MOI UNIVERSITY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF MEDICINE

DEPARTMENT OF PATHOLOGY

COURSE: HAEMATOLOGY

COURSE CODE: MSP 301

ASSIGNMENT: HAEMATOLOGY PRACTICAL REPORT

PRESENTED TO: MR. MACHARIA

Practical one: Hb estimation

Introduction

Hemoglobin (Hb) is a blood substance containing iron and protein. Hb is responsible for carrying
oxygen from lungs to the various parts of the body through blood. It is also responsible for
carrying carbon-di-oxide from various parts of the body to the lungs. The normal level for
hemoglobin in the blood is as given below. Women 12 - 16 g/100 ml blood 2) Men 14 - 18 g/100
ml blood 3) Newborn 14 - 20 g/100 ml blood. Anemia is an unhealthy condition that develops in
human beings when Hb level in their blood is below the normal level of the blood.

A) Cyanmethaemoglobin method

Sample: Both capillary and venous blood may be used for this test.

Principle

Blood is diluted in a solution containing potassium cyanide and potassium ferricyanide. The
solution hemolysis blood and then converts oxyhemoglobin and carboxyhaemoglobin and not
sulphaemoglobin to haemoglobincyanide (cyanmethaemoglobin)-HiCN. The absorbance of this
solution is measured at a wavelength of 540nm.

Method

 Reagent and equipment for Cyanmethaemoglobin method:

 Diluent (Drabkin’s solution)
 5 ml pipette.
 Cuvettes.
 Test tube.
 20 micro liter pipettes. Potassium ferricyanide 200mg
 Potassium cyanide 50mg
 Potassium dihydrogen phosphate 140mg
 Nonionic detergent 1ml
 Dissolve the components in 1 litre of water
 Hemoglobin standard

Procedure
 20ul blood + 4ml diluent HiCN.
 Mix, 5-10min sample Measured by spectrophotometer at 540nm standard Use the
calculator:
 Hb (g/dl)= Absorbance of test X Conc of standard Absorbance of standard
 Measure 4ml of the reagent into a clean bottle or tube into this solution add 20µl of
anticoagulated blood and mix
 Let the solution stand at room temperature for 3-5 minutes
 Adjust the wavelength of the spectrophotometer to 540nm
 Using blank reagent adjust the reading of the spectrophotometer to read zero
 Using another cuvette read and record the absorbance of the standard
 Similar read and record the absorbance of the test solution

Calculation

The value of test Hb=Absorbance of test sample x conc. Of standard Absorbance of standard.

Results

Discussion

B) Sahli’s method

Introduction

Sahli’s method, also known as the acid hematin method, is a visual comparator method for
estimating hemoglobin1. It was first performed by Hermann Sahli. However, it’s important to
note that visual comparison may lead to unacceptable imprecision and accuracy. As a result,
this method is not recommended nowadays and the use of spectrophotometric methods like
Cyanmethemoglobin method is preferred

Principle

The principle of Sahli’s Method or Acid hematin method is quite easy that when the blood is
added to N/10 Hydrochloric acid (HCl), the hemoglobin present in RBCs is converted to acid
hematin which is a dark brown colored compound. The color of the formed acid hematin
 complex corresponds to the Hemoglobin concentration in the blood and is matched with the
standard which is a reference brown glass given in the Sahli’s apparatus by diluting with
N/10 hydrochloric acid or distilled water until the color of acid hematin complex match with
the color of the standard.

Reagents

N/10 hydrochloric acid (It is prepared by diluting concentrated hydrochloric acid 0.98 ml in
distilled water and volume is made up 100 ml).

 Distilled water

Apparatus

 Sahli’s Apparatus
 Hemoglobin pipette (0.02 ml or 20 µl capacity)
 Sahli’s graduated Hemoglobin tube
 Thin glass rod Stirrer for Hemoglobin Tube
 Sahli’s Comparator box with brown glass standard
 Spirit swab
 Blood Lancet
 Dry cotton swab
 Pasteur pipette

Procedure

1. Fill the tube with 0.1N Hydrochloric Acid (HCl) up to 2 marks1.

2. Place the tube in the Hemoglobin meter2.
3. Suck blood into Sahli’s pipette up to 20 marks and drop this blood into the graduated
tube2.
4. Mix the solution with a stirrer and wait for 3-5 minutes

Results

Discussion

Practical two: PCV test

Introduction

Packed cell volume (PCV), also known as hematocrit (HCT), is a measure of red blood cell
mass. It measures how much of the blood consists of cells. For example, a PCV of 50% means
that 50 ml of cells are present in 100 ml of blood. The PCV test is used to diagnose conditions
such as anemia, polycythemia or dehydration in patients. It is generally done along with a full
blood count test and can be used to estimate the need for any blood transfusions and monitor the
response to treatment.

Principle

The principle of PCV estimation using a centrifuge involves placing heparinized blood in a
capillary tube, filling it to 75% of its length, sealing it with plasticine and then centrifuging it in a
microhematocrit centrifuge at 10,000 RPM for five minutes1. This separates the blood into
layers. Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the
red cells. Centrifugation is done in a special microhematocrit centrifuge until packing of red cells
is as complete as possible.

Procedure

The procedure for capillary method of PCV estimation using a centrifuge involves placing
heparinized blood in a capillary tube, filling it to 75% of its length, sealing it with plasticine and
then centrifuging it in a microhematocrit centrifuge at 10,000 RPM for five minutes1. This
separates the blood into layers. The materials required for this procedure include a centrifuge,
whole blood in heparin or EDTA tube, microhematocrit tubes, plasticine tray, paper towel or
tissue, microhematocrit reader and gloves.

Results

Discussion

References