Beruflich Dokumente
Kultur Dokumente
Practical :
Learning Objectives
At the end of the practical, students should be able to :
Identify the parts of a haemacytometer, and areas where cells are counted under the microscope. Explain the purpose of performing total RBC and platelet counts. State the important properties of diluting fluid for counting RBC and platelets. Perform manual cell counts for RBC and platelets Calculate the results using the general formula for calculating cell counts. List the precautions to be observed in performing manual cell count for RBC and platelets. Name the pathological conditions that are associated with increased or decreased RBC or platelet counts.
Units Reported
Methods for counting cells are designed to obtain the number of cells in 1 L of whole blood. SI unit recommended by ICSH : per 1 L. Unit formerly used : per 1 mm3.
1 mm3 = 1 L = 1 x 10-6 L
ICSH = International Council for Standardisation in Haematology
8
Basic procedures :
1. Diluting the blood quantitatively. 2. Determining the no. of cells. 3. Converting the no. of cells (in diluted sample) to the no. of cells in 1 L of whole blood.
1 mm3 = 1 L = 1 x 10-6 L
1 L = 1 x 106 L
9
Blood Sample
Blood capillary blood, EDTA venous blood. diluted to enable cells to be counted.
Prevent lysis or gross changes in cells to be counted (i.e. isotonic, fixative). Ideally destroy unwanted cells. Contain an anticoagulant. Be free from debris.
10
Haemocytometer
0.1 mm depth
0.1 mm
Single Net
Double Net
NonBright-Line
14
Neubauer Ruling
WBC = A + B + C + D = 4 mm2
Care of Haemocytometer
Before use.
- haemocytometer held by sides and bottom to avoid fingerprints. - coverglass handled by edges. - wiped with lens papers dipped in 70% or 95% alcohol. - dried and polished with gently with lens papers.
After use.
- disinfected by soaking in 10% chlorine bleach for 10 mins. - stored in container to keep dirt and dust off, to protect ruled areas from scratches. - placed gently on secure surface.
17
Time-consuming. Inaccurate (large error - 20% due to uneven cell distribution in chamber) Limited use in routine practice (reference method is automated).
18
RBC Diluting fluid : Dacies fluid / Hayems solution 30% trisodium citrate + formalin (formal-citrate) Preserve morphology. Prevent clumping or clotting.
estimation on blood film A well-prepared blood smear can be used to estimate platelet count. Is still necessary if there is a significant proportion of giant platelets.
21
PLT Diluting fluid : Ammonium oxalate (1%) Prevent clumping or clotting. Completely lyse RBC. Leaves PLT and WBC intact. Other PLT diluent :
30% trisodium citrate + formalin (formalcitrate) does not lyse RBC. Gives incorrect results when platelet count is low.
22
24
Count the cells using 40X dry obj. or 10X eyepieces. Count as many as possible increased accuracy. Count cells in 80 smallest squares (centre) 0.2 mm2 or 0.02 mm3. Include cells touching top and left hand margin. Minimum 500 cells to be counted.
25
(per L)
TRBC =
R X
1 0.02
X 200
Volume of blood counted = 0.02 mm3 (L) Dilution factor = 200 (1:200 dilution) Final TRBC count = ______ x 1012/L
26
(per L)
TPLT =
P X
1 0.02
X 100
Volume of blood counted = 0.02 mm3 (L) Dilution factor = 100 (1:100 dilution) Final TPLT count = ______ x 109/L
27
End
Left and top lines counted (solid circles) Right and bottom lines not counted (open circles)
Each group of squares is separated by triple lines. The middle one is the boundary. Triple lines show clearly which cells lie within the counting area and eliminate guesswork.
31
Tally Counters
33
34
35