A&P II Lab #2 Name: _______________________

BLOOD HISTOLOGY

INTRODUCTION

Blood is a type of connective tissue that exists as a complex suspension of formed elements in
a liquid medium called blood plasma. Blood plasma is a solution containing many particles,
including proteins, minerals, and atmospheric gasses. These tiny solutes are dissolved in the
aqueous medium of plasma and cannot be observed through a microscope, although they can
be detected and quantified using a variety of routine chemical analytical methods.

The formed elements of blood are cellular in nature and are many times larger than the tiny
particles dissolved in the plasma. Hence, formed elements can be isolated and visualized by
allowing blood to sediment under gravity or centrifugation. Following such treatment,
sedimentation of whole blood results in the separation of three distinct layers or phases, with
clear-yellow plasma on top and two layers of cellular material underneath. The plasma phase
consists of ~55% of the volume of whole blood, with formed elements constituting the
remainder. The majority of formed elements are erythrocytes, or red blood cells (RBC’s), so
called because of their bright red coloration resulting from their hemoglobin content. This
fraction sediments at the bottom of the tube because these cells have the highest density. This
is due to the heavy mass of iron found in hemoglobin protein. The % volume of blood
composed of erythrocytes (typically ~45%) is reported as “hematocrit”, a clinically important
value that is routinely measured in diagnostic blood analysis.

The top phase of formed elements, which is known as the “buffy coat” is small (only ~1% total
volume of whole blood) and is composed of leukocytes and thrombocytes (or platelets).
Leukocytes, or white blood cells (WBC’s), are named because they have a whitish appearance
when viewed without staining. This can be seen in the white appearance of the buffy coat
sediment of blood or in lymphatic tissue with large aggregates of leukocytes, such as the “white
pulp” of spleen. When viewed under the microscope, leukocytes are typically stained and
therefore have a variable appearance depending on the type of cell, as there are five major
types of leukocyte.

Modern differential WBC counts rely on automated methods of estimating the relative
numbers of each type of leukocyte found in a blood sample. This can provide clinically useful
information about a patient, such as indicating whether a bacterial infection is likely or not.
Clinically normal ranges of WBC counts varies depending on a number of patient demographic
factors, including age, gender and race. However, the average overall distribution of WBC
counts for humans >1 year of age based on previous studies is reported by the CDC1 as follows:

Type of leukocyte Mean % of total leukocytes (approx.)

Neutrophils 56%
Lymphocytes 35%
Monocytes 6%
Eosinophils 3%
Basophils 0.4%

Despite the implementation of automated cell counting methods in medicine, performing cell
counts manually on a prepared slide is a great way to study the composition of blood, and is the
basis for this lab exercise. In this lab, you will visually examine commercially prepared
specimens in which human blood has been smeared across a glass slide to form a monolayer of
blood cells. This type of preparation is known as a blood smear, and is routinely performed as a
diagnostic blood test in clinical laboratories. Blood smears are typically stained with some
variant of Wright’s stain, a modification of the Romanowsky stain. Background information
about the history and development of these stains is widely available on the internet or can be
found in histology textbooks.

The advantage of Wright’s stain is that it allows for relatively easy differentiation of the five
major types of white blood cells. Three of the leukocytes are called granulocytes because the
cytoplasm of these cells are filled with granules containing a variety of different chemicals. The
color of these cytoplasmic granules following Wright’s staining is different for the three types
of granulocytes. The cytoplasmic granules of eosinophils take up the acidic portion of the stain
(eosin) and stain red. In basophils, the granules take up the basic portion of the stain
(methylene blue) and stain blue. In neutrophils, the granules are largely colorless because they
are chemically neutral and stain poorly since they do not have a high affinity for either stain.
The other two types of leukocytes lack cytoplasmic granules and are thus called agranulocytes.
The two types of agranulocytes are lymphocytes and monocytes. Since these cells don’t have
cytoplasmic granules, other characteristics, such as the size of the size and shape of the nuclei
are used to identify them.

It should be noted that nuclei of all cells are acidic and therefore basophilic. This makes the
nuclei in basophils somewhat difficult to discern from the cytoplasmic granules in these cells,
although the nuclei and granules usually stain a different shade of blue and can be discerned
with careful observation. In eosinophils the nuclei are much easier to discern since their blue
coloration contrasts sharply with the red staining (eosinophilic) granules. Neutrophilic nuclei
are also very easy to discern since their cytoplasm stains very poorly. In addition to using the
color of stained cells, the morphology, or shape of the nuclei is an important visual cue to
distinguish between different types of leukocytes. Lymphocyte and monocyte nuclei have
distinct appearances, which you will describe in the lab exercise below.
LAB PROCEDURE

In this lab you will use the microscopes to examine two different pre-prepared blood smear
slides. It is a good idea to look at more than one slide since some cells (particularly basophils)
are rare and may not be found on all slides. Additionally, the quality of slides sometimes varies
and cells may appear somewhat different on separate slides.

In the following exercises you will answer questions, make illustrations, and learn to identify all
the different types of cells that make up the formed elements of blood. After you learn to
identify the different cell types you will then perform a differential WBC’s count and assess the
result.

Students are encouraged to use their textbooks, lab guides, their instructors, the internet, or
any other available resource to identify blood cells and answer questions as they work their
way through this lab exercise.

Exercise 1 – Visualizing the cell monolayer

Observe the slide with the 4x objective in place and adjust the focus until you have a clear view
of the cell monolayer. Then switch to the 10x objective and make minor adjustments. Don’t
forget to use the ocular focus adjustment!

Pan around the slide at 10x magnification and describe what you see:

What are most of the cells you see? What else did you observe?

Exercise 2 – Erythrocytes and platelets

Examine the blood smear at 400x total magnification and illustrate at least one example
of an erythrocyte and one platelet here:
 Describe the shape of an erythrocyte. Do erythrocytes have nuclei?

Are erythrocytes eosiniphilc or basophilc?

What is the function of erythrocytes?

Describe the shape of a platelet compared to an erythrocyte.

What are platelets? Are they cells? What is their function?

Erythrocytes are also called: _____________________.

Another name for platelets is: _____________________.

Exercise 2 – Identifying Leukocytes

Continue using the 40x objective to observe the blood smear. This time you will be trying to
identify the different leukocytes. Correct identification usually requires some practice, so take
your time, make careful illustrations and be sure to ask your lab instructor if you need
assistance.

You are welcome to use the 100x objective if you would like to view the cells at higher
magnification, although this is not necessary. Using the 100x objective requires oil immersion--
If you want to do this you will need to ask your lab instructor to provide oil and directions for
viewing and cleanup.
 What are the three types of granulocytes? Draw and label one example of each type of
granulocyte you observe in the space below.

What are the two types of agranulocytes? Draw and label one example of each type of
agranulocyte your observe in the space below.

Use the space below to list the different types of leukocytes in order of most to least
common.

Once you are confident that you can identify the different types of leukocytes, you can proceed
to next exercise.
Exercise 3 – Differential cell count

Use the microscope to count and identify 100 different leukocytes on a single blood smear
slide. Try to do this as randomly as possible and be careful not to ID the same cell twice. Then
repeat this procedure using a second different slide. Report your results in the data table
below:

SLIDE #1 SLIDE #2
Cell # % total Cell # % total

Neutrophils ________ / ________ Neutrophils ________ / ________

Eosinophils ________ / ________ Eosinophils ________ / ________

Basophils ________ / ________ Basophils ________ / ________

Monocytes ________ / ________ Monocytes ________ / ________

Lymphocytes ________ / ________ Lymphocytes ________ / ________

Did you get similar results on the two slides? Explain why you think that is.

Are your results consistent with clinically normal cell counts? If not, explain why you
think that could be.

.
**When you have finished answering all the questions and completed all the drawings for this
lab exercise, make sure your name is written on the top page of this lab handout and turn your
work into your lab instructor before you leave lab. If you do not complete this lab exercise you
will not receive credit for this lab assessment.

REFERENCES
1
Hollowell JG, van Assendelft OW, Gunter EW, Lewis BG, Najjar M, Pfeiffer C; Centers for
Disease Control and Prevention, National Center for Health Statistics. Hematological and iron-
related analytes--reference data for persons aged 1 year and over: United States, 1988-94. Vital
Health Stat 11. 2005 Mar;(247):1-156.