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 REDIf bound to CO2 iron content

appears BLUEO2 Bound = OxygenatedNot 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. Cotton4. Tapes.5. Drabkin solution: consist of

1. Potassium ferricyanide 200mg2. Potassium cyanide 50 mg3. Potassium dihydroen phosphate 140 mg4. Triton-X 1 ml5. Distilled water to 1 L

6. Colorimeter or spectrophotometer.7. Test tubes.

Cyanmathemoglobin Method

Hemoglobin (Fe+²)K+ Ferricyanide

Oxidation Methemoglobin (Fe+³)

+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

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

MECHANICAL SOURCES OF ERROR:

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

Place

Spin

RBC INDICES

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RBC INDICES

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

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MCV

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

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MCH

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

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MCHC

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

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MCHC

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