HEMOGLOBIN

DETERMINATION

Cyanmethemoglobin Method
INTRODUCTION

 Hemoglobin (Hb) is the primary constituent of

RBC.
 The primary function of hemoglobin is to transport
oxygen (O2) from the lungs to the tissue cells of
the body and to carry carbon dioxide (CO2).
 INTRODUCTION

 Hemoglobin has 2 parts as its

name indicates:
 Heme----Globin
 Heme: iron-containing non-protein
group (4 in one hemoglobin
molecule, each one binds reversibly
with one O2)
 Globin: a protein consist of four
subunits
INTRODUCTION

 Hemoglobin:
 If bound to O2  iron content appears RED
 If bound to CO2  iron content appears
BLUE
 O2 Bound = Oxygenated
 Not Bound to O2 = Deoxygenated
INTRODUCTION

 Each gram of Hemoglobin can carry 1.34 ml of O2

 The oxygen combining ability of the blood is in direct
proportion to the hemoglobin concentration, rather than
the # of RBC
MATERIAL
1. Alcohol wipes.
2. Hb pipettes.
3. Cotton
4. Tapes.
5. Drabkin solution: consist of
1. Potassium ferricyanide 200mg
2. Potassium cyanide 50 mg
3. Potassium dihydroen phosphate 140 mg
4. Triton-X 1 ml
5. Distilled water to 1 L
6. Colorimeter or spectrophotometer.
7. Test tubes.
Cyanmathemoglobin Method

K+ Ferricyanide
Hemoglobin (Fe+²) Methemoglobin (Fe+³)
Oxidation
+
K+ Cyanide

Cyanmethemoglobin (HiCN)
CYANMETHEMOGLOBIN
METHOD

 Compare the color of (Hemiglobincyanide:HiCN) with the

standardat 540 nm
 Conventionally used in most routine labs
METHODOLOGY

1. Add 5 ml of Drabkin reagent to a test tube.

2. Add 20ul blood to the test tube using Hb pipette or
micropipette.
3. Mix blood with Drabkin’s and wait for 5 to10 minute
for the reaction to take place.
4. Measure the absorbance of the sample against blank
(drabkin’s reagent) at 540 nm by spectrophotometer
NORMAL HB VALUES ARE:

 Adult males: 14-18gm

 Adult females: 12-16 gm
 Newborn: 17-19 gm
SIGNIFICANT OF THE TEST:
 The measurement of blood hemoglobin is one of
the most common clinical laboratory tests.
 The hemoglobin test is used to indirectly evaluate
the oxygen-carrying capacity of the blood. This
makes it an important aid in detecting and
evaluating blood loss and diagnosing and treating
anemia.
SIGNIFICANT OF THE TEST:
 Physiological Variation:
 HB values are affected by age, sex, pregnancy, and altitude.

 Pathological Variation:
 Decreased level of HB:
 Blood loss.
 Bone marrow suppression
 Increased level of HB:
 Are found in any condition in which the # of RBC rises above
normal
:MECHANICAL SOURCES OF ERROR

 Pipetting error.
 Use of dirty or scratched cuvettes.
 Use of deteriorated reagent.
 HEMATOCRIT

(Packed Cell Volume of Whole Blood)

HEMATOCRIT (HCT)

 Hct or “ Packed cell volume (PCV) Is a proportion of

packed red cells to whole blood in terms of %
 Provides information on the amount of red blood cells
(RBC) present in the blood.
 Decreased levels means anemia from hemorrhage, parasites,
nutritional deficiencies or chronic disease process, such as liver
disease, cancer, etc.
 Increased levels are often seen in dehydration or polycythemia
BLOOD COMPOSITION
 MATERIALS
 Blood sample : EDTA or Finger stick blood sample
 capillary tube:
 heparinized for fingersticks (red tip)
 or plain for anticoagulanted blood (blue tip)
 Microhematocrit centrifuge
 microhematocrit reader
 Clay-type tube sealant
 Gauze
 Alcohol swab
 Lancets for capillary puncture
 (MICRO)HEMATOCRIT
TUBE
 Glass or plastic
 Plain (Blue-ringed) or
heparin (Red-ringed)
coated
 Characteristics:
75 mm long, 1 mm
diameter
 Sealant
MICROHEMATOCRIT
CENTRIFUGE
 Procedure
 Fill blood sample up to 3/4 the length of
the tube (use 2 tubes for each sample, plain
blue-ringed tube for anticoagulated blood,
heparinized red-ringed tube for finger
stick).
 Procedure

 Seal one end with sealant of clay at a 90°.

Procedure
 Place the filled and sealed capillary tubes
into the centrifuge. The sealed ends should
point toward the outside of the centrifuge.
 Centrifuge at (11000 rpm) for 5 min
 Read Hct in % by using microhematocrit
reader
 NR :
 F 37 - 47%
 M 41 – 51%
 new born 53%- 65%
SEQUENCES OF HCT
DETERMINATION
Spin

Place
RBC INDICES
 RBC INDICES
27

 RBC indices include:

 Mean Corpuscular Volume (MCV)
 Mean Corpuscular Hemoglobin (MCH)
 Mean Corpuscular Hemoglobin Concentration
(MCHC)
RBC INDICES

 Color index = Hgb %

RBC x 20

Normal range = 0.9- 101

 MCV
29

 Mean cell volume

 MCV is average size of RBC
 MCV = Hct x 10
RBC (millions)
 If 80-100 fL, normal range, RBCs considered
normocytic
 If < 80 fL are microcytic
 If > 100 fL are macrocytic
 Not reliable when have marked anisocytosis
 MCH
30

 MCH is average weight of hemoglobin per RBC.

 MCH = Hgb x 10
RBC (millions)
 It’s the weight of Hb in RBCs in the individual blood
sample
 Normal range:
 27 -32 picogram
 >27 : pg Microcytic or normocytic
 <32 pg macrocytic
 MCHC
31

 MCHC is average hemoglobin concentration per

RBC
 MCHC = Hgb x 100
Hct (%)
 If MCHC is normal, cell described as
normochromic
 If MCHC is less than normal, cell described as
hypochromic
 There are no hyperchromic RBCs
 MCHC
32

 Normal values:
 33 -38 %
 < 38 Not found
 > 32 hypochromia