Biology 212: Anatomy and Physiology II
Lab #2: BLOOD/HEMATOLOGY
Blood is one of the most important components of the human body. It transports materials (i.e., oxygen,
carbon dioxide, hormones, waste products, nutrients, etc.) from one area of the body to another and allows
tissues in distant areas to communicate with one another. In addition, blood protects us from harmful things
like bacteria and viruses via the coordinated actions of a variety of different types of leukocytes (a.k.a. white
blood cells). The average human body contains roughly 5 liters of blood that is constantly being forced
through our arteries and veins by the heart.
Many pathologies of the human body involve blood.. For example, anemia can result from excess blood loss
or the inability to form red blood cells and deliver oxygen to the body or parts of the body. Heart disease risk
is associated with the large amounts of LDL (bad) cholesterol, low HDL (good) cholesterol, and high levels of
triglycerides in the blood. Blood clotting (i.e., hemostasis) of blood are associated with inappropriate blood
clot formation (i.e. stroke) and an inability to form blood clots (hemophilia).
Blood is technically a type of connective tissue that essentially consists of two fractions:
1. Formed elements – the cells and cell fragments found in blood
a. Erythrocytes (red blood cells)
b. Leukocytes (white blood cells)
c. Platelets
2. Plasma – the “liquid/matrix” portion of blood. Blood plasma contains a great deal of water,
ions, proteins, antibodies, and clotting factors.
The analysis of blood and its components are very important clinically since the body often changes the
composition of blood in response to various conditions (changes in physiology or disease). The clinical study
of blood and its components is known as HEMATOLOGY and CLINICAL CHEMISTRY. A Clinical Laboratory
Scientist completes these types of blood analyses. Part of the job of an RN is to explain what the clinical
values mean to their patients and what it means in terms of their disease diagnosis
In this lab exercise, we will draw a small blood sample from a
finger (not the arm). We will examine the different components of
blood as well as look at some of the basic diagnostic tests
commonly used in hematology.
Working safely with blood
In this lab exercise, we will be working with human blood (your
own). If you are not comfortable with blood donation, if you
know you are sick, if you have history of fainting, if you consider
yourself at risk for transmitting a blood born disease, etc., then
please do not draw your own blood. Work to safely support your
peers in your laboratory group. While it is somewhat unlikely that
someone in this class carries an infectious agent in their blood
(bacteria, viruses), it is common practice in the lab to simply
assume that every blood, tissue, or fluid sample you deal with is
tainted with something harmful. As a result, it is important to
1
remember to wear disposable gloves whenever you are handling blood or any materials that have come into
contact with blood (even if it is your own). Disinfecting solutions will be supplied by the department and will
be in the lab for your use. Anything that comes in contact with blood must be disinfected and materials that
will be disposed of (microscope slides, gloves, lancets) must be placed in designated “biohazard” receptacles
and NOT the regular trash! Wash your hands before leaving the lab.
Blood Plasma:
It is important to understand some of plasma’s properties, even though it is not the major focus of this lab.
Plasma makes up approximately 55-60% of our blood volume and is nearly 90% water. The remaining 10% of
plasma consists of ions (i.e., electrolytes), many different proteins, antibodies, and hormones. When fresh
blood is allowed to settle, the formed elements sink to the bottom and a yellow-tan (straw colored) fluid
rises to the top. This yellow fluid is the blood plasma. Blood plasma contains many important molecules such
as antibodies needed to fight infections as well as critical clotting factors. These molecules are very much
sought after for therapeutic/clinical applications, which explains why plasma centers often pay cash for poor
college student’s blood plasma!
