IMMUNOLOGY PRACTICAL: Differential Blood Cell Counts

AIM:

To examine a prepared blood smears from humans in order to identify and count different types of white blood cell. Also to analyse and view the data, taken during the course of a parasitic infection (on mice).

INTRODUCTION

The total number of white blood cell does not necessarily indicate the severity of a disease, since some serious ailment may show a low white blood cell count. For this reason, a differential white blood cell count is performed.

A differential white blood cell count consists of an examination of blood to determine the presence and the number of different types of white blood cells. This study often provides helpful information in determining the severity and extent of an infection, more than any other single procedure used in the examination of the blood.

The role of white blood cells, or leukocytes, is to control various disease conditions. Although these cells do most of their work outside the circulatory system, they use the blood for transportation to sites of infection.

There are five different types of white blood cells, which are normally found in the circulating blood. They are 1) lymphocytes, 2) neutrophils, 3) monocytes, 4) basophils and 5) eosinophils.

Lymphocytes constitute 20-40% of the body’s white blood cell and 99% of the cells in the lymph. These lymphocytes continually circulate in the blood and lymph and are capable of migrating into the tissue spaces and lymphoid organs, thereby integrating the immune system to a high degree.

To perform a differential cell count, you must be able to identify the different types of white blood cells. The ability to properly identify the different types of white blood cells is not difficult to develop, but require a thorough knowledge of staining characteristics and morphology (the study of the form and structure of organisms). This knowledge can be gained only by extensive, supervised practice.

METHOD

For this practical you have to produce a blood smear of an infected animal, but due to not being able to handle blood containing parasites, you will be provided with a prepared blood smear slide. In this practical no parasites are involved. You are only working with the blood of the infected animals so there is no infection risk.

The methodology will still be provided in order to gain understanding of the procedure.

You have been provided with clean microscope slides on which you prepare a blood smear. For this practical you should work in pairs, one person from each pair should do a smear from the infected animals; the other should do a smear from the control animals. Take your slides to the demonstrator who will give you a drop of blood, make a smear.

The stains used will stain your skin and are also potentially harmful if ingested. Standard laboratory procedure applies.

Staining of Blood Smears; fix the cells on the smear in 95% methanol for 2 min. then Immerse the slide in buffer for 5 min (remember which side of the slide the cells are on). Shake the droplets off slide into a waste pot. Then stain in Giemsa solution for 15 - 20 min. After 15-20 min have gone by, rinse in buffer by immersing the slide several times. Put a drop of buffer and a cover slip on the slide and examine using the X40 objective on the microscope. If the cells are over-stained (too blue) remove the cover slip and allow them to stand in buffer for 5 min.

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). ...

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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

RESULTS

Cell Count:

Table 1: Results from my observation

Table 2: Results of class data

Total white cell counts

Table 3

DISCUSSION AND ANALYSIS

A column bar chart is used below to view the range of different leukocytes produced over the different field of view:

Graph 1:

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:

Table 4

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:

Graph 2:

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.

Graph 3:

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.

Table 5

Graph 4

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.

Leukocytes

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).