Unit 3.3 HEMOGLOBIN DETERMINATION Flashcards
is the main component of red blood cells that carries oxygen from the lungs to the body tissues and returns carbon dioxide from the tissues back to the lungs.
Hemoglobin
Measurements of hemoglobin from[?] aids in the detection of a variety of conditions that alter the normal hemoglobin concentration of the blood (e.g. anemia or polycythemia)
venous or capillary blood
METHODS OF HEMOGLOBIN DETERMINATION
- GASOMETRIC Method (Van Slyke Oxygen Capacity)
- CHEMICAL Method
- GRAVIMETRIC Method
- COLORIMETRIC Method
Principle: A given sample of blood can be equilibrated with oxygen under standard conditions of Temperature and Pressure. Hemoglobin can then be derived using the Hufner constant.
- GASOMETRIC Method (Van Slyke Oxygen Capacity)
Limitation: Measures active hemoglobins only
- GASOMETRIC Method (Van Slyke Oxygen Capacity)
A. Direct Visual Colorimetric
B. Photoelectric Colorimetric
Principle: Based on the total iron content of blood
CHEMICAL Method
CHEMICAL Method Equation:
1 gm hb = 3.47 mg iron
Iron is made to form ferric thiocyanate:which is then measured spectrophotometrically
Wongs’ Method
Based on specific gravity
GRAVIMETRIC Method
Uses a standard CuSO4 solution (in 40 tubes) with sp. gr. of 1.035 to 1.075 at interval of .001
GRAVIMETRIC Method
A drop of patient’s blood is placed into each tube. The drop of blood becomes coated with copper proteinate and for about 12 seconds, the blood drop may either sink (if heavier/denser), float (if lighter/less dense), or remain (if it has the same density asd the CuSO4 solution) in the suspension.
GRAVIMETRIC Method
GRAVIMETRIC Method
Female:
Male:
1.053 = 12.5 g/dL hb
1.055 = 13.5 g/dL hb
Patient’s undiluted blood is absorbed unto an absorbent pad and the color is compared with a lithographed color scale representing values from 10 - 100%.
Tallquist Mtd
Inaccurate, gives as much as 50% error
Tallquist Mtd
blood is drawn by capillary action between 2 glass plates and the color of blood is matched with a rotating disc of red tinted glass with varying thickness and color.
Dare’s hemoglobinometer
Inaccurate, gives as much as 30% error
Dare’s hemoglobinometer
Principle: Hemoglobin is converted into acid hematin in the presence of an acid.
Acid Hematin
Limitation: does not measure inactive forms
Acid Hematin
Methods: Sahli-Hellige; Sahli-Adams; Sahli-Hayden Haydenn-Hausser; Newcomer; Osgood-Haskin
Acid Hematin
Principle: hemoglobin is converted into alkali hematin upon addition of an alkali. Abnormal hb are converted to alkali hematin and are thus measured
Alkali Hematin
Produces a more stable pigment but is not ideal for infants and children
Alkali Hematin
Methods: Wu; Klegg & King
Alkali Hematin
0.02 ml whole blood is reacted with 5 ml of 0.07N NH4OH / 0.1% Na2CO3 the mixed for proper oxygenation. Absorbance is read at 540 nm against reagent blank.
Oxyhemoglobin Mtd
Oxyhemoglobin Mtd
- 415 nm (Soret band: 400 430 nm)
Harboe
- hb catalyzes the rapid oxidation of benzidine by H2O2
Naumann
- more sensitive but less accurate
Naumann
Most accurate
Cyanmethemoglobin Mtd
Principle: When Blood is mixed with a solution of potassium cyanide and potassium ferricyanide, the erythrocytes are lysed thereby producing hemoglobin. Potassium ferricyanide oxifizes hemoglobin into methemoglobin that combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin) whose absorbance is measured at 540 nm against the reagant blank.
Cyanmethemoglobin Mtd
Advantage: all type of hemoglobin is transformed to cyanmethemoglobin (except sulfhemoglobin),
Cyanmethemoglobin Mtd
Drabkins Reagent Composition
Non-ionized Detergent
Potassium Ferricyanide (K3Fe(CN)6)
Potassium Cyanide (KCN)
Dihydrogen Potassium Phosphate (KH2PO4)
Sodium Bicarbonae (orig. Drabkins)
Quality Control of the Reagent:
Keep reagent in an [?]and store at RT
Reagent should be pale yellow with a pH of[?]
