A&P 2 Blood Lab Guide In-Lab Exercises Have someone in your group read the following out loud, while the others read along: In this "Lab Guide", we will be looking at the blood and some common blood tests. These labs change greatly from semester to semester. Sometimes the student draws blood; other times they do not. Ask your instructor if you need to follow special precautions this semester. For each of these, an explanation is followed by a short lab. For several of these, your group will be drawing a small amount of blood. Hepatitis is a group of extremely contagious diseases. Please be sure to dispose of all materials properly. Do not come in contact with another person s blood. And, before drawing blood, make sure your group has the following: Surgical gloves Paper towels (LOTs of them) Antiseptic table-top cleaner Alcohol Swabs NOTHING GOES INTO REGULAR TRASH! All disposable items must be thrown away in: 1. one of the Sharps containers (glassware or other breakables) 2. the plastic "autoclave" bag
All non-disposable materials that are exposed to blood must be placed in the BLEACH BATH at the back of the room (by the sink). NOTE: we no longer have a Coulter Counter in lab. However, I still expect you to be able to read and interpret a Coulter Printout (example later in this guide). The Steps found in this first "Pre-lab Guide" do NOT have to be done in the order they are found. However, please do all the questions within a step before moving on to another step! Note: For all of the blood tests we mention, know the normal values. If there is a slight discrepancy between the lab book, lecture book and some other source, STICK TO THE NUMBERS FOUND HERE. We will be talking about some blood tests that we won t actually preform. However, we ll look at the results of these tests on the Coulter Exam. At the end of this lab, we will be looking at the Coulter Counter Readout.
Step 1. Blood Typing #1 Read Me Review Key Concepts from the book and videos Have someone in your group read this, while the others follow along: We will be typing blood for the ABO and Rh blood groups. This is done by collecting a small amount (a few drops) of blood and adding to it a drop of known antibody preparation. If the antibody causes agglutination, then the matching antigen must have been present. If the antibody fails to cause agglutination, then the matching antigen must have been absent. Pay Attention! #2 In this exercise, the student must be able to: 1. Explain the basis of ABO blood typing, explaining the terms antigens, antibody, and agglutination. 2. Explain the basis of Rh blood typing. 3. Identify the ABO type and Rh of a blood sample. Get prepared for drawing blood. Before beginning, bring to your station several brown paper towels (5 or so), and several toothpicks.
#3 Let s collect data! We will be using blood sample in the back of lab. There are 4 samples, each from a different person. You will determine the blood type of each sample, and determine which antigens & antibodies the person has in their blood. Do This! 1 PUT ON SURGICAL GLOVES! 2 Obtain a blood sample tray as seen: 3 Read Me To test both ABO and Rh blood types three pools of blood are required from each patient; one for testing with antibody A, one testing with antibody B, and one for testing with the Rh antibody (anti-d). We have blood samples from 4 patients in the back of the room, on a mixing tray as show here (the apparatus may look different, as we have two!).
Do This! Use a dropper to put three small pools (a few drops) of blood from a patient. Do not contaminate blood from 1 vial with the blood of another vial! Use this image, following the steps (1, 2 & 3): 4 Do This! Also in the back, in an ice bath, are 3 flasks containing antibodies (Anti-A, Anti-B, and Anti-D). Bring them to your desk, add antibody to your blood sample as described below, and then get them back to the ice bath because THEY GO BAD!
5 Add antibody to the blood sample, following the image below: Anti-A is added to pool #1, Anti-B is added to pool #2, and Anti-D (Rh antibody) is added to pool #3. Read Me Note: Agglutination reactions with A or B antibodies is an immediate and relatively obvious reaction. Rh agglutination is less obvious and many take several minutes to be noticeable.