Since plasma is 90% water, it is a major reservoir for water in our bodies. When we become dehydrated our
tissues pull water from our plasma and our overall blood volume drops. If we are over-hydrated, the extra
fluid volume is added to the blood stream (plasma) where it can eventually be removed by the kidneys and
excreted as urine. In addition to acting as a reservoir for water, plasma also plays an important role in
maintaining the pH, or acid/base, balance of the body. Plasma contains buffer molecules which allow the
blood and most other tissues of the body to maintain a relatively constant pH of roughly 7.4. Finally, plasma
plays an important role in maintaining body temperature near 98.6oF. Water has a great capacity to absorb
and release heat. As a result on a hot day, plasma supplies the water in sweat that helps cool our bodies. On
a cold day, delivery of warm plasma from our body’s core to our extremities (i.e. fingers) hopefully keeps
them from freezing when we are outside enjoying the Minnesota winter! It is important to be aware of the
composition of plasma as we study our hematocrits later on in lab . . .
Exercise #1: Formed elements of blood:
Keep in mind that the formed elements of blood consist of erythrocytes, leukocytes, and platelets.
In this exercise, we will be studying the formed elements of blood using the blood smear slides found in your
slide boxes (Slide #6). Since these are prepared slides and have been sterilized, you won’t be needing gloves
for this exercise. Our focus will be on the morphology of erythrocytes as well as the various different types of
leukocytes found in human blood. NOTE: the identification of most of the different types of leukocytes is
most easily accomplished by looking at the relative size of the leukocyte and also at the structure of the
nucleus (remember, erythrocytes don’t have a nucleus). The vast majority of the cells that you see on a
blood smear will be erythrocytes (no nucleus), so when identifying the different leukocytes, just look for cells
that have a dark-stained nucleus.
That being said, you won’t be able to identify EVERY cell that has a nucleus since some cells will have been
damaged during the processing of the slide. As you proceed, make sure you look at the images in your text
book to familiarize yourself with what a “typical” WBC looks like and search your slide for that rather than
simply picking a random cell and trying to determine its identity. Make sure everyone in your group can
2
identify the various white cells. Check out Chapter 18 in Saladin for images and descriptions of the
different leukocytes (Figure 18.1) or use one of the Histology textbooks in the lab.
The appearance of the slide and the cells are shown above. The clinician has to determine the percentage
of each cell type and determine if this is a normal or abnormal relative to what was expected. They use
cell size determinations and also the shape of the nucleus of individual cells to discern cell type.
Erythrocytes: By far, erythrocytes are the most numerous of the cells you will see on a blood smear. From
your Bio 211 experience, you will remember that erythrocytes are small (7.5 microns in diameter), round,
and have an indented center. Basically, they look like little doughnuts that lack a nucleus.
Platelets: Platelets will often have an irregular shape and be much SMALLER (1-2 microns in diameter) than
an erythrocyte. Platelets also lack a nucleus since they are really only fragments of a cell. They tend to stain
dark in color.
Leukocytes: There are two main categories of leukocytes in human blood, GRANULOCYTES and
AGRANULOCYTES. Granulocytes will possess visible stained granules (look like speckles within the cell), while
agranulocytes will not. Additionally, granulocytes tend to possess a nucleus which is more lobed in
appearance (often makes them look like they are multinucleated), while agranulocytes tend to possess a
more rounded nuclei that we commonly expect to see in most cells.
Granulocytes:
1. Neutrophils – the most common granulocyte. These cells should be pretty easy to identify based
on their sheer numbers. Neutrophils are phagocytic cells which play an important role both in the
bloodstream as well as in tissues where infections may occur. Their numbers will increase
dramatically when an individual is sick with a viral or bacterial infection. This phenomenon is
commonly referred to as an “elevated white cell count” by clinicians and is confirmed through a
blood test. However, neutrophils are not the only cells which increase in number in response to
an infection. Neutrophils typically possess a nucleus consisting of three or more “lobes” that often
can be seen connected by thinner strands. However, some cells may simply appear to have more
than one nucleus, even though in reality there is only one.
2. Eosinophils – much less common than neutrophils, and you may need to spend a few minutes to
identify one. Eosinophils are phagocytic cells like the neutrophils but contain large granules that
tend to stain a distinctive orange or red due to the fact that the granules absorb and bind a dye
known as EOSIN. Eosinophils possess a nucleus that consists of two large lobes.
