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Blood Collection and Analysis
BIO 1008 Anatomy & Physiology II
Illinois Valley Community College

Blood samples are usually collected by a phlebotomist (phlebo= vein), who draws the sample from the median cubital vein. For our purposes, it will not be necessary to draw this much blood. Each student will use a sterile lancet, which looks like a large thumbtack, to initiate bleeding from the fingertip. An understanding and familiarity with the hematology lab procedures outlined in the next several pages is necessary for students to complete these labs successfully.  The first laboratory practical will include specifics from this lab.  Be sure to pay close attention to the equipment used for each test, the information obtained from each test (particularly normal blood cell numbers and/or percentages), and have a basic understanding of the clinical significance of abnormal values.

   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:

  1. 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;
  2. If you are having trouble perforating your finger, your laboratory instructor will assist you;
  3. 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);
  4. Before perforating your finger, disinfect the area with an alcohol wipe;
  5. 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;
  6. 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;
  7. Spray your tabletop with VIREX disinfectant.  Leave for 10 minutes and wipe dry with a paper towel.

Note:  The normal ranges given for the following tests come from Exercise 19 in the Sarikas lab manual:  Laboratory Investigations in Anatomy and Physiology (Cat Version), Pearson-Benjamin Cummings (First Ed., 2007).

Introduction to Blood, a Highly Specialized Connective Tissue

A.  Composition of Whole Blood

1.  Formed Elements (makes up ~45% of whole blood)

a.  Erythrocytes - red blood cells (RBCs)

b.  Leukocytes - white blood cells (WBCs)

c.  Platelets - formerly called "thrombocytes"

2.  Plasma (~55% of whole blood) - matrix of blood composed of water (90%), proteins, electrolytes, nutrients

B.  Types of Leukocytes

1.  Neutrophil (40-70%)

2.  Eosinophil (1-4%)

3.  Basophil (less than 1%)

4.  Lymphocytes (20-45%)

5.  Monocytes (4-8%)

Don't Forget:  Never Let Monkeys Eat Bananas!!


Test One:  Differential White Blood Cell Count

The purpose of the Differential WBC Count is to examine a sample of blood and determine the relative percentages of each type of white blood cell in the sample.  In doing so, the clinician is able to determine if there are any WBC deficiencies or excesses, which are indicative of certain illnesses or disease processes. 

Differential White Blood Cell Count Procedure

1)  Obtain a prepared blood cell smear and scan the slide on low power (10x) to find an area where blood cell distribution is best (cells are evenly spread out and not clustered together).  Avoid the outside edges of the smear where, in some slides, the cells have an abnormal appearance.

2)  Bring the slide in focus under oil immersion (see your laboratory instructor if you've forgotten how to do this!).  Scan the slide in a systematic manner (see Fig. 1) until you've counted and identified 100 leukocytes. 

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Figure 1.  Pattern for locating and identifying 100 leukocytes on a prepared slide for the White Blood Cell Differential.

3)  Record the results of your differential on the following table:

Type of Leukocyte Predicted Value Out of 100 cells Tally

Neutrophil
 
   

Lymphocyte
 
   

Monocyte
 
   

Eosinophil
 
   

Basophil
 
   

Note:  Although most blood tests performed in hematology labs are computer automated, when abnormal results are obtained,  a lab technician will make blood smears and check for the appearance of abnormal blood cells under the microscope.  This is the practice at Illinois Valley Community Hospital's hematology lab. 


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

1 lancet 1 Tallqvist Scale Booklet (2 per table)
1 heparinized capillary tube (with a red tip) 1 capillary tube (nonheparinized)
1 Seal-ease (2 per table) 1 wax pencil
1 small piece of Tallqvist paper 1 Clean microscope slide
2-3 alcohol wipes (draw 3 circles and label A, B and Rh)

Test Two:  Hematocrit Measurement (VPRC):

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 using 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 (percentage of RBCs in the sample) 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 fresh 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:____

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Figure 2. ADAMS Microhematocrit Reader

Test Three:  Hemoglobin Measurement:

Hemoglobin (abbreviated Hb) is the oxygen-carrying protein within 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

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 Test Four 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:________________________


Test Four:  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.

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

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


Test Five:  Blood Typing:

The surfaces of the RBCs contain numerous glycoprotein markers known as antigens (Ag).  There are many different types of antigens, but the most common with respect to blood typing are the A, B, and D antigens (D is also known as the Rh Factor).  The presence or absence of these antigens determines the blood type of the individual.  In addition, about six months after birth, lymphocytes begin to produce certain antibodies (abbreviated Ab), which are proteins that circulate in the blood plasma.  Each antibody type is specific for a certain antigen.  When an antibody combines with the antigen it is specific for, it forms an antigen-antibody complex.  This reaction is the basis for determining a person's blood type.  The antibodies from human plasma are isolated and used as anti-sera in the laboratory.  Mixing the anti-sera with whole blood stimulates the formation of antigen-antibody complexes, a process called agglutination (clumping).  For example, mixing anti-A serum with type A blood will cause the anti-A antibodies in the serum to "stick to" the A antigens on the erythrocytes (clumping occurs).  This Ag-Ab reaction will give the slide a beaded appearance (see Fig. 4).  Mixing anti-B serum with type A blood will NOT show agglutination.  This is a negative test for the B antigen.  

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Figure 4.
Anti-A serum has been added to the blood in the left well causing agglutination, which gives the mixture a "beaded" appearance.   When anti-B serum was added to the right well, agglutination did not occur.   For this particular sample of blood, this test indicates that the A antigen is present and the B antigen is not present; therefore this individual has type A blood.

The above example shows a positive test for the A antigen due to agglutination of the RBCs, thus the blood would be type A.   Q:  What type of antibodies make up Anti-B serum?____________________________.  

In this exercise, you will be using a sample of your blood to determine your blood type.  Use the following table to fill in the characteristics of the four major blood groups.  You will be responsible for this information on your first lab practical.

BLOOD TYPE ANTIGEN PRESENT
(on the RBC)
ANTIBODY PRESENT
(in the plasma)
A    
B    
AB    
O    
Rh Positive    
Rh Negative    

*Note:  Be sure to have a clean microscope slide with three circles drawn in wax pencil and the circles labeled A, B, and Rh (or D) available BEFORE you stick yourself with the lancet!*

Blood Typing Procedure:

1)  Continue to stimulate bleeding.  On a prepared microscope slide with three circles drawn with wax pencil and labeled A, B and Rh (or D), place a small drop of blood in each circle (make sure you have enough blood, but do not completely fill the circle...if you do, you have too much blood on your slide).

2)  Add anti-A serum to the circle labeled A, anti-B serum to the one labeled B, and anti-D serum (or Anti-Rh) to the circle labeled D.  Mix the contents of each circle with a clean toothpick (Fig. 5). 

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Figure 5.  Mixing the anti-serum with the blood sample to determine blood type.

Check for agglutination (you may need an Rh-Typing box to see a positive reaction for the Rh factor, which requires higher temperatures).   Place your slide on a white piece of paper to better view the agglutination process.  What is your blood type?______________________


Test Six:  Total Blood Cell Counts (demonstration only):

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).  

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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

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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).

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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/mm3*
Normal total RBC count for Adult Males:  4.5-6.0 million cells/mm3*

Normal total WBC count Males and Females:  6,000-8,000 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 06/08/07

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