The White Blood Cells (WBCs) also known as Leucocytes are round nucleated cells in blood, that provide protection against foreign invaders. There are many types of White Blood cells found in blood, and identified as Granulocytes (Neutrophils, Eosinophils & Basophils) and Agranulocytes (Monocytes and Lymphocytes). The size of White Blood Cells can vary between 10 to 20 um(microns). Each type of White Blood Cell has a specific function in the body.

The life span of white blood cells ranges between a few days to a few years. Memory cells can last for several years and serve as defense against the various infectious agents.

What’s a White Blood Count (WBC)?

White blood counts determines the amount of white blood cells that are present in your blood. White blood cells form part of your immune system. They aid your body in fighting against infections and other illnesses.

If you are in a state of illness, your body creates greater numbers of white blood cells in order to combat viruses, bacteria or other foreign compounds which cause the disease. This can increase the number of white blood cells in your body.

Other illnesses could cause your body to produce more white blood cells that what you need. This can lower the number of white blood cells in your. Conditions that could lower your number of white blood cells include kinds of cancer as well as HIV/AIDS, which is a virus that targets white blood cells. Certain medications, such as chemotherapy, can reduce the amount of white blood cells.

There are five main types of blood white cells.

• Neutrophils
• Lymphocytes
• Monocytes
• Eosinophils
• Basophils

The white blood count is a measure of the totality of cells present in your blood. Another test, referred to as the blood differential, determines the amount of each kind of white blood cells.

Alternative names are: WBC count, white cell count White blood cell count,

The Aim / Purpose Of Performing Total Wbc Count

The goal of performing Total White Blood cell (WBC) count is to determine whether or not you’re being affected by leucocytosis (i.e. the rise in the number in White Blood Cells (to more than 11,000/mm3) also known as Leucopenia (i.e. the reduction in the number of White Blood Cells (to less than 1500/mm3). The count of Total Leucocytes (TLC) of White Blood Cells (TLC) is performed to determine the health of the Bone Marrow.

Principle Of Total Leucocyte Count Using Hemocytometer

A huge amount of White Blood Cells , also known as Leucocytes are found within the blood Specimen. In reality, calculating this huge quantity of White cells using a microscope is virtually difficult. Therefore the Leucocytes are counted using chambers that are specially designed that is designed to count the number of blood cells within the specimen. This is known by the name of Hemocytometer also known as Neubauer’s chamber.

To do this purpose, the blood sample is diluted (usually in a ratio of 1:20) with the aid of WBC diluting liquid (commonly known as the Turk’s fluid) that protects, stains and fixes the White blood cells as well as Lysis, the Red Blood Cells. The Turk’s fluid is isotonic with it’s White blood cells. It is not causing any harm to them.

After diluting the sample, the sample is then charged onto Hemocytometer/ Neubauer’s chamber. Then, they are counted within the regions that are specifically designed for the WBC count.

The composition of WBC diluting Fluid (Turk’s Fluid)

COMPONENTS	QUANTITY
Glacial Acetic Acid	2 ml
Gentian Violet (1% Aqueous)	1 ml
NaCl	0.9 gram
Distilled Water	97 ml

The pH of the final solution (at 25°C) can vary from 2.0 to 2.4 depending on the formulation and the company that manufacture it.

Certain Labs & Companies add the NaCl (Sodium Chloride) for isotonicity and Thymol crystals in order to avoid formation of Mould development in solution, which could differ according to their standard operating procedures (SOPs).

Hemocytometer / Neubauer’s Chamber

It is a specific kind of chamber made of glass that is utilized for cell count particularly for Blood cells. Nowadays, the Improved Neubauer’s Chamber is utilized in certain laboratories. However different chamber types are also utilized, like Burkers chamber Levy’s chamber, Burkers chamber, Fusch Rosenthal chamber. Rosenthal chambers, among others.

It is believed that the Neubauer’s Chamber has ruled the space of 9 square millimeters and the depth is 0.1 millimeters when the coverslip is put on the top of the chamber that counts and the gap between the top of the glass and the bottom of the grooved area is 0.1 millimeters in depth.

From the 9 squares The 4 corners are used to calculate counting the White Blood Cell number. Each corner square is then divided into 16 smaller squares, which is the total number. of squares to be counted in the WBC Count is –

16 × 4 = 64 small squares of the 4 large corner squares

Two Methods have been devised to estimate the manual estimation of the Total Leucocyte Count using Hemocytometer and Neubauer’s chamber

1. Microdilution Method
2. Macrodilution Method

In this article, I’ll go over each method, but the Microdilution method has not been popular anymore because of the use of mouth pipettes. Let’s begin with the Microdilution and later shift to the Macrodilution technique.

Microdilution Method For The Estimation Of Total Leucocyte Count (Tlc) Using Hemocytometer

Materials Required 

• Blood sample (Capillary blood or EDTA anticoagulated specimen)
• WBC diluting fluid (preferably Turk’s fluid)
• Gauze piece or Cotton
• WBC pipette
• Hemocytometer a.k.a. Neubauer’s Chamber
• Coverslip
• Microscope

WBC Pipette

WBC pipette has a graduated design which provides a dilution of 1:20. It has two marks at the bottom , 0.5 and 1. The uppermost part of the pipette has a mark 11. It features a circular bulb, which houses the White bead that mixes the blood specimen with the diluting liquid. On top, an elastic tube is connected to the pipette that allows sucking blood along with diluting fluid.

When the blood is sucked up to 0.5 mark and diluting liquid up to 11 , provides the 1:20 dilution for Blood Diluting fluid. When the blood is sucked to 1 mark, and the diluting fluid is up to 11, it gives the 1:10 dilution of blood; Diluting fluid that is not as frequently is used. Following the sucking of Specimen and diluting fluid the contents are gently mixed by turning the pipette around its long axis, ensuring complete mixing of diluting fluid and blood.

Note: Nowadays , Mouth pipetting is prohibited in a majority of the labs due to the danger of being infected by extremely contaminated samples of patients. Therefore, instead of the Microdilution method methods, Macrodilution is used at Laboratories.

Procedure of the Total Leucocyte Count (TLC) Count by Microdilution Method

1. The WBC pipette filled up to the 0.5 mark using the blood sample and then wipe the pipette with a cotton/tissue paper to prevent falsely high result.
2. The same pipette should be filled with WBC diluting liquid (preferably Turk’s Fluid) up to 11 mark.
3. Take care to ensure that there will be no air bubbles in the bulb of the pipette.
4. Mix the diluting fluid and blood into the pipette, rotating this pipette (horizontally) between your palms.
5. Remove the Neubauer’s chamber/Hemocytometer from the case and wipe it using clean gauze or swabs. Clean the cover glass , and then place it on top of the grooved portion of the Hemocytometer.
6. Note: Here , a particular kind of glass cover is employed that measures 0.4 millimeters thick, with a very uniform thickness and smooth surfaces. This means that the gap between the grooved portion in the chamber as well as the the cover glass is precisely 0.1 millimeters.
7. Then, place the WBC pipette into the container, mix the solution inside it and then take out 1 or 2 drops of the pipette before charging your chamber.
8. Simply press the rubber tube into the WBC pipette until the next drop of fluid is in a the hanging position.
9.  Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45deg approximately.  
10. Allow a tiny amount of fluid from the pipette enter the chamber. This is caused by the Capillary action. Don’t overcharge the chamber and there shouldn’t be any air bubbles in the Chamber.
11. After charging, let it sit for 3 to 5 minutes so that the cells settle into the chamber. After that, you can place the chamber in microscopes to count White Blood Cells.

Macrodilution Method For The Estimation Of Total Leucocytes Using Hemocytometer

Materials Required

• Blood sample (Capillary blood or EDTA anticoagulated specimen)
• WBC diluting fluid (preferably Turk’s fluid)
• Hb pipette or Micropipette (0.02 ml or 20 ml)
• Hemocytometer / Neubauer’s Chamber
• Gauze piece or Cotton swab
• Graduated Pipette (5 ml)
• Test tubes
• Cover Slip

Procedure of the Total Leucocyte Count (TLC) Count by Macrodilution Method

1. Take 0.38 milliliters WBC diluting fluid into a dry, clean and grease free Test tube.
2. Note: if you don’t have a variable pipette in your lab that can be used to measure 0.38ml or the equivalent of 380 ul diluting fluid, then measure 400 ul of diluting fluid using a micropipette inside the test tube. Discard 20 ul of the fluid with the micropipette, or Hb pipette.
3. Add 0.02ml or 20 ul of Blood Specimen into the diluting fluid tube by using a Micropipette and WBC pipette.
4. Alternately, take 1.9 milliliters of WBC Diluting Fluid into a tube and mix 0.1 milliliters of blood specimen to it.
5. Mix well for a few minutes before preparing your Hemocytometer or Neubauer’s chamber.
6. Remove the Neubauer’s chamber/Hemocytometer from the case and wipe using an swab or gauze. Clean the glass cover and put it on top of the grooved portion of the Hemocytometer.
7. Then, remove the WBC pipette, fill it with diluted Specimen. Mix the solution thoroughly and remove 1-2 drops from the pipette prior to charging the chamber.
8. Press the rubber tube gently on the WBC pipette to ensure that the next drop of fluid is in a the hanging position.
9. Make sure to touch the tip of the pipette by placing the hanger against the edges of the coverslip , creating an angle of around 45deg.
10. Allow a tiny amount of liquid from the pipette to be absorbed into the chamber, which happens through the Capillary effect. Make sure that the chamber is not charged too much and there should not be an air bubbles in the Chamber.