3. Basophils – MUCH LESS common than neutrophils and eosinophils. The granules of these cells
possess histamine and heparin which is released in response to inflammation and tissue injury. As
3
a result, these cells spend more time within a tissue than in the bloodstream and may be difficult
to find. Eosinophils are not phagocytic and contain many dark-staining granules that may hide the
nucleus. When you can see one, the nucleus of a basophil often appears U- or S-shaped. You may
have to look at a couple of slides before you see one, but at least be sure you are familiar with
their appearance by looking at a photo in your textbook!!
Agranulocytes:
1. Lymphocytes – fairly common in the bloodstream. Since lymphocytes are agranulocytes, you
won’t be able to see any granules in the cytoplasm of the cell. Lymphocytes are a very diverse
group but tend to be categorized as small and large lymphocytes. Lymphocytes can be identified
by their very large rounded nucleus that sometimes may have a slight indentation. The nucleus
actually is so large that you often will only be able to see a very small, thin ring of cytoplasm
surrounding the nucleus. This is in contrast to most other human cells we’ve seen that have a
smaller nucleus and a more expansive cytoplasm.
Aside from the size variation, there are at least two different types of lymphocytes known as “B”lymphocytes (or “B” cells) and “T”-lymphocytes (“T” cells). A major function of “B” cells is to
produce and secrete antibodies needed by the immune system to fight infections. “T” cells play a
slightly different role by secreting factors which recruit phagocytes and macrophages to an
infection site, while some specialized “T” cells are also able to directly attack damaged or infected
cells. “B” cells and “T” cells are pretty much indistinguishable from one another on a microscope
slide so don’t try to tell them apart. You may learn more about these cell types when the immune
system is discussed.
2. Monocytes – relatively large agranulocytes, much less abundant than lymphocytes, but you
should still be able to identify them on your slides. Monocytes have a smaller nucleus than
lymphocytes which often has a large indentation and has a kidney bean appearance. Monocytes
eventually will leave the bloodstream and enter tissues where they will mature into macrophages
that phagocytose bacteria, viruses, and damaged cell debris.
Exercise #2: Making a Differential White Blood Cell Count
In this exercise, we will be performing what is known as a differential white blood cell count. The goal of this
clinical test is to assess the relative numbers of different leukocytes in a given blood sample. This is an
important diagnostic test since the numbers of certain leukocytes will rapidly change in response to an
infection or inflammation of a tissue.
Differential counts are important for the diagnosis of many diseases. A
high differential count for lymphocytes may be suggestive of
lymphocytic leukemia. A low differential count for neutrophils is often
suggestive of radiation exposure or chemotherapy. A high differential
count for eosinophils often results from intestinal parasitic infection.
Allergic reactions often are associated with high differential count for
basophils. A chronic infection such as tuberculosis often results in a
high differential count for monocytes.
Again using your blood smear slide (slide #6), you will start at one edge
of the slide and systematically keep track of the numbers of different leukocytes you see. A good way to do
4
this is to count all the different cells in one field of view and then move the slide either directly across or
directly down and count the cells in the adjacent field of view, making sure you are not re-counting the same
cells. You can ignore all of the erythrocytes and platelets you see since we are only interested in the
leukocytes. Keep moving to new adjacent regions of the slide until you have counted and identified 100
leukocytes. Keep track of the numbers in the table below. (You want a total of 100 when you are done.)
The figure on page 4 illustrates a pattern for how you should scan up and down over a blood smear to
identify white blood cells for this differential count.
Total Number of Cells Counted:_____________
Exercise #3: Characterizing Erythrocytes and Oxygen Carrying Capacity
Erythrocytes (red blood cells) contain the protein hemoglobin. There is an iron ion (Fe2+) as a co-factor at the
center of the porphyrin ring for each heme. Oxygen is carried from the lungs to the body bound to the iron
of the heme group. Persons who do not have very many erythrocytes or whose erythrocytes do not have
enough hemoglobin cannot deliver enough oxygen to meet the demands of the body. These persons have a
poor ability to deliver oxygen, grow tired quickly, and are typically diagnosed as anemic. Persons who have
too many erythrocytes (polycythemia) can have so many cells that the blood becomes too viscous and forms
clots that can lead to heart attack or stroke.
