BIOL 212: HUMAN ANATOMY & PHYSIOLOGY

PHYSIOLOGY OF THE BLOOD

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Physiology of Blood
INTRODUCTION

Overview: There are several fluid compartments in the human body (i.e., blood, lymph, synovial fluid, cerebrospinal fluid and urine). While we consider each individually in lecture, each fluid is partially dependent on other body fluids for its composition. Change in one fluids composition is typically reflected in a change in the composition of the other body fluids. Our laboratory exercise focuses on the blood component of body fluids and the formed elements found in it. Because of its many functions, knowledge of the physiological properties of blood is essential to the understanding of the complex workings of the healthy human body. Changes in the relative frequencies of blood formed elements; as well as chemical changes in the composition of the plasma, quickly reflect changes in an individual's homeostatic state. The established norms and some pathologies associated with deviations from these norms will be discussed in this exercise. SAFETY PRECAUTIONS 1.

down on the floor. Do not attempt to leave the classroom. If you are about to faint and attempt to leave the class, you may make it past your desk, into the front of the room or into the middle of the hallway prior to going down (we have all had it happen before). In any of these cases, your lab instructor will be all over you. If you fall and crack your head, you will either be escorted to the health center for complete evaluation or an EMT team will be called. The easiest way to minimize the risk to your body, if you do faint, is to minimize the distance your body must travel to reach the floor. That is why you are asked to sit down. It is partly the responsibility of the lab partners to help monitor this situation. Everyone needs to talk to the blood donor and make sure that they are "in the moment" and responding to your discussion. Introduction to Blood Collection Methods: Venipuncture is the most commonly performed blood collection technique used by health care professionals. You will not be doing this in Anatomy and Physiology 212. Skin puncture is most commonly employed method whenever small amounts of blood (like a couple of capillary tubes full) are required for analysis. Three sites are most commonly used and include the pad of the fingers, the lobe of the ear, or in infants, the heel of the foot. The finger pads are used for the exercises described in this lab. Follow the procedures as outlined below. a. The Unistik is the lancet that allows tissue penetration by a small, but sharp lancet and then automatically retracts the blade into the device for safety reasons. See figure below. b. Once the site of puncture has been selected -- middle finger typically for starters -- the subject should improve circulation of blood to that site. This can be accomplished by having the subject immerse that hand in warm/hot water for about five minutes followed by vigorous drying with a towel. c. The site, the fingertip, is then swabbed/dabbed with an alcohol swab and allowed to dry. Do not blow on the finger.

Like all other body fluids, blood can house viruses and bacterial agents -- pathogens that can cause disease. AIDS (HIV) and Hepatitis are just two examples of such contaminants that you are probably familiar with. You need not worry about exposure to these contaminants from your own blood -- you are already exposed. But you must be much more diligent when working with the fluids from someone else. You must wear gloves. Do not assume a sample is disease-free even from your best friend, instead assume it is contaminated. Do not re-use any lancet, needle or object that has been exposed to a blood sample. Dispose of all materials according to the lab instructions by placing the lancets and other sharp items in the RED Sharpee container, and place all blood contaminated paper towels, alcohol swabs, Kim Wipes into the autoclave bags. Throughout the course of the semester, you will be collecting small samples of your blood. If you already know that you cannot do this, you will not be forced to participate. If at anytime during the course of the lab you feel dizzy or faint then sit

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Now the finger is punctured using the UNISTIK assembly. Cock the lancet -- Push the plunger end of the covered lancet into the body of the Unistik until it clicks. Now twist off and discard the protective cap. This is the tip you will place on your fingertip. You will find that one determined press of the release button works best. It will make a click sound and the lancet will strike the finger and then retract. Let the blood well up naturally out of the puncture on the fingertip. Try not to squeeze the finger, but if you must, squeeze the fingertip with gentle pressure. Excessive milking of the finger will cause tissue cells to be damaged and the blood will be diluted interstitial fluid from the surrounding area. 1) Helpful tips for blood collection: Keep your finger/hand below the level of heart and let a drop of blood form. 2) The harder you milk the area, the more bruised you will be tomorrow and the sorer your fingertips will be.

