White Blood Cell Differential Count Using Wright Stain
Title: White Blood Cell Differential Count using Wright Stain
The objective of the experiment was to prepare a fresh blood smear and distinguish different types of blood cells using wright’s stains.
Blood is an important part of a human body. Blood is the fluid in our body. The blood contains cellular elements. The cellular elements are serum and plasma. Serum is the blood fluid after thrombin and other clotting factors have been removed. Plasma is the blood fluid with the clotting factors and other substances in it. There are two types of blood cells. They are Erythrocytes and leukocytes.
Erythrocytes or Red blood Cells main purpose is to transport oxygen throughout the cells of the body. Oxygen binds to hemoglobin. Hemoglobin is an iron-containing protein, which facilitates transportation of oxygen by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. Arterial blood carries oxygen from inhaled air to the tissues of the body, and venous blood carries carbon dioxide, a waste product of metabolism produced by cells, from the tissues to the lungs to be exhaled.
Leukocytes or white blood cells are responsible for resisting infections that enter the body. There are five types of white blood cells. They are neutrophils, basophils, eosinophils, monocytes, and lymphocytes. Each blood cell protects the body from specific infections. They can be identified by their different morphology. Some have noticeable granules whereas others don’t. WBC’s are placed into two different categories. The WBC’s with visible granular are called granular whereas WBC’s without visible granular are called a-granular. WBC’s that are a-granular include lymphocytes and monocytes. Granular WBC’s include neutrophils, basophils, and eosinophils.
Neutrophils make up to 50-70% of all of the white blood cells. It is the most abundant white blood cell. It is highly phagocytic during the infection process. It is usually the first WBC to arrive during an infection. The average half-life of a non-activated neutrophil in the circulation is about 4-10 hours. Upon activation, they migrate into tissues, where they survive for 1-2 days. These phagocytes are normally found in the blood stream. However, during the acute phase of inflammation, particularly as a result of bacterial infection, neutrophils leave the vasculature and migrate toward the site of inflammation in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance. Neutrophils react within an hour of tissue injury and are the hallmark of acute inflammation. They can internalize and kill many microbes, each phagocytic event resulting in the formation of a phagosome into which reactive oxygen species and hydrolytic enzymes are secreted. Neutrophils have two types of granules primary and secondary. The primary granules are large and dense granules that are made out of a lysosome. The secondary granules are very small granules that contain enzymes. Both primary and secondary granules are used to fuse with phagocytomes and kill the pathogen. Neutrophils are phagocytes, capable of ingesting microorganisms or particles. Neutrophils stain with a neutral color. It stains dark blue. The cytoplasm usually stains lilac with fine granules.
Basophils make up 0.5-1% of all white blood cells. It is the least abundant of all of the white blood cells. Basophils have histamine that is located in the nucleus. Basophils surface during allergies. Basophils have a nucleus that stains pale blue with a cytoplasm that stains pink. It contains a coarse equal sized deep bluish-purple granule. It stains with basic dyes.
Eosinophils make of 1-5% of white blood cells. Eosinophils can be phagocytic and is associated with a parasitic infection. Eosinophils usually have two-lobed nucleus that stains blue-red or just blue. The cytoplasm stains pale lilac with deep pink granules that are equal in size. The granules are very large and red. Eosinophils usually stain with acid stains and contain a very large acidic granule.
Monocytes make up 2-6% of all white blood cells. It surfaces as a first line of defense along with neutrophils. Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissues monocytes mature into different types of macrophages at different anatomical locations. Monocytes are responsible for phagocytosis of foreign substances in the body. Monocytes can perform phagocytosis using intermediary proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody, termed antibody-mediated cellular cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter. Monocytes only remain in the body for a short period of time. Monocytes are the precursor to tissue macrophages. Monocytes are able to differentiate into the dedicated phagocytic macrophage cell after migrating from the bloodstream into tissues. Monocytes have a bean shaped nucleus that stains pale blue. The cytoplasm stains gray-blue with non-specific granules that are not easily seen. Monocytes are known to be larger than it’s a granular counterpart lymphocyte.
Lymphocytes make up 20-30% of all white blood cells. It is the second most abundant white blood cell. There are several types of lymphocytes some include B cells and T cells. T cells and B-cells are the major cellular components of the adaptive immune response. T cells are involved in cell-mediated immunity whereas B cells are primarily responsible for humeral immunity. The function of T cells and B cells is to recognize specific “non-self” antigens, during a process known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells. B cells respond to pathogens by producing large quantities of antibodies which then neutralize foreign objects like bacteria and viruses. In response to pathogens some T cells, called helper T cells produce cytokines that direct the immune response whilst other T cells, called cytotoxic T cells, produce toxic granules that induce the death of pathogen infected cells. Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an animal these memory cells will “remember” each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. They protect the body from some bacterial and viral infections. Lymphocytes have a round nucleus that is not easily distinguished from the cytoplasm. The cytoplasm stains a dark blue and is small. The granules are not visible. Lymphocytes morphology is similar to monocytes however it is small in size unlike monocytes.
A blood smear was prepared using a microscope slide. A subject was picketed with a lancet to obtain a blood sample. The blood was evenly spread across the slide at a 45 degree angle with a spreader slide. Once the blood smear was dry, 11 drops of Wright stain was place on the blood smear. After 10 minutes, 11 drops of Wright buffer was placed on the blood smear. After 10 minutes was finished, the stained slide was cleansed with water and blotted dried with bibulous paper. Once the slide was dried, the slide was places under the microscope for observation. Once the microscope was focused, the WBC was tallied by their different morphology.
Table 1: This table shows each WBC morphology and how many of each was counted by 100.
Neutrophils Basophils Eosinophils Monocytes Lymphocytes
60 1 2 6 27
60% 1% 2% 6% 27%
The results shown that each WBC has a different morphology and because of that morphology, they have different functions. For instance, a person with a high count of basophils suggests that they are suffering from an allergic reaction. Some white blood cells have more than one granule inside the cell. An example of that is a neutrophil. The neutrophil can have up to three granules inside the cell. Other white blood cells do not have a visible granular inside. Examples of those are Monocytes and lymphocytes. Another observation shown from the results were that each of the WBC stained differently. For instance, a neutrophil stains a neutral color with the cytoplasm staining lilac. If that was to be compared to a monocyte, another phagocytic blood cell, it would show that monocytes typically stain pale blue with non specific granules that are not visible. During the observation through the microscope, all of the WBC’s were visible. The subject was able to distinguish each WBC based on their different morphology. In conclusion, based on the percentage of the entire total WBC’s it was determined that this subject has a typical white blood cell count.
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Puget Sound Blood Center. (2006). Retrieved February 7, 2007, from Puget Sound Blood Center: