Lab Activity 19.1:  Identifying Blood Cells and Lab Activity 19.2:  Identifying White Blood Cell Types. 
Follow the instructions in your lab manual for completing these two activities.  Answer any questions included in the lab manual.  You will need a microscope and a prepared blood smear slide for this exercise.

Lab Activity 19.3:  Differential WBC Count. 
Follow the instructions in your lab manual for completing this activity.  Answer any questions to consider included in the lab manual.  The class may share their results upon completion.  You will need a microscope and a prepared blood smear slide for this exercise.  If time permits, you may also look at blood smears from individuals diagnosed with infectious mononucleosis or leukemia.

IMPORTANT:  Before completing the next activity, be sure to read the precautions for dealing with human blood in the box below!

Lab Activity 19.4:  Determining Your Blood Type
After your lab instructor provides the background information regarding blood typing, follow the instructions in your lab manual for completing this activity.  Answer any questions to consider included in the lab manual.  The class may share their results upon completion.  Note:  Information on distribution of blood types in the U.S. is FYI (for your information) and will not be included on the lab practical.

Materials needed for this activity:  (1) sterile glass microscope slide, (2) wax pencil, (3) lancet (to be used only once, then discarded properly!), (4) alcohol swab, and (5) anti-A serum, anti-B serum, and anti-D (Rh) serum.

PRECAUTIONS:  Working with blood in the lab poses little risk provided students are   careful, they work only with their own blood, and everyone takes the following precautions seriously: Lay out one or two layers of brown paper toweling at your lab station before starting the tests.  Wash your hands with soap and water before and after doing the tests; If you are having trouble perforating your finger, your laboratory instructor will assist you; Use lancets only one time and dispose of in the red "sharps container," not the wastebasket! (location of this container will be pointed out by your laboratory instructor); Before perforating your finger, disinfect the area with an alcohol wipe; When loading the centrifuge or using other equipment to take blood measurements, be sure to wear rubber gloves in order to prevent contact with blood from other students; At the end of the laboratory period wash all equipment with soap and water and dispose of microscope slides, pipettes and other things that contacted blood (including paper toweling) appropriately; Spray your tabletop with VIREX disinfectant.  Leave for 10 minutes and wipe dry with a paper towel.

Terms/Information to Know:

Antigen (agglutinogen) - glycoprotein on the RBC membrane whose presence (or absence!) determines the blood type

Antibody (agglutinin) - protein secreted by lymphocytes involved in the immune response; can be isolated from plasma

Agglutination -

Antigen-Antibody reaction -

Antigens and Antibodies comprising the following blood types:

BLOOD TYPE	ANTIGEN PRESENT	ANTIBODY PRESENT
A
B
AB
O

Rh Factor (D Antigen) -

Purpose of the antisera in this experiment -

 

Before you begin these tests, you will need to collect the following materials:

Experiment:  Hematocrit Measurement (VPRC)

Note:  Pay attention to the words in bold and italics for the remaining experiments.  They may show up on your lab practical.

Background information: 

The hematocrit (also called volume of packed red cells, VPRC, or packed cell volume, PCV) is a measure of the relative percentage of blood cells (mainly erythrocytes) in a given volume of whole blood.  This test is included in the complete blood count (CBC) ordered by physicians.  Whole blood is centrifuged in a special hematocrit centrifuge.  The cells, which are dense, settle to the bottom of a heparinized capillary tube.  The straw-colored plasma is on top.  What is the function of the heparin lining the capillary tube? ________________________

The hematocrit is measured using an ADAMS Microhematocrit Reader.  Abnormal values may suggest either deficiencies (anemias) or excesses (erythrocytosis or polycythemia).

Normal hematocrit for Adult Females:  37-47% (ave. 42%)
Normal hematocrit for Adult Males:  42-52% (ave. 47%)
 

Hematocrit Procedure:

1)  Using a sterile lancet, lance the tip of your middle finger (on the side) to stimulate bleeding.  Raising the opposite arm will promote increased blood flow to the bleeding finger.  Be sure to have your opposite hand gloved!

2)  Load the red end of a clean heparinized capillary tube until it is approximately 1/2 to 3/4 full of blood.  Note: When loading the capillary tube, hold the opposite end downward to allow gravity to facilitate loading of the tube.  After loading the tube with blood, gently press the red end of the tube straight down into Seal-ease, a clay-like substance that closes off the loaded end of the capillary tube.  Important:  Wear latex gloves when sealing your tube, as many other people will be using the same seal ease and it will be contaminated with blood!

3)  Set aside tube and continue to the next experiment (Test Three) before completing this test.

4)  When instructed to do so, take your loaded heparinized capillary tube to the hematocrit centrifuge.  An instructor will load your tube in a specific numbered section of the centrifuge.  Record your number here _______.

5) After centrifugation, take your capillary tube to the ADAMS Microhematocrit Reader (AMR, see Fig. 2).  Place tube in the groove with the top of the seal-ease (bottom of the packed cells) lined up with the bottom line of the AMR.  Slide the plastic holder to line the top of the plasma with the 100% marker of the AMR.  Move the slider bar (with black knob on right of AMR) to the border between the packed red cells and the plasma.  Read the percentage in the window at the top of the AMR.  Record your hematocrit value here:____

Figure 2. ADAMS Microhematocrit Reader

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Experiment:  Hemoglobin Estimate

Background information:

Hemoglobin (abbreviated Hb) is the oxygen-carrying protein inside red blood cells.  One component of the hemoglobin molecule is iron.  Normal ranges of hemoglobin for males and females are good indicators of normal oxygen-carrying capacity.  Deficiencies of hemoglobin are indicative of iron-deficiency anemia.  An instrument called a Hemoglobinometer is able to measure the amount of hemoglobin in a blood sample.  Tallqvist paper may also be used to give a rough estimate of hemoglobin levels, though it is no longer used today. 

