1. Estimation of Hemoglobin(Hb) Content
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Salman bin Abdul-Aziz University College of Pharmacy Physiology-I (practical part)
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Estimation of Hemoglobin(Hb) Content

2. After completing this lecture, you should be able to:
1. Define hemoglobin and explain its structure. 2. Mention the various functions of hemoglobin. 3. Determine the Hb level by the Sahli‘s acid hematin method. 4. Indicate normal levels of Hb in different age groups and sexes. 5.Name the common causes of increased and decreased levels of Hb.
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3. Erythrocytes- Hemoglobin
Hemoglobin, a carrier protein which gives form to 95% of erythrocytes within the body, is involved almost explicitly with oxygen transport. Although this protein also acts as a carrier for carbon dioxide.
Hb molecule consists of :
1- Non-protein pigment [heme]: each heme contains an iron ion {Fe++} that combines reversibly with one molecule of O2
2- Globin protein which consisting of 4 polypeptide chains, each chain can bind to one heme molecule.
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4. Erythrocytes- Hemoglobin
Hb in its oxygenated state it is called oxyhemoglobin.
 is bright red.
In the reduced state it is called deoxyhemoglobin.
 is purple-blue.
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5. 3-D structure of hemoglobin
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6. Hemoglobin functions
1. Carriage of oxygen. It carries O2 from the lungs to all the tissues. Each iron atom loosely binds one molecule of O2 at the 6th covalent or coordination bond. 2. Carriage of CO2. Hb carries about 23 % of the total CO2 carried by the blood from the tissues to the lungs. The CO2 reacts with the amino radicals of the goblin to form carbaminoHb. 3. Regulation of blood flow and blood pressure. In addition to the key role of Hb in carriage of O2 and CO2, Hb also plays a role in the regulation of local blood flow and blood pressure by the effect of nitrous oxide.
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4. Homeostasis of pH (buffer action of Hb) Buffer systems convert strong acids and bases (which ionize easily and contribute H+ or OH–) into weak acids and bases that do not ionize as much to contribute H+ or OH–. As H+ or OH– are formed in red cells, or enter them, they are readily accepted by Hb (Hb + H = HbH) that gives up its oxygen. Hb is an excellent buffer and is responsible for 75% of the buffering power of blood.
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8. Principle
The Hb present in a measured amount of blood is converted by dilute hydrochloric acid into acid hematin, which in dilution is golden brown in color. The intensity of color depends on the concentration of acid hematin which, in turn, depends on the concentration of Hb. The color of the solution (i.e. its hue and depth), after dilution with water, is matched against golden-brown tinted glass rods by direct vision. The readings are obtained in g%.
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9. Apparatus and materials
A. Sahli (Sahli-Adams) Hemoglobinometer (Hemometer). The set consists of: 1. Comparator. 2. Hemoglobin tube. 3. Hemoglobin pipette. 4. Stirrer. 5. pipette. 6. Distilled water. 7. hydrochloric acid (0.1 N HCl) solution. 8. Materials for skin prick. • Sterile lancet/needle • cotton swabs • 70% alcohol pads.
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10. Procedure
1. Using a dropper, place amount of 0.1 N HCl in the Hb tube up to the mark 20% or 3 g and set it aside. 2. Get a finger prick under aseptic conditions, wipe away the first 2 drops of blood. then draw blood up to the 20 cmm mark (0.02 ml). Carefully wipe the blood sticking to the tip of the pipette with a cotton swab. 3. Without any waiting, immerse the tip of the pipette to the bottom of the acid solution and expel the blood gently. Rinse the pipette 3–4 times by drawing up and blowing out the clear upper part of the acid solution till all the blood has been washed out from it.
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4. Withdraw the pipette from the tube, then Mix the blood with the acid solution with the flat end of the stirrer by rotating and gently moving it up and down. 5. Put the Hb tube back in the comparator and let it stand for 6–8 minutes. During this time, the acid ruptures the red cells, releasing their Hb into the solution (hemolysis). The acid acts on the Hb and converts it into acid hematin which is deep golden brown in color.
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6. Diluting and matching the color. The next step is to dilute the acid hematin solution with distilled water till its color matches the color of the standard tinted glass rods in the comparator. 7. Take the Hb tube out of the comparator and add distilled water drop by drop, stirring the mixture each time and comparing the color with the standard. 8. Hold the comparator at eye level, away from your face, against bright but diffused light.
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Note: The color of acid hematin does not develop fully immediately, but its intensity increases with time, reaching a maximum, after which it starts to decrease. An adequate time, usually 6–8 minutes, must be allowed before its dilution is started. Too little time and all Hb may not be converted into acid hematin. And, waiting too long, may result in fading of color. In either case, the result will be falsely low.
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14. Observations and results
Take the average of 3 readings as shown below, and report your result as: Hb = ……….g/dl. 1st reading, when the color is slightly darker than the standard:………….…..g/dl. 2nd reading, when, after adding a few drops of distilled water, the color exactly matches the standard: ………..……… g/dl. 3rd reading, when, after adding some more drops, the color becomes a little lighter than the standard:………..…….. g/dl. For report. Express your result as: Hb= ....….g/dl.
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15. Normal Values The average levels and their ranges are as follows:
i. Newborns : 18–22 g/dl
ii. At 3 months : 14–16 g/dl
iii. 3 months–1 yr : 13–15 g/dl
iv. Adult males : 14.5 g/dl (13.5–17 g/dl)
v. Adult females : 12.5 g/dl (11.5–15.5 g/dl).
There may be some decrease after age 60 years.
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16. High Hb level causes High hemoglobin count may occur because:
Your red blood cell production increases to compensate for chronically low blood oxygen levels due to poor heart or lung function.
You have a bone marrow dysfunction that results in increased production of red blood cells.
Smoking.
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17. Low Hb level causes
A low hemoglobin count can be associated with many diseases and conditions that cause your body to have too few red blood cells. This can occur if your body produces fewer red blood cells than usual, if your body destroys red blood cells faster than they can be produced, or if you experience blood loss.
Diseases and conditions that cause your body to produce fewer red blood cells than normal include:
Aplastic anemia
Cancer
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18. Do You Have Any Questions?
Thank You
For Your Attention
Do You Have Any Questions?
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