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ZOOLOGY 142L BLOOD LAB
I.General Blood Background Information
Blood consists of the fluid (mainly water) and cells contained in the vascular system of the body. The elements of the blood smaller than albumin proteins freely exchange with the interstitial fluid between the cells . The three main functions of the blood are transportation, regulation, and protection.
As blood is pumped through the body by the heart it transports all the different substances between cells that place the materials into the blood, to cells that extract the materials. A major example is the transport of oxygen (O2), on the hemoglobin in red blood cells, from the lungs to the tissue cells where it is taken in to metabolize nutrient molecules (proteins/amino acids, carbohydrates/glucose, and fats/fatty acids) for energy (ATP). The waste products of this metabolism are carbon dioxide (CO2), acid (H+), and heat, which are transported by the blood to the lungs and kidneys for elimination from the body. Other categories of substances include electrolytes (e.g. Na+, Cl-), proteins (e.g. albumin, fibrinogen, antibodies), nutrients, and other cellular waste products.
Blood participates in the regulation of physiological conditions such as acidity ([H+]~pH), body temperature, and pressure. The blood contains chemicals (like H2PO4--) that buffer the excess H+ from metabolism, and prevents large changes in the [H+] while it is being transported to the kidneys for excretion. Body temperature is kept at about 37 degrees centigrade (C) because excess heat produced by metabolism is transported from the cells to the skin, where it is transferred to the cooler areas outside the body. The solutes in the blood (mainly Na+, Cl-, and albumin) hold the water volume in the vessels and the volume stretches the walls to produce pressure. Along with the pressure produced by pumping of the heart blood is pushed through the vascular system.
Blood protects itself from lose, and protects against most pathogens (germs). Clotting proteins and cell fragments (platelets) form clots to plug small holes in vessels so that blood does not leak out. There are a variety of white blood cells (WBC) of the immune system that recognize and eliminate pathogens by lethal attacks and phagocytosis. One particular WBC makes antibody proteins that bind and incapacitate specific invading microbes. Each different WBC defends against different pathogens.
II.Hematocrit
The percent red blood cell (RBC) volume in a whole-blood sample is called hematocrit (Hct). The amount of RBCs in blood depends on the balance between how fast they are produced compared to how fast they breakdown. The blood is mainly plasma and cells. The plasma is mostly water (~91%), proteins (~7%), and other solutes (~1.5%). The blood cells are mostly RBCs (~99%+), and some white blood cells (~>1%). The percent of the volume of RBCs of the blood is called the hematocrit. Each person has about 5 liters of blood volume.
Athlete “Blood Doping”
The process of “Blood Doping” requires that a volume (~500ml) of blood be drawn and the red blood cells (RBC) preserved for about a month while the body replaces the blood. The RBCs are reinfused several days before an aerobic (oxygen using), endurance athletic event to produce an extra advantage. This is illegal in professional sports.
%RBCs of the volume = × 100
The RBCs transport oxygen, so the amount of oxygen transported depends on the % RBCs, and the total volume of blood (ℓ = liter). ). Always convert percent numbers into a decimal numbers before using it in a calculation (%/100 = decimal fraction).
× volume of blood = volume of RBCs → volume of oxygen
!Unexpected End of Formula × ℓ blood = ℓ RBCs
× zℓ RBCs = ℓ O2
(x, y, & z are the specific number of liters of each material, O2 or RBCs)
A normal hematocrit depends on gender and age.
Adult females: 38-46% (42% average)
Adult male:40-54% (47% average)
This means that for males there is an average of 0.470 l of RBCs (z) in every 1.0 ℓ of blood (Hct = 47%). There is 0.20 ℓ of O2 (x) per 0.47 ℓ of RBCs (y), so how much O2 is there in 1.0 ℓ of blood?
× zℓ RBCs = normal
What would happen to the hematocrit if an extra 0.13 l (q) of RBCs was reinfused into each 1.0ℓ of blood volume? (=blood doping)
Normal:
× 100 = 47.0%
Blood Doping:
100 = × 100 = %
How much O2 would there be in each liter (ℓ) of blood that has been “doped”?