Normal Range of Hemoglobin values:  12-18 g/100 mL

Normal values for Adult Females:  12-16 g/100 mL
Normal values for Adult Males:  14-18 g/100 mL

Hemoglobin Estimate Procedure:

1)  After loading your heparinized capillary tube, continue to stimulate bleeding by massaging your hand down toward your fingertip.   Using a piece of Tallqvist paper, gently roll your bleeding finger over the paper as if to blot dry. 

2)  Continue on to the next test and load the capillary tube for blood coagulation before proceeding to step three (but do not wait until the Tallqvist paper dries!).

3)  Compare the slightly wet Tallqvist paper coloration with the Tallqvist scale by placing the paper under the round holes (Important:   Make sure you are wearing gloves when handling the Tallqvist booklet as it may be contaminated with blood from others!).

Record your estimated hemoglobin value here:________________________

Experiment:  Coagulation Time

Blood coagulation is a complex process involving over 30 substances.   Platelets are responsible for releasing many of these substances.   Can you name one vitamin and one mineral that is crucial to the clotting process?   ______________________  ______________________ When the enzymes involved in the process of hemostasis (stoppage of bleeding) are activated, they trigger a chain of events that leads to the formation of insoluble fibrin strands which form a meshwork that traps blood cells and seals the damaged vessel.

Coagulation Time Procedure:

1)  Continue to stimulate bleeding by massaging your hand toward your fingertip.   Load a capillary tube (nonheparinized) to about 1/2 full.   Set aside and continue to the setup for Test Five before completing #2 below.

2)  After about one to five minutes (time varies considerably), take the loaded capillary tube between your thumb and forefingers and gently break in half (see Fig. 3).  Slowly pull the ends apart to view the insoluble fibrin strands.  If you don't see any fibrin, wait a little longer and break the tube again.  Don't forget to dispose of the broken tube in the SHARPS CONTAINER.

Figure 3. Breaking the capillary tube to view insoluble fibrin strands.

Normal Range of Platelets for Adults:  150,000-500,000 platelets/mm³

Demonstration:  Total Blood Cell Counts

Note:  Although you will not be performing the following experiments, you will be required to have an understanding of the procedure, equipment used, how blood cell values are calculated, normal blood cell numbers, and significance of the test results.

Background information:

While knowing relative percentages of each of the blood cell types is helpful, other important clinical values that can be revealing are the Total RBC Count and the Total WBC Count.  A deficiency of RBCs is the cause of one form of anemia.  When the RBC count is elevated, the condition is called erythrocytosis or polycythemia.  Deficiencies of leukocytes are referred to as leukopenia, while elevated levels in general are described as leukocytosis.  Although hematology labs use automated equipment to count the number of cells in a given sample, it can also be done manually using a special blood cell counting slide called a hemacytometer (microscopic view shown below).  

The hemacytometer is divided into sections.  WBCs are counted in the outer four sections (each section contains a 4x4 grid with 16 total squares in each section).   To determine the number of leukocytes per cubic millimeter, use the following formula:

Total WBCs in all four sections x 50 = Total WBCs/mm³

The center section of the hemacytometer contains a 5x5 grid.  RBCs are counted in the four corner squares and the central square (each square contains a 4x4 grid, also).  To get the total number of RBCs per cubic millimeter (or microliter), use the following formula:

Total RBCs in all five squares x 10,000 = Total RBCs/mm³

Your laboratory instructor will perform these two tests.  You are responsible for recognizing the equipment and solutions used for these tests (and their basic functions) and for knowing the normal range of total blood cell values for adult males and females. 

Materials Used:
Red Blood Cell Diluting Pipette
Red Blood Cell Diluting Fluid
White Blood Cell Diluting Pipette
White Blood Cell Diluting Fluid
Hemacytometer
Microscope

Figure 6.1  Blood is drawn for the Total RBC count using a red blood cell diluting pipette (note the [red] crystal in the dilated portion of the pipette).

Figure 6.2  Next, red blood cell diluting fluid is drawn and mixed with the blood sample.  This fluid functions to lyse the WBCs in the blood sample.

Total Blood Cell Count Ranges:

Normal Total RBC count for Adult Females:  4.0-5.5 million cells/mm^3*
Normal total RBC count for Adult Males:  4.5-6.0 million cells/mm^3*

Normal total WBC count Males and Females:  6,000-9,000 cells/mm³

Author:  Susan M. Caley Opsal, Anatomy/Physiology Instructor, Illinois Valley Community College, Copyright 2000.  This lab may be copied and distributed for educational purposes.

*Note:  With the exception of the total RBC count (above), blood cell averages and ranges taken from Sarikas" "Investigations in Anatomy & Physiology (Cat Version)" laboratory manual, Pearson-Benjamin Cummings, 1st Edition, 2007.

Last Updated 01/14/07

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