Hemoglobin Determination PDF

} Also known as: Hgb; Hb; H and H (Hemoglobin and Hematocrit) } Formal name: Hemoglobin The hemoglobin test is often used to check for anemia, usually along with a hematocrit or as part of a complete blood count (CBC). The test may be used to screen for, diagnose, or monitor a number of co...

} Also known as: Hgb; Hb; H and H (Hemoglobin and Hematocrit) } Formal name: Hemoglobin The hemoglobin test is often used to check for anemia, usually along with a hematocrit or as part of a complete blood count (CBC). The test may be used to screen for, diagnose, or monitor a number of conditions and diseases that affect red blood cells (RBCs) and/or the amount of hemoglobin in blood. Hemoglobin is the iron-containing protein found in all red blood cells that enables RBCs to bind to oxygen in the lungs and carry it to tissues and organs throughout the body. } Screen for, diagnose, and measure the severity of anemia (low RBCs, hemoglobin and hematocrit) or polycythemia (high RBCs, hemoglobin and hematocrit) } Monitor the response to treatment of anemia or polycythemia } Help make decisions about blood transfusions or other treatments if the anemia is severe } In certain conditions were RBC production in the bone marrow may cause an increase or decrease in the number of mature RBCs released into the blood circulation. } If there is increased destruction of RBCs (hemolysis) or loss of RBCs through bleeding and/or the bone marrow is not able to produce new ones fast enough, then the overall number of RBCs and hemoglobin will drop, resulting in anemia. } This test can indicate if there is a problem with red blood cell production and/or lifespan, but it cannot determine the underlying cause. } the tests may be performed at the same time or as follow up to establish a cause include a blood smear, reticulocyte count, iron studies, vitamin B12 and folate levels, and in more severe conditions, a bone marrow examination.  The hemoglobin test may be ordered during a general health examination or when a person has signs and symptoms of a condition affecting red blood cells such as anemia or polycythemia. Some signs and symptoms of anemia include: } Weakness or fatigue } Lack of energy } Fainting } Paleness (pallor) } Shortness of breath Some signs and symptoms of polycythemia include: } Disturbed vision } Dizziness } Headache } Flushing } Enlarged spleen  Low hemoglobin with low RBC count and low hematocrit indicates anemia. Some causes include: } Excessive loss of blood from, for example, severe trauma or chronic bleeding from sites such as the digestive tract (e.g., ulcers, polyps, colon cancer), the bladder or uterus (in women, heavy menstrual bleeding, for example) } Nutritional deficiencies such as iron, folate or B12 deficiency } Damage to the bone marrow from, for example, a toxin, radiation or chemotherapy, infection or drugs } Bone marrow disorders such as aplastic anemia, myelodysplastic syndrome, or cancers such as leukemia, lymphoma, multiple myeloma, or other cancers that spread to the marrow } Kidney failure—severe and chronic kidney diseases lead to decreased production of erythropoietin, a hormone produced by the kidneys that stimulates RBC production by the bone marrow. } Chronic inflammatory diseases or conditions } Decreased hemoglobin production (e.g., thalassemia) } Excessive destruction of red blood cells, for example, hemolytic anemia caused by autoimmunity or defects in the red blood cell itself; the defects could be hemoglobinopathy (e.g., sickle cell anemia), abnormalities in the RBC membrane (e.g., hereditary spherocytosis) or RBC enzyme (e.g., G6PD deficiency).  High hemoglobin with a high RBC count and high hematocrit indicates polycythemia. Some causes include: } Lung (pulmonary) disease—if someone is unable to breathe in and absorb sufficient oxygen, the body tries to compensate by producing more red blood cells. } Congenital heart disease—in some forms, there is an abnormal connection between the two sides of the heart, leading to reduced oxygen levels in the blood. The body tries to compensate by producing more red blood cells. } Kidney tumors that produce excess erythropoietin } Smoking—heavy smokers have higher hemoglobin levels than nonsmokers. } Genetic causes (altered oxygen sensing, abnormality in hemoglobin oxygen release) } Living at high altitudes (a compensation for decreased oxygen in the air) } Dehydration—as the volume of fluid in the blood drops, the hemoglobin artificially rises. } Polycythemia Vera—a rare disease in which the body produces excess RBCs inappropriately  is measured as oxyhemoglobin. Some methods measure oxyhemoglobin indirectly by converting it to compounds like acid hematin, cyanmethemoglobin, alkali hematin, and carboxyhemoglobin. Other indirect methods determine hemoglobin levels by detecting the concentrations of oxygen and iron carried by the hemoglobin molecule as well as by the blood’s specific gravity. } Measure hemoglobin accurately using various methodologies } Compare the various methods of hemoglobin measurement. } Enumerate the causes of increased and decreased hemoglobin values. } EDTA blood } 0.1N HCl – Hydrochloric acid } Distilled water } Suction device/Aspirator/ Sampler } Hemoglobinometer ◦ Comparator clock ◦ Sahli’s tube ◦ Sahli’s or square tube ◦ Dropper ◦ Stirring rod } Wassermann tube } Drabkin’s reagent } Cyanmethemoglobin } Spectrophotometer  The principle involved in the acid hematin method is the conversion of hemoglobin to acid hematin by using 0.1N HCl. The yellowish brown solution is compared to the color standard in the comparator block. 1. Deliver 0.1N HCl up to the 2 mark of the Sahli’s or square tube. 2. Aspirate 0.02 mL of blood using the Sahli’s pipette or sampler. 