#4 Interpret the results on the DATA ANALYSIS SHEET below! We will be using blood sample in the back of lab. There are 4 samples, each from a different person. You will determine the blood type of each sample, and determine which antigens & antibodies the person has in their blood. The pattern of agglutination (or non-agglutination) indicates both ABO and Rh blood types. REMEMBER: agglutination indicates a POSITIVE result (the antigen is present). For example, if agglutination occurs with the addition of all three antibodies, then all three antigens are present and the person is AB(+). Or, if none of the antibodies causes agglutination, then all three antigens are absent and the person is O(-). Know the basic trends as seen on this table for the exam: DATA/ANALYSIS SHEET Do This! Write down the subjects' blood types: Subject A: Subject B: Subject C: Subject D:
#5 Assess your understanding of what you ve just done by answering the following questions. This may be done outside of lab. Use another sheet of paper to answer these so you can study them for the exam. Q1. You test a friend. Subsequently, agglutination occurs in pool #1, and pool #3. What is the patient's blood type? Which antigens do they produce naturally? Which antibodies do the produce naturally? Q2. DISCUSS THIS and take notes: When you cut yourself, would you expect an agglutination reaction to occur at the bleeding site? Explain why or why not. Q3. DISCUSS THIS and take notes: It is important that we do these tests outside of the body. It would be dangerous to apply the antibodies in the patient's bloodstream. Why? Q4. DISCUSS THIS and take notes: Explain why erythroblastosis fetalis is considered an incompatibility between mother and fetus by answering these questions: What is incompatible? Which antigen(s) is/are involved? What must mom s type be for this to be a possibility? Which antigen is present: Which antibody might be present: What must the fetus type be for this to be a possibility? Discuss why this is only a danger for the second (or subsequent) pregnancies:
Step 2. Hematocrit (Hct) #1 Review Key Concepts from the book and videos Have someone in your group read this, while the others follow along: Read Me Your hematocrit is the percent by volume of your blood that is cellular (as opposed to plasma), technically the percent by volume of RBC's. In a sample of WHOLE BLOOD, males have an average hematocrit of 38-54%, females 36-47%. Readings outside of these normal ranges can indicate a blood abnormality, or might simply be due to environmental reasons. For example: Anemia: Lowered ability to deliver oxygen to the tissues. There are many causes. One form of anemia is due to a low hematocrit, independent of why. This may be due to a bone marrow problem, dietary deficiencies, etc. Polycythemia: High Hematocrit. Most cases of this are benign (not a problem). For example, if you move to a higher elevation, there is less oxygen in the air. Your blood will not have enough RBC to deliver O2 to the tissues, despite the fact that the RBC count is normal (for lower elevations). After a few months, your body will respond by making more RBCs. You will have polycethemia (High hematocrit), although it will be perfectly normal! This will last for a few months, which is why athletes like to train at high elevations. It gives there blood a high oxygen capacity when they compete at sea level. Blood analyzers, such as the Coulter Counter, estimate hematocrits in a fraction of a second. However, we no longer have a Coulter Counter. Do this! Please make sure you see where to find the hematocrit value on the Coulter readout. Read Me An alternative method is to centrifuge blood that has been collected in a capillary tube, and then estimate hematocrit using a "reader" device of some sort. Hematocrits taken by use of a centrifuge are referred to as "spun" hematocrits.
#2 Use of a blood analyzer to read hematocrit will be demonstrated in lab. Read Me The technique for taking a "spun" hematocrit will be demonstrated in lab. Use each of the "readers" provided to read a spun hematocrit and check the accuracy of your reading with your instructor. Some semesters, we ll be drawing blood and reading the hematocrit from the sample(s). Other semesters, we ll be using fake blood in pre-prepared tubes. In either case, know how to use both hematocrit readers, and know what a centrifuge is. Do This!
The spun sample has various important features. Do This! Read Me Now, we are ready to take the patient s hematocrit, which is the percentage of RBCs in the entire sample. Remember this from the previous page: Do This! To determine the percentage of the red area, we can use one of several Hematocrit Readers. Use all in the lab room, because you do not know which will be on the lab practical: Directions for use of the spun hematocrit readers: Hematocrit Reader #1 -- Place the bottom of your red layer (top of the wax) on the zero line. Move the lever in a circular manner until the top of the plasma hits the 100 line. Look straight down at the tip of the RBC layer and read the number.
Hematocrit Reader #2 -- Place the bottom of your RBC layer (top of the wax) on the red line found on the plastic plate. Make sure the bottom of the RBC layer is on the lowest black line on the scale ( zero ). Now, you need to make sure that the top of the sample is on the upper-most black line (100). Slide the capillary tube over until this is the case. Read Me #3 Hematocrits (Hct) are always given in PERCENT! Average Hematocrit Males = 38-54% Average Hematocrit Females = 36-47% Let s collect data! There are 4 test hematocrit vials. Write down what you read the hematocrit as for each: Hematocrit 1: Do This! Hematocrit 2: Hematocrit 3: Hematocrit 4:
#4 Assess your understanding of what you ve just done by answering the following questions. This may be done outside of lab. Use another sheet of paper to answer these so you can study them for the exam. Q5. DISCUSS THIS and take notes: If a person was suffering from an anemia, would you expect their hematocrit to be high or low? Explain. Q6. DISCUSS THIS and take notes: Why is it important for males to have higher average hematocrits, RBC counts and hemoglobin readings than females?
