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Red Blood Cells Count (RBCs count) 5

الكلية كلية طب الاسنان     القسم  العلوم الاساسية     المرحلة 2
أستاذ المادة وسن نجم عبد السادة الربيعي       21/02/2019 19:03:43
RED BLOOD CELLS COUNT
(RBCs COUNT)

Red blood cells RBCs are biconcave disks, they are 8 microns in diameter and 2 microns thickness. The name of red blood cells reflects the bright red colour of the cell that occurs when oxygen is attached to the haemoglobin. Because the cell dose not has a nucleus its life span is limited by its energy supply.
The purpose of RBCs count is to determine the number of circulating red blood cells in the body. Since RBCs carry O2 to all tissues, any change in RBCs count in the body must be diagnosed immediately.
RBCs count is decreased in case of “anaemia”, and increased in case of “polycythemia”.

THE PRINCIPLE
The blood is diluted 200 times, with the RBCs diluting fluid, then counted in a haemocytometer (the apparatus used for blood cells counting).

HAEMOCYTOMETER
Haemocytometer: consists of
a. counting chamber
b. thick cover glass
c. pipette for diluting blood
d. Rubber tube with plastic mouth piece for drawing the fluid into a pipette.

COUNTING CHAMBER:
It is a heavy glass slide, with two chambers extending across the slide, set parallel to each other, and when the coverglass rest upon the center platform, it producing the depth 0.1 mm of the counting chamber.
The counting chamber covers an area of 9mm2 divided into squares of 1 mm2 each. The four corner squares are divided into16 squares each with an area of 1/16 th of a mm2 used for counting WBCs. The central ruled area of 1 mm2 is divided into 25 large squares by sets of triple lines. These large squares are further subdivided into 16 small squares by single lines, each with an area of 1/400 of 1 mm2 used for RBCs count (Fig.1).

Thoma Red Cell Pipette:
It has a narrow glass stem, the stem is lined with two lines marked 0.5 & 1.0 another line above the bulb (that contains a red glass bead which helps in mixing) is marked 101 (Fig.2).











































DILUTING FLUIDS
1. Hayem ‘ s fluid:
mercuric chloride 0.25 gm sodium chloride 0.5gm
sodium sulphate 2.5 gm D.W. 100 ml
2. Formal citrate
sodium citrate 3 gm
formaldehyde 1 ml
D.W. 100 ml

PROCEDURE:
1. Examine the chamber under the low power objective of microscope, without the coverslip, in order to understand the ruling.
2. Clean the counting chamber and the coverslip, place the chamber on a flat surface, and then put the coverslip on the surface of the chamber.
3. Draw blood into red cell pipette up to the 0.5 mark. This will give final dilution of 1 in 200. Make sure that there are no air-bubbles. If the blood drawn above 0.5mark, the end of the pipette should be touched against the hand, to withdraw the excess.
4. Wipe the out side of pipette with a piece of clean gauze.
5. Draw the diluting fluid up to the 101 mark, don’t allow any of the blood sample to leak out into the diluting fluid, because any count performed afterward would be incorrect. The count would be too low because the blood is too diluted..
6. Withdraw the pipette from the diluting fluid, and wipe the outside with a piece of clean gauze. Close the tip of the pipette with the thumb, remove the sucker, place the middle finger over the top and mix well by shaking for at least 2 min, and discard about a quarter of the mixture to get rid of the unmixed suspention.
7. Fill the chamber by holding the pipette at an angle of 45 and lightly touching the tip against the edge of the coverslip; allow the diluted blood to flow evenly and slowly under the coverslip by cappilary action. It is important that the fluid is not allowed to overflow into the channels; this will cause the chamber to overfill. Overfilling results in an inaccurate cell count. If this occur, the chamber shoul be cleand and refilled.
8. Leave the chamber for another 2 min., for the red cells to settle down.
9. Place the chamber on the microscope stage, count the cells in squares of the central portion of improved Neubauer (4 corner and 1 in the middle) of the whole chamber.




CALCULATION:
1. You count the No. of RBCs in 80 small squares.
2. The area of one small square is :
1/20 x 1/20 = 1/400 mm2
3. Depth of small sq. is 1/10 mm3
4. Volume of fluid in each small sq. is :
1/400 x 1/10 = 1/4000 mm3
5. Volume above 80 small sq. is:
1/4000 x 80 = 1/50 mm3
6. (N) Is No. of cells in 80 small sq. so the No. of RBCs in 1 mm3 is:
(50 x N)
7. diluted blood is 1/200, so 1 mm3 of blood contains (200 x 50 x N)

RBCs count = N x 10000 cells/mm3 of blood

-Normal values are shown in tab.*

SOURCE OF ERRORS:
Low Counts
1. Squeezing the site of puncture when filling the pipette.
2. Insufficient blood being draw into the pipette.
3. Too much diluent being draw into the pipette.
4. Insufficient mixing.
5. Using the first expelled from the pipette; it contains only diluting fluid without cells.
6. Insufficient filling the containing chamber.
7. Not well distribution of the cells in the counting chamber.
8. Faulty counting technique.
9. Errors in calculation.

High Count
1. Using the first drop expelled from the site of the puncture.
2. Too much blood being drawn into the pipette.
3. Insufficient diluent drawn into the pipette.
4. Insufficient mixing of the blood diluent suspension.
5. Overfilling the counting chamber, this may raise the cover glass, causing variation in the depth.
6. Not well distribution of the cells in the counting chamber.
7. Errors in calculation.
8. Contaminated diluting fluid.
9. Wet or dirty pipettes.
10. Drying of the dilution in the counting chamber may occurs.


المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .