Blood and Bone Marrow Lab

Learning Objectives

  • Identify the component cells of a typical blood smear
  • Identify the hematopoietic precursor cells of a typical bone marrow smear
  • Correlate the light and electron microscope images of red and white blood cells
  • Distinguish the different classes of white blood cells and the conditions under which each would be expected to dominate
  • Identify some key pathological examples related to bone marrow and peripheral blood

Pre-Lab Reading

Introduction

Blood provides a mechanism by which nutrients, gases, and wastes can be transported throughout the body. It consists of a number of cells suspended in a fluid medium known as plasma. Serum refers to plasma after clotting factors and fibrin have been removed.

Peripheral Blood Smear

The cells of the blood are important because they are a readily accessible population whose morphology, biochemistry, and ecology may give indications of a patient's general state or clues to the diagnosis of disease. For this reason, the complete blood count (CBC) and the differential white cell count are routinely used in clinical medicine. It is very important to be able to recognize normal blood cells and to distinguish pathological cells from the normal variants.

The identification of blood elements is based primarily on observations of the presence or absence of a nucleus and cytoplasmic granules. Other helpful features are cell size, nuclear size and shape, chromatin appearance, and cytoplasmic staining. The chart at the end of this section explains what to look for in the effort to identify the component cells of a blood smear.

Component Cell of the Blood Smear

A blood smear is created by placing a drop of blood near the end of a clean glass microscope slide. Another slide is held at an angle, backed into the drop, and then smoothly dragged forward to spread the blood film along the slide. The blood must then be fixed, stained, and washed.

When you view a properly prepared blood smear of a healthy individual, there are several populations of cells that you will notice. Keep in mind that these are all mature cells. The next section will discuss the identification of the immature cells of the bone marrow.

  • Erythrocytes, or red blood cells, are by far the predominant cell type in the blood smear. They are anucleate, non-granulated, eosinophilic cells that are uniform in shape (biconcave discs) and size (7.2 microns). Red blood cells have a central concavity that appears pale under the light microscope. These cells contain hemoglobin and are responsible for the transport and delivery of oxygen. Erythrocytes have a lifespan of 120 days.
  • Reticulocytes are immature red blood cells that are released from the bone marrow. They mature into erythrocytes after 1 to 2 days in the peripheral blood. There should be about one reticulocyte for every 100 red blood cells in a normal blood smear. These cells stain with a light blue tint because they still have RNA-containing organelles like free ribosomes.
  • Thrombocytes, or platelets, are the smallest elements of the blood and are responsible for the formation of clots through a complex, highly regulated cascade that you will study in Physiology and Immunobiology. Platelets are between 2 and 5 microns in diameter and appear ovoid and anucleate with purple granules.
  • Leukocytes, or white blood cells, are cells of the immune system that are present in both blood and interstitial fluid. There should be about 1 leukocyte for every 1000 red blood cells. They can be classified into two groups according to their nuclear pattern and the presence of cytoplasmic granules.

Monomorphonuclear leukocytes are cells with round, non-lobed nuclei These include:

  • Small lymphocytes, which are about the same size as erythrocytes and have deeply stained nuclei with a thin rim of cytoplasm. This population includes both B-cells and T-cells.
  • Large lymphocytes, which appear similar to small lymphocytes, but with larger nuclei and a greater amount of cytoplasm. This population also includes both B-cells and T-cells. Lymphocyte counts are raised in response to viral infections.
  • Monocytes, which are larger than lymphocytes and have less-clearly demarcated nuclei that are usually not centered in the cell. These nuclei appear horseshoe-shaped and the cytoplasm contains fine granules that give it a muddy gray color. These granules contain lysosomal enzyme and peroxidase. Monocytes are phagocytic cells that are important in the inflammatory response. They are the precursors to the tissue macrophages that you studied in the Laboratory on Connective Tissue.

Polymorphonuclear leukocytes are cells with lobed nuclei and cytoplasmic granules. While these cells share the same primary (nonspecific) or azurophilic granules, they are named based upon the characteristics of their secondary (specific) granules.

  • Neutrophils are by far the most numerous of the leukocytes. They are characterized by a nucleus that is segmented into three to five lobes that are joined by slender strands. The cytoplasm of neutrophils stains a pale pink. Its primary granules contain acid hydrolases and cationic proteins, and its secondary granules contain a variety of antimicrobial substances used to destroy bacteria that they phagocytose during the acute inflammatory response.
  • Eosinophils are larger than neutrophils and are distinguished by large red or orange granules of uniform size. These granules contain major basic protein, which is released to kill organisms too large to phagocytose, such as parasites and helminthes (worms).
  • Basophils are intermediate in size between neutrophils and eosinophils and have simple or bilobed nuclei. They contain many coarse purple granules that can vary in size or shape. These granules contain histamine, which is released to cause a vasoactive response in hypersensitivy reactions, and heparin, which is an anticoagulant. Basophils are not phagocytic.

