Blood Analysis and Hemostasis

Objective:

To explain in a short essay or a diagram the importance of hematocrit, hemoglobin determination, clotting time and blood typing, at the level of 85% proficiency for each student.

In order to achieve this objective, you will need to be able to:

1. Conduct the following blood tests: hematocrit, hemoglobin determination, clotting time, and ABO and RH blood typing and to list the norms and importance of each.

Explain the reason for transfusion reactions resulting from the administration of mismatched blood.

Materials:

General Supply:

Models and charts of blood cells

Clean microscope slides

Sterile lancets

Glass stirring rods

Alcohol swabs (wipes)

Absorbent cotton balls

Test tubes

Test tube racks

Disposable gloves

Pipette cleaning solutions-- (1) 10% household bleach solution, (2) distilled water, (3) 70% ethyl alcohol, (4) acetone

Bucket or large beaker containing 10% household bleach solution for slide and glassware disposal

Disposable autoclave bag

Spray bottles containing 10% bleach solution

Because many blood tests are conducted in this exercise, separate supply areas are set up for the various tests

Hematocrit supply area:

Heparinized capillary tubes

Micro-hematocrit centrifuge and reading gauge (if the reading gauge is not available, millimeter ruler may be used)

Seal-ease (Clay Adams Co.) or modeling clay

Hemoglobin determination supply area:

Sahli hemoglobin hemometer kit

Mechanical suction device

Coagulation time supply area:

Capillary tubes (non-heparinized)

Fine triangular file

Blood typing supply area:

Blood typing sera (anti-A, anti-B, and anti-Rh [D])

Rh typing box

Wax marker

Toothpicks

Clean microscope slides

Group Materials:

microscope

Hematocrit

The hematocrit, or packed cell volume (PCV), is routinely determined when anemia is suspected. Centrifuging whole blood spins the formed elements to the bottom of the tube, with plasma forming the top layer. Since the blood cell population is primarily RBCs, the PCV is generally considered equivalent to the RBC volume, and this is the only value reported. However, the relative percentage of WBCs can be differentiated, and both WBC and plasma volume will be reported here. Normal hematocrit values for the male and female, respectively, are 47.0 ± 7 and 42.0 ± 5.

Methods:

The hematocrit is determined by the micro-method, so only a drop of blood is needed. If possible, all members of the class should prepare their capillary tubes at the same time so the centrifuge can be properly balanced and run only once.

Obtain two heparinized capillary tubes, Seal-ease or modeling clay, a lancet, alcohol swabs, and some cotton balls.
Clean and prick the finger to produce a free flow of blood. Wipe away the first few drops and, holding the red-line-marked end of the capillary tube to the blood drop, allow the tube to fill at least three-fourths full by capillary action. If the blood is not flowing freely, the end of the capillary tube will not be completely submerged in the blood during filling, air will enter, and you will have to prepare another sample.
Plug the blood-containing end by pressing it into the Seal-ease or clay. Prepare a second tube in the same manner.
Place the prepared tubes opposite one another in the radial grooves of the micro-hematocrit centrifuge with the sealed ends abutting the rubber gasket at the centrifuge periphery. This loading procedure balances the centrifuge and prevents blood from spraying everywhere by centrifugal force. Make a note of the numbers of the grooves your tubes are in. When all the tubes have been loaded, make sure the centrifuge is properly balanced, and secure the centrifuge cover. Turn the centrifuge on, and set the timer for 4 or 5 minutes.
Determine the percentage of RBCs, WBCs, and plasma by using the micro-hematocrit reader. The RBCs are the bottom layer, the plasma is the top layer, and the WBCs are the buff-colored layer between the two. If a micro-hematocrit reader is not available, use a millimeter ruler to measure the length of the filled capillary tube occupied by each element, and compute its percentage by using the following formula:

Height of the column composed of RBCs (mm) x 100
Height of the original column of whole blood (mm)

Height of the column composed of WBCs (mm) x 100
Height of the original column of whole blood (mm)

Results and Discussion:

Record your calculations below.

