Materials Included In Kit

Additional Materials Required

Prelab Preparation

Cut the test paper into 1 cm x 3 cm strips and place on a clean piece of paper towel.

Disinfecting solution: A freshly prepared 10% bleach solution or a freshly prepared Lysol^® solution made from the concentrate work well. Please read all labels before choosing a disinfectant.

Safety Precautions

Follow all universal precautions for working with human blood: wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Do not come into contact with any blood during this laboratory. As a precaution wear chemical-resistant gloves during the lab. If the gloves become contaminated, discard them in the biohazard container and don a clean pair of gloves. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Items contaminated with blood must be sterilized prior to disposal. Flinn Scientific Biological Waste Disposal Type Ia, Autoclaving, or Type Ib, Chemical Sterilization, are two possible disposal methods. Please check all local regulations before conducting this activity.

Lab Hints

• Enough materials are provided in this kit for 30 students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before this laboratory activity.
• Review all bloodborne pathogen rules and recommendations required by your school prior to beginning this activity. Review appropriate laws such as OSHA’s Bloodborne Pathogen Regulation 1910.1030, accessible online at http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051 (Accessed March 2009), and the CDC’s Universal Precautions accessible online at http://www.cdc.gov/ncidod/dhqp/bp_universal_precautions.html (Accessed March 2009).
• Ask the school nurse to discuss universal precautions before conducting any lab in which human blood or other cells will be used.

Further Extensions

• Extend this activity by completing other hematology activities, such as blood typing. One such kit is the ABO Blood Typing Kit, Flinn Catalog No. FB1229. Another is Hemocytometer Lab, Flinn Catalog No. FB1863 using simulated blood.
• Allow students to research the mission of the World Health Organization.

Answers to Prelab Questions

1. Polycythemia can be caused by a genetic mutation. One treatment method for polycythemia is phlebotomy which is the removal of up to a pint of blood by a trained medical professional. Give reasons why this treatment is successful for many people with polycythemia.

   Polycythemia is an abnormally high number of red blood cells. Phlebotomy reduces the volume of blood and therefore red blood cells in the body, making it easier for the remaining blood to function properly within the body.

2. Many illnesses and diseases are transmitted by direct contact with contaminated blood. In this lab you will be working with your own blood. What precautions are necessary to ensure no blood is touched by anyone other than the donor?

   The work area will be disinfected twice, once before the lab and once after. All work will be completed on a clean piece of paper towel. Each student will perform his or her own test using only his or her own blood. All blood contaminated objects will be disposed of in a biohazard container that will be disposed of appropriately by the instructor. Chemical splash goggles and chemical resistant aprons will protect the students from getting blood into their eyes or onto their clothes.

Sample Data

Observations
Most students should fall within the “normal” range. Borderline anemia is typical during pregnancy, lactation, heavy menstruation, rapid growth phases and an iron deficient diet. Do not use this laboratory as a clinical test but if indicated refer students to his or her physician for a clinical evaluation.

Answers to Questions

1. What estimated hemoglobin concentration would be expected if someone had iron-deficiency anemia?

   Borderline anemia, 70–80% on the Hemoglobin Scale.

2. The colorimetric test for hemoglobin was developed by the World Health Organization for use by field doctors in extremely poor areas of the world. What are some of the benefits of using a colorimetric test in these areas?

   Anemia is common in poor areas of the world where electricity is rarely present. The colorimetric test allows doctors to determine, in general, the hemoglobin level in patients who present themselves for treatment in these areas.

3. Describe two simple steps everyone can take to be sure they get enough iron.

   Diet (red meat) and vitamin pills.

4. Name two common medical treatments that may be used to increase hemoglobin levels in the blood.

   Ingest more red meat, vitamin and mineral (i.e., iron) supplements, blood transfusions, bone marrow transplant and prescription medication are all methods used to treat anemia.

5. People with borderline anemia often complain of being tired, lethargic and fatiguing easily. Explain these symptoms based on the role of iron and red blood cells.

   A lack of red blood cells or a reduction in the amount of hemoglobin within the red blood cells leads to a decrease in the oxygen-carrying capacity of blood, which can cause a decrease in blood flow to tissues and sometimes cell death.

Teacher Handouts

10970_Teacher1.pdf

References

Lewis, S. M.; Stott, G. J.; Wynn, K. J. An inexpensive and reliable new haemoglobin colour scale for assessing anaemia. J Clin Pathol. 1998; 51:21–24.

Introduction

Medical professionals use blood tests as one way to determine the health of a patient. Many different types of blood tests are available. The colorimetric method is used to estimate the amount of hemoglobin in a small sample of blood. This allows doctors to determine whether or not a patient is anemic.

