Hemalab: Hemoglobin Determination

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is the major constituent of the red cytoplasm, accounting for approximately 90% of the dry eight of mature cells

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Hemoglobin

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Hemoglobin composition

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Heme and Globin

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carry oxygen to and carbon dioxide from the tissue

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Hemoglobin

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Methods of Determination

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Colorimetric method, direct or visual, Gastrometric methods, specific gravity methods, chemical methods, Hemoglobin electrophoresis

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Under Colorimetric method

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Cyanmethemoglobin method and Oxyhemoglobin method

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Blood is diluted in alkaline Drabkin's reagent

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Cyanmethemoglobin

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Drabkins reagent

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potassium ferricyanide, potasium cyanide, sodium bicarbonate and a surfactant

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Maximal absorption of cyanmethemglobin

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540 nm

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is the reference method approved by Clinical and Laboratory Standard Institue because all forms of hemoglobin are converted to cyanmethemoglobin except sulfhemoglobin

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Cyanmethemoglobin

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HgB is then conver tes to this by reaction with ammonia and the color of the solution is measure directly in spectrophotometer

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Oxyhemoglobin

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Direct pr Visual Categories

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Sahli' s or Acid Hematin Method

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Blood is mixed with 0.1 N HCL in the conversion of Hgb to acid hematin

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Sahli's or Acid Hematin Method

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Color in Shali's or Acid Hematin Method

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Yellowish brown

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The solution is diluted until color matches with the briwn colored glass of comparator box

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Sahli's Acid Hematin Method

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It gives true estimate of Hgb and is not affected by the preesence of plasma protein and lipids

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Alkaline Hematin Method

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Alkali in Alkaline Hematin Method

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Sodium hydroxide

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it is not used routinely as it is less accurate than the cyanmethemoglobin method and some forms if Hgb such as HbF are resistant to alkali denaturation

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Alkaline Hematin Method

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Unde Gasometric Method

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Van Slyke Oxygen capacity

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This is a measure of the Hgb Function

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Van Slyke Oxygen capacity

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Oxygen binding capacity of blood in Van Slyke Oxygen Capacity

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1.34 mL Oxygen per gram of hemoglobin

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it is not used routinely as it is not practical and gives result 2% lower than the other methods

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Van Slyke Oxygen Capacity

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Thi is a qualitative screening test based on specific gravity

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Specific gravity methods

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The density of blood is directly proportional to rhe amount of Hgb it contains

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Specific gravity method

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Specific gravoty

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1.052 to 1.054

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If blood sinks Oxygen level is

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Greater than 12.5 mg/dL

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Iron found within the RBCs bind with Hgb

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Chemical Method or Iron content of Hgb

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It can be directly related to the Hgb Concentration

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Iron

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Formula of Iron converted to Hgb

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0.347 mg of iron 100g Hgb

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Chemical Methods 2

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Kennedys and Wongs

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Hgb Electrophorrsis usss

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Cellulose acetate

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Alkaline pH in Hgb Electrophoresis

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8.2 to 8.6

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Screening test for detecting variant or abnormal Hgb

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Hgb Electrophoresis

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Further Hgb electrophoresis is confirmes using

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Citrate agar electrophoresis