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. 2. 3. 4. 5.

Alcohol wipes. Hb pipettes. Cotton Tapes. Drabkin solution: consist of
1. 2. 3. 4. 5.

Potassium ferricyanide 200mg Potassium cyanide 50 mg Potassium dihydroen phosphate 140 mg Triton-X 1 ml Distilled water to 1 L

6. 7.

Colorimeter or spectrophotometer. 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. 2.

3.

4.

Add 5 ml of Drabkin reagent to a test tube. Add 20ul blood to the test tube using Hb pipette or micropipette. Mix blood with Drabkin¶s and wait for 5 to10 minute for the reaction to take place. 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 ³ 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 

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