BIO211 Weekly Guide #4
BLOOD;
BLOOD CHEMISTRY
After completing this laboratory you should be able to:
1) Identify and distinguish between the major blood cell types in blood smears
2) Identify the hematopoeitic tissues where new blood cells are produced
3) Describe the processes and organs involved in breakdown of blood cells
4) Describe the relative cellular concentrations of each blood cell type and provide the major known functions for each
5) Describe the mechanisms involved in blood clotting processes
6) Conduct simple blood measures such as cell counts, hematocrit, hemoglobin, blood typing, clotting times, and blood glucose level
Guide to Gross Anatomy Guide to Histology Guide to Physiology
Outline
I. Blood Constituents {FAP Spotlight, Fig 19-1, Table 19-3}
A. Blood functions {FAP 19-1}
transport and exchange
ECF homeostasis
defense
B. Plasma {FAP 19-2}
fluid, salts, proteins
serum = plasma-fibrinogen
C. Cells
origin - precursors in marrow
Erythrocytes - 5x106/mm3 {FAP 19-3}
structure - 7΅, biconcave, hemoglobin, carbonic anhydrase,
no organelles
function - transport of O2, CO2
Leukocytes - 3x103/mm3 {FAP 19-5}
Granulocytes
Neutrophils - 70%
structure - 9-12΅, 3-5 lobed nucleus, neutrophilic staining
function - phagocytic
Eosinophils - 3%
structure - 9-14΅, 2 lobed nucleus, acidophilic staining
function - antiparasitic, anti-inflammatory
Basophils - <1%
structure - 8-10΅, S-shaped nucleus, basophilic staining
function - inflammatory response
Agranulocytes
Lymphocytes - 20-30%
structure - 7-8΅, large central nucleus, pale cytoplasm
function - 2 populations
B-cells - humoral immunity (-->plasma cells)
T-cells - cellular immunity
Monocytes - 3-8%
structure - 12-20΅, C-shaped nucleus, pale cytoplasm
function - macrophagocytic (-->macrophages)
Platelets - 250,000/mm3 {FAP 19-6}
structure - 2-3΅, anucleate, granular
origin - megakaryocytes
function - clotting
II. Blood Chemistry, Blood Physiology, and Assessment
A. Oxygen Transport {FAP 19-3}
hematocrit
hemoglobin
B. Blood Chemistry {FAP 19-2}
osmolarity and electrolytes
glucose
C. Blood Clotting {FAP 19-7}
clotting proteins and cascade mechanisms
extrinsic mechanism
intrinsic mechanism
soft and hard clots
D. Blood Typing {FAP 19-4}
RBC antigen-antibody reactions and agglutination
ABO system
Rh system
Gross Anatomy List
There is no unique gross anatomy associated with this week's topic
Guide to Gross Anatomy
There is no unique gross anatomy associated with this week's topic
Guide to Histology
Blood and Blood Cell Types {FAP Spotlight 19-1, Tabe 19-3; APL Exercise 20.1}
Blood is a rather specialized type of connective tissue. The extracellular material consists of fluid, dissolved ions and gases, and proteins. We will concern ourselves here principally with the mature cellular components, which are:
Erythrocytes (red blood corpuscles - RBCs)
Thrombocytes (platelets)
Leukocytes (white blood cells - WBCs)
Granulocytes
Neutrophils
Eosinophils
Basophils
Agranulocytes
Lymphocytes
Monocytes
For each of these cell types you should be able to identify it, state its relative abundance in the blood, and state its primary function(s). WBC Fig. 3.20 is a partial guide. Identification of blood cell types can be done easily on just two loan collection slides. You may want to use oil immersion for better cellular detail. NOTE: Always clean both lens and slide with lens paper and cleansing solution after using immersion oil!
a) Erythrocytes (RBCs)
These are by far the most numerous cells in the smear. They are small, of uniform diameter (7΅), and readily identifiable by the pale pink color, lack of a nucleus, and "doughnut" appearance. In clinical medicine the combined volume of the red cells relative to that of the blood is quantified by the "hematocrit".
- Identify erythrocytes and compare their numbers with the number of leukocytes in the smears. Note that erythrocytes are technically not cells, in that the nucleus and most of the intracellular machinery has been lost during maturation.
- Where are RBCs formed in the adult? In the fetus? What does the presence of reticulocytes (RBCs retaining some ribosomal RNA) in an adult imply? What is anemia? What can cause it?
