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					SUPP-L12

                             RED BLOOD CELL EXAMINATION

Introduction:
Blood is approximately 55% plasma and 45% cells and cell fragments. The plasma
consists mainly of water, along with plasma proteins, ions, and nutrients. The cellular
portion of blood consists of red blood cells, white blood cells and cell fragments called
platelets. Red blood cells, containing hemoglobin, transport oxygen and other molecules
involved in cellular respiration. Red blood cells are disk-shaped bags, lacking a nucleus,
and having a bi-concave shape to increase surface area.

Common indices (plural for index) used to evaluate red blood cells include the:
1. red blood cell (rbc) count - # of red blood cells in a mm3
2. hemogloblin (Hgb) concentration – grams of Hgb per dL
3. hematocrit (Hct) - % of the whole blood that is cellular

These are easily measured in the laboratory. From these measurements other indices
can be calculated (corpuscle is an alternative name for cell):
1. mean corpuscular volume            MCV = Hct X 10 / rbc count
2. mean corpuscular hemoglobin concentration        MCHC = Hgb X 100 / Hct

Normal values for adults for the above indices are listed:
                                 Male                      Female          Units
                                                                                        3
Red Blood Cell count:            4.5 – 6                   4 – 5.5         Million / mm
Hemoglobin:                      13 – 16                   12 – 15         g / dL
Hematocrit:                      40 – 54                   37 – 47         %
                                                                               3
MCV                                              M or F 82 – 92            um
MCHC                                             M or F 32 – 36            %

If red blood cell physiology is abnormal, there may be too few cells or they may be small
and not contain enough hemoglobin. A reduction in rbc count and/or hemoglobin and/or
hematocrit is known as anemia (literally “without blood”). Anemia can be caused by a
variety of illnesses or nutritional deficiencies. Anemias can result from less red blood
cells made or more being destroyed. Either way, anemia causes the oxygen carrying
capacity to be reduced, which reduces aerobic capacity and increases the risk for
ischemia (inadequate oxygen for the need of the tissue) and tissue damage.

Procedure for Blood Handling:
1. Set up your biohazardous worksite (placemat, gloves, etc) according to biohazard protocols.
Everything in contact with your worksite will be considered biohazardous.
2. Obtain and arrange all the materials and equipment you need for the entire lab prior to
obtaining your blood. Wrap equipment that needs to remain non-contaminated (microscope,
pencil, book, etc). Put away all nonessential items to avoid contamination.
2. Have your worksite and materials checked by the instructor.
3. Each student can only work with their own blood and may not assist other students.
4. Clean up materials appropriately (sharps, non-sharp biohazards, non-contaminated).
5. Understand that deviation from appropriate biohazard protocols will result in loss of laboratory
privileges, ejection from the lab, and loss of lab credit.

A. Red blood cells are formed in the bone marrow, derived from blood stem cells. Red
blood cell production is stimulated by erythropoietin, a hormone secreted by the kidneys.
After release, rbcs live approximately 120 days, then are broken down by phagocytes in


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the spleen, liver and bone marrow. The number of red blood cells is determined by the
balance of rbc production and destruction.

Procedure for Red Blood Cell Count:
Materials needed: micropipettor and disposable
tip, diluting reservoir or test tube of saline,
hemocytometer, disposable cover slip, kimwipe,                    Hemacytometer
capillary tube, microscope, hand counter
1. Examine the hemocytometer, noting that it
has 2 shiny platforms, upon each of which a grid                                                cover slip
                                                                                                covers grid
is etched. Place the hemocytometer under the
microscope and find the grids, practice focusing.
Locate the appropriate squares. Close the iris
diaphragm somewhat to get more contrast.                                                               1    Setting
2. Place the disposable tip on the micropipettor                                                       0    for 10ul
                                                                                                            volume
and set it to 10ul. Cover completely with Saran.
                                                                      notch in slide                   0
Practice your pipetting technique (see procedure below).
3. Mix your tube of whole blood gently by rocking it back and forth (or prepare for finger stick).
Remove the rubber stopper, using the kimwipe to prevent splatter.
4. Depress the micropipettor plunger to the first stop, insert the tip into the blood and gently
release the plunger. This will be exactly 10 ul of whole blood (if there are no bubbles, etc.). Wipe
off the outside of the pipette tip on the blood tube rim.
5. Open and hold the reservoir in your left hand and place the micropipettor tip against the inside
                                                                                     nd
of the tube, above the fluid level. Eject the blood by pressing down to the 2 stop on the plunger.
Withdraw the micropipettor before releasing the plunger. Cover the reservoir and mix well.
6. Fill a glass capillary tube with the diluted blood (it fills by capillary action) and place 1-2 drops
into the V-shaped notch of the hemocytometer, with the cover slip already in place (see above).
Make sure the diluted blood has completely spread under the cover slip, covering the grids
etched on that side of the slide. Place the slide on the microscope (without contaminating the
microscope) and view that grid under low power. Locate the appropriate squares (the 5 small
squares marked in the diagram), focusing well and
adjusting your lighting. Go to higher power (whatever you
feel is necessary to be able to count the red blood cells).
Using the hand counter, count the cells in these 5 squares.
Don’t count cells that fall on the outermost boundary line
and do count cells that are on the innermost line. For cells
that fall on the middle boundary line, include ½ of them in
your count (for instance, include the ones that fall on the
top and left boundaries, not the bottom or right). If you
delayed looking at your slide, the count will be incorrect, so
wipe out the slide, fill again, and count promptly.