After collecting the blood for use in the measurement of Clotting time, Hematoctit and Blood type, the finger should be cleansed with a fresh alcohol swab. You may even want to put a band-aid on the area, although you will stop bleeding within a few minutes. f. Cleanup of the area is essential. Once blood has been collected, the gray Unistiks -- the sharp objects -- need to be placed into a SHARPS CONTAINER, A RED BOX located at the washing area or on adjacent counters. All lancets must be put into this container and not in the trash. The alcohol swabs, and any other material with blood on it should be placed in the red/tan labeled dishpans over by the cleanup area. There are usually orange autoclave bags in this each of the bins. All bloody materials need to be autoclaved for safe disposal. ______________________________________________________________________________________

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Exercise 1: Determination of Clotting Time Platelets (i.e., thrombocytes) play an integral role in homeostasis (stoppage of bleeding). These are the smallest of the formed elements in the blood such that they are literally cell fragments of megakaryocytes with some lysosomes, ER and Golgi complex, and vesicles (i.e., granules). In a normal blood sample, there are approximately 130,000 - 500,000 platelets per microliter of whole blood. These small cell fragments have diverse functions that all directly relate to hemostasis (i.e., maintenance of blood volume). All vertebrates have developed mechanisms that minimize exsanguination (i.e., blood loss). Hemostasis is kind of a balancing act, you want to have the capability to stop bleeding, but you do not want clotting to occur when it is not necessary. How does our body accomplish this? Numerous enzymes tightly regulate the overall process. The physical act of the platelets encountering a tear in a blood vessel causes one set of enzymes to be activated. This first enzyme activates an entirely different enzyme that activates a third enzyme and so on until a plasma-soluble protein (i.e., fibrinogen) is converted to an insoluble protein (i.e., fibrin) and a clot is created. The time it takes for the entire process to occur is important. Too fast, and the system may be over-responding to a minor situation. If your clotting time is too slow, you may have already lost enough blood to compromise your health and wellbeing. Three processes are involved in prompting the stoppage of blood flow: vasoconstriction, platelet plug formation, and clot stabilization. In a typical human population, clotting times range from 6 -12 min. Problems with clotting are common causes of disease in the heart and brain. Inappropriate formation of blood clots in the arteries of the heart or brain can cause a heart attack or stroke because the clot can block the supply of oxygen rich blood from reaching dependent tissues. If the blood cannot clot normally, you could have excessive bleeding from a cut (common in hemophiliacs) or excessive bleeding in the brain (cerebrovascular accident [CVA] and a stroke). In the case of a CVA, blood accumulates around the break in the vessel and this puts pressure

on the brain tissues that can cause physical trauma to the cells. The break in the vasculature disrupts blood flow to the tissues downstream from the cut and these tissues undergo hypoxia and potentially cell death (necrosis). Blood samples can be collected with agents to prevent clotting (anticoagulants such as heparin, sodium citrate and EDTA) or they can be collected without such agents. When the samples are collected without anticoagulants, clotting will occur and the plasma protein fibrinogen will be converted to sticky fibrin. Fibrin polymers create sticky nets that catch and attach to the wall of the vessel and to any formed elements, especially platelets and other blood cells that are rushing out the hole. Stabilization of the clot occurs when the sticky fibrin monomers polymerize into a larger net and becomes filled with cellular debris. When blood samples have been allowed to clot and the fluid component is separated, the solution is devoid of fibrinogen and is called serum. Hematocrit capillary tubes are very thin walled glass tubes that classically will contain no more than a good drop or two of blood. We will use non-heparinized hematocrit tubes (be sure to check the label usually in blue) to determine the clotting time and heparinized hematocrit tubes (again check the label-- usually in red) to determine the hematocrit values. Procedure for Determining Clotting Time:

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6. This is generally a very hard test for students to complete, in part because it requires 2-3 capillary tubes full of blood. As an alternative lets complete the online section presented by McGill Medical School. Follow this link: http://www.medicine.mcgill.ca/physio/vlab/bloodlab/he mostasis_n.htm Once at this site, click on the clotting time video. Watch the video. After you have watched the video and if you still want to try this on your own, follow the directions below. Check with your lab instructor prior to beginning. 1. Obtain a sterile alcohol swab, UNISTIK assembly, and 2 non-heparinized