Reagent must give a reading of[?]
Standard: corresponds to[?]; Stable in a polythene container at (?)
amber bottle
7.0 -7.4
0 at 540 nm
15 g/dl; 2-8oC
QUANTITATION OF FETAL HEMOGLOBIN (HbF)
A. Alkali Denaturation Method (Betke Method; Singer Method)
B. Acid Elution Method (Modification of Kleihauer and Betke by Shepard)
Quantitation of the percentage of HbF in the blood
Alkali Denaturation Method (Betke Method; Singer Method)
A hemolysate is alkalinized and then neutralized, and the denatured adult Hb is precipitated by ammonium sulfate.
Alkali Denaturation Method (Betke Method; Singer Method)
A filtrate will then contain only alkali-resistant Hb, which is measured spectrophotometrically and expressed as a percentage of the total
Alkali Denaturation Method (Betke Method; Singer Method)
Reference value (Modification by Betke):
RV: 0.2 % - 1.0 %
Recommended by NCCLS for Hb F quantitation in the range of 2 -40%
Alkali Denaturation Method (Betke Method; Singer Method)
RID if Hb F is less than 2%
Alkali Denaturation Method (Betke Method; Singer Method)
Alkali Denaturation Method (Betke Method; Singer Method)
Column Chromatography if Hb F is
more than 40%
HPLC
Alkali Denaturation Method (Betke Method; Singer Method)
Elevated Hb F is observed in
Alkali Denaturation Method (Betke Method; Singer Method)
: some hemoglobinopathies
: B-thalassemias
: hereditary persistence of fetal hemoglobin (HPFH)
To assess whether the distribution of HbF in all red cell is the same
Acid Elution Method (Modification of Kleihauer and Betke by Shepard)
Principle: Hemoglobins other than Hb F are eluted from the red cells
Acid Elution Method (Modification of Kleihauer and Betke by Shepard)
Acid Elution Method (Modification of Kleihauer and Betke by Shepard) Stain
Erlich acid Hematoxylin & counterstained w/ Erythrosin.
Results:
RBCs with HbF =
RBCs w/o HbF =
stained bright pink-red
ghost cells
: the Hb F is distributed evenly among red cells.
H ereditary Persistence of Fetal Hemoglobin (PFH)
: the distribution o f the Hb F is heterogenous among cells.
Thalassemia and Hemoglobinopathy
For variant identification
HEMOGLOBIN ELECTROPHORESIS
For the detection and preliminary identification of both normal and abnormal Hbs particularly, Hbs A, F, S and C
Cellulose Acetate Electrophoresis (pH 8.4 8.6)
A small quantity of red cell hemolysate is placed on the cellulose acetate membrane between the center and the cathode. An electrical field is created in the chamber through the use of a power supply.
Cellulose Acetate Electrophoresis (pH 8.4 8.6)
Hemoglobin molecules have a net NEGATIVE charg e at alkaline pH, and therefore migrate towards the anode.
Cellulose Acetate Electrophoresis (pH 8.4 8.6)
Owing to the variations in amino acid content of different Hbs, the net charge of each varies and this determines their rate of mobility in the electrical field.
Cellulose Acetate Electrophoresis (pH 8.4 8.6)
For the separation of Hbs that migrate together on cellulose acetate
Citrate Agar El ectrophoresis (pH 6.0 6.2)
Also useful in detecting small amounts of either HbA or F in the presence or large amounts of the others, and in revealing small amounts of adult Hbs A and S present at birth in cord blood.
Citrate Agar El ectrophoresis (pH 6.0 6.2)
Hbs are separated based on the interactions among Hb variants, agar and citrate buffer ions in addition to the altered electrical charge of the various Hbs at acid pH.
Citrate Agar El ectrophoresis (pH 6.0 6.2)
Hbs are identified by their mig ration toward the anode and cathode and comparing the migration to that of known control samples
Citrate Agar El ectrophoresis (pH 6.0 6.2)