In this exercise, we will be performing some of the very common clinical tests including determination of
hematocrit, average red cell size (MCV=Mean Corpuscular Volume), hemoglobin concentration per red cell
(MCH=Mean Corpuscular Hemoglobin), and the amount of hemoglobin relative to the size of the red cell
(MCHC=Mean Corpuscular Hemoglobin Concentration) using your own blood. Please work in groups of 2-4
for this part of the lab. Again -- If you are not comfortable with blood donation, if you know you are sick, if
you have history of fainting, if you consider yourself at risk for transmitting a blood-borne disease, etc.,
then please do not draw your own blood. Work to safely support your peers in the laboratory group to
examine their blood.
A hematocrit measures the volume of cells (formed elements) in a given sample of blood. The other
measures (MCV, MCH and MCHC) are collectively called RBC indices and are values that describe the size and
hemoglobin content in red cells. These measures are used in a variety of clinical diagnostics, many specific to
anemia. The MCV is the average volume of a red blood cell and is a measure of red cell size. Small red blood
cells have low MCV values while large red cells have high MCV values. The way this value is determined is to
divide the hematocrit by the RBC count and then multiply by 10 (with units of fL). MCHC is the mean
5
corpuscular hemoglobin concentration in the red cells. This measure categorizes red blood cells based on
their hemoglobin content and typically indicates if the cells might be swollen or shrunk (i.e., crenated). This
value is determined by dividing the sample’s hemoglobin concentration by the hematocrit and then
multiplying by 100 (with units in the g/dL). The MCH is the hemoglobin concentration of the red cells and
correlates with the MCV. This value is determined by dividing the hemoglobin concentration by the red
blood cell count and then multiplying by 10 (units are pg/cell).
PATIENT SAMPLE ID:
Measure
Normal Values
Hematocrit (Hct)
37 – 42 women
%
42—48 men
Red Cell Count from Sheet
5 x106 cells/liter
HGB (hemoglobin conc.)
11.5 – 16.5 g/dL
Calculations for Patient
Blood
(Hct/RBC)x10 = MCV
MCV
80--100 fL
([Hb]/Hct)x100 = MCHC
MCHC
32 – 36 g/dL
([Hb]/RBC)x10 = MCH
MCH
27 – 31 pg/cell
(1 liter is
1x10-6
liters)
PART 1: Hematocrit
In order to obtain your own blood, you will be performing a “finger stick” using a lancet with a VERY small
needle. If you happen to be diabetic, you are probably a pro at this since diabetics need to test their blood
sugar levels several times a day. For this exercise, you will need an alcohol swab, capillary tube with a
colored band on ONE end, a lancet, and a paper towel.
1. Wash your hands in warm water and dry vigorously with a paper towel. Hold the hand that you plan
to obtain blood from below your waist for a minute or so to allow more blood to flow to the tips of
your fingers.
2. Using an alcohol swab, clean the side of your ring or middle finger.
3. With your hand still below your waist, prepare the lancet (instructor will demonstrate) and press it
firmly against the side of your finger about halfway down your distal phalange (basically off to the
side of your fingernail).
4. Lance your finger and wipe away the first small drop of blood that appears with a paper towel.
5. Have one of your team members place the capillary tubes on the edge of the table and have that
person hold the tubes in place with their finger. As the second drop appears, move your finger with
the drop of blood on it up to the capillary tube – it will automatically flow into the tube by capillary
action. You want the tube to be about 1/2 to 2/3rds of the way full. [You may have to squeeze your
6
finger a bit to keep the blood flowing (not too hard though). NOTE: If you are planning on doing the
blood typing experiment, you may want to harvest some blood at this time in order to avoid having to
stick yourself a second time. For the blood typing exercise, you will want to place two small drops of
blood on a clean microscope slide. It is important to space these two drops apart on the slide (one
drop on each half of the slide).
