Practical :

Blood Cell Counting I

Tay SP Department of Pathology

Faculty of Medicine & Health Sciences

Universiti Malaysia Sarawak (UNIMAS)

MDP 10307: Blood & Immunology

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.

2

Blood Cell Counting (1) Counting the various cell types (white cells,

red cells, platelets) found in the blood. Is a fundamental procedure in haematology

laboratory. Procedures used for cell enumeration :

a. Microscope + counting chamber (haemocytometer).

b. Electronic counting devices.

Nowadays, most blood cell counts are done by electronic devices.

3

Blood Cell Counting (2)

Manual blood cell counts are limited to white cell count and platelet count.

Manual red cell count is almost never done manually because of high degree of error inherent and time-consuming.

Advantages of electronic counting devices : a. Avoid human errors. b. Statistically more accurate. c. Count many more cells than manually.

4

Principles :

a. Selection of a diluting fluid.

− will dilute the cells manageable numbers may be counted.

− will destroy cellular elements that are not to be counted.

b. Use of a haemocytometer or electronic cell counter.

− number of cells per unit volume of fluid can be counted.

5

Blood Cell Counting (3)

Automated vs. Manual Blood Cell Counting

Advanced instrumentation with computer-assisted technology

automated routine haematology.

Certain investigations – manually, visually.

Manual procedures

reference methods, calibration.

6

1. Reference method for calibration.

2. Platelet count – reference technique.

3. WBC , PLT with instruments –

confirmation.

4. Failure – instruments.

5. Absolute counts of certain cell types –

e.g. eosinophils.

6. Counting of cells in other body fluid –

e.g. CSF, urine.

Manual cell counting is still important :

7

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.

ICSH = International Council for Standardisation in Haematology

1 mm3 = 1 L = 1 x 10-6 L

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

Criteria of a suitable diluting fluid :

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.

Blood – capillary blood, EDTA venous blood.

diluted to enable cells to be counted.

10

Haemocytometer (Neubauer Counting Chamber)

0.1 mm depth

0.1 mm

Counting Chambers (1)

Single Net

Double Net

Bright-Line

14

Non- Bright-Line

Counting Chambers (2)

RBC / PLT = 1 + 2 + 3 + 4 + 5 = 0.2 mm2

WBC = A + B + C + D = 4 mm2

Neubauer Ruling

Bright-Line Counting Chambers (Metallized Surface)

Advantages :

1. Allows the ruling to appear brightly illuminated.

2. Provides sharp contrast to the bright lines and cells.

3. Helps the user with accurate counts.

4. Reduces eyestrain.

5. Difference in surface tension characteristics between metalic surface (on chamber) and polished cover glass assures smooth capillarity for precise sample loading and even cell distribution.

16

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

Had been virtually eliminated from most routine laboratory tests because :

Time-consuming.

Inaccurate (large error - ± 20% due to uneven cell distribution in chamber)

Limited use in routine practice (reference method is automated).

Manual RBC Count

18

Other RBC diluent :

NaCl (0.85 g/dL) or Saline Solution.

Gower’s Solution.

Toison’s Solution.

Rees-Ecker Solution.

RBC Diluting fluid : Dacie’s fluid / Hayem’s solution

30% trisodium citrate + formalin (formal-citrate)

Preserve morphology.

Prevent clumping or clotting.

19

Platelets (PLT) are difficult to count because :

Small and difficult to discern.

Confuse with extraneous dirt.

Have adhesive character and become attached readily to glassware or particles of debris in diluting fluid.

Clump easily and not evenly distributed in blood.

Disintegrate easily and difficult to distinguish from debris.

Sticky nature tend to adhere to other platelets in clumps.

Manual Platelet Count (1)

20

Time-consuming.

Error involved can be very large.

Must be verified by performing a platelet 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.

Manual Platelet Count (2)

21

Other PLT diluent :

30% trisodium citrate + formalin (formal-citrate) – does not lyse RBC.

Gives incorrect results when platelet count is low.

PLT Diluting fluid : Ammonium oxalate (1%)

Prevent clumping or clotting.

Completely lyse RBC.

Leaves PLT and WBC intact.

22

Procedures – Manual RBC/PLT Count (1)

1. Clean haemacytometer and cover glass with alcohol and tissue paper.

2. Place cover glass on the haemacytometer.

3. Pipette 0.02 mL (20 L) of blood sample and wipe off excess blood on the outside of the pipette with gauze or tissue paper.

4. Deliver into a tube containing 4 mL / 4000 L (for RBC) or 1.98 mL / 1980 L (for PLT) of dilution fluid, and mix thoroughly.

5. For PLT count, leave the mixture for 10 min to allow the RBC to be lysed completely.

23

6. With aid of glass capillary tube or Pasteur pipette, fill the counting chamber with the diluted sample, taking care not to overfill the chamber.

24

Procedures – Manual RBC/PLT Count (2)

7. Place the chamber inside a moistened petri dish for 5 min (RBC) or 15 min (PLT) to allow the cells to settle.

8. Using x40 objective or x10 eyepieces, count the cells in at least 80 smallest squares.

9. 500 cells are the minimum number to be recorded.

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

If R cells are counted in squares 1, 2, 3, 4, 5,

TRBC = R X 1

X 200 0.02

Volume of blood counted = 0.02 mm3 (L)

Dilution factor = 200 (1:200 dilution)

(per L)

Final TRBC count = ______ x 1012/L

26

If P cells are counted in squares 1, 2, 3, 4, 5,

TPLT = P X 1

X 100 0.02 (per L)

Volume of blood counted = 0.02 mm3 (L)

Dilution factor = 100 (1:100 dilution)

Final TPLT count = ______ x 109/L

27

Left-to-right, right-to-left counting pattern.

Begin

End

Rules of Manual Cell Counting - 1

Left and top lines counted (solid circles) Right and bottom lines not counted (open circles)

Rules of Manual Cell Counting - 2

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.

Rules of Manual Cell Counting - 3

31

Tally Counters

33

34

35

Precautions & Technical Errors 1. Poor collection of samples.

2. Inadequate mixing of samples.

3. Haemocytometer / coverslip – dust / fingerprints may cause difficulty in distinguishing the cells.

4. Diluting fluid – free of contaminants.

5. Chamber must be charged properly (not over-/under-filled) – uneven flow of sample results in an irregular distribution of cells.

6. After the chamber is filled, allow the cells to settle for 1-2 minutes before counting – a pause longer than that allows fluid to start evaporating.

36