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BIO 202 Blood Lab Goals for this activity: 1. Safely acquire and analyze a sample of a few drops of blood 2. Use a centrifuge to separate out the components of blood, including hematocrit. Understand why that works and what you are seeing in the capillary tube with your blood in it. 3. Type your own blood and appreciate what blood type means in regard to the antigens on your blood cells and what types of donors can be used for transfusions to what types of recipieints. 4. View your own blood under the microscope and identify its various cellular components. Getting the BloodFinger Prick You will need three drops of blood for three purposes Hematocritto see volume of cells and plasma in your own blood Blood smearto view your own blood under microscope Blood typingto see antigen reactions for typing your own blood (A,B,O,Rh) Be sure you have the following ready before you prick your finger Sterile lance Alcohol pad Hematocrit or capillary tube Two microscope slides (for smear) Blood typing card Helper wearing gloves Read the entire lab, get everything ready and then do these steps quickly in this order: 1. FINGER PRICK: Wipe with alcohol, let dry, prick with lanceuse side of fourth finger. 2. DROP ON MICROSCOPE SLIDE. Apply blood to microscope slide and smear with other slide as shown below 3. HEMATOCRIT TUBE. Touch hematocrit tube to drop on finger so that capillary action sucks it into tube. Then, holding pricked finger over end of tube, press other end into play-dough capping material to cap tube. Give tube to helperonly helper should wear gloves. 4. DROPS ON TYPING CARD: Apply three blood drops to blood typing card. Have helper add Anti-A, Anti-B and Anti-D (Rh) reagents and mix right away. Use a separate toothpick to mix each reagent with the blood on the card. Read results in two minutes. 5. STOP BLEEDING. Wipe again with alcohol swab and then cover with cotton gauze or band-aid if needed.

Part I: Blood Typing ABO Blood typing is clinically important because if an antigen and an antibody of the same letter are mixed an agglutination reaction will occur. For example, if a patient with A antigen on the surface of their RBCs receives a type B blood transfusion (containing anti-A antibodies) the donor cells will clump together (agglutinate). This clumping is called a transfusion reaction and the clumped cells can clog small blood vessels cutting off blood supply to different regions of the body. As some of the RBCs begin to break down due to the action of phagocytes, hemoglobin is released and may block kidney tubules leading to kidney failure. Furthermore, ruptured RBCs can no longer carry O2. If the reaction is severe enough, the patient receiving this mismatched blood transfusion can die.

Rh blood typing is also important as once an individual receives mismatched blood (e.g. an Rh- patient receives Rh+ blood), the patient will develop anti-D antibodies which will lead to a transfusion reaction if this same mistake is ever repeated. The most common concern, however, is erythroblastosis fetalis. In this case, an Rh- mother carrying an Rh+ baby may produce anti-D antibodies if some of the fetal blood crosses the placenta and enters her circulation. Upon a second pregnancy with an Rh- fetus, the anti-D antibodies can cross the placenta and destroy the fetal RBCs leading to fetal death. To prevent this, all Rh- mothers are given RhoGAM, a serum derived product, which can block the mothers immune system from generating anti-D antibodies by agglutinating the Rh factor that enters her blood stream. It is therefore given to pregnant mothers who experience bleeding, at about 28 weeks of pregnancy, and within 72 hours after a birth, miscarriage, abortion, or amniocentesis. For two minutes after mixing, watch for agglutination or clumping of your blood/reagent mixture and record your results:

Anti-A Serum

Anti-B Serum

Anti-D (Rh) Serum

Then compare to determine what type of blood you have. Blood type: ___________

Part II: Hematocrit This is the normal % of erythrocytes in a whole blood sample. RBCs normally make up ~45% of the total volume of a blood sample. Leukocytes make up less than 1% of the total sample. Plasma makes up the rest of the volume (~55%). These percentages can be determined by centrifuging a sample of whole blood which will cause the red blood cells to settle to the bottom. 1. Put your tube in the same slot as the number on your microscope slide. All samples from the whole class can be spun at once. Spin the tubes in the centrifuge for about five minutes. 2. Estimate the percentage of blood volume that is RBCs or erythrocytesthis is your hematocrit. Can you see a coat of leukocytes or white blood cells?

The Standard method for taking a hematocritreading involves lancing a finger and then pulling a blood sample into a capillary tube. The tube is then spun in a centrifuge. The amount of red blood cells per unit volume is defined as the hematocrit level.

Hematocrit tube after centrifugation from a cow with acute lymphoblastic leukemia. The sample had a white blood cell count of 290,000/l. The buffy coat is easily recognized in this tube; it constitutes nearly half of the cellular mass.

Part III: Blood Smear under Microscope 1. Stain your blood smear with a drop or two of Wrights stain. 2. Observe the slide under the microscope highest power. You can even use the oil lens to get great views of the cells. Ask for help if you have not done this before. 3. Be sure you identify all the cell types: a. Erythrocytes or RBCsmost abundant cells b. Platelets: are these really cells? c. Leukocytes or white blood cellsgranulocytes (have distinct granules) and agranulocytes (with indistinct granules)

DISTINCT GRANULES Neutrophils: Most numerous WBCs. Multi-lobed nucleus, pale cytoplasm, fine granules. (considered neutral because they take up both acidic and basic stains). Immunologically, these are active phagocytes. Eosinophils: Less numerous and hard to findlet others know if you find one. Bi-lobed nucleus, reddish/orange granules in the nucleus, stains red because absorbs acidic stain eosin. These phagocyte-type cells are involved mostly in immune signaling and will burst releasing signal proteins. Also involved in allergic and inflammatory responses. Basophils: Least numerous and harder to findlet others know if you find one. Nucleus large and U-shaped, absorbe basic-staining dye and have large bluish-black granules with histamine. Also contain heparin. WITHOUT DISTINCT GRANULES Lymphocytes: Most numerous of agranulocytes and second-most common WBCs (after neutrophils). Nucleus is large and round and fills cell. Most lymphocytes are outside blood in lymph tissues. These are the B-cells and T-cells of the specific immune response. Monocytes: Largest WBCs. Kidney-shaped nucleus and light gray-blue cytoplasm. Wandering phagocytes that can leave blood and move into tissues. Can become fixed macrophages in tissues (e.g. Langerhans cells in skin or microglial cells in CNS)

Things to Identify for Blood Lab FROM BLOOD TYPING Antigens: A, B, D (Rh) Antibodies: Anti-A, Anti-B, Anti-D (Rh) Blood Types: A, B, O, Rh+/ Agglutination reactions What blood types are compatible? What happens when someone is given blood from a non-compatible donor? When is Rh factor (+/-) most important and why? FROM HEMATOCRIT Percentage or fraction Hematocritnormal range Plasma, WBCs or buff coat What might affect hematocrit? How does centrifuge separate out components of blood?

FROM BLOOD SMEAR Erythrocytes or RBCs Platelets Leukocytes or WBCs o Neutrophils o Eosinophils o Basophils o Monocytes o Lymphocytes Know each WBC function in immune response