microhematocrit capillary tubes -- ones with blue rings around one end. Place the hematocrit tubes on the side of the desk. Collect a droplet of blood on your fingertip using the procedure described above. Have a lab partner hold the capillary tubes so that about the tube is suspended off of the table. (Your lab partner will have to keep a finger on the tube for blood collection.) Once you have a large drop of blood pooled on the tip of your finger, move your finger to the open end of the suspended of the hematocrit tube. Blood will automatically be drawn into the tube by capillary action. Fill the tube to almost full. The more sample you have the better your results. Carefully put your finger over the end of the tube while keeping the tube horizontal. With your finger over the end of the tube, plug the other end with the clay sealant provided. ONLY seal one end of the tube. Then remove your finger from the top of the tube and twist the tube out of the clay. You should only seal one end of the hematocrit tube! You must have two full capillary tubes to complete this exercise. Have your lab partner note the time when you have filled the capillary tubes. At two minutes, using your thumbs and forefingers, break off a small piece of a capillary tube and carefully, and slowly, pull the two broken pieces apart. Look for sticky threads of clotted blood between the broken pieces of the tube. Continue to break small pieces off of the capillary tubes every 30 sec until fibrin threads (the above sticky threads) are clearly visible in the sample and you can confirm that clotting has occurred. Record this as your clotting time. (usually between 3-5 min) Report your clotting time to your lab instructor. They will determine the average and standard deviation for the class data. Then compare these values to those of other class members and then compare your value to the standard number provided in your text.

Once the procedure is complete you need to analyze your class data and summarize your findings. Follow the directions on the exercise sheet (See Back of Lab).

Remember that hematocrit tubes are made from glass, if they are broken they can be SHARP! Place them and any other potentially sharp objects in the RED SHARPS CONTAINERS.

Exercise 2: Procedure for Determination of Hematocrit: Blood is this wonderful body fluid and is maintained at a given level in our body. For most individuals, this volume is about 8% of their total body weight although this varies with age and for the sexes. At any moment in time within the vascular system about 70% of that volume may be found in the veins, 20% in the arteries and about 5% in the capillaries. (The remaining 5% is the error of these measurements.) One of the many blood parameters routinely measured is the hematocrit. The hematocrit is the percentage of blood volume that is composed of formed elements (mostly erythrocytes). The hematocrit can also be called the packed cell volume. It is determined on a venous sample of blood and normally about 44% for an adult. So why worry about this number? This value gives a physician a small bit of information about the composition of your blood and therefore provides information concerning a component of your entire cardiovascular system. Erythrocytes, specifically the hemoglobin inside them, carry oxygen to our body. Therefore, a low hematocrit means a low oxygen carrying capacity to our body. Changes in the bloods ability to carry oxygen may be related to many things, but for our discussion we will focus on cell number and hemoglobin concentration in each red cell. Too few erythrocytes and therefore a low hematocrit will mean a low oxygen carrying capacity. But a second, and sometimes confounding factor is the actual concentration of hemoglobin inside of the red cells. If you do not have enough iron in your body to create hemoglobin or you do not have enough hemoglobin in your red blood cells, you are anemic. The physical number of cells in your blood can also influences the viscosity of your blood. Viscosity relates to the bloods ability to flow. Physiologically, blood viscosity is related to three hematology properties: the blood-cell content, the plasma-protein concentration, and temperature. There are a number of situations that can alter these components, although temperature effects can be minimized based on the idea that body temperature rarely fluctuates more than a couple degrees, even with a fever. However, if you are dehydrated the relative volume of plasma in the blood becomes less and the blood becomes more viscous. So under severe dehydration, you would have a high hematocrit and your heart has to work harder to pump the blood through the body. The same thing also occurs if your body makes too many cells (i.e., polycythemia). Any change in the blood viscosity changes the resistance in the cardiovascular system and alters blood flow. Let's determine your hematocrit and then compare your results with that of the class. 1. Obtain a sterile alcohol swab, UNISTIK assembly, and two heparinized capillary tubes -ones with a red ring around one end. (Heparin is an anticoagulant that prevents the clotting of blood. You want it to be in these capillary tubes so that your sample does not coagulate prior to centrifugation.) Collect a sample of blood from your finger using the procedure described previously. Once you have a large drop of blood pooled on the tip of your finger, submerge one end of the hematocrit tube into the droplet of blood. When almost full, seal one end of the tube with clay sealant. Place your tube in one of the numbered slots in the hematocrit centrifuge. Make sure that the sealed end of the capillary tube is facing out, away from the middle of the tube holder. Your lab instructor will start the centrifuge when the tray is full, or near full. The centrifuge will be run for 5 minutes and then you can determine your hematocrit. Place your hematocrit tube next to the centimeter scale on a small ruler. Slide the tube along until the top of the sealant is at the zero point of the scale. Measure to the top of the packed cell volume in millimeters and record that number. Now determine the total blood volume to the top of the plasma and record that number. The hematocrit is the percentage (packed cell volume)/(total volume) X 100. Example: total volume of hematocrit tube filled with blood =200 mm, part of tube filled with formed elements 66.7 mm, part of tube filled with plasma (133 mm). The hematocrit is 66.7/200 X 100% = 33%. This hematocrit would indicate that the person, no matter if male or female, has too few red cells and more than likely suffers from anemia and potentially experiences poor oxygen delivery.

