1. (Hemocytometry and hemoglobin)
Practical
(Hemocytometry and hemoglobin)
Physiology
232 BMS
Dr/Nahla Yacout
2016/2017

2. Objectives At the end of this lecture, students should be able to:
Define Hemocytometry, and explain its principle
Identify Hemocytometer and its set
Explain steps of hemocytometry
Define Hemoglobin and enumerate its functions
Explain different varieties and derivatives of hemoglobin
Define Hemoglobinometry and its different methods

3. Hemocytometry
Procedure of counting the number of cells in a sample of blood
Red blood cells (RBCs), white blood cells (WBCs), & platelets are counted separately
It is assumed that blood cells are homogenously mixed in the plasma in all regions of the body

4. Principle
How to overcome this problem?????
The sample of blood is diluted in a special pipette, & then placed in the counting chamber in the cell counter
By knowing the dilution, the number of cells in the undiluted blood can be easily calculated
Since the number of blood cells is very high, it is difficult to count them even under microscope

5. Units for counting Cells/cubic mm Example:
RBCs count: 5.0 million/c.mm
c.mm = µl (Micro liter)

6. Hemocytometer
Is the apparatus used for cell counting Hemocytometer set consists of:
1. Two diluting pipettes
One for counting RBCs & the other for WBCs
Each having a bulb
2. Counting chamber
4. RBCs & WBCs diluting fluids
3. Cover slips

7. Hemocytometer

8. Diluting pipette

9. Difference between RBC &
WBC pipettes
RBC pipette
WBC pipette
Calibrations are 0.5 & 1.0 below the bulb & 101 above the bulb
Calibrations are 0.5 & 1.0 below the bulb & 11 above it
Bulb is large
Bulb is small
The volume of the bulb is 100 times the volume of the stem
The volume of the bulb is 10 times the volume of the stem
The dilution can be 1 in 100 or 1 in 200
The dilution can be 1 in 10 or 1 in 20

10. Steps of hemocytometry
Keeping all the equipment ready
Getting a sample of blood
Pipetting: Filling the pipette with blood & diluting it
Charging: Filling the counting chamber with diluted blood
Counting the cells & reporting the results

11. Hemoglobin
Is the protein molecule in the red blood cells that carries oxygen from the lungs to the tissues, & return carbon-dioxide from the tissues back to lungs
It consists of 4 subunits
Each subunit consists of Heme + Polypeptide chain
The four polypeptide chains
make up the protein part
called Globin
2 alpha chains
2 beta chains

12. 1. Carriage of O2 2. Carriage of CO2
Functions of Hb:
1. Carriage of O Carriage of CO2
from lungs to tissues from tissues to lungs
3. Stabilization of tissue PO2, in which sufficient
amount of O2 is delivered between PO2 of
20 – 40 mmHg

13. 4. Regulation of blood flow & blood pressure
Heme has high affinity to nitric oxide (NO) …… A local vasodilator
SO, decrease in the amount of Hb ….. Leads to decrease in NO action …… And so vasoconstriction & decrease in blood flow

14. Different varieties of Hb:
Adult Hb (HbA): 95% of Hb in adults is HbA
2 alpha + 2 beta
2. HbA2: 2-5% of Hb in adults is HbA2
2 alpha + 2 delta
3. Fetal Hb (HbF): In fetus till 5 months
2 alpha + 2 gamma

15. Difference between adult & fetal Hb:
Adult Hb (HbA)
Fetal Hb (HbF)
Contains 4 polypeptide chains (2 alpha & 2 beta)
Contains 4 polypeptide chains (2 alpha & 2 gamma)
At birth, 20% of total Hb is HbA
At birth, 80% of total Hb is HbF
Life span: 120 days
Life span: 2 weeks
Has the normal affinity for O2
Has higher affinity for O2

16. Derivatives of Hb OxyHb Reduced Hb CarbaminoHb Hb + O2
Release of O2 from OxyHb
Hb + Carbon - dioxide
CarboxyHb
Methemoglobin
Hb + Carbon – monoxide (CO)
Normally CO is present in small amounts
Bu, if present in large amounts as in smokers …… it will impair O2 transport
Ferrous iron (Fe2+) of Hb is converted into ferric iron (Fe3+)

17. Is the measurement of the amount of Hb in blood
Hemoglobinometry
Is the measurement of the amount of Hb in blood

18. Hemoglobinometry methods
Gasometric method Hb is saturated with O2 & then its amount is measured Visual color comparison Amount of Hb is measured by changing to another color

19. Hemoglobinometry methods
Spectrophotometric method Hb is converted to other compounds, & then by using definite wavelengths of light each of these compounds can be measured Electronic Hematology Analyzer Can give Hb concentration automatically

20. References
CL Ghai MBBS MD, “A Textbook of Practical Physiology”; 7th edition