Component Cells of a Bone Marrow Smear

While the peripheral blood smear indicates the status of mature blood cells, the bone marrow smear can be used to assess the process of hematopoiesis, or blood cell formation.

Active bone marrow appears highly cellular. The majority of the developing cells will become erythrocytes, which confer a red color to the marrow. For this reason, active bone marrow is also known as red bone marrow. Over time, the marrow becomes less active and its fat content increases. It is then referred to as yellow bone marrow.

Once again, there are several important characteristics to take into account when viewing a bone marrow smear. These include:

  • Size of the cell
  • Cytoplasm to nucleus volume ratio
  • Shape of the nucleus
  • Degree of chromatin condensation
  • Presence or absence of nucleoli
  • Cytoplasmic staining
  • Presence of cytoplasmic granules

The blast cell is a pluripotent stem cell from which erythrocytes, granulocytes, and lymphocytes originate. Erythrocytes develop from erythryoblasts, granulocytes from myeloblasts, and lymphocytes from lymphoblasts. These cells, however, all appear identical - they are large with round or ovoid nuclei, a distinct nuclear membrane, visible nucleoli, and an abundant blue cytoplasm. As the blast cells differentiate, the resultant cells can be assigned to a particular cell line.

Erythropoiesis is the development of red blood cells. There are several recognizable steps in this lineage:

  • The erythroblast develops into a proerythroblast, which is only slightly smaller than the blast, but has a more basophilic cytoplasm.
  • The basophilic erythroblast forms when the proerythroblast loses its nucleolus. These cells are much smaller than the blast cells and have an intensely basophilic cytoplasm that results from the accumulation of ribosomes.
  • The polychromatophilic erythroblast has a darkly staining nucleus and its cytoplasm stains a grayish-green color due to the accumulation of hemoglobin.
  • In the orthochromatic erythroblast, or normoblast, the nucleus becomes smaller and darker and the cytoplasm becomes pinker. Nuclear expulsion occurs at the end of this stage through an asymmetric division of the orthochromatic erythroblast. The portion that contains the cytoplasm and organelles becomes the reticulocyte, while the portion containing the nucleus is destroyed by macrophages.
  • The reticulocyte contains cytoplasm, cytoplasmic organelles, and many ribosomes. It is released from the bone marrow and develops into a mature erythrocyte after spending 1 to 2 days in the peripheral blood.

Granulopoiesis is the process by which white blood cells develop. The myeloid series has the most characteristic cell lineage:

  • The myeloblast differentiates into a promyelocyte that becomes irreversibly committed to the neutrophilic cell line. This cell is large, with a large round nucleus, prominent nucleoli, and purple azurophilic granules. These granules are primary, nonspecific granules. Promyelocytes also give rise to eosinophils and basophils
  • The myelocyte stage is characterized by the production of secondary, specific granules. Myelocytes can vary in cell size and nuclear shape. They contain both the purple staining azurophilic granules and lilac staining specific granules. As they develop, they decrease in size, their nucleus becomes indented, and there is a shift toward more specific granules. There is also a reduction in the number of organelles, which results in decreased basophilia of the cytoplasm.
  • The metamyelocyte has a flattened nucleus with condense chromatin.
  • The band cell has a horseshoe-shaped nucleus that is "immature." As development continues, it will mature into a segmented nucleus with multiple lobes. It will then be a mature neutrophil.

Eosinophils and basophils undergo sequential stages of differentiation in a very similar manner to those of neutrophils. Their specific granules are also produced during the myelocyte stage.

The platelet lineage is similar. Large, multilobed promegakaryocytes develop into megakaryocytes, which are the largest cells of the bone marrow (30 to 40 microns). Platelets form through the segmentation of these cells.

Monocytes develop from promonocytes and lymphocytes develop from prolymphocytes. These elements are difficult to distinguish in normal bone marrow smears.

