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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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