A.

Gasometric Method
B. Chemical Method
C. Gravimetric Method
D. Colorimetric Methods
 A given sample of blood can be equilibrated
with O2 under standard conditions of TO & P
 Equation: (Hufner’s factor)
1 gm Hb = 1.34 ml O2
 Limitation: Measures active hemoglobins only
 Equation: 1 gm hb = 3.47 mg iron
A. Wong Method
 Iron is liberated by H2SO4 and potassium
persulfate. Proteins are precipitated by
tungstic acid.
 Iron (PFF) is measuredvia ferric thiocyanate
reaction
B. Assendelft Method
 Based on specific gravity
 Commonly used 1.053 for mass screening
DIRECT VISUAL COLORIMETRIC

1. Tallquist Mtd:

 10 - 100%
 Error: 50%
DIRECT VISUAL COLORIMETRIC

2. Dare’s hemoglobinometer:
 2 glass plates
 red tinted glasses
 Error: 30%
3. Acid Hematin:

 Hb + acid  Acid Hematin

 Acid hematin is then diluted
with d. H2O

 Limitation: does not measure

inactive forms
Methods:
Sahli-Adams Haydenn-Hausser
Newcomer Sahli-Hellige
Sahli-Hayden Osgood-Haskin
4. Alkali Hematin:

 Hb (N & Abn) + alkali  alkali hematin

 More stable
 Limitation: Not ideal for infants & children

Methods: Wu; Klegg & King

PHOTOELECTRIC COLORIMETRIC
1. Oxyhemoglobin Mtd:
 0.02 ml Blood + 5 ml 0f 0.07N NH4OH or
0.1%Na2CO3
 Shake (proper oxygenation)

 Product is: oxyhemoglobin

 540 nm against reagent blank

Methods:
Harboe – 415 nm Naumann
2. Cyanmethemoglobin Mtd/

DRABKIN’S Composition
 Non-ionized Detergent (Sterox [Harleco] or
Triton)
 (K3Fe[CN]6)
 KCN
 KH2PO4 (NaCO3 - orig. Drabkin’s)
 Procedure:
 1:251 or 1:301 blood-reagent
 3 minutes @ RT
 Absorbance at 540 nm against Reagent B.
 Determine concentration:
 standard curve
 calculate (Beer’s Law)
Quality Control of the Drabkin’s Reagent:

 Use fresh reagent

 Keep reagent in an amber bottle & store at RT
 Reagent should be pale yellow with a pH of
7.0 – 7.4
 A = 0 @ 540 nm
Standard: 15 g/dL
: Stable in a polythene at 2 - 8oC.

Precautions:
 Toxic
effects can be seen if swallowed in the
quantity range of 4 – 16 L.
 Poisonous cyanide (HCN) gas is released if the
sink has an acidic solution.
 TECHNICAL ERRORS

 Inaccuracy of the pipet

 Pipeting error
 Use of unmatched cuvet
 Deteriorated reagent
 Oxidation of the reagent
 PHYSIOLOGIC ERRORS

Turbidity
 Lipemic blood
R: Use patient blank

 High Leukocyte count

R: Centrifuge and use supernate
 PHYSIOLOGIC ERRORS

 Abnormal Hb
 R: dilute sample 1:1

 Easily ppted CHONS (M. Myeloma &

Waldenstrom Macroglobulinemia)
 0.1 gm of K2CO3/L Orig. Drabkin’s
A. ALKALI DENATURATION

A. Betke Method
B. Singer Method

 Quantitation of the percentage of HbF in

the blood
 (+) control: fetal umbilical blood
A. ALKALI DENATURATION

 RBC hemolysate + Drabkin’s + NaOH,

+ (NH4)2SO4 - ppt denatured HbA

 Filtrate (HbF) - measured

spectrophotometrically
A. ALKALI DENATURATION
 Reference value (Modification by Betke) :
0.2 % - 1.0 %
 Recommended by NCCLS for Hb F
quantitation in the range of 2 – 40%
 PERFORM RIA if Hb F is less than 2%
 PERMOFROM Column Chromatography if
Hb F is more than 40%
 Elevated Hb F:
 some hemoglobinopathies
 B-thalassemias
 Hereditary persistence of fetal
hemoglobin.
B. ACID ELUTION (Modification of Kleihauer and
Betke by Shepard)

 To assess whether the distribution of HbF in

all red cell is the same
 Determines the presence of fetal red cells
in the maternal circulation
 Useful in the dx of HDN
B. ACID ELUTION (Modification of Kleihauer and
Betke by Shepard)

 Hemoglobins other than Hb F are eluted

from the red cells on an air-dried blood film
by a citric acid-phosphate buffer (pH 3.3).
 Only Hb F remains in the fixed cells
 Stain - Erlich acid Hematoxylin
Counterstain - Erythrosin.
B. ACID ELUTION (Modification of Kleihauer and
Betke by Shepard)

 RBCs with HbF = stained bright pink – red

 RBCs w/o HbF = ghost cells
normal adults: almost all red cells appear as
ghosts
:1% - 5% of red cells contain
residual Hb F.
B. ACID ELUTION (Modification of Kleihauer and
Betke by Shepard)

Clinical Significance:
 HPFH: even distribution of HbF among red
cells.
 Thalassemia and Hemoglobinopathy:
heterogenous distribution of the Hb F
among cells.
A. Cellulose Acetate Electrophoresis
(pH 8.4 – 8.6)

 Forthe detection and preliminary

identification of both normal and abnormal
Hbs.
 Hbs A, F, S and C
A. Cellulose Acetate Electrophoresis
(pH 8.4 –8.6)
Principle:

 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)

A2
Cathode SFC
A A2 S F A Anode
ORIGIN C D
E
D (+)
(-) E G
G
C O
B. Citrate Agar Electrophoresis (pH 6.0 – 6.2)

 Detects small amounts of either Hb A or F in the

presence or large amounts of the others;

 Reveals small amounts of adult Hbs A and S

present at birth in cord blood.

 Separates hb that migrate together on cellulose

acetate.
B. Citrate Agar Electrophoresis (pH 6.0 – 6.2)
Principle:
 Separation of Hbs: interactions among Hb
variants, agar and citrate buffer ions; altered
electrical charge of the various Hbs at acid pH
B. Citrate Agar Electrophoresis (pH 6.0 – 6.2)

A2
Cathode Anode
A
C
S
F
(+)
(-)

ORIGIN
 DITHIONITE SOLUBILITY TUBE TEST

 Screening test: sickling hemoglobin

 Red cells are lysed by saponin allowing hb to
escape.
 Sodium dithionite binds with oxygen
 Deoxygenated hb S polymerizes and forms a
precipitate
 The tactoids make the solution turbid.
 DITHIONITE SOLUBILITY TUBE TEST

 Turbidity: against a newsprint/reader card with

thin black lines.

 Turbidity: (+) _______

1. Isopropanol Pptn Test

2. Heat Denaturation/Instability Test

(50OC for 3 hrs.)

3. Heinz body staining