CBP is one of the common medical laboratory procedures which are done in identifying, confirming, analyzing and reporting any abnormality in the blood sample. It is one of the most basic laboratory procedures and hence it is often heard. (1)
What is the Purpose of the Complete Blood Picture (CBP) Lab Test?
- The purpose of complete blood count is to perform quantitative enumeration of various elements of blood and hemoglobin in whole blood.
- It is done by automated cell counter based on the principle of Impedance Variation.
- Complete Blood Count test is intended for enumeration of cellular elements of blood to find out cell morphology through stained smears.
- Counter Method is being used in recent times to perform Complete Blood Count test. (2)
What Does Complete Blood Count include?
As the name suggests, it is the complete count of blood elements. Hence it includes various other tests like:
- Total RBC count
- Total WBC count
- Platelet count
- MCHC count
- MCH count
- MCV count
- Hematocrit count
- Hemoglobin count
What is Coulter Method?
- Coulter Method is a procedure through which it is possible to detect and measure changes in electrical resistance when a small particle (like a cell) in conductive liquid passes through the minute aperture.
- When each of the cells passes through the aperture, it impedes current and results in the measurable pulse. The height of each such pulse is directly proportional to the size of the particle. That means, bigger the cell taller will be the pulse.
- Also, it is possible to find out the number of cells (particles) by counting the number of pulses.
- The height of the electrical pulse produced by a cell is characterized as it is directly proportional to the cell volume.
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How to Do the Coulter Method?
- The sample is first diluted i.e. 1:250 for WBC count and 1:6250 for RBC count indices & platelet count.
- Then the diluted sample is directed to 2 sides of instrument and particles that are bigger than 36 fl are considered as RBCs.
- Cells ranging from 2 to 20 fl size are considered as platelets.
How WBCs are Counted Using Coulter Method?
First RBCs are counted and then they are lysed using ionic or non-ionic detergent (which do no lyse WBCs). So the sample will now contain only WBCs and the system is processed again to count the number of WBCs.
- Lysing agent is added to the sample after RBC count and platelet count is taken.
- Now the sample will be subjected to differential shrinking of WBCs, so as to allow them to get separated into partial differential i.e. 3 part differential.
- White cells are shown in 3 groups. They are:
- lymphocytes of 35 to 90 fl
- mononuclear cells of 90 to 160 fl
- granulocytes of 160 to 450 fl
How HCT is Calculated in Coulter Method?
RBC pulse height value is considered while calculating Hct. It is calculated based on the principle that the height of the impulse generated by passage of particles through micro-aperture will be directly proportional to the volume of that particle.
After RBCs are lysed, the freed hemoglobin will combine with potassium cyanide and forms cyanmethemoglobin. It can be measured by Spectrophotometry at 525 nm.
How MCH & MCHC are Calculated?
For calculating MCH and MCHC, we need to calculate RBC count, Hb and Hct.
Hct: it is calculated from RBC & MCV
MCH: it is calculated from HGB and RBC
MCHC: It is calculated from Hgb and Hct
MCV: it is calculated from RBC histogram
MPV & PLT: it is calculated from PLT histogram
Size of Different Particles for Identification
|Cell||Linearity||Measurement||Minimum Detection Range|
|RBC (x 106 cells / µL)||7.0||0 – 7.0||0|
|HGB (g / dL)||25||0 – 25||0|
|WBC (x 103 cells / µL)||99.9||0 – 99.9||0|
|Platelets (x 103 cells / µL)||999||0 – 999||0|
How to Take Sample for Complete Blood Count (using Coulter Method)?
- Draw blood sample from the subject/patient using standard procedures
- Take whole blood as sample
- Collect the sample in K2EDTA
- Use homemixer for mixing the sample well, for at least 5 minutes
- Make sure to process the collected samples within 2 hours of collection
- Contaminated/decomposed samples should NOT be used
- Clotted samples are to be rejected
Reagents / Chemicals used for Complete Blood Count Test (using Coulter Method)
For performing a complete blood count test using Coulter method, following consumables / reagents / chemicals will be necessary:
- cell diluent
- lytic reagent
- shutdown diluent
Procedure to Perform Complete Blood Count Test (Using Coulter Method)
Here is the step-by-step procedure to perform a complete blood count test using Coulter method(3):
- Background values & QC values are to be checked if they fall within the specified range
- Check if the system is working within the acceptable range, by running any of the 3 levels (low, normal & high) of controls
- [Lab reference numbers are to be entered and saved in this stage, if applicable]
- Mix blood samples thoroughly in hemomixer for at least 5 minutes
- Position sample tube under sample probe
- Press the key/button present behind the sample probe
- The LCD screen will display the results once the sample is processed.
Normal Values of RBCs / WBCs / Platelets
|RBC Count||4.5 to 6.5 x 106 cells / cu.mm (in males)
3.9 to 5.6 x 106 cells / cu.mm (in females)
|WBC Count||10,000 to 25,000 cells / cu.mm (at birth)
6,000 to 18,000 cells / cu.mm (infants)
6,000 to 15,000 cells / cu.mm (4 to 7 years)
4,500 to 13,500 cells / cu.mm (8 to 12 years)
4,000 to 11,000 cells / cu.mm (adults)
|Platelet Count||15,000 to 450,000 / cu.mm|
|Hemoglobin||13.6 to 19.6 g% (at birth)
11.3 to 13.0 g% (at 1 year)
11.5 to 14.8 g% (10 to 12 years)
13.5 to 18.0 g% (adult males)
11.5 to 16.5 g% (adult females)
|Hematocrit||44 to 63 % (at birth)
35 % (at 1 year)
37.5 % ( at 10 years)
40 to 54 % (adult males)
30 to 40 % (adult females)
|MCH||27 to 32 pg|
|MCV||76 to 96 µm3|
|MCHC||30 to 35 %|
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What are the Factors that Affect Counting in the Complete Blood Count Test?
While performing a Complete Blood Count test using Coulter Method, many factors may affect the counting, this may lead to inaccurate results. Hence these factors are to be taken care of:
- RBC lysing doesn’t occur completely
- Presence of cryoprecipitate & micro clots in the sample
- Improper calibration of the analyzer
- Inadequate dilution of the sample
- Diluting solution getting deteriorated
- Dust particles present in the diluent
- Obstructions (either partial or complete) in aperture
- Improper threshold setting of the instrument
- Left overs/ carry overs from one instruments to the other
- Electric current fluctuations