Pre-Lab Quiz

  1. What is the difference between blood, plasma, and serum?
  2. Answer:
  3. Draw how a red blood cell would appear under the electron microscope from above and in cross section. How would you imagine that a reticulocyte would appear in EM cross section? Indicate the diameter of these cells.
  4. Answer:
  5. In your infectious disease sub-internship, you see a patient who has just returned from a whitewater rafting trip in Uganda on which she spent a significant amount of time walking barefoot along the banks of the Nile. She has been vomiting, but has not had a fever. You suspect that she has picked up a helminthic infection. What do you expect to see in her white blood count?
  6. Answer:
  7. Your next patient has been experiencing fevers and fatigue. His white cell count comes back and you see that his neutrophil levels are elevated. What type of infection do you suspect?
  8. Answer:
  9. Write the name of the mature cell that is associated with the lineage including each of the following cells:
    • megakaryocyte
    • band cell
    • normoblast
    • promyelocyte
    • reticulocyte
    • promonocyte

Slides

Please select whether to view the slides in study mode or quiz mode. In study mode, the images will contain labels and a description. In quiz mode, labels and description will be hidden.

  1. Erythrocytes
  2. Erythrocytes EM
  3. Platelets EM
  4. Neutrophil
  5. Neutrophil EM
  6. Eosinophil
  7. Eosinophil EM
  8. Basophil
  9. Basophil EM
  10. Small Lymphocyte
  11. Large Lymphocyte
  12. Monocyte
  13. Blast Cell
  14. Erythropoiesis
  15. Granulopoiesis
  16. Megakaryocyte

Virtual Microscope Slides

  1. Blood Smear
  2. Identify each of the following cell types in the blood smear:
    • erythrocyte
    • neutrophil
    • platelet
    • eosinophil
    • lymphocyte
  3. Bone Marrow Smear
  4. Identify cells in the different stages of erythropoiesis and granulopoiesis.

Pathology

Please select whether to view the slides in study mode or quiz mode. In study mode, the images will contain labels and a description. In quiz mode, labels and description will be hidden.

  1. Sickle Cell Anemia
  2. Acute Lymphoblastic Leukemia
  3. Chronic Myelogenous Leukemia

Quiz

  1. Name the cell type.
  2. Answer: Monocyte
  3. Name a condition in which these cells will be elevated.
  4. Answer: Allergy or Parasitic infection (Eosinophil).
  5. Name a condition in which these cells will be elevated.
  6. Answer: Bacterial Infection (Neutrophil)
  7. What is contained in the granules?
  8. Answer: Histamine and Heparin (Basophil)
  9. What structure causes the blue staining pattern in the cytoplasm?
  10. Answer: Ribosomal RNA (Small lymphocyte)
  11. Name the cell type and its primary function.
  12. Answer: Platelet - Blood Clotting
  13. What changes will take place in the nucleus of this cell?
  14. Answer: Band Cell. Become segmented
  15. Why is the cytoplasm becoming pink?
  16. Answer: Reduction in ribosomes (blue) and increase in hemoglobin (orange).
  17. Identify the large cell.
  18. Answer: Large cell is a promyeloblast.
  19. Identify theses cells.
  20. Answer: Metamyelocytes
  21. Identify A, B, C, D, E, F, G in this image.
  22. Answer: A = band cell, B = monocyte, C = lymphocyte, D = eosinophil, E = neutrophil, F = basophil, G = erythrocyte
  23. What is abnormal about this blood smear? Which type of disease does this blood smear suggest?
  24. Answer: The number of small lymphocytes is greatly increased, suggesting a B-cell or T-cell leukemia.
  25. This x-ray is from a 9-month old who was diagnosed with beta-thalassemia major. A mutation in which gene causes beta-thalassemia major? Explain why a mutation in that gene would lead to the change in the structure of the skull seen in the x-ray.
  26. Answer: Beta-thalassemia major is caused by mutations in both beta-globin genes that results in reduced or no synthesis of beta-globin. The lack of beta-globin leads to erythrocytes with reduced oxygen-carrying capacity and much shorter life span. One consequence is a greatly expanded bone marrow trying to compensate for fewer erythrocytes. The expanding bone marrow erodes the cortical bone and induces new bone formation leading to the appearance in the x-ray.
  27. Frustrated phagocytosis is a process that occurs when neutrophils are unable to phagocytose a pathogen and instead release their granular contents into the extracellular space rather than into phagosomes. What effect might you expect this process to have on the pathogen and on the host?
  28. Answer: In frustrated phagocytosis, the azurophilic and specific granules release their contents into the extracellular matrix. These granules contain lysosomal enzymes and microbicidal agents that will kill the pathogen but damage the extracellular space in the process. This will cause tissue damage to the host.