White blood cell:- Part 2 – Total Leukocytes Count Procedure, TLC Solution Preparation

Sample
- EDTA blood is needed.
- Oxalate or citrated blood can also be used.
Purpose of the test (Indications)
- To differentiate between acute and chronic infection.
- To follow the patient with chemotherapy.
- To find the effect of drugs.
Pathophysiology
- White blood cells develop from the stem cells in the bone marrow.
- Stem cells differentiate:
- Granulocytic series cells.
- Non-Granulocytic cells.
- The granulocytic series consists of:
- polymorphonuclear leucocytes (Neutrophils).
- Eosinophils.
- Basophils.
- Monocytes.
White blood cells are divided into:
- Phagocytic cells:
- Polys.
- Eosinophils.
- Basophils.
- Monocytes.
- Immunocytic cells:
- B- lymphocytes.
- T-lymphocytes are:
- T-helper cells.
- T-cytotoxic cells.
- T-effector cells.
Preparation of TLC solution:
- Principle of the TLC method:
- Blood is diluted with a fluid that causes the RBCs’ hemolysis, but WBCs remain intact, and then these are counted in the Neubauer chamber.
- Gentian violet lightly stains the leucocytes and allowing those to be counted.
- Chemicals needed are:
- Glacial acetic acid = 2 ml.
- Gentian violet (1%) = 1 ml.
- Distal water = 97 ml.
- This is basically a 2% solution of acetic acid.
- Add 2 ml of glacial acid 2 + gentian violet (1 %) 1 ml to 97 ml of distle Water to make up to 100 ml solution.
- TLC or WBC pipette:
- This pipette (also called Thoma pipette) long stem is divided into two parts:
- The long stem is marked with 0.5 and 1.0
- While the short arm after the bulb is marked 11.
- Its central portion is a bulb or a globular shape with one white bead in it.
- Rubber tubing is attached to suck the blood.
- Ultimately the dilution of the blood to the TLC fluid is 1:20.
- This pipette (also called Thoma pipette) long stem is divided into two parts:
- Procedure:
- Take the TLC pipette, which has a white bead inside.
- Fill the blood into the make and then add the TLC solution.
- Fill the blood to 0.5 in the pipette.
- Fill the pipette with the TLC solution to point 11.
- Remove the rubber tubing.
- Seal both ends or hold in between two fingers.
- OR can put this pipette on the mechanical device to shake it.
- Shake for 1 minute or preferably for 2 minutes.
- Shaking is important before filling the Neubauer chamber.
- After thorough mixing, discard the first few drops and then gently fill the chamber until the platform is filled.
- The capillary action will draw the fluid.
- Allow the chamber on the microscope stage for 2 to 3 minutes, till the cells are settled.
- Tube method:
- Take 0.02 mL blood and mix it with diluting fluid.
- Take TLC dilution fluid 0.38 mL in a small tube and mix it with the blood.
- Mix them very well.
- This tube method is more accurate than the Thoma pipette technique.
- In case of low WBC count, fill the pipette to mark 1, and this will give dilution of 1:10, OR
- Take 0.1 mL blood and 0.9 mL diluent solution.
- In the case of high WBC count, then make higher dilution.
Calculations:
- Count the cells in the Neubauer chamber. These are counted in the four large corner squares labeled as WBC and if the number is Y.
- One large area is 1 x 1 mm, and the depth is 0.1 mm.
- Total area counted in 4 large squares = 4 x 1 x o.1 = 0.4 µL (4/10).
- Y x 10/4 is the total WBC in the cell in 1 µL.
- Now dilution is 1:20.
- Number of cells in 1µL = Y x 10 x 20/4 = Y x 50 = Total WBC count.
- Total TLC = counted cells Yx 50 = TLC/cmm.
- Source of errors are:
- If there are microclots in the sample.
- If inadequate mixing is done.
- Improper filling of the chamber.
- If the dilutions are improper.
- Mistakes in the calculations.
Normal values of total leucocytes are:
Source 2
- Adult /child = 5000 to 10,000 /cmm
- Child ≤2 years = 6200 to 17000 /cmm.
- Newborn = 9000 to 30,000 /cmm
Other sources
- At birth = 10,000 to 25,000/cmm
- Infants = 8000 to 15,000/cmm
- Adults = 4000 to 10,000/cmm
- Pregnant ladies = 12,000 to 15,000/cmm
Increased TLC (Leucocytosis) is seen in:
- Mostly in the case of infections that may be bacterial or viral.
- In the case of leukemias.
- After the strenuous exercise.
- Pain and anorexia.
- Epileptic seizures.
- Emotional reaction.
- Mild leucocytosis in pregnancy.
Decreased leucocytosis (neutropenia) is seen in:
- This may be seen in fever, malaise, and chills.
- Infections.
- Hypersplenism.
- Deficiency of vitamin B12.
- Malignant infiltration of the bone marrow.
- Bone marrow aplasia.
- Bone marrow depression by radiations.
- Autoimmune diseases like SLE.
- Critical value = <2500 or >30,000 /cmm.
- Please see for more details in complete blood count (CBC) part 1.
Very essential information
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You made this topic very easy for me
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It is so helpful for me
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It is 100% easy than to read it from university manual.
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Perfectly OK sir
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What is 10 in wbc count formula?
Total area counted in 4 large squares = 4 x 1 x o.1 = 0.4 µL (4/10).
x10 comes from above formula.