Hemoglobin (Hb) determination Flashcards by snow riego

A tetramer containing two pairs of similar polypeptide chains called

globin chains

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Hemoglobin is a conjugated protein that comprises of

90% of dry weight of RBC

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Indications for Hemoglobin Estimations

Determine the presence and severity of anemia
Screening for polycythemia
Assess response to specific therapy in anemia
Estimation of red cell indices
Selection of blood donors

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N/10 HCl converts hemoglobin into soluble unstable acid hematin. The colour intensity of the acid hematin after dilution is compared with standard brown glass in the comparator.

Sahli’s Method

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which converts hemoglobin to acid hematin

N/10 HCl

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N/10 HCl converts hemoglobin into

soluble unstable acid hematin

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it is graduated on both sides. One side is graduated in gram percentage from 2-22 (g%). Other side is graduated as percentage from 20-140 (%

Hemoglobin Tube

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central slot accommodates hemoglobin tube and on either side non-fading brown tinted glass pieces for colour matching are present.

Comparator

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has one mark indicating 20 cc (no bulb)

Hemoglobin Pipette

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Thin glass rod for stirring the solution.

Stirrer

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To make 500 ml of N/10 HCl mix:

4.5 mL of concentration HCl + 500 mL distilled water

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outline the Sahli’s Method procedure:

Add N/10 HCl to the hemoglobinometer tube up to its lowest mark - 2 g%

Take blood up to 20 cc mark on the pipette and transfer it to the hemoglobinometer tube containing N/10 HCl

Leave the solution for 10 minutes (for conversion of hemoglobin to acid hematin)

After 10 minutes add distilled water drop by drop and mix it by stirrer until the color matches with the color of comparator. While matching the color, glass rod should be removed from the solution.

The lower meniscus of the solution should be taken as the result which expresses hemoglobin content as g%

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Blood is mixed with hypotonic solution like distilled water to produce hemolysis of RBCs.

Haldane

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Carbon monoxide is added to the mixture.

Haldane

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In this method small glass chamber is filled with whole blood by capillary action.

Dare

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The glass chamber is illuminated by:

battery bulb

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drop of blood is placed on filter paper

Tallquists

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high degree of errors are possible

Tallquists

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Colour of the blood is matched with standard after seeing through eye piece.

Dare

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based in comparing the color of drop of blood absorbed on a particular type of chromatography paper against a printed scale of color corresponding to different levels of hemoglobin ranging from 4 to 14 gm/dL

WHO hemoglobin color scale method

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color of drop of blood absorbed on a particular type of:

chromatography paper against a printed scale of color

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different levels of hemoglobin ranging from

4 to 14 mg/dL

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the color of diluted oxyhemoglobin is matched visually.

Spencer

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This method is less accurate than Sahli’s method.

Spencer

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It is more difficult for the human eye to accurately grade and match small differences in red color than brown color of acid hematin.

Spencer

When the blood is mixed with a solution, containing potassium ferricyanide and potassium cyanide, all types of hemoglobins except sulfhemoglobin is converted to cyanmethemoglobin. The intensity of color is proportional to hemoglobin concentration and it is compared with a know cyanmethemoglobin standard at 540 nm (green filter) in a photoelectric colorimeter.

Cyanmethemoglobin Method

MOST ACCURATE METHOD IN SPECTROPHOTOMETRIC METHOD

Cyanmethemoglobin Method

Drabkins reagent:

Distilled water - 1000 mL Potassium ferricyanide - 200 mg Potassium dihydrogen phosphate - 140 mg Potassium cyanide - 50 mg Non-ionic detergent

Drabkins reagent: pH: COLOR: STORAGE: TEMPERATURE: ABSORBANCE: WAVELENGTH:

Drabkins reagent pH: 7-7.4 COLOR: clear and pale yellow STORAGE: brown borosilicate bottle TEMPERATURE: ref temp 2-8 C ABSORBANCE: 0 WAVELENGTH: 540 nm

Drabkins solution should not be used if:

1. pH is outside the range 2. Fluid is turbid 3. Its absorbance in spectrophotometer is not zero at 540 nm

Cyanmethemoglobin standard solution reading obtained corresponds to:

to 15 g/dL of hemoglobin for dilution of 1:250

outline the Cyanmethemoglobin Method procedure:

20 µl of blood is added to 5mL of Drabkins reagents and mixed well. Leave it for 5 minutes. Now measure the absorbance of this solution and standard (in spectrophotometer at 540 nM after adjusting the OD at 0 by using Drabkins solution as blank).

write the calculation of cyanmethemoglobin method

calculation

Visual error is absent because

there is no color matching

Cyanmethemoglobin is stable so

it does not fade away quickly.

