been provided with a pre-stained slide of human blood. Examine under the X40 objective and identify the following cell types in your slides; lymphocytes, neutrophils, monocytes, eosinophils and basophils. Also carry out a differential cell count as described below.
Cell Counts; for differential cell counts count the number of each leukocyte cell type in the field of view of the microscope. Look at least 5 - 10 fields of view over the slide. (White cells tend to occur most near the edges of the smears). Add up all of the cells that you have counted and calculate percentages for each cell type.
In order to relate percentages of white cells to actual numbers, it is necessary to know the total number of leukocytes/ml of blood. For this it is necessary to isolate the leukocytes from the blood.
Prepare a slide of the mouse cell using a haemocytometer slide; the demonstrator will place a drop into the slot for you to observe the slide at x 40 objective.
The cover slip on the slide allows a small amount of the cell suspension to run under the cover slip and examine under the microscope (X40 objective). Count the cells and estimate the number of white cells/ml blood for the infected animal.
Number of cell/ml = Total Number of Cells counted X 25 X 104
Number of triple ruled squares
Table 1: Results from my observation
Table 2: Results of class data
Total white cell counts
DISCUSSION AND ANALYSIS
A column bar chart is used below to view the range of different leukocytes produced over the different field of view:
In this chart we can see that there is a generally high population of neutrophils and a fairly consistent amount of lymphocytes and monocytes. There is very small population of eosinophils and basophils.
For a normal human blood you should expect the following:
According to the class data (see Table 2), the differential cell count for the normal human blood sample compared to the human blood sample used contains neutrophils below the range of 50-70 (decreased), monocytes outside the range of 1-6% (increased), eosinophils are outside the range of 1-3% (increased) and also basophils are outside the range of <1 (increased). This is illustrated in a column bar chart below.
Comparisons of the class data with the standard expected results for a normal human blood are shown below in a column bar chart:
In this chart we can see that lymphocytes fall in between the expected range. Neutrophil fall below the range and monocytes, eosinophils and basophils are above the expected range value. Reasoning is given below, to why might there be an
increase or decrease in leukocytes.
In addition to looking at differences in % of different white blood cells, we will also consider estimating total numbers of each type of cell / ml of blood.
Number of cell/ml = Total Number of Cells counted X 25 X 104
Number of triple ruled squares
Number of cell/ml = 53 X 25 X 104
Number of cell/ml = 2.6 x 10
Table below shows the data from a different infection which includes blood counts from different periods post infection.
This chart shows the percentage of cell count over a period of post infection; we see that there is an increase in neutrophils, more than the expected range in weeks 1, 8 and 16. Week 0 and 4 fall in betwwen the expected range. Generally lymphocytes monocytes, eosinophils and basophils range between their expected range. Reasoning related to the function of leukocytes are given below.
Lymphocytes produce antibodies and destroy the toxic products of protein metabolism. Lymphocytes fight viral infections and some bacterial infections. Certain lymphocytes directly attack invading microorganisms; others produce antibodies that attack and destroy microorganisms and other foreign material. Large lymphocytes, called atypical lymphocytes, are seen during infectious mononucleosis and other illnesses.
The cytoplasm of a lymphocyte is clear sky blue, scanty, with few unevenly distributed, azurophilic granules with a halo around them. The nucleus is generally round, oval, or slightly indented, and the chromatin (a network of fibers within the nucleus) is lumpy and condensed at the periphery.
Neutrophils account for the largest percentage of leukocytes found in a normal blood sample, and the function by ingesting invading bacteria. Neutrophils increase in response to bacterial infection. They destroy bacteria by enveloping and digesting them, a process called phagocytosis. When many neutrophils are needed, they are released from the bone marrow as immature cells, called bands or stab cells.
On a stained blood smear, the cytoplasm of neutrophils has numerous fines, barely visible lilac-coloured granules and a dark purple or reddish purple nucleus. The nucleus may be oval, horseshoe, or “U”- shaped, or segmented (lobulated).
