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

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

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

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

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

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

Hemocytometer

Diluting pipette

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

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

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

1. Carriage of O2 2. Carriage of CO2 Functions of Hb: 1. Carriage of O2 2. 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

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

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

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

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+)

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

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

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

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