Total rbc in 5 squares = ____________________
(How many do you expect to find? There are 5 small
squares, each with 16 very small squares within it. See the math below and calculate how many
cells you would expect to find in the total of the 5 squares, given that a normal rbc count is 4-6
            3
million/ mm .)


7. Your blood was diluted 1:200 (10 ul of blood in the micropipetor diluted in 2.0 ml total volume
                                                                                             3
in the reservoir). The volume of diluted blood contained in the 5 small squares is 0.02 mm (the
                                 2
grid is 0.1 mm deep X 0.04 mm surface area per small square X 5 squares). (Go slow and don’t
be intimidated by the math.) So, to convert your count of this small diluted sample, just reverse
the dilution factor and volume factor: 200 (the inverse of 1/200) X 50 (the inverse of 0.02) =



                                                                                                         2
                                                                             3
10,000. Multiple 10,000 X your count of rbcs in 5 squares = # of rbc in mm of your whole blood.
Record the total # of red blood cells in the report.

B. Hemoglobin is the oxygen-carrying molecule within red blood cells. Each
hemoglobin molecule consists of 4 proteins (2 alpha chains and 2 beta chains) and 4
organic pigmented heme molecules (each containing Fe++). Each heme bonds to one
oxygen molecule when oxygen concentration is high (called loading) and releases it
when oxygen levels are lower (called unloading). Hemoglobin is also able to bond H+,
CO, and CO2. Hemoglobin is more bright red in the presence of oxygen (oxyhemoglobin)
and more dusky red if oxygen content is low (deoxyhemoglobin).

The color of any clear, colored solution can be measured by a spectrophotometer (AKA
colorimeter). Beer’s law states that the color intensity is directly proportional to the total
solute concentration. However, because of the variable color of hemoglobin, this
method would not be accurate. If hemoglobin is converted to only one form, as happens
when it is oxidized by Drabkin’s (POISONOUS!!) solution to cyanomethemoglobin, it has
a uniform absorption at 540 nm.

Procedure for Hemoglobin
Materials needed: test tube, parafilm, Drabkin’s solution, 5 ml pipette, micropipettor (wrapped
with Saran) and disposable tip, spectrophotometer, Hgb standard
1. Label a clean, new test tube with your name at the top. Pipette 5.0 ml of Drabkin’s into your
test tube. Drabkin’s is poisonous! Do not contaminate the reagents and pipettes and keep the
test tube in a non-contaminated test tube rack (off your worksite).
2. Set the micropipettor to 20 ul volume and cover it with Saran wrap to keep it from being
contaminated. Or, if you already have it set to 10ul, then pipette twice to get 20ul.
3. Gently mix your tube of whole blood and safely remove the stopper. Withdraw 20 ul of whole
blood and expel it into the Drabkin’s solution. Eject the disposable tip. After taking off your
gloves and washing your hands, cover the test tube with parafilm and mix. Place in the test tube
rack and wait 15 minutes.
4. Follow the directions on the spectrophotometer. Turn on (use the “%T I/O” knob) and set
the wavelength to 540 nm. Adjust the reading to 0 by using the same knob. Change the “Mode”
to absorbance. Test tubes labeled “blank” (contains Drabkin’s and no hemoglobin) and “standard
hemoglobin” (contains purchased hemoglobin of a known 18g/dL concentration) will be next to
the spectrophotometer for your use. Wipe all tubes with a kimwipe to remove dust and
fingerprints prior to using (why?) Insert the blank tube into the chamber, close the cover and
adjust absorbance to zero, using the knob on the right (labeled “100% T/O A”). Make no further
adjustments!! Now place the standard tube in the chamber and record the absorbance.
Absorbance standard = ______________               Concentration standard = 18g/dl (from its label)
Lastly, place your unknown tube in the chamber and read the absorbance.
Absorbance (your) unknown = __________________
5. Calculate your hemoglobin concentration and record:

        Conc of Your Unknown Sample = Conc Standard X Absorbance Unknown
                                           Absorbance Standard

C. Hematocrit is the % or proportion of the whole blood that is cellular. This is
measured by centrifuging whole blood in glass capillary tubes. The centrifugation
“packs” the red blood cells at the bottom and leaves clear yellowish plasma at the top.

Procedure Hematocrit:
Materials needed: 3 capillary tubes (red fire polished on one end), sealing clay, 3 critocaps,
hematocrit reader card (saran wrapped)




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1. Mix your tube of blood gently and remove the stopper.
2. Fill the capillary tube by placing the clear (not red) end into the blood at an angle and allowing
the tube to fill by capillary action. The tube needs to filled ½ or more.
3. Quickly invert the tube to insert the red marked end into the tray of clay. This will seal it with a
plug of clay. Repeat with the other tubes so that you have at least 3.
4. As an additional seal, insert a critocap gently into the same end as the clay. Be careful not to
break the tube while inserting the cap.
5. Place your capillary tubes into the microcentrifuge with the caps toward the outside, write
down the numbers of the slots your tubes are in and allow the instructor to run the centrifuge.
6. Place your tube on the hematocrit reader card and move it side to side along the diagonal
lines until it fits, with the bottom of the blood at the “zero” line and the top of the plasma at the
“100%” line. The directions are on the card. Once it is in the proper position, determine which
line marks the junction between the plasma and the packed red blood cells. This line
corresponds to the hematocrit.

D. Mean corpuscular volume (MCV) and Mean Corpuscular Hemoglobin Concentration
(MCHC) are calculated values, giving information about red blood cell size and fullness,
respectively. Anemias can be sometimes categorized by their MCV and MCHC levels,
such as iron deficiency anemia, which has a low MCV (meaning small cells) and low
MCHC (meaning not full of hemoglobin).

MCV = _____hematocrit X 10____
           rbc count (in millions, e.g. 4.5 or 5.2)

MCHC = __hemoglobin (g/dl) X 100__
           Hematocrit (in whole numbers, e.g. 42 or 53)




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                             LABORATORY REPORT L12
                           RED BLOOD CELL EXAMINATION

Name __________________________________                Lab Section ___________________

RESULTS
1.     Red Blood Cell count _______________ per mm3
       Hemoglobin ___________________ g/dL Hematocrit __________________%
       MCV _________________________ um3 MCHC _____________________ %
2. Are your results normal? What could be some reasons for abnormal results?



QUESTIONS
1. Match the following:
_____ red blood cell count                    a.   the % of blood that is packed cells
_____ MCV                                     b.   typically 4-6 million per mm3
_____ hemoglobin                              c.   indicates the fullness of a rbc
_____ MCHC                                    d.   responsible for oxygen carrying capacity
_____ hematocrit                              e.   best indicator of rbc size

2. Match the following:
_____ oxyhemoglobin                           a.   hemoglobin bound to carbon dioxide
_____ deoxyhemoglobin                         b.   oxidized hemoglobin
_____ carboxyhemoglobin                       c.   hemoglobin bound to oxygen
_____ carbaminohemoglobin                     d.   hemoglobin bound to carbon monoxide
_____ methemoglobin                           e.   hemoglobin without oxygen

3. A high red blood cell count, called polycythemia, occurs at high altitudes. Identify its
benefit __________________________________________ and its adverse effects
________________________________________________.

4. Define anemia.


Give an example of an anemia that would be due to:

a. Increased blood loss/destruction ________________________________________

Why does this cause anemia? ____________________________________________

b. Decreased blood production/synthesis ___________________________________

How would you treat this anemia? _________________________________________

5. List the series of events that leads to jaundice in a newborn premature baby (short
phrases OK). Start with increased red blood cell destruction occurs after birth →
excessive hemoglobin is released →




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