% RBC _________ % WBC _________ % plasma _________

Usually WBCs constitute 1% of the total blood volume. How do your blood values compare to this figure and to the normal percentages for RBCs and plasma?

________________________________________________________________________

As a rule, a hematocrit is considered a more accurate test for determining the RBC composition of the blood than the total RBC count. A hematocrit within the normal range generally indicates a normal RBC number, whereas an abnormally high or low hematocrit is cause for concern.

Hemoglobin Concentration

As noted earlier, a person can be anemic even with a normal RBC count. Since hemoglobin is the RBC protein responsible for oxygen transport, perhaps the most accurate way of measuring the oxygen-carrying capacity of the blood is to determine its hemoglobin content. Oxygen, which combines reversibly with the heme (iron-containing portion) of the hemoglobin molecule, is picked up by the blood cells in the lungs and unloaded in the tissues. Thus, the more hemoglobin molecules the RBCs contain, the more oxygen they will be able to transport. Normal blood contains 12 to 16 g hemoglobin per 100 mL blood. Hemoglobin content in men is slightly higher (l4 to 18 g) than in women (12 to 16 g).

Several techniques have been developed to estimate the hemoglobin content of blood, ranging from the old Sahli method to expensive colorimeter methods, which are precisely calibrated and yield highly accurate results. Directions for the Sahli method are provided here.

Methods

The Sahli hemometer method utilizes the conversion of hemoglobin into acid hematin which has a brown color in solution. The intensity of the color is related to the amount of hemoglobin in the blood sample. Water is added to dilute the brown solution until it matches that of a standard. The more hemoglobin, the more water required to obtain a color match. Hemoglobin values are read at the meniscus of the brown solution.

Obtain a Sahli hemometer kit, 0.1 N HCl, alcohol swab, and blood lancet and bring them to your bench.
Place 5 drops of 0.1 N HCl in the square calibrated Sahli tube.
Place the tip of the pipette into the blood drop and allow the blood to flow to the 20 mm³ mark. If necessary use the mechanical suction device.
Carefully wipe the blood from the outside of the pipette. Be very careful to avoid touching the bore of the pipette. Place the tip below the surface of the acid in the bottom of the Sahli tube, and gently expel the blood into the acid. Flush the pipette twice by drawing up and expelling the acid and blood solution.
Place the tube in the comparator and add distilled water drop by drop until the color exactly matches that of the glass filter standard. The solution should be thoroughly mixed after each drop of distilled water is added.
After 10 minutes read the scale on the tube at the level of the fluid meniscus. Some tubes are calibrated directly in gm/100 mL; others are calibrated in percent of a standard hemoglobin concentration.

Results and Discussion:

Record the hemoglobin as gm/100 mL

Hemoglobin _________ gm/100 mL

Generally speaking, the relationship between the PCV and grams of hemoglobin per 100 mL blood is 3:1. How do your values compare?

________________________________________________________________________

Coagulation Time

Blood clotting, or coagulation, is a protective mechanism that minimizes blood loss when blood vessels are ruptured. This process requires the interaction of many substances normally present in the plasma (clotting factors, or procoagulants) as well as some released by platelets and injured tissues. Basically, hemostasis proceeds as follows: The injured tissues and platelets release thromboplastin and PF3 respectively, which trigger the clotting mechanism, or cascade. Thromboplastin and PF3 interact with other blood protein clotting factors and calcium ions to form protbrombin activator, which in turn converts prothrombin (present in plasma) to thrombin. Thrombin then acts enzymatically to polymerize the soluble fibrinogen proteins (present in plasma) into insoluble fibrin, which forms a meshwork of strands that traps the RBCs and forms the basis of the clot. Normally, blood removed from the body clots within 2 to 6 minutes.