Using Micropipette instead of WBC pipette for charging the Hemocytometer

• It is also possible to use a micropipette instead WBC pipette to charge the Hemocytometer. If you use a micropipette draw about 20 ul of the dilute sample. Press the knob of the pipette to make a hanging drop at the tip of the micropipette.  
• Place the tip of the pipette against the edges of the glass covering it and, if needed, gently expel the liquid until the chamber is filled. This is accomplished through Capillary action, however take care not to fill the chamber too full. A quantity of 10 ul is enough to fill the chamber for counting.
• After charging, let it sit for three to five minutes, so that cells can settle into the chamber.

Counting The White Blood Cells (Wbc) Under Microscope

• Focus the ruling with the 10x lens and then count the WBCs on four large corner squares in the manner previously described with the 40x objective lens.
• Check the cells that are located on the lower and right line of 4 Corners, but not on the opposite lines. If marginal cells are present you can count them on the “L” line which are or is on the right and lower lines or left and upper lines.

CALCULATIONS FOR THE TOTAL WBC COUNT USING HEMOCYTOMETER

After taking the count of cell under microscopes, we can determine the number of WBC in the 4 Corner squares. We can think of it as the “N” no. of cells. Now, the volume of the fluid within the chamber is a result of the area and depth of the Hemocytometer or Neubauer’s chamber.

4 × 1 × 1 × 1/10 = 2/5

The Hemocytometer’s depth measures 0.1 (1/10) mm, as explained above in a brief description of the Hemocytometer. Use the formula below to calculate the total White Blood Cell Number

Thus, 2/5 mm3 Contains = N × Dilution

Then, 1 mm3 Contains = N × 20 × 5/2

Total WBC count = N × 50 / mm3

Utilizing the formula above, we can determine the total No. of White Blood Cells found on the Blood Specimen.

Normal Values Of White Blood Cells

AGE OF PERSON	WBCs / Cu-mm OF BLOOD
immediately after birth	9,000 to 30,000
1 to 7 days	9,400 to 34,000
8 to 14 days	5,000 to 21,000
15 days to 1 month	5,000 to 20,000
2 to 5 months	5,000 to 15,000
6 months to 1 year	6,000 to 11,000
2 years	5,000 to 12,000
3 to 5 years	4,000 to 12,000
6 to 11 years	3,400 to 10,000
12 to 15 years	3,500 to 9,000
Adults	3,500 to 10,500

The usual percentages of different kinds of WBCs that make up the overall Leucocyte count typically fall in the following levels:

TYPE OF WBC	NORMAL PERCENTAGE IN BLOOD
Neutrophil	45% to 75%
Eosinophil	2% to 6%
Basophil	0% to 1%
Monocytes	2% to 10%
Lymphocytes	20% to 40%

Precautions To Be Taken While Performing Total Leucocyte Count By Hemocytometer

• The use of mouth pipettes (WBC pipette) is prohibited in many countries. If you must make use of it, you should be aware to not ingest the diluting fluid, or the Specimen.
• Make sure you accurately measure the amount of specimen as well as Diluting Fluid so that there is no errors in the results.
• If you’re conducting this test with the microdilution technique, Mix the sample and diluting fluid according to the procedure by gently turning the pipette the palms of your hands.
• Be sure to ensure that you charge your Hemocytometer/Neubauer’s chamber so that it is not overcharged and should not have any air bubbles in it.

What is it used for?

The white blood count often used to identify diseases that can be caused by high white blood cells count or a low the number of white blood cells.

Conditions that are associated with having high levels of white blood cells are:

• Autoimmune and inflammatory conditions, which cause an immune system system that attacks healthy tissue
• Viral or Bacterial infections
• Cancers such asleukemiaandHodgkin disease
• Allergy reactions

The most common disorders that result from having low white blood cell count are:

• Immune system diseases like HIV/AIDS
• Lymphoma, a type of cancer of the bone marrow
• Liverorspleen-related diseases

A white blood count will reveal if the quantity of white blood cells are too excessive or too low, but it’s not able to determine a diagnosis. Therefore, it’s usually performed alongside other tests, for example, an entire blood count or Blood smear, differential blood or bone marrow tests.

What are the reasons I should have the White blood cell count?