Monocytes are the largest of the normal white blood cells and its function is destroying bacteria, foreign particles, and protozoa. Monocytes increase during severe infections, and other conditions. They remove debris and microorganisms by phagocytosis.
Its colour resembles that of a lymphocyte, but its cytoplasm is a muddy gray-blue. The nucleus is lobulated, deeply indented or horseshoe-shaped, and has relatively fine chromatins structure. Occasionally the cytoplasm is more abundant than in the lymphocytes.
Basophils are believed to keep the blood from clotting in inflamed tissue. Basophils increase in response to allergic reactions and parasitic infection.
They are scattered large, and have dark- purple/blue granules that are darker than the nucleus. The granules may overlay the nucleus as well as the cytoplasm.
Eosinophils aid in detoxification, they also break down and remove protein material. Eosinophils increase in response to allergic reactions and parasitic infection.
The cytoplasm of eosinophils contains numerous coarse, reddish-pink granules, which are lighter coloured than the nucleus.
Table 6: Blood cell counts from three different patients
Table 7: Comparing the differential cell count in percentages
A white blood cell (WBC) count determines the concentration of white blood cells in the patient's blood. A differential determines the percentage of each of the five types of mature white blood cells.
This test is included in general health examinations and to help investigate a variety of illnesses. An elevated WBC count occurs in infection, allergy, systemic illness, inflammation, tissue injury, and leukemia.
A low WBC count may occur in some viral infections, immunodeficiency states, and bone marrow failure. The WBC count provides clues about certain illnesses, and helps physicians monitor a patient's recovery from others. Abnormal counts which return to normal indicate that the condition is improving, while counts that become more abnormal indicate that the condition is worsening. The differential will reveal which WBC types are affected most. For example, an elevated WBC count with an absolute increase in lymphocytes having an atypical appearance is most often caused by infectious mononucleosis. The differential will also identify early WBCs which may be reactive (e.g., a response to acute infection) or the result of a leukemia.
In response to an acute infection, trauma, or inflammation, white blood cells release a substance called colony-stimulating factor (CSF). CSF stimulates the bone marrow to increase white blood cell production. In a person with normally functioning bone marrow, the numbers of white blood cells can double within hours if needed. An increase in the number of circulating leukocytes is rarely due to an increase in all five types of leukocytes. When this occurs, it is most often due to dehydration and hemoconcentration. In some diseases, such as measles, pertussis and sepsis, the increase in white blood cells is so dramatic that the picture resembles leukemia.
Reviewing the case study:
For case 1 there is a slight difference, but not significant, we say this by look at the total WBC count as it is in range to a normal human blood sample (see Table 7). Monocytes are out of the range by 0.4%.
As for the same as case 1, case 2 has a slight difference, but not significant, as the total number WBC count is in range (see Table 7). Monocytes are out of the range by 1.03%, eosinophils by 3.5%, and basophils by 0.8%
Case 3 shows Abnormal Results because the total number of WBC count is below the range of 4.5-11.0 x 106/ml; there is a significant difference of 3.39 x 106/ml. A low number of WBCs is called leukopenia. This can be due to bone marrow failure (for example, due to infection, tumor, or abnormal scarring), collagen-vascular diseases (such as lupus erythematosus), disease of the liver or spleen or radiation. Looking at the individual percentage of leukocytes also show a significant difference: neutrophils are below the range by 12.4%, lymphocytes out of range by 12.9%, and monocytes are out of the range by 0.4% (see Table 7).
- Goldsby, Richard Allen. (2000). Kuby Immunology. 4th ed / Richard A. Goldsby, Thomas J. Kindt, Barbara A. Osborne.New York: W.H. Freeman.
- Differential White blood cell count. http://www.tpub.com/content/medical/14295/css/14295_283.htm. [Accessed on 19th December 2008].
White Blood Cell Count (WBC) and . Images available at:
. From home page [Accessed on 19th December 2008].