Step 3. Blood Cell Identification #1 Review Key Concepts from the book and videos Have someone in your group read this, while the others follow along: Read Me Recall from Hematocrit discussion: Blood is formed of 2 components namely blood plasma and blood cells. Out of these plasma constitutes 55% of the blood while 45% is contributed by blood corpuscles or cells. Out of the 45% that are cells, about 92-94% are RBCs, 0.2% are WBCs, and 6.7% are platelets. Easier to remember: ~94% = RBC ~6% = platelets <1% = WBCs A magnification of 200 times is enough to allow you to observe and identify the different types of cells. If you use a higher power, you can also see the cells details better. You can use the highest objectives on your microscopes or with the oil immersion technique (if you know how). You will be tested at medium to high power. NOTICE: there is more than 1 type of blood slide in the lab. Regular blood smears do not have a lot of white blood cells on them. To see WBCs, see the slides marked Basophilia and Eosinophia, which have a lot of those otherwise rare cells. For each cell type: 1. Be able to identify under the microscope. 2. Know their relative amounts under normal circumstances. Sometimes your book gives you amounts in Cells/mm3 (ul), sometimes as a percent of total. Here, we will stick to percentages of blood cells (which are easier numbers to remember), unless the table gives you other numbers. The Coulter Counter Readout at the end will tell you normal values in Cells/mm3 (ul), so be able to read the readout! 3. Know their role in he bloodstream. There is a summary table for each cell type!
#2 Get a microscope and some blood smear slides. We will learn about them while we look at them! 1 ERYTHROCYTES look at slides Normal Blood Smear and Sickle Cell Amemia. The red cells are very numerous in the blood (see a in the image below). They shape is described as a biconcave discs, annucleate, and strictly carry gasses (oxygen and carbon dioxide). Usually, they measure 6 8 μm in diameter. In the observation field of the microscope, you will see a lot of erythrocytes and, sometimes, some isolated leukocytes. Under the microscope, the RBCs look like pink discs clearer in the middle. As we saw, the red cells can also have different shapes from those we described. Sometimes, this is normal, while at other times, this is due to diseases or to defective process of preparation and staining of the smear. Description Presence in blood Role Annucleate Biconcave discs Pinkish under scope 94% of cells (hematocrit tells us the % in whole blood of the patient) 4.2-5.0 million /mm3 (ul) Transport of respiratory gases.
Look at the slide that says Sickle Cell Anemia. THIS SLIDE WILL NOT BE N THE EXAM! See if you can find any sickled cells. They can be difficult to find! RED Blood Cell Count The red blood cell (RBC) count is a blood test which determines the number of red blood cells, or erythrocytes, in a sample of blood. This test also evaluates the shape and the size of the red blood cells. All of this information is then used to determine the number of red blood cells per microliter of blood. Red blood cell count values vary according to the age and the sex of a patient. The RBC count ranges from 4.2-5.0 million red blood cells per microliter of blood for women and 4.6-6.0 million for men. 2 PLATELETES (thrombocytes) cell fragments containing clotting chemicals Hemostasis.- the ability of the blood to form a clot protects the individual from excessive bleeding from minor wounds. Summary of clotting (more detail in lecture): Clotting involves two plasma proteins, prothrombin and fibrinogen, both of which are synthesized in the liver. To form a clot prothrombin must first activate to thrombin. Thrombin in turn causes fibrinogen to convert to active fibrin. Fibrin is a protein that organizes into long, sticky threads that form the basic meshwork of the clot. Bleeding stops when the fibrin mesh has become fine enough to trap RBC's, thus forming a plug of fibrin and RC's which blocks the wound. Description Presence in blood Role Discoid cell fragments About 6% of the cells 150,000-400,000/mm3 (ul) Hemostasis (clotting)
The reactions that lead to prothrombin activation involves: 1. calcium ions 2. a substance called thromboplastin 3. blood platelets and chemicals they contain which we will call platelet factors 4. a group of chemical compounds which we will call the accessory factors. 5. Vitamin K is also important for clotting in that it is needed for the synthesis of prothrombin by the liver. Intravascular clotting within healthy vessels is normally prevented by an anticoagulant called heparin which is present in the plasma. Heparin is thought to be produced by mast cells found in the tissues of various organs. Coumarin is the most common "anticoagulant" drug. It is a vitamin K inhibitor. Some causes of clotting problems: Hemophilia is the inability to make one of the clotting factors. Calcium deficiencies often express themselves as clotting problems. Any systematic antibiotic that kills the bacteria in the gut that provide us with vitamin K can lead to clotting problems. A malignancy of the bone marrow, leading to lowered platelet formation. Testing for Clotting Neither of these two tests are tests we have current capability to do in lab at Madison College. Know their names and significance for the lab practical. Historically, there are a number of different test procedures that have been used to estimate a person's ability to clot. Look for them on the Coulter Counter readout later! Two of the more sophisticated are: 1. PT Test = Prothrombin Time Test This test determines the amount of prothrombin in the blood and is a test of the extrinsic clotting pathway. It may be used to follow the effects of coumarin, or other Vitamin K inhibitors, since Factor VIII which is part of the extrinsic pathway, but not part of the intrinsic pathway, is most sensitive to Vitamin K. 2. PTT Test = Partial Thromboplastin Time Test This test is a test of the intrinsic pathway. If both PT and PTT test times are prolonged, then the problem is with the common pathway.