3. Expel the said volume of blood to the Sahli’s tube containing the 0.1N HCl. 4. Rinse the pipette with distilled water three times adding all the washings to the mixture of blood and diluted acid. Mix and stand for at least 10 minutes. 5. Add distilled water to the mixture drop by drop, ensuring the thorough mixing after each addition which the use of stirring rod. Continue adding distilled water until the color of the solution matches that the standard on the comparator block. 6. Take the reading of the lower meniscus. 7. Report the hemoglobin level in gm% or gm/dL or gm/100 mL (CU) and in gm/L (SI). a. Sahli – Hellige b. Sahli – Adam’s c. Sahli – Haden d. Haden – Housen e. Newcomer’s f. Osgood – Haskin  Drabkin’s reagent is used to measure hemoglobin through the cyanmethemoglobin method. It consist of sodium bicarbonate, potassium cyanide, and potassium ferricyanide.  The ferricyanide in Drabkin’s reagent converts the iron in the hemoglobin molecule from the ferrous to the ferric state, forming methemoglobin or hemiglobin or ferrihemoglobin. The product then combines with the potassium cyanide to produce cyanmethemoglobin cyanide.  The cyanmethemoglobin method is the best method of manual hemoglobin determination because all forms of hemoglobin are converted to cyanmethemoglobin except sulfhemoglobin. The procedure uses a very stable standard. 1. Deliver the reagent, standard, and blank on Wassermann tubes as indicated in the table below. Blank Standard (15 g/dL) Unknown Drabkin’s reagent 5 mL – 5 mL Whole blood – – 0.02 mL Cyanmet standard – 5 mL – 2. Mix unknown by inversion. 3. Let the mixture stand for 10 minutes. 4. Read absorbance at 540 mm against blank. 5. Compute for the value hemoglobin. Formula for computation : Au * Cu = As * Cs 1. The pigment cyanmethemoglobin is stable even in a diluted solution. 2. The method measures all hemoglobin except sulfhemoglobin. 3. Certified cyanmethemoglobin standard is commercially available. 4. The spectral curve of cyanmethemoglobin allows the use different types of spectrophotometers. Composition of Drabkin’s reagent: – Sodium bicarbonate ------------------ 1.00 gm – Potassium cyanide -------------------- 0.05 gm – Potassium ferricyanide -------------- 0.20 gm Dissolve these compounds in 1000 mL distilled water.  Hemoglobin standard Drabkin’s Tube Concentration (g/dL) Dilution (mL) Reagent (mL) 1 1 4 1:5 2 2 3 2:5 3 3 2 3:5 4 4 1 4:5 5 15 5 0 Undiluted 1. Mix the contents of the tubes and let the mixture stand for 10 minutes. 2. Read absorbance at 540 mm. Record the results. 3. Plot the absorbance reading at the y – axis and the hemoglobin concentration at the x – axis using graphing paper. 4. Construct a standard table of readings and the corresponding hemoglobin values from the graph. Conversion Normal value:Factor - 10 CU (g/dL, g/mL, gm/100 SI (g/L) mL) Male 14 – 18 140 – 180 Female 12 – 15 120 - 150 I. Colorimetric Method A. Direct Matching 1. Tallquist Scale This method is based on color comparison. It uses a very simple chart consisting of different hues of red as a comparator. Blood is collected and a drop is placed on a piece of thick absorbent paper. The color of slightly dried blood on the paper is then compared to the color chart. Hemoglobin is reported in terms of percentage (%). However, it has a percentage error of 30% - 50%. 2. Dare Hemoglobinometer The Dare hemoglobinometer consist of a glass plate and an eyepiece. It has percentage error of 20% - 30%. 3. Alkaline Hematin The use of an alkaline solution for hemoglobin determination produces a true and relatively stable solution of hematin. Procedure: 1. Place 5 mL of NaOH on a reaction tube. 2. Deliver 0.05 mL of blood to the tube. 3. Heat the tube in a boiling water bath for 4 – 5 minutes. 4. Cool and read against an appropriate standard. Disadvantage. The blood of newborns and young infants contains alkali – resistant fetal homoglobin determination when using the alkaline – hematin method. B. Indirect Method 1. Oxyhemoglobin a. Sodium carbonate A photometric determination of hemoglobin is done by measuring oxyhemoglobin. It is simple and quick but there is no possibility of preparing a stable HbO2 standard. b. Photoelectric oxyhemoglobin Pulse oxygen saturation and pulse rate can be measured through finger using a photoelectric oxyhemoglobin monitor.  2. Carboxyhemoglobin Carboxyhemoglobin test is not done as a routine examination. It is performed only when carbon monoxide poisoning is suspected. II. Gasometric Method A. Van Slyke Oxygen Capacity This method measures the amount of oxygen using a Van Slyke manometric apparatus. The level of hemoglobin is determined by computation. NOTE: 1 gm of hemoglobin = 1.34 mL O2 III. Specific Gravity Method The specific gravity method employs copper sulfate with known specific gravity (1.052 and 1.054). A drop of blood is added to the copper sulfate solution. If the drop falls, the level of hemoglobin is said to be acceptable. This method is used in screening potential blood donors and not for patients. NOTE: The specific gravity of blood is 1.053 II. Chemical Method The chemical methods of hemoglobin determination measures the amount of iron present in the blood. One gram of hemoglobin carries 3.47 mg of iron. This is based on the fact that most of the iron is found in RBCs and combines with the hemoglobin molecule. A. Kennedy’s B. Wong’s III. Hemoglobin Electrophoresis Hemoglobin electrophoresis using cellulose acetate at an alkaline pH (8.2 to 8.6) is a screening test for detecting variant or abnormal hemoglobin. Further confirmation can be achieved by using citrate agar electrophoresis.