Methods:

Obtain a non-heparinized capillary tube, a lancet, cotton balls, a triangular file, and alcohol swabs.
Clean and prick the finger to produce a free flow of blood.
Place one end of the capillary tube in the blood drop, and hold the opposite end at a lower level to collect the sample.
Lay the capillary tube on a paper towel. Record the time.
At 30-sec intervals, make a small nick on the tube close to one end with the triangular file, and then carefully break the tube. Slowly separate the ends to see if a gel-like thread of fibrin spans the gap. When this occurs, record below and on the data sheet the time for coagulation to occur. Are your results within the normal time range?
Dispose of the capillary tube and used supplies in the disposable autoclave bag.

Results and Discussion:

Time for coagulation to occur. _______ sec

Are your results within the normal time range?

Blood Typing

Blood typing is a system of blood classification based on the presence of specific glycoproteins on the outer surface of the RBC plasma membrane. Such proteins are called antigens, or agglutinogens, and are genetically determined. In many cases, these antigens are accompanied by plasma proteins, antibodies or agglutinins, that react with RBCs bearing different antigens, causing them to be clumped, agglutinated, and eventually hemolyzed. It is because of this phenomenon that a person's blood must be carefully typed before a whole blood or packed cell transfusion.

Several blood typing systems exist, based on the various possible antigens, but the factors routinely typed for are antigens of the ABO and Rh blood groups which are most commonly involved in transfusion reactions. Other blood factors, such as Kell, Lewis, M, and N, are not routinely typed for unless the individual will require multiple transfusions. The basis of the ABO typing is shown in the chart.

Individuals whose red blood cells carry the Rh antigen are Rh positive (approximately 85% of the U.S. population); those lacking the antigen are Rh negative. Unlike ABO blood groups neither the blood of the Rh-positive (Rh+) nor Rh-negative (Rh-) individuals carries preformed anti-Rh antibodies. This is understandable in the case of the Rh-positive individual. However, Rh-negative persons who receive transfusions of Rh-positive blood become sensitized by the Rh antigens of the donor RBCs and their systems begin to produce anti-Rh antibodies. On subsequent exposures to Rh-positive blood, typical transfusion reactions occur, resulting in the clumping and hemolysis of the donor blood cells.

Methods:

Obtain two clean microscope slides, a wax marking pencil, anti-A, anti-B, and anti-Rh typing sera, toothpicks, lancets, alcohol swabs, and the Rh typing box.
Divide slide 1 into two equal halves with the wax marking pencil. Label the lower left-hand corner "anti-A" and the lower right-hand corner "anti-B." Mark the bottom of slide 2 "anti-Rh."
Place one drop of anti-A serum on the left side of slide 1. Place one drop of anti-B serum on the right side of slide 1. Place one drop of anti-Rh serum in the center of slide 2.
Cleanse your finger with an alcohol swab, pierce the finger with a lancet, and wipe away the first drop of blood. Obtain 3 drops of freely flowing blood, placing one drop on each side of slide 1 and a drop on slide 2.

Quickly mix each blood-antiserum sample with a fresh toothpick. Then dispose of the toothpicks, lancet, and used alcohol swab in the autoclave bag.
Place slide 2 on the Rh typing box and rock gently back and forth. (A slightly higher temperature is required for precise Rh typing than for ABO typing.)
After 2 minutes, observe all three blood samples for evidence of clumping. The agglutination that occurs in the positive test for the Rh factor is very fine and difficult to perceive; thus if there is any question, observe the slide under the microscope. Record your observations in the chart.
Put the used slides in the bleach-containing bucket at the general supply area; put disposable supplies in the autoclave bag.

Results and Discussion:

blood typing

clumping	Observed (+)	Not observed (-)
with anti-A
with anti-B
with anti-Rh

Interpret your ABO results.

__________________________________________________________________
Interpret your Rh results.

__________________________________________________________________
Determine your blood type.

__________________________________________________________________

Hematologic Test Summary

Hematocrit (PCV):

RBC % of blood volume

WBC % of blood volume

Plasma % of blood volume

Hemoglobin content:

g/100 mL blood; %

Ratio (PCV/grams Hb per 100 mL blood):

Coagulation time:

sec

Blood typing:

ABO group Rh factor