- Note that the ubiquity (in every capillary) and uniform size (7΅) of RBCs makes them a handy "anatomical micrometer" for almost any histology slide.
b) Thrombocytes (platelets)
Platelets appear as clumps of small (2-3΅), irregular shaped, flattened, anucleate cells.
- Identify blood platelets among the erythrocytes on the smear. Again, these are not true cells, but rather cellular fragments of a much larger precursor cell of the bone marrow, the megakaryocyte (see below).
- What substances do these fragments contain that contribute to blood clotting? What difficulties are associated with thrombocytopenia?
c) Granulocytes (polymorphonuclear leukocytes)
Granulocytes are named for the granular cytoplasm, containing both "primary" granules (lysosomes, common to all granulocytes) and "specific" granules (secretory vesicles unique to each of the three types of granulocytes). The nuclei of these cells have multiple lobes, connected by very thin nucleoplasmic bridges. Granulocytes are 8-14΅in diameter, somewhat larger than RBCs. The three types, named for their respective cytoplasmic staining characteristics, are neutrophils, eosinophils, and basophils.
- What is the site of production of granulocytes in the adult? What is leukemia? Why is it harmful?
1) Neutrophils
Neutrophils are the most common white blood cell found in blood, about 65% of the total number of white blood cells. The nucleus has 3-5 distinct lobes and only the primary cytoplasmic granules stain in standard preparations, producing a lightly stained pinkish cytoplasm. The specific granules, which contain phagocytins and alkaline phosphatase, require special stains to differentiate.
- Identify several neutrophils. What is their primary function?
2) Eosinophils Eosinophils are considerably rarer than neutrophils, about 2% of the total WBCs. They are increased in pathological conditions of allergy or parasitic infestation. Nuclei are typically bilobed, and the cytoplasm is marked by dense red (eosinophilic) granules that contain histaminase. Eosinophils may be found in tissue, especially in the gut.
- Locate an eosinophil. What is the primary function of this cell type?
3) Basophils
The rarest WBCs of all, basophils form 1% or less of the total population of WBCs. Bilobed nuclei often form an "S" shape, but are usually obscured by dense, purple staining (basophilic) granules in the cytoplasm. These specific granules contain heparin and histamine.
- Try to locate a basophil. Their scarcity means that you may have to scan 100 to 500
white blood cells before you find one. Scan at low power, then switch to high power to
make a positive identification. What is the function of basophils, i.e. what tissue processes
do heparine and histamine promote?
- What resident cell type of connective tissue has the same basic function (but NOT a
common origin)?
d) Agranulocytes
The remaining two types of WBCs are called agranulocytes because the cytoplasm does not appear granular with conventional stains, but rather stains a uniform pale blue. The nuclei are not lobed.
1) Lymphocytes
Lymphocytes are marked by round nuclei with a pale rim of bluish cytoplasm. They are the smallest WBCs (7-8΅) and are relatively common, about 30% of the total WBCs. Lymphocytes produce antibodies and other substances used in the immune response. They are frequently seen in the tissue, either a transient free wandering lymphocytes or as resident plasma cells.
- Identify several lymphocytes. What are the two types of lymphocytes and what is the
function of each? (Note that these types are indistinguishable in our slides). Where are
lymphocytes produced in the adult?
- What resident cell type of connective tissue develops from lymphocytes?
2) Monocytes
Monocytes are the largest WBC's (12-20΅), and are characterized by the large C-shaped or indented "horseshoe" nucleus and abundant cytoplasm, which may contain phagocytic debris. Monocytes make up about 5% of the total number of WBCs and are precursors to the tissue macrophages (histocytes).
- Identify several monocytes. What is their function in the blood? Where are monocytes
produced in the adult?
- What resident cell type of connective tissue develops from monocytes?
Bone Marrow {FAP Fig 19-5; Table 19-10}
The fetal bone marrow in the developing bones of this slide is a site of hematopoiesis. The precursors of the mature cells which you have just studied in the blood smear may be found here, in red marrow.
a) In the adult, the red marrow (myeloid tissue) is found primarily in the skull, vertebrae, ribs, sternum, clavicle, pelvis, and proximal portions of the humerus and femur. The medullary cavities of most adult long bones are filled with yellow marrow, consisting mainly of fat cells.
- In the fetus what additional organs are capable of red blood cell production?