× =
How much more O2 is there in each liter (ℓ) of the “doped” blood compared to normal blood?
- =
So each liter of “doped” blood would hold more/less oxygen than a liter of normal blood? So, each liter of “doped” blood that is pumped through an athlete’s muscles would deliver more/less oxygen? Would there be any possible advantage to blood “doping” for athletes in an endurance sport (like long-distance running), which uses aerobic metabolism (oxygen requiring)? yes/no
Hypothesis About Determination Of “Blood Doping”
What would predict the condition of “blood doping”?
Experimental Design
What two factors (variables) would need to be measured to determine if blood “doping” has occurred?
What factor would be the controlled (independent) variable?
What factor would be the observed (dependent) variable?
What would be the “control condition”?
How could the control condition” be established in the lab class?
What would be measured for the two factors (see procedures below)?
What calculations would be needed to analyze the measurements (data, see procedures below)?
Hematocrit Procedure
To measure a hematocrit a small volume of blood is drawn by a “finger stick”, under sterile conditions, so that pathogens are not introduced into the person (see blood drawing procedure in reference section). The blood sample is often drawn into a glass capillary tube, which is usually about 50 mm long, and has a 1mm wide internal tube. A clay plug is jammed into one end of the capillary tube, then it is placed in a centrifuge and spun at several thousand revolutions-per-minute (rpm) for several minutes. Since the RBCs are more dense than the plasma they are compressed to the bottom of the tube and the plasma is separated to the top.
Male Athlete A:
////RRRRRRRRRRRRRRRPPPPPPPPP
Female Athlete B:
////RRRRRRRRRRPPPPPPPPPPPPPPP
Male Athlete C:
////RRRRRRRRRRRRPPPPPPPPPPPPP
Results
The length of the tube is proportional to the volume, so the length of the red RBC region and the overall length of the blood sample are measured in millimeters (mm, which includes the clear-yellowish plasma region, P). Measure the length of RBCs (R), and the overall length of the blood sample (R+P) for the three athletes above, and place the values in the table below. Do not include the clay plug in the measurements (//). Calculate the hematocrit for each athlete and place the resulting value in the table below.
Calculation/Analysis Method:
%RBCs = × 100 = × 100 = hematocrit = Hct
Results and Analysis Table:
Athlete Type / length RBC / length blood sample / HctGender / mm / mm / (mm/mm)x100
Male A
Female B
Male C
Discussion and Conclusions
How do the results compare to the hypothesis?
How accurate are the measurements?
Is the accuracy good enough to make decisions about a sample being normal or not?
How could the normal value for Hct be determined by authorities in the field of hematology?
Should the hypothesis be accepted or rejected with respect to each athlete?
Which athlete would be suspected of “blood doping”?
Effects of “Blood Doping” and Health
A high hematocrit increases the concentration of red blood cells and so makes the blood more viscous (thicker, similar to polycythemia, see below). This may actually slow the blood flow (at a certain Hct) and so how much oxygen gets to the tissues may be less than normal. Higher blood pressure is needed to shove the blood through the vessels and thus may increase the possibility of a heart attack or stroke in people with weakened blood vessels (like with atherosclerosis). [Principles of Anatomy and Physiology, Tortora and Derrickson, 11th edition, pp. 669-670]
Anemia and Polycythemia
If the hematocrit is lower than normal then oxygen delivery is low, causing fatigue and cold sensitivity due to lack of metabolic energy and associated heat production. This can be due to lack of iron in the diet to make hemoglobin (the O2 carrying molecule in the red blood cells), or lack of vitamin B-12 (pernicious anemia) or folic acid for blood cell production, or even excess RBC breakdown (hemolytic anemia, as with thalassemia or sickle cell anemia).
Polycythemia occurs when there are too many RBCs and hence a higher than normal hematocrit (>55%). A certain type of bone marrow cancer can cause excess RBC production, compared to how fast the RBCs are broken down, producing a high Hct. Polycythemia may also be due to excess erythropoeitin (EPO), a hormone that stimulates more rapid RBC production (greater number of RBCs produced per minute). The excess may be due to a problem with the juxtaglomerular cells of the kidney or injections of EPO.