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Once the procedure is complete you need to analyze your class section data and summarize your findings. Refer to the exercise sheet for helpful questions that will guide you through the thought process.

Exercise 3: Determination of Blood Type and Antigen-Antibody Reactions: All cells have glycoproteins on their outer surface (review the structure of the plasma membrane focusing on integral and peripheral proteins). The particular combination of proteins is the same for all of your body cells, and is used by your body to distinguish self from cells of any other individual or organism. These protein markers are called antigens. One of the jobs of your immune system is to perpetually scan cells in your body to make sure they are your cells and not the cells of an invading pathogen. (Any bacteria or virus, is a pathogen and will have a distinct set of antigens on its membrane.) In most cases, the introduction of foreign antigens into the body causes activation of the immune system and results in the production of sensitized lymphocytes -WBC's that attack and destroy the pathogen directly, and/or by activating plasma cells (other immune system cells) to produce antibodies to specific antigen combinations causing agglutination and pathogen destruction. In RBC's these antigens are mucopolysacchrides (Figure 18.12, Human Anatomy and Physiology, Saladin, 2006). RBC antigens are called agglutinogens and are the basis for the various blood types found in the human population. The presence of these agglutinogens is genetically determined and varies among ethnic groups. The most common blood grouping is the ABO blood group. In this case there are four blood types: A, B, AB and O. And your blood type is determined by the agglutinogens on your cells. You may have type A agglutinogens, type B agglutinogens, both type A and B agglutinogens, or express no aggluitinogens on your cell surface. (Note that with type AB blood there are two different mucopolysacchrides present on the surface of every red cell.) Aside from these surface antigens, your body also spontaneously makes antibodies for the mucopolysacchride not expressed on your red blood cells. These antibodies (plasma proteins) are called agglutinins. By definition, the anti-A agglutinins react with A agglutinogens and anti-B agglutinins react with B agglutinogens creating congealed masses. It is this binding of the agglutinins with the agglutinogens that is the basis for transfusion reactions. It causes severe blood clumping and clot formation. A second type of blood group antigens that you may be familiar with is the Rh blood group. This is what gives your blood type the + or determination. Again this blood type is genetically determined and is distinct from the antigens of the ABO group. Like the ABO group, the frequency of occurrence of this group 2. 3. of antigens varies with ethnicity. The difference between the groups is that anti-Rh agglutinins are not normally present in the blood, they are not spontaneously produced. They are formed only in Rh(-) individuals (one that would never express the antigen on the surface of their red cell) and only once that individual is exposed to Rh(+) blood (red cells covered with these antigens). This means the first exposure of an Rh(-) individual to Rh(+) blood will initiate an immune response. But it takes time for the body to build these new plasma antibodies, these agglutinins. So the person is in little danger the first time of exposure. It is the second time, and every time thereafter, when a life-threatening situation can occur. This is extremely important during pregnancy when an Rh(-) mother is carrying an Rh(+) child (see Pgs 695-698 of Saladin to understand what happens after this second exposure). Let's determine your blood type and compare your results with that of the rest of the class. 1. Obtain a sterile alcohol swab, Unistix assembly, 2 clean glass microscope slides, a wax marking pencil and a couple of toothpicks. Hold the clean slide by the edges like you were going to examine the contents of the slide and with the pencil, draw a line vertically through the center of one of the microscope slides thereby dividing it into two equal halves. Label the upper left corner A and the upper right corner B. On the second slide write Rh in the upper right corner. Collect a sample of blood from your finger using the procedure described previously. Once you have a drop of blood pooled on the tip of your finger, touch the tip of your finger to the left side and then the right side of the first slide. A second drop of blood needs to be added to the Rh slide. Perform Steps 5 and 6 QUICKLY so the blood sample does not dry out. Add a drop of Anti-A serum to the left side of the first slide (the divided slide) and Anti-B serum to the right side of the slide. Then add Anti-Rh serum to the second slide. (Sometimes this preparation needs to be warmed slightly to work. If this is the case, put the slide on a light box for a brief time and then remove and agitate the slide.) Use a toothpick to mix the anti-serum with the drop of blood. Use a different toothpick for each sample. Place the slide on a white background and observe changes in the texture of the mixture. If there is clumping of the cells -- the mixture appears grainy, or blotchy; this is a positive reaction and means your cells contain that specific agglutinogen. If there is no clumping of cells and the mixture looks homogenous, it is a negative reaction and that means you lack that agglutinogen. See Saladin Fig 18.13-15 to