Concepts

• Hemoglobin
• Anemia
• Erythrocytes

Background

Hematology is the study of blood. Blood has three main functions: transportation, regulation and protection. Blood supplies cells with oxygen and nutrients while removing carbon dioxide and other cellular waste products. Blood may look like a homogeneous red fluid but upon closer analysis three major types of cells and a complex fluid called plasma are visible. The three major types of cells found in blood are the red blood cells or erythrocytes, the white blood cells or leukocytes and the platelets or thrombocytes.

Erythrocytes are biconcave, disc-shaped cells (see Figure 1). Their unique shape provides a large surface area, gives them flexibility so they can bend easily and flow through capillaries, and allows the cell membrane to easily contact the oxygen-carrying molecules found in the inside the cell. The oxygen-carrying portion of a red blood cell is the small metalloprotein called hemoglobin (see Figure 2). Each hemoglobin molecule is made up of four protein chains, two alpha (α) chains and two beta (β) chains, and four metal-carrying heme groups. Each protein chain holds a heme group containing a single iron atom. Oxygen reversibly binds to the heme group in the lungs and transports the oxygen to oxygendepleted areas of the body. Each red blood cell is composed of roughly 35% hemoglobin by volume, corresponding to several million protein molecules within each cell. Since hemoglobin provides the mechanism for transport in the blood, the amount of hemoglobin present is a good indicator of the oxygen-carrying capacity of the blood. Measuring the number of red blood cells per cubic milliliter of blood is called a red blood cell count. In order to complete a red blood cell count, a small sample of blood is introduced into a very expensive calibrated glass chamber and each cell is counted using a light microscope. A faster method is to estimate the number of red blood cells and therefore hemoglobin molecules using a colorimetric test developed for the World Health Organization (WHO). A series of colors that match the color of known concentrations of red blood cells is printed on paper to create a colorimetric scale. When the color of a drop of blood is compared to the colorimetric scale, an approximate concentration of red blood cells is determined and the hemoglobin level is obtained. Hemoglobin concentration is measured in grams per 100 milliliters g/100 mL of blood. The normal range for an adult male is 13–18 g/100 mL. The normal range for an adult female or for a child is 12–16 g/100 mL. Values below the normal levels are considered anemic.

Anemia can be due to either a lack of red blood cells or a reduction in the amount of hemoglobin contained within the red blood cells. Both causes lead to a decrease in the oxygen-carrying capacity of blood, which can cause cell death. An abnormal increase in the number of red blood cells can also occur. The condition, called polycythemia, can increase the viscosity of the blood, which can lead in turn to a decrease in blood flow to tissues and also result result in cell death.

Experiment Overview

Determining hemoglobin levels is just one factor involved in a full blood profile. This activity is not a clinical diagnosis of health problems. The purpose of this activity is to compare the color of blood versus a standardized color scale to determine the approximate concentration of hemoglobin in blood.

Materials

Prelab Questions

1. Polycythemia can be caused by a genetic mutation. One treatment method for polycythemia is phlebotomy, which is the removal of up to a pint of blood by a trained medical professional. Give reasons why this treatment is successful for many people with polycythemia.
2. Many illnesses and diseases are transmitted by direct contact with contaminated blood. In this lab you will be working with your own blood. What precautions are necessary in a clinical setting to ensure no blood is touched by anyone other than the donor?

Safety Precautions

Review and follow all universal precautions for working with human blood: Wear chemical splash goggles and a chemical-resistant apron. Disinfect all surfaces that may have come in contact with blood. Do not contact anyone else’s blood unless you are wearing chemical-resistant gloves, goggles and apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

1. Clean the desk or table with the disinfecting solution.
2. Place a piece of paper towel on the disinfected area. This will be used as the work surface.
3. Roll up and secure long sleeves and tie back long hair. Put on chemical-splash goggles and a chemical-resistant apron.
4. Using soap and warm water wash your hands thoroughly for two minutes. Use clean paper towels to completely dry your hands.
5. Select a puncture site on the side of a fingertip on your non-writing hand. Scrub the finger with a clean cotton ball saturated with 70% isopropyl alcohol. Allow the finger to air dry.
6. Puncture the side of the fingertip using a clean lancet.
7. Use a clean cotton ball to wipe away the first drop of blood. Increase blood flow to the puncture by gently “milking” the finger from its base toward the puncture site. Note: Do not squeeze directly at the puncture site as this will force tissue fluids instead of blood out of the puncture. Allowing the punctured fingertip to dangle below the level of the heart will also increase blood flow to the puncture site.
8. Touch the edge of the test paper to the next two drops of blood.
9. Place an adhesive bandage over the puncture site.
10. Wait 30 seconds and then compare the color of the blood stain on the test paper to the colors on the Hemoglobin Scale Card. Record the data on the Estimating Hemoglobin Concentration Worksheet.
11. Place all items with blood contamination into the biohazard container. Clean and disinfect the desk or table and wash your hands thoroughly.

Student Worksheet PDF

10970_Student1.pdf