- In the bone marrow slide focus on one of the giant, pink staining megakaryocytes (visible even on low power). These cells bud off cytoplasmic fragments that become blood platelets.
b) Different blood cell or "formed element" types each develop pass through a complex set of recognizable intermediate stages during differentiation. FAP Fig 19-10 is a good guide to blood cell lineages and precursors.
- Try to identify immature (nucleated) red blood cells and immature granular leukocytes. You are not responsible for the specific sequence and appearances of precursors for each cell type, but you should develop a general appreciation for blood cell origins and the changes that occur during the maturation process of each blood cell type.
- What resident cell type of connective tissue develops from monocytes?
Guide to Physiology
Obtaining Blood Samples and Safe Handling Practices
This part of the laboratory will require you to obtain several small samples of your own blood. Participation in this part of the laboratory is entirely optional. You may choose not to participate and may stop participating at any time.
If you choose to participate, before you can begin you must:
1) Set up a unique blood sampling station which only you will use. Your station will consist
of an absorbent pad, a clean and sterilized lancet device, several sterile lancets, several
alcohol pads, several bandaids.
2) Read entirely through the Guide to Handling and Testing Blood handout.
3) Make sure that you understand the methods and precautions for drawing and handling
blood and have all of your questions answered.
4) Sign and submit the Informed Consent.
The primary rules for handling human blood are:
1) Make sure that any device which breaks the skin is STERILE.
2) Do not come in contact with any blood other than your own own.
3) Do not expose anyone else to your blood.
4) Dispose of all sharp objects in a proper Sharps container.
5) Dispose of all non-sharp blood-contaminated materials in an ORANGE autoclave bag.
Producing a Blood Smear and Performing a Differential WBC Count
1) Obtain 2 clean microscope slides.
2) Follow the handout instructions to prick your finger and deposit a 4mm drop of blood near one end of one slide.
2) Before the blood can coagulate or dry (within 3 minutes) follow the instructions at the slide preparation station to create, dry, dehydrate, and stain a blood smear.
Google Staining a Blood Smear - entry 1 Making and Staining a Blood Smear
3) Under high power (400X) systematically scan the smear and record WBC types for at least 100 WBCs. Calculate percentages for each WBC type by dividing the number of cells of that type by the total number of WBCs, then multiplying the result by 100. You may use the table below.
Cell Type Neutrophils Eosinophils Basophils Monocytes Lymphocytes Total Number
of cells
Percentage
100%
Hematocrit
1) Obtain a clean capillary tube.
2) Follow the handout instructions to prick your finger and express a drop of blood. Transfer enough blood to fill at least half of the tube. Gently tamp the tube until the blood contacts the plug at the bottom.
3) Follow the instructions at the microcentrifuge station to spin down your blood sample and measure the hematocrit. Record you value here:
Hct =
Blood Glucose
1) Obtain a blood glucose meter and at least one test strip. Insert the strip into the meter. The meter should automatically turn on, initialize itself, and display a small droplet symbol, indicating that it is ready to measure a blood sample.
2) Follow the handout instructions to prick your finger and express a drop of blood. Carefully touch the exposed end of the test strip to the drop of blood and wick blood into the test strip.
Record your meter reading here:
Blood Glucose Level = mg/dl
Blood Typing {FAP Fig 19-6; APL Exercise 20-2}
1) Obtain an Eldoncard set.
2) Follow the handout instructions to prick your finger and express several drops of blood. Follow the instructions accompanying the Eldoncard to transfer individual drops of blood to each of the four antibody test circles. Add a drop of water to each and stir each with its own stick. Carefully tilt the card a few times to distribute the blood in each circle. Compare your card to the Endoncard guide to determine your ABO and Rh blood type and record the result here:
Blood Type =
3) If you have time, work through APL Exercises 20-1, 20-3, and 20-4 to explore further the theory and practice applications of blood typing information.
Cleanup
1) Transfer all used or contaminated lancets to a sharps container.
2) Immerse all contaminated microscope slides to a beaker of bleach.
3) Immerse your lancet device in a beaker of alcohol. Make sure the lancet has been removed.
4) Wrap all non-sharp waste and contaminated products in the absorbent pad. Place the pad in an orange autoclave bag.
5) Wipe all table surfaces down with a dilute bleach solution. Carefully wipe any contaminated surfaces on the microscope, centrifuge, and glucose meter with an alcohol pad.