You might need this test if there are symptoms of an inflammation, infection or an autoimmune condition. The symptoms of infection are:

The symptoms of autoimmune and inflammation are different, based on the location of inflammation and the type of disease.

It is also possible to take this test if an illness that affects your immune system, or you’re taking medication which reduces your immune response. If the test indicates that your white blood count being reduced the doctor might be able to modify the treatment.

Your infant or older child might be screened as part of routine screening or if they exhibit signs of an illness of the white blood cells.

Purpose of a white blood cell count

The WBC count is usually performed as part of the CBC. The tests can be scheduled to be part of a blood test routinely.

The white blood cells are also known as leukocytes are a vital component in your immune system. They are derived from bone marrow, but they move through the bloodstream. They fight off infections by attacking viruses, bacteria and other germs that infiltrate the body.

A count of white blood cells will reveal hidden infections in the body. It can also help doctors identify non-diagnosed medical conditions like autoimmune diseases or immune deficiencies. It can also be used to diagnose blood conditions. The test can also help doctors evaluate the efficacy of radiation therapy, chemotherapy treatments, and other treatments for cancer patients.

A high or low WBC count

If the results of your test are not normal, it could be a sign that your values are greater or less than normal of your age.

A high or low WBC count could indicate an underlying blood disorder or medical issue. To determine the root of an elevated or low WBC count, your physician will take a variety of aspects into consideration including your current medication list as well as any signs you are experiencing or have experienced, as well as your medical background.

Low white blood cell count

Leukopenia can be a medical term to describe lower WBC count. It can be caused by:

• HIV/AIDS
• autoimmune disorders
• bone marrow problems or damaged
• lymphoma
• serious infections
• liverandspleendiseases, such as an enlarged spleen
• lupus
• radiation therapy
• certain medications, for instance, chemotherapy and antibiotics.
• Alcoholism
• malaria

The term “neutropenia” can also refer to the presence of a low blood count. The term refers to a small amount of neutrophils, however since this kind of white blood cells makes about 55-70 percent of your total white blood cells the condition of leukopenia is usually caused by a small amount of neutrophils.

Being diagnosed with neutropenia can increase your risk of developing illness, infection and even death. It is because you may lack enough white blood cells that can protect yourself from bacteria and germs. If you are suffering from this issue It is crucial to take preventive measures like wearing a face mask when in public places to minimize your exposure to germs and decrease the risk of contracting infection.

High white blood cell count

Leukocytosis is a medical term that refers to an elevated WBC count. The cause of this can be:

• infections such astuberculosis,sepsis, or septic shock
• The fever
• burns, or injury
• pregnancy
• Allergies
• asthma
• a recent vaccination
• certain medications, including corticosteroids and epinephrine. other non-steroidal anti-inflammatory (NSAID) medications
• stress
• tissue damage
• acute hemolysis
• hemorrhage
• leukemoid reaction
• heart attack
• surgery
• An increased number of white blood cells can also be chronic or lasting in the following circumstances:
• smoking
• Tumors in bone marrow
• Leukemia
• Conditions that cause inflammation, like arthritis, bowel disease
• obesity
• vasculitis or inflammation of blood vessels

The doctor could run additional tests to find out the reason of the abnormal White blood cell count.

After identifying the reason behind an excessive or low WBC count and then recommending an appropriate treatment plan Your physician will frequently review your WBCs through other tests for blood.

If your WBC count is either low or high, this could indicate that your health has become worse. Your doctor could utilize this information to alter the treatment you receive.

When your WBC count is within an average range it usually means that the treatment is effective.

Symptoms of an abnormal WBC count

The signs of the low WBC count are:

• The body is aching
• febrile
• chills
• headaches

The high WBC counts aren’t usually the cause of symptoms, however the underlying circumstances that cause the increased count can trigger the symptoms themselves.

The signs of an insufficient WBC count can make your doctor recommend an WBC count. It’s also common for doctors to request an CBC and test your WBC number during your annual physical examination.

Refrences

• https://allmedtests.com/differential-leukocyte-count-procedure/
• https://biologyreader.com/wbc-count-method.html
• https://labpedia.net/white-blood-cell-part-2-total-leukocytes-count/
• https://www.medicine.mcgill.ca/physio/vlab/bloodlab/WBC_count.htm
• https://paramedicsworld.com/hematology-practicals/total-white-blood-cell-count-wbc-total-leucocyte-count-tlc-using-neubauer-chamber/medical-paramedical-studynotes
• https://eclinpath.com/hematology/tests/wbc-count/
• https://ashpublications.org/blood/article/13/7/677/73150/A-Rapid-Method-for-Estimation-of-the-Total
• https://labmonk.com/estimation-white-blood-cell-count-tlc-total-leucocyte-count