Dynamic Maintenance of Hematocrit
The number (amount) of RBCs in the blood at any time depends on the balance of the rate of production (number of RBCs produced per minute) and the rate of destruction (number of RBCs broken down per minute).
For a simple (but effective) example consider a volume of blood that has 100 RBCs in it. If the RBC production rate is 20 per minute (X#), and the rate of breakdown is 20 per minute (Y#), one minute later there would be no change in the number of RBCs.
100 RBCs + (rate of production – rate of breakdown) × (1.0 minute) = # RBCs
100 RBCs + ()× (1.0 minute) = # RBCs
100 RBCs + () × (1.0 minute) = 100 RBCs
What would be the number of RBCs after one minute, if the rate of production was 25 RBCs produced per minute (X#) and the rate of breakdown was 20 RBCs per minute (Y#)?
100 RBCs + () × (1.0 minute) = RBCs
Hematocrit, Exercise, and Fitness
EPO production is normally elevated by conditions that cause chronically lower oxygen in the tissues than needed (hypoxia). If the conditions have an effect on the hematocrit slowly (weeks, vs. minutes for blood doping) the cardiovascular system can fully adapt to the change and so this allows a safe adjustment. What conditions could cause this?? (work through the critical thinking questions below)
If someone exercised more intensely that usual the oxygen would be used by the muscles faster than it could be delivered from the lungs, by the cardiovascular system.
In the case of intense exercise the amount of oxygen in the muscle tissues (and the blood) would be more/same/less than normal?
This condition would be called hyperoxyia/normoxyia/hypoxia?
The secretion of erythropoietin (EPO) by the kidneys would decrease/stay the same/increase?
The rate of RBC production (number of RBCs produced per minute) would decrease/stay the same/increase?
The hematocrit (Hct) would decrease/stay the same/increase?
The ability of the person to exercise at higher intensities (fitness) would decrease/stay the same/increase?
Hematocrit, Altitude, and Fitness
If an athlete lived up on Mauna Kea (14,000 feet elevation) for two months, and went down to Kona (sea level) to exercise for only six hours each day, what would happen to their hematocrit? (work through the critical thinking questions below)
Since the pressure at 14,000 feet is only 88% of the pressure at sea level the amount of oxygen in the air is only 88% of the amount at sea level.
At 14,000 feet elevation the amount of oxygen the body tissues receive would be more/the same/less than at sea level (with no change in respiration or hematocrit)?
This condition would be called hyperoxyia/normoxyia/hypoxia?
The secretion of erythropoietin (EPO) by the kidneys would decrease/stay the same/increase?
The rate of RBC production (number of RBCs produced per minute) would decrease/stay the same/increase?
The hematocrit (Hct) would decrease/stay the same/increase?
The athletes capacity to exercise at sea level would decrease/stay the same/increase (compared to if they had lived and exercised at sea level for two months)?
III.Blood Typing
Transfusions
Transfusions are needed when there is significant blood loss, like in severe car accidents or surgery. Without the proper volume of blood, the blood pressure and flow will decline to the point that tissues are not supplied with enough oxygen and nutrients, and cannot dispose of enough carbon dioxide and other waste products of metabolism. The blood to be given to a recipient needs to match their own blood as closely as possible, or the immune system can produce a full-body transfusion-reaction, called anaphylaxis. Severe anaphylaxis can kill a person if untreated. What needs to match are the special identification proteins on the membrane of red blood cells.
Blood Type Background Information
The special identification proteins on the membrane of red blood cells are called antigens (in immunology) or agglutinogens (in hematology). There are only two major types of identification proteins, A and B. There are four blood types consisting of different combinations, or the absence of the two identification proteins on the RBC membrane (A,B,AB,O). (Figure 1 & 1a below)
The blood automatically has immune proteins, called antibodies (in immunology) or agglutinins (in hematology), which recognize, bind to, and agglutinate (clump together) any antigens that are not normally present for each blood type (Figure 1 & 1a below). For example, a blood-type A person would automatically have type B antibodies, which would recognize, bind to, and agglutinate any B antigens that were introduced with transfused blood. Agglutination is a completely different process than coagulation/blood clotting.