3 LEUKOCYTES look at slides Normal Blood Smear for the more common ones, and then neutrophilia and basophilia There are 5 types of WBCs, although each type can have many sub-types (we won t look at that here). Leukocytes play important roles in the immune system. They are in the blood so they can be transported to areas of infection or damage. Many can then leave the bloodstream in order to perform their role in immunity within the tissues Unlike red cells, leukocytes have a nucleus. It is easily visible under the microscope. The nucleus of these cells can show multiple lobes, or be indented or kidney-shaped. Usually, the shape of the nucleus of various kind of leukocytes is typical, and can be used to ID the different WBCs. There are 2 broad classifications of WBCs, based on the presence or absence of granules. Leukocytes are divided into granulocytes and non-granulocytes (lymphoid cells). A Total WBC count looks at how many there are all together, without classifying them. We can also count the individual types, which may help deduced what the patient is infected with. Description Presence in blood Role Lymphocytes (WBCs) < 1% of whole blood, 5,000-10,000/mm3 (ul) is a Total WBC Count (all of them counted together) Immunity (see below for specifics) Granulocytes (i) neutrophils 54 62% of all white blood cells Phagocyize bacteria in acute infections (ii) basophils. <1% of all white blood cells Release histamine and heparin, among other chemicals. They become Mast Cells when they leave the bloodstream (iii) eosinophils. 1 6% of all white blood cells Destroy parasitic worms, involved in inflammation during an allergic response.
Agranulocytes (i) leukocytes 28 33% of all white blood cells (ii) monocytes 2 10% of all white blood cells Several types with several roles. More in the immunity chapter. For now, know that 1 type, B-lymphocytes, make antibodies. During CHRONIC INFECTIONS (lasting more than 1-2 years), monocytes migrate from the bloodstream to other tissues and differentiate into tissue resident macrophages. Distinguishing features: 1. Neutrophils ( b in image below left): The neutrophil are the more common leukocytes. The cytoplasm is transparent because its granules are small and faintly pink colored, so you may mistake it for an agranulocyte at first...but that multi-lobed (4 or 5 lobes!!) nucleus is VERY typical. The image on the right shows lots and lots of neutrophils (condition called neutrophilia )
2. Eosinophil: ( c in image on previous page) The eosinophils are quite rare in the blood. They have the same size as the neutrophils. Generally their nucleus is bi-lobed. The cytoplasm is full of granules which assume a characteristic pink-orange color. As with the neutrophil, the nucleus is still easily visible. The nucleus is described as telephone-handle shaped, for those of you old enough to remember old telephone receivers! 3. BASOPHIL Basophils are the rarest leukocytes: less than 1 %. However, a lot of people are a little high in their number as so many people have slight allergic responses. Cytoplasm is very rich in granules which take a dark purple color. The nucleus is bi- or tri-lobed, but it is hard to see because of the number of granules which hide it.
4. LYMPHOCYTES - Lymphocytes ( d on lower image left) are quite common in the blood: 20-40%, 8-10 Im in diameter and generally they are smaller than the other leukocytes but they are still a few larger than red cells. The cytoplasm is transparent. The nucleus is round and large in comparison to the cell and it occupies most of it. Often, the cell is mostly nucleus, with the cytoplasm appearing as a halo around the nucleus. Notice they are about the same size as the RBCs (see image on right, high power). 5. MONOCYTES Monocytes are the biggest leukocytes. They have a great kidney-shaped or horseshoe-shaped nucleus, in some cases even bi-lobed. The cytoplasm is transparent, but with an appearance of "ground glass". Monocytes become macrophages during chronic infections, although you won t be seeing them in the blood stream.
Step 3. Blood Cell Identification #1 Review Key Concepts from the book and videos Have someone in your group read this, while the others follow along: Read Me In most health-care situations, most of the blood tests will be done by a machine. One of these is a Coulter Counter; but there are many, and their readouts look similar. Here, we learn what to look for on a read out. After watching the videos online covering the Coulter Counter readout, answer the questions below. Make sure you can: 1. Recognize the common blood test abbreviations, and be able to read their normal expected values. 2. Be able to read the patient s values. 3. ID the units for each test. For A&P lab, you can ignore MCV (mean cell volume), MCH (mean corpuscular hemoglobin), MHCH (mean corpuscular hemoglobin concentration), and RDW (I m not even sure what that means!!). Notice that there is a column indicating if the exam is higher or lower than expected.
Q7. Is there any sign of infection? Allergies? Q8. Which of the above values are not in the normal range? Why did you choose them? Q9. Are some of them questionable results (that is...way out of range)? Why might they be questionable? What might you ask the patient? (HINT: think about what we learned in STEP 2 ).