6. Place your finger over the end of the capillary tube to create a seal. Now using the tray of clay, push
the capillary tube into the clay to seal off the open, non-colored end of the capillary tube and place it
flat on the paper towel.
7. Clean up any waste materials and disinfect contaminated objects. Do not forget to spray disinfectant
on the area where you were working! Be Smart - Be Safe.
In order to determine the proportion of formed elements versus plasma in your blood, we will spin the
capillary tubes in a special centrifuge (instructor will do this). After 3-5 minutes of centrifugation the heavier
formed elements will accumulate at the bottom of the capillary tube and a tan/yellow fluid will be seen at
the top (this is the plasma). The vast majority of the formed elements seen at the bottom of the tube will be
erythrocytes, with a smaller thin layer of leukocytes and platelets in between the erythrocytes and plasma.
You are now ready to determine the percentage of formed elements versus plasma in your blood. This can
be accomplished simply by using a millimeter ruler or a fancier hematocrit reader (instructor will
demonstrate).
Using a millimeter ruler:
Percentage of elements in blood = Height of the layer being measured (mm formed elements or plasma) X
100
Overall height (mm) of the total sample
% of formed elements =
% of plasma =
_______________ = hematocrit
_______________
Is your hematocrit within the normal range?
You may also want to try to be a little more specific and identify the proportions of individual formed
elements as well. To do this, you will need to be able to identify the thinner middle layer that contains the
leukocytes and platelets (often appears as a distinct layer that is not as red as the lower erythrocytes, and
more reddish than the above plasma).
% of erythrocytes =
% of platelets and leukocytes =
% of plasma =
_____________
_____________
_____________
“Normal” ranges of a hematocrit are given in the reference table. Small deviations are common especially in
young women. But all values may be a little low if you had to squeeze your finger to obtain a blood sample.
An abnormally high hematocrit reading indicates an excessive number of erythrocytes and is known as
POLYCYTHEMIA. A low hematocrit is indicative of ANEMIA.
7
Part 2: Red Cell Indices:
Go up to the front desk and obtain a handout of the
differential blood count for a patient. Take this sheet
back to your desk and review the contents. The page can
be divided into three sections: Patient Information,
Tables of Results, Graphs used by machine to determine
differential counts. You will want to focus your attention
on the first two sections: Patient Information and
Results.
1. Record your patient sample number into the
blank above the table.
2. Transfer the RBC count, HGB concentration
and HCT from your patient sheet into the
table. As you do so note if the values are
within the normal range.
3. Now use these values to calculate MCV, MCH
and MCHC. Use the formulas given in the
table.
4. Check with another group for their patient
information and run through the calculations a
second time.
Microcytic anemia (low MCV) is common with chronic illness, low iron in diet or inherited anemia (e.g.
thalassemia). Macrocytic anemia is present in B12 and folic acid deficiencies. Low MCHC will be common in
thalassemia (a hemoglobinopathy) and/or in low iron diet conditions. MCH should correlate with MCV
values for the various pathologies.
Exercise #4: Determination of Blood Type (ABO and Rh-factor)
In this exercise, we will be testing your blood type. Most cells in our bodies possess a characteristic group of
glycoproteins on the cell membrane that specify “self” versus “non-self”. As a result our immune systems will
react and attempt to destroy cells which it views as being foreign. This is a common problem when an organ
from one person is transplanted into another person. These
glycoproteins on the cell surface are known as ANTIGENS. Every person
in this room has a slightly different set of antigens on their cells, making
each of us unique. Erythrocytes also contain many antigens on their
surfaces, but luckily for us there are only a couple of different antigens
that are clinically most important. There are many classes of
erythrocyte surface antigens, but the two most important are the ABO
and Rh blood groups.
The ABO blood group describes a type on antigen found on
erythrocytes in human blood. A person with Type A blood will have
erythrocytes that only possess the “A” antigen, Type B blood only
possesses the “B” antigen, Type AB blood contains both types of antigen, and Type O blood does not possess
either “A” or “B” antigens.
