Hemoglobin

بـسم الله الرحمن الرحيم

Practical Clinical Pathology

I-Introduction

II-Clinical Hematology

III-Clinical Biochemistry

IV- Urinalysis

V-Clinical Parasitology

Clinical Hematology

1. Introduction to Clinical Hematology

2. Hemoglobin

3. PCV

4. ESR

5. Blood film and staining

6. Bone marrow examination

7. Reticulocyte counting

8. RBCs (count, morphology and indices)

9. WBCs (count, morphology and differential count)

Clinical Hematology

1. Hemoglobin estimation by Sahli’s method

2. Hemoglobin estimation by Drabkin’s method

3. RBCs count

4. WBCS count (Total Leukocytic count)

5. Differential Leukocytic Count (DLC)

Hemoglobin estimation

Direct matching methods

1. Tallquist method

2. Dare hemoglobinometer

Indirect methods

1. Sahli method (Acid hematin)

2. Drabkin method (Cyanomethemoglobin)

(Photometric method)

Direct matching methods1-Tallquist method

Direct matching methods2-Dare hemoglobinometer

A drop of blood is placed in a capillary space

between two glass pieces

The red color is compared through an eye piece

with red graded glass standards

Dare hemoglobinometer

Direct matching methodsAdvantages and Disadvantages

Advantages Simple,

Rapid

Inexpensive

Disadvantages Error ranges from 10% to 40%

Comparison of red color is difficult

Hemoglobin estimationIndirect methods

Sahli method(Acid hematin)

Sahli apparatus

• Two lateral standard tubes

• One central bigraduated tube

• Sahli pipette (20 µl)

Central bigraduated tube2 scales

Gram/dl scale Percent % scale

Sahli pipette Single mark pipette

Sahli method

9 items in most practical experiments

Principle

Sample

Reagents

Procedures

Wavelength

Reading

Calculation

Normal values

Interpretation

Sahli methodPrinciple

Hemoglobin + 0.1 N HCl

The resulting brown colour is matched with brown glass standard

Globin

Acid hematin (brown colour)

Sahli methodSample

Whole blood

=

Anticoagulated blood

Sahli methodReagents

0.1 N HCl Prepared by diluting 0.8 ml conc. HCl with 99.2 ml D.W.

Distilled Water (D.W.)

Sahli methodProcedures

1. Place 0.1 N HCl into the central bigraduated tube till the mark 2 on the gram scale

2. Draw the blood into Sahli pipette exactly to the mark 20 Wipe off excess blood adhering to the pipette

3. Rinse the pipette by drawing the acid in the tube several times

4. Mix, let stand for 10 minutes

5. Add distilled water (drop by drop with mixing) until it matches the color of the standard tubes

6. Take the reading in g/dl scale

Sahli methodWavelength

-----

Sahli methodReading

Hb conc. = ----- g/dl

Sahli methodCalculation

-----

Sahli methodNormal values

Hb conc. = 11 -18 g/dl

In Equines

Sahli methodInterpretation

Increased values (>18 g/dl)Physiological

High attitude

Young age

PathologicalDehydration

Sahli methodInterpretation

Decreased values (<11 g/dl)Physiological

Fluid therapy

Pathological Anemia

Hemorrhage

Blood parasites

Malignant tumors

Sahli methodAdvantages and Disadvantages

AdvantagesError may be 5-10% by careful technique

DisadvantagesDifficult colour matching

Colour fading by time

Hemoglobin estimationIndirect methods

Drabkin method(Cyanmethemoglobin)

Drabkin method

9 items in most practical experiments

Principle

Sample

Reagents

Procedures

Wavelength

Reading

Calculation

Normal values

Interpretation

DrabkinPrinciple

Hemoglobin + Ferricyanide Methemoglobin

Cyanide ions

Cyamethemoglobin

- Stable red compound- Measured colorimetrically-Its intensity is directly proportional to the amount of hemoglobin in the sample

Drabkin methodSample

Whole blood

=

Anticoagulated blood

DrabkinReagents

Drabkin reagent

Potassium ferricyanide

Potassium cyanide

Sodium Bicarbonate

Sterox (Detergent)

DrabkinProcedures

1. Add 2.5 ml of the reagent into both the blank and (test) tubes

2. Add 10 µl of blood into the (test) tube

3. Rinse the pipette several times

4. Mix well and allow to stand for 5 minutes

5. Use the blank tube to adjust the colorimeter to zero optical density at 546 nm wavelength.

6. Read the optical density of the test

Blank Test

0 -

1 –

2 –

3 –

4 –

5 –

0 -

1 –

2 –

3 –

4 –

5 –

0 -

1 –

2 –

3 –

4 –

5 –

0 -

1 –

2 –

3 –

4 –

5 –

Drabkin reagent

Blank’ Test

0 -

0.01 –

0.02 –

0.03 –

0.04 –

0.05 –

0.06 –

0.07 –

0.08 –

0.09 -

0 -

0.01 –

0.02 –

0.03 –

0.04 –

0.05 –

0.06 –

0.07 –

0.08 –

0.09 -

Blood

0 -

0.01 –

0.02 –

0.03 –

0.04 –

0.05 –

0.06 –

0.07 –

0.08 –

0.09 -

Blank’ Test

Blank’ Test

Blank’ Test

DrabkinWavelength

546 nm

DrabkinReading

OD = 0.xxx

DrabkinCalculation

Drabkin methodNormal values

Hb conc. = 11 -18 g/dl

In Equines

Drabkin methodInterpretation

Increased values (>18 g/dl)Physiological

High attitude

Young age

PathologicalDehydration

Drabkin methodInterpretation

Decreased values (<11 g/dl)Physiological

Fluid therapy

Pathological Anemia

Hemorrhage

Blood parasites

Malignant tumors

Drabkin methodAdvantages and Disadvantages

AdvantagesAccurateRapidInternationally adapted

DisadvantagesCyanide is toxic solution

Lipemia may falsely increase the Hb values

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