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understand what clumping looks like and refer to the table below. 8. Common Blood Types Type O Type A Type B Type AB Rh positive Rh negative

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Based on your results, determine your blood type.

Agglutinogens on RBC's None A B A&B Rh None

Agglutinins in Plasma Anti-A & anti-B anti-B anti-A None None anti-Rh

What is the universal Donor blood type? What is the universal Acceptor blood type?

Once the procedure is complete you need to analyze your data and summarize your findings. Refer to the exercise sheet for helpful questions that will guide you through the thought process.

The American Red Cross has an excellent website for this: http://www.redcrossblood.org/learn-about-blood/bloodtypes

Exercise 4: Cell Morphology and Identification (Complete this in Open Lab if you are unable to complete it in your lab section time.) 10X and move the stage down very slowly. You should be able to see a "carpet" of pale red blood cells dotted with distinct blue leukocytes. Blood smears are created using a couple of drops of blood and literally smearing and preserving the blood to the slide. The cells are then dyed with Wrights stain and the individual cell types can be differentiated. Whole blood can be divided into two components: formed elements that are enclosed structures surrounded by a plasma membrane and plasma, the fluid in which the formed elements are suspended. The formed elements, in turn, can be divided into two categories: the platelets that are tiny fragments of a larger cell (megakaryocyte) and all the other blood cells (i.e., red blood cells and white blood cells). Unlike normal body cells, platelets are bags of enzymes necessary for coagulation. The blood cells are also divided into two groups: red blood cells (erythrocytes) which contain oxygenbinding and iron-rich hemoglobin (which makes them dense so they undergo sedimentation/they sink), but no nucleus at maturity; and white blood cells (leukocytes) which have nucleus at maturity. The purpose of this part of the lab is to teach you how to distinguish between the different blood cells on stained blood smears. You should be able to identify the different cell types on a slide based on their appearance. Before you start, clean your slide with lens paper. It is often hard to locate the thin layer of blood cells on the slide, you will focus right through them. Try starting at When you have found the layer of blood cells, scan the slide, looking for an area where the cells are spread out and where the cell outlines are round, rather than angular or clumped together. If you are having trouble finding the cells on your slide, try looking at your partner's slide to see what the cells should look like. (If you are still having trouble, ask your lab instructor for assistance.) When you can see the cells under low magnification, switch up to 40X to see them in more detail. A hint: when you are looking for a particular kind of leukocyte, it is often helpful to search for candidates at 10X and then check them out at 40X. We will not use the 100X objective that requires immersion oil that can damage the other lenses if used improperly. The most numerous cells on the slide are the erythrocytes. They may appear pinkish or bluish depending on the stain, see picture below. They often appear lighter colored in the middle due to the biconcave shape of the cells. (Biconcave means that both sides of the central part of the cell are pushed in toward the center of the cell.) They are also lighter in color since they lack a nucleus at maturity.