8
In order for our bodies to keep track of “self” versus “non-self” cells, plasma from a person with Type A
blood will contain antibodies that will detect the presence of cells possessing the “B” antigen. Plasma from a
person with type B blood will contain antibodies against the “A” antigen, a person with type O blood will
have antibodies against both “A” and “B” antigens, and a person with type AB blood will not contain any
antibodies against either antigen. This is summarized in the table to the left.
Administration of the wrong blood type to a person often has devastating consequences. For example if a
person with Type A blood receives Type B blood their anti-B plasma antigens will stick to the transfused Type
B blood cells and cause them to clump together. This is called AGGLUTINATION. Large clumps of the
transfused cells will create blood clots that quickly occlude blood vessels, often leading to death.
In this exercise we will be using commercial antibodies against either the “A” antigen or “B” antigen to
determine your blood type. Type A blood will form clumps in the presence of antibodies against the “A”
antigen (these antibodies come from plasma of a person with Type B blood), Type B blood will clump in the
presence of “B” antibodies, Type AB blood will clump in the presence of either antibody, and finally Type O
blood will not clump at all in the presence of either antibody.
1. Remember to wear your gloves when dealing with someone else’s blood.
2. Using a wax pencil label one side of a clean microscope slide “A” and the other half “B”
3. Place one drop of blood on each half of the microscope slide.
4. On the “A” side, place one drop of the Anti-A antibody NEXT to the blood drop, not on top of it. Do
the same on the other side with the Anti-B antibody
5. Using a clean toothpick carefully mix the drop of blood with the antibody
6. After several minutes, examine the slide to see if the blood exposed to either antibody has clumped.
If after 5 minutes you cannot detect any clumping in the presence of either antibody, it can be safe to
assume that you have type O blood. (The blood should not be dried on the slide.)
7. Remember to clean up your work area and place your used microscope slides in the appropriate
biohazard container OR in a container of bleach/water.
Since Type O blood cells do not contain either the “A” or “B” antigen, they can be transfused into any person.
So, a person with Type O blood is considered a “universal donor”. Blood cells from a person with Type AB
blood will have both “A” and “B” antigens on their surface. Consequently, people with Type AB blood do not
have antibodies against either type of antigen. So, people with Type AB blood are considered “universal
recipients” since they can safely receive type A, B, AB, or O blood! Draw your results in the circles below.
Anti-A
Anti-B
Anti-D
Control
The Rh-factor is the second type of blood group antigens that you may be familiar with. This blood group is
genetically determined and is distinct from the antigens of the ABO group. Like the ABO group, the
frequency of occurrence of this group varies with ethnicity. The difference between the groups is that antiRh agglutinins (Anti-D) are not normally present in the blood. They are formed only in Rh(-) individuals (one
9
that would never express the antigen on the surface of their red cell) only after exposure to Rh(+) blood (red
cells covered with these antigens). This means the first exposure of an Rh(-) individual to Rh(+) blood will
initiate an immune response that generates antibodies. But it takes time for the body to build these new
plasma proteins, these agglutinins (anti-Rh antibodies). So the person is in little danger with the first time of
exposure. It is the second exposure to this different blood type that a life-threatening situation can
occur. This is extremely important during pregnancy when an Rh(-) mother is carrying an Rh(+) child. The
plasma proteins from the mother cross the placenta and create a condition called hemolytic disease of
newborn (or erythroblastosis fetalis). In a clinical setting, this situation is prevented by the administration of
Rhogam to an Rh(-) woman to suppresses Rh antibody production.
In this lab, put a drop of your blood on the slide and a drop of the anti-Rh solution. You may want to hold the
slide in the palm of your hand to warm the slide slightly. This enhances the pace of the reaction. If the
mixture takes on a grainy appearance, the person has Rh and would be AB+, A+, B+ or type O+. If no
clumping is observed, the person is Rh-negative (they do not have any Rh-factor antigens on the cell surface).
10