TOTAL ERYTHROCYTE COUNT (MANUAL METHOD)

Published in Hemotology
Tuesday, 18 July 2017 13:44
Erythrocyte (Gr. erythros, red; kytos, cell) or red blood corpuscles are circular, anucleated, highly flexible, biconcave disc-shaped cells with high edges. The sixe of each cell averages 7.2 micrometer in diameter and 2.1 micrometer in thickness. It is 1.0 micrometer thick in the center. A complex membrane surrounds it, which is a bimolecular layer of protein. There is an inner most structure, called stroma, which is composed of lipids and proteins in the form of a fibrous protein. The cell contents are 90% hemoglobin. There are two methods for estimation of erythrocyte count:
 
  • Manual or microscopic method
  • Automated method
 
MANUAL METHOD

Equipment

Hemocytometer with cover glass, compound microscope.
 
 
Hayem’s diluting solution is prepared as follows:

HgCl2               0.05 gm
NaSO4               2.5 gm
NaCl                  0.5 gm
Distilled water     100 ml
 
Specimen
 
EDTA anticoagulated venous blood or blood obtained by skin puncture is used.
 
Method
 
  1. Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate blood to 0.5.
  2. Aspirate diluting Hayem’s solution to the 101 mark. It will give 1:200 dilution of the blood.
  3. Hold the pipette horizontally and role it with both hands between finger and thumb.
  4. Place the counting chamber, absolutely free from dust and grease, on the table and lay the cover glass in place over the ruled area.
  5. Discard the first two or three drops from the pipette. Charge the counting chamber by holding the pipette in an inclined position. Allow 3 minutes for the cells to settle.
  6. Locate the central square, which is divided into 25 medium sized squares. Each of the medium sized squares is further divided into 16 smallest squares.
  7. Count the erythrocytes in medium sized squares (80 smallest squares) using high power objective.
  8. In order to avoid confusion in counting, count all cells wihich touch the upper and left outer double line of the group of 16 squares as if they were inside the square. Neglect all those cells, which touch the lower and right inner line.
 
Calculation
 
You may calculate total number of erythrocytes per cu mm of the blood as shown in the following.
 
Supose number of erythrocytes counted in 5 intermediate squares
 
= E
 
Area of each of the five squares in which cells are counted
 
= 1/25 sq mm
 
Therefore, total area counted
 
= 1/25 sq mm x 5
= 1/5 sq mm
 
Depth of chamber = 1/10 mm
 
Therefore, the volume in which cells are counted
 
= Area x Depth
= 1/5 sqmm x 1/10 mm
= 1/50 cu mm
 
Now, in 1/50 cu mm of diluted blood, the number of erythrocyte counted = E
 
Number of erythrocyte in one cu mm in diluted blood = E x 50
 
Since the dilution of the blood is 1 in 200, the number of erythrocytes in one cu mm of undiluted blood
 
= E x 50 x 200
 
GENERAL NOTES

(1) Increased in numbers of RBC called polycythemia it is due to
 
Congenital heart disease
• Cor pulmonale
Dehydration
• Pulmonary fibrosis
• Polycythemia vera
 
(2) Decreased in numbers of RBC is due to
 
• Anemia
Bone marrow failure
• Erythropoietin deficiency (2ndry to kidney disease)
Hemolysis (RBC destruction) from transfusion reaction
Hemorrhage
• Leukemia
• Multiple myloma
• Nutritional deficiencies of (Iron, Copper, Folate, Vit B12, B6)
 
REFERENCE RANGES

• Newborns: 4.8-7.2 millions
• Children: 3.8-5.5 millions
• Adult ( male): 4.6-6.0 millions
• Adult (Females): 4.2-5.0 millions
• Pregnancy: slightly lower than normal
 
REFERENCES
 
  • Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
  • Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
  • Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
  • Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
  • Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
  • Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.

Useful Sites

  • NCBI

    National Center for Biotechnology Information
  • LTO

    Lab Tests Online® by AACC
  • ASCP

    American Society for Clinical Pathology
  • ASM

    American Society for Microbiology
  • The Medical Library®

    Project of BioScience.pk
Advertisement

Connect With Us

Contact Us

All comments and suggestions about this web site are very welcome and a valuable source of information for us. Thanks!

Tel: +(92) 302 970 8985-6

Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Website: https://www.bioscience.pk

Our Sponsors

The Physio ClubB2BPakistan.com

By using BioScience.pk you agree to our use of cookies to enhance your experience on this website.