Blood

Blood is a liquid connective tissue. It is composed of a variety of cells circulating in a fluid, plasma. We are not interested in plasma in this lesson, only in the cells, both white and red blood cells. Blood cells are in three functional classes: red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes). All three are formed in the bone marrow but have vastly different appearances and functions.  

Red Blood Cells (Erythrocytes)

Red blood cells (RBCs) are the most numerous cell type in the blood (4.8-5.4 million RBCs/mL of blood). The cells are modified structurally to carry oxygen. The cells are biconcave disks approximately 8m in diameter (a doughnut without a hole) with no nucleus or metabolic machinery. The absence of cellular organelles allows the internal space of the cell to be available for O2 carrying. The interior of the RBC is filled with Hemoglobin (Hgb), a protein that functions primarily to carry O2. A typical RBC has approximately 280 million Hgb molecules in it due to the lack of cellular organelles.

White Blood Cells (Leukocytes)

White blood cells (WBCs) are much less numerous than RBCs (5,000 - 10,000 WBCs/mL blood) with a RBC/WBC ratio of approximately 700:1. WBCs work to protect the body from infection. WBCs are divided into two main groups based on cytoplasmic appearance: agranular leukocytes - lymphocytes and monocytes which have relatively clear cytoplasm; and granular leukocytes neutrophils, eosinophils, and basophils whose cytoplasm is filled with granules.

Granular Leukocytes

Neutrophils

The most abundant granulocyte, and most abundant WBC, is the neutrophil comprising 60-70% of all WBCs. They are approximately 10-12 m diameter with very fine, pale lilac granules in the cytoplasm. The nucleus has from 2-5 finelyconnected nuclear lobes that are rarely uniform in size. The multiple lobed nucleus and lightly stained cytoplasm are the most identifiable characteristics of the this cell. Neutrophils respond rapidly by chemotaxis to bacterial damage. They then phagocytize pathogens and release lysozymes, strong oxidants and defensins to help fight the infection. Click image to enlarge.

Eosinophils

Eosinophils represent only 2-4% of all WBCs. They are similar in size to neutrophils, 10-12 m diameter, with red/orange, large uniform granules, that do not block the nucleus. The nucleus usually has 2 but may have 3 connected nuclear lobes that tend to be more rounded and uniform than found in neutrophils. The red/orange color and more uniformly shaped lobed nucleus are the keys to identifying this cell. Eosinophils combat histamine in allergic responses, phagocytize antigen-antibody complexes and destroy some parasitic worms. Click image to enlarge.

Basophils

The least common WBC is the basophil comprising only 0.5-1% of all WBCs. Basophils are 8-10 m diameter with large blue-black round granules in the cytoplasm that block the bilobed nucleus. The dark granules are the most easily identifiable characteristic of this cell. Basophils exit capillaries and enter tissue fluids where they release heparin, histamine and serotonin during hypersensitivity (allergic) reactions to stimulate inflammation.Click image to enlarge.

 Agranular Leukocytes

Lymphocytes

Lymphocytes make up 20-25% of all WBCs. These cells vary in size with small lymphocytes being 6-9m and large lymphocytes 10-14m in diameter. The nucleus stains dark and is round or slightly indented with the cytoplasm appearing as a rim around the nucleus. The round, uniform nucleus and small amount of cytoplasm surrounding it are the best identifying characteristics for this cell.  Lymphocytes are involved in the specific immune response including antigen-antibody reactions. Click image to enlarge.

Monocytes

Monocytes comprise 3-8% of all WBCs. They are the largest of the WBCs, 12-20 m in diameter. The nucleus is indented or kidney shaped, not rounded, and surrounded by foamy cytoplasm. Their size is the most easily identifiable characteristic.  Monocytes are slow to respond to infection but arrive in larger numbers. When stimulated they enlarge and differentiate into wandering macrophages that clean up cellular debris and microbes following infection. Click image to enlarge.