Look carefully to see if your slide has numerous tiny specks between the erythrocytes. If so, these are likely to be platelets. Other slides may not have visible platelets because these fragile structures were destroyed by the staining procedure. Now let's dive into the leukocytes. For purposes of identification it is useful to split the leukocytes into two groups: those with compact rounded nuclei and those with "lobed" nuclei -- where the nucleus consists of two or more interconnected, irregular chunks. Let's look first at the rounded group consisting of the lymphocytes and monocytes. Perhaps the easiest of all the leukocytes to identify are the lymphocytes. They have a completely round nucleus. The other rounded leukocytes, the monocytes are much larger than typical lymphocytes and usually have an indentation in an otherwise smoothly-rounded nucleus. Their nucleus is often described as being "C"-shaped, when viewed from the side. You may have to scan your slide at 10X to find this relatively rare form of cell. The lobed leukocytes consist of the neutrophils, eosinophils and basophils. Basophils are very rare, and we will not worry about them. You probably will not find one on your slide. (If you are curious, they are dark cells with many dark granules that obscure the nucleus. You probably saw them in areolar connective tissue as mast cells. Mast cells in the blood are called basophils.) The neutrophils have a nucleus with several lobes connected by thin strands. They are quite numerous. A second form of the neutrophil exists, the band neutrophil. It has a multilobed nucleus, but the lobes are connected together by broad connections into a linear structure. It is often confused with a monocyte if the ends of the band are bent into a "C" shape. To distinguish between band neutrophils and monocytes, look for the irregular edge of the band nucleus as

opposed to the smooth edge of the monocyte nucleus. Eosinophils are the final leukocyte we will view. These cells have a bilobed nucleus. To verify the identity of a suspected eosinophil, look for the pinkish granules in the cytoplasm. Verification by granules is a good idea because a monocyte or band neutrophil viewed from the edge may appear to have two lobes. Eosinophils, like monocytes, are rare so consider scanning at 10X if you are having trouble finding them. A Differential WBC Count is used to examine how common each type of WBC is relative to the total number of WBCs observed. For examples if you observed 1000 white blood cells and 20% of the WBCs observed were neutrophils this would be clinically significant because neutrophils should represent 60-70% of all WBCs in an average person. A low (-penia or poor) neutrophil differential count would indicate neutropenia (neutrophil counts below 2000) and a poor ability to fight against bacterial infections. Another View of the formed elements: (Tortora and Graboswski, 2003): Identify which cells have each of the following key characteristics: 1) Clear cytosol and a round nucleus that fills almost all or most of the small-sized cell, 2) Clear cytosol and a large cell with a horse-shoe (or lucky) shaped nucleus, 3) Grainy cytosol with a chain-like nucleus, 4) Grainy and very dark purple, 5) Grainy orange-pink cytosol, 6) Very tiny pale anucleate objects, and 7) Anucleate biconvar cells. In the diagram below macrophages are what monocytes become when they patrol places like the surface of your lung phagocytosing bacteria. Consider T- and B- lymphocytes as the same for the purpose of this lab. For your own curiosity remember that plasma cells are the active lymphocytes that produce antibodies when you have a viral or bacterial infection.

One of the easiest ways to learn the blood cells is to make a dichotomous key. But the only tried and true way is to spend several hours looking through a microscope at a slide.

Celluar elements without a nucleus and numerous on smear Cellular element red/pink in appearance. Circular in appearance Cellular elements that are very small compared to other not circular Cellular elements with a nucleus Cell noticeably grainy in appearance (red or purple specks in cell obvious difference) Cell with bilobed nucleus, grains typically red in color Cell very grainy, appears to almost have no nucleus but it is present Cell not noticeably grainy in appearance Cell with multilobed nucleus (usually 3-5 lobes) Cell with intact nucleus Nucleus U shaped or horse shoe shaped Nucleus mostly spherical almost filling cell, maybe flattened

..RBC ..Platelest

..Eosinophil ..Basophil ..Neutrophil ..Monocyte ..Lymphocytes

On the Lab Exam you will have one or two microscopes with a peripheral blood smear slide on it and a blood cell at the end of the pointer, you will need to identify the cell based on the characteristics you observe). This takes time to master and will require time spent in the open lab sessions.

Virtual Blood Lab online http://www.medicine.mcgill.ca/physio/vlab/default.htm

Additional information: Venipuncture is typically performed upon the antecubital vein on the side of handedness of a subject. The vein can be palpated, and in most instances, the vein can be made more prominent if the subject opens and closes his/her fist. Once the vein is located, a soft rubber tourniquet is applied to the arm approximately 3 inches above the elbow. The arm surface is then prepared for venipuncture by swabbing the antecubital fossa with a sterile alcohol swab. Blood is taken with either a conventional syringe or with commercially-available evacuated tubes fitted with an appropriate needle.