There is a third important antigen that is on the membranes of RBCs, called the Rh factor. A person that has the Rh antigen is referred to as Rh-positive (Rh+), and so does not have an antibody in their blood that recognizes the Rh antigen (Ab-Rh). A person without the Rh antigen is called Rh-negative (Rh-), and may have some small amount of Ab-Rh.
Monoclonal antibodies have been produced that are able to recognize each of the two different antigens on red blood cells. Antibody-A (Ab-A) recognizes and binds to A antigens, Antibody-B (Ab-B) recognizes and binds to B antigens, Antibody-Rh (Ab-Rh) recognizes and binds to Rh antigens, and they all cause agglutination when the antibody binds to the antigen. Each of the antibodies can be put into a solution and stored in a dropper bottle, called anti-sera A (contains Ab-A), anti-sera B (containing Ab-B), and anti-sera Rh (containing Ab-Rh).
Figure 1
Figure 1a
Figure 2
Sample 1 – top slide
Sample 2 – second slide down
Sample 3 – third slide down
Sample 4 - fourth slide down
Hypothesis About Determining Blood Type With ThreeAntibodies (Agglutinins)
If you had two antibody solutions (A and B) what would you predict about the capacity to determine and differentiate the four blood types, and why?
Blood Typing Procedure
Two drops of an unknown blood type are placed on a card (sample number 1-4)(Online lab students use the example slides above as your experiment samples). A drop of antibody-A solution (blue) is combined with one of the drops of blood, and a drop of antibody-B solution (yellow) is combined with the other. The two drops are stirred with a pick, of a corresponding color (blue and yellow) so they are not mixed up. The card is then gently rocked back-and-forth to mix the solution and sample for about a minute (not so much as to make the drops run). The agglutination reaction (clumping) is most easily seen where the solution is thinner when the fluid is moving away. The agglutination looks slightly different with the two antibody solutions. See figure 2 (above) that shows examples of agglutination in the two different solutions (no Rh reaction shown). Place the results of your observations about the reactions in the table, next to your unknown sample number. The results for the Rh antigen are already filled in on the table. Fill in the rest of the table by looking at the reactions for the other unknown samples from other lab groups (or the other sample slides, for online students).
Results
Antisera -> / Anti-A / Anti-B / Anti-D / BloodAntigen detected / Antigen A / Antigen B / Antigen Rh / Type
Sample # / Agglut. Y/N / Agglut. Y/N / Agglut. Y/N / A,B,AB,O,+/-
1 / Y
2 / N
3 / Y
4 / N
Discussion and Conclusions
Did the results support the hypothesis?
Were the agglutination reactions clear enough to be confident that a reaction was distinguishable from a non-reaction?
What authoritative source could verify the observations?
Is the hypothesis to be accepted or rejected?
Transfusion Considerations
In each sample of blood there are red blood cells with specific antigens and there are also specific antibodies, which recognize antigens other than the one present. If an antibody recognizes an antigen it will start the allergic-inflammatory-anaphylactic-like, transfusion reaction mentioned in the introduction. When a recipient takes a blood transfusion they must take blood that their antibodies will not react to. For example a Type A person has A antigens (that is what makes the blood type A), and B antibodies, so when they take a transfusion B antigens cannot be present in the donated blood. Remember that the donated blood also has antibodies in it.
When properly matched the donated blood antigens are acceptable for the recipient, but are the antibodies in the donated blood always compatible with the antigens of the recipient’s blood?
Under what conditions might this be a problem?
IV. White Blood Cells
White Blood Cells Background Information
There are mainly red blood cells and white blood cells in normal blood. The red blood cells mainly carry oxygen and some carbon dioxide. The white blood cells have a number of very different types of cells. The categories are; neutrophils, eosinophils, basophils, lymphocytes, monocytes, and platelets (table 19.3,Tortora, 12th ed./table 18.7,Saladin, 4th ed.,p.698). Each different white blood cell (WBC) is involved in a different type of defense against damaged tissue and/or pathogens (table 19.2, Tortora, 12th ed., Saladin, 4th ed.,p.698). When a specific type of pathogen attacks, a specific WBC responds by increasing in number and then counter-attacking.