All forms of hemoglobin except _______ are converted to cyanmethemoglobin

Sulfhemoglobin

Potassium cyanide is

poisonous and should not be pipetted by mouth.

Rate of conversion of blood containing _______ is long (30 minutes)

carboxy hemoglobin

Blood with abnormal plasma proteins and high leukocytes count may cause _______ on dilution of blood.

turbidity

Spectrophotometric Methods are:

1. Cyanmethemoglobin method 2. Oxyhemoglobin method 3. Alkaline hematin method

___________ is added to blood which causes hemolysis of red cells in Oxyhemoglobin

0.007 N Ammonium hydroxide

0.007 N Ammonium hydroxide is added to blood which causes hemolysis of red cells and converts hemoglobin to oxyhemoglobin.

Oxyhemoglobin

Oxyhemoglobin method does not give satisfactory results in the presence of

1. methemoglobin 2. sulfhemoglobin 3. carboxyhemoglobin

Hemoglobin is converted to ________ by adding alkali such as sodium hydroxide.

alkaline hematin

wavelength of spectophotometric methods:

540 nm

Standard solution used in Alkaline Hematid Method are:

Gibsons and Harrisons solution

This is mixture of _____, ______, and _____ in aqueous solution.

1. Chromium potassium sulphate 2. Cobaltous sulphate 3. Potassium dichromate

This standard solution Alkaline Hematid Method is equal in color to 1 in 100 dilution of containing:

16.0 g of Hb g/dL

It gives true estimate of total hemoglobin including sulfhemoglobin and methemoglobin. It is not affected by plasma proteins and lipids.

Alkaline Hematid Method

Gasometric Method is an indirect method of estimation of hemoglobin by using ___________

Van Slyke Apparatus

This method is based on the assumption that 1 gram of hemoglobin carries 1.34 mL of oxygen

Gasometric Method

Gasometric Method is not routinely used method because:

it is time consuming and has complex process.

It is used for research purpose and for preparation of standard solutions.

Gasometric Method

Specific Gravity Method

Copper Sulfate Method

Specific gravity of copper sulfate solution is:

1.053 = 12.5g% of hemoglobin

outline Specific Gravity Method - Copper Sulfate Method procedure

Drop 1-drop of blood into the copper sulfate solution. dense = float denser = sink to bottom

It is an indirect method which is based on the assumption that 0.347 mg of iron is compared with 100g of hemoglobin.

CHEMICAL METHOD

chemical method is an indirect method which is based on the assumption that _______ is compared with 100g of hemoglobin.

0.347 mg of iron

The principle of the impedance technology is ___________ in which a known dilution of cells in suspension passes through a small orifice.

electrical resistance (or impedance)

The electrolyte containing diluent serves as a __________ of a constant electrical current between two electrodes

conductor

what serves as a conductor that has constant electric current

electrolyte (w/ diluent)

In this method blood is diluted using reagent diluent and then Sodium lauryl sulphate (SLS) hemoglobin reagent is added.

AUTOANALYZERS

has surfactant which lyse RBC’s and release hemoglobin. It also reduces the turbidity developing from plasma lipids and cell membrane

Sodium lauryl sulphate (SLS) reagent

SLS converts:

Ferrous (Fe+2) to ferric (Fe +3) forming methemoglobin.

Methemoglobin combines with SLS forming:

SLS hemichrome molecule

The absorbance of this molecule is measured at what wavelength

555nm

False elevation is noted when there is high WBC count which is:

>30,000/uL

CONDITIONS WITH ELEVATED HEMOGLOBIN:

1. Chronic obstructive pulmonary disease 2. Congenital cyanotic disease of heart 3. Smokers polycythemia 4. Renal cell carcinoma - due to ectopic secretion of EPO 5.Pheochromacytoma 6. Polycythemia vera 7. People living in high altitude

CONDITIONS WITH FALSELY-ELEVATED HEMOGLOBIN:

1. Burns 2. Severed dehydration 3. Immediately after acute hemorrhage 4. Blood taken during the intravenous infusion of iron containing drugs.

CONDITIONS WITH FALSELY ANEMIA

1. Pregnancy - increase in plasma volume leading to fall of 1-2 g/dL 2. Hypervolemia - disproportionate increase in plasma volume and RBC volume

CONDITIONS WITH DECREASED HEMOGLOBIN CONCENTRATION

Anemia due to: 1. Blood loss 2. Parasitic infection 3. Drugs and lead poisoning 4. Dietary deficiency (iron, copper, vitamins) 5. Malabsorption on nutrients 6. Chronic diseases (diseases of liver, kidney and cancer)

Physiological Variations

1. Strenuous physical exercise 2. Diurnal variation - highest in morning and lowest in evening. 3. High altitude - increase with increase altitude