 

Thrombocytes

Thrombocytes, or platelets, are numerous (150,000-400,000 platelets/mL blood) with a short 5-9 day life span. These cell fragments are small (2-4 m diameter) with many vesicles and no nucleus. They often appear as spots or "dirt" between the RBC's. Platelets function to plug small holes in vessels and stop bleeding. The granules contain many factors involved in blood clotting, including clotting factors, platelet derived growth factor (PDGF), Ca++, ADP, ATP, Thromboxane A2, vasoconstrictors, and clot promoting enzymes. Click image to enlarge.

 

Blood Cell Counts

A common physiological assessment of blood is the determination of the number of blood cells present. In this lab, you will perform two types of cell counts: 1) Hematocrit a measurement of the number of red blood cells currently in the blood; and 2) Differential white blood cell count determination of the percentage of each type of white blood cell in the blood. Your data for this portion of the lab along with the blood typing data will be recorded on the Blood Lab Worksheet. Please print out a copy of the worksheet to be turned in with your results.

 

General Protocol

You will be assigned a number between 1 and 24. This number will determine the set of slides you use for your cell counts. Record this number on your Blood Lab Worksheet. Use the same number for both the red and white blood cell counts. At the end of these instructions there is a link to the Cell count home. Once you have accessed the Cell count home use the pull-down menu under your assigned number to select either the red (Hematocrit) or white (Differential white blood cell) blood cell count option.

 

Hematocrit Protocol

Red blood cell counts employ a special microscope slide called a hemocytometer.  A hemocytometer is a special glass slide with a small chamber, or counting area, for counting cells in a given volume.  The counting area has a grid on it to allow for easier counting of the cells.  This technique allows for an estimation of the total number of red blood cells present in a sample. The large numbers of red blood cells present in blood (discussed above) make it impossible to count every cell. Instead, you will use a simulated hemocytometer to estimate the number of RBCs present.

After you select your "Red blood cell count, you will be presented with 4 images, or "slides", of blood cells. Each image has a grid superimposed on it, much like the grid found on a hemocytometer slide. The grid divides the area to be counted into 16 smaller sections, allowing you to count the red blood cells in smaller, manageable groups.  Count all of the red blood cells in each of the grid squares. DO NOT count a cell if ANY part of the cell touches ANY part of the OUTER grid lines. If a cell touches or overlaps any of the INNER grid lines, count the cell but be sure to count it only once.  There may be white blood cells present in these samples. The white blood cells are not to be counted. Count only the red blood cells.  Repeat this procedure for each of the four images.

 

Count and record the number of red blood cells in each square and, ultimately, the entire area of the grid.  Record each of your cell counts in the table on the Blood Lab Worksheet. Add up your counts from all 4 slides and record the total on the "Total" line of the worksheet. Once you have added all of your counts, multiply your total by 2500 (Note: DO NOT use 10,000 as indicated in the lab manual). Record this value, your RBC estimate, in the appropriate blank and answer the following questions.

 

Question 1

As described earlier, assuming a ratio of 700 RBCs to 1 WBC, use your hematocrit count to determine the number of white blood cells you would expect to see in this same sample of blood. Record this on the worksheet.

Question 2

Using the information in Chapter 19 of your textbook, identify a condition that might generate an abnormally high or low hematocrit count. Explain why this effect occurs.

 

Differential White Blood Cell Count Protocol

Select the appropriate White blood cell count. You will be presented with a series of blood smear slides. For each slide, you will identify and record the number of each of the 5 different types of leukocytes present.  Record your white cell count data on Blood Lab Worksheet.

 

For each slide, identify the WBCs present and place a tick mark next to the name of the white blood cell you see. When you have counted 100 TOTAL white blood cells (all WBCs together, NOT 100 of each type of WBC) STOP! Count the number of ticks for each cell type and determine the percentage of each type of leukocytepresent in your sample. Record the percentages in the table on your worksheet.

When you are finished with your white blood cell count, answer the following question.

 

Question

Using the information in Chapter 19 of your textbook, identify two (2) types of disorders that would generate an abnormal differential white blood cell count. In each case, specify which white blood cells are affected and how they are affected.

 

Cell count home