Hemocytometry

  • Introduction 
  • RBC count
  • WBC count
  • Platelets count 
  • Eosinophil count
  • Reticulocyte count 

  1. Introduction

Counting of cells found in blood is done under hematometry.

1. Counting chamber is used in this technique and in this technique blood friendly diluting fluid is used

2. Counting of RBC, WBC, Platelets, Eosinophile, Reticulote is done by this technique.

3. Manual counting of total cells is done with the help of microscope.


RBC Count


Clinical Significance: 

1. Total RBC at the time of birth 6.5 to 7 million and after some time it comes to the normal level.

2. RBC Count is increase in the condition of Cholera, Diarrhea, Vomiting, Heart disease, Polycythemia.

3. RBC Count is decrease in the condition of Pregnancy, Old Age , Anemia.

Principal: 

When blood specimen is diluted with RBC diluting fluid at 1:200 dilution and cells are counted at high power objective (40x).


Normal Value : 

Male.        4.5 to 6 Million/ cumm
Female.    4 to 4.5 Million/cumm



Specimen: 

Capillary blood or well mixed anticoagulant blood.

Note :
Fasting sample is not required for this test.


Requirement: 

  1. Microscope
  2. Neubauer Counting Chamber 
  3. RBC Pipette 
  4. RBC Diluting Fluid.

Composition of RBC Diluting Fluid 

  1. Sodium Citrate.        3gm 
  2. Formalin.                   1ml 
  3. Distill water.             100ml

Procedure : 

  1. RBC takes blood up to 0.5 mark in pipette and cleans the extra blood on the side of pipette with the help of cotton.
  2. The RBC pipette takes the RBC diluting fluid up to the 101 mark.
  3. Mix the pipette in a horizontal position for 5 minutes.
  4. After 5 minutes, a few drops are drained with a pipette and a small drop of fluid is placed between the cover slip and the counting chamber.
  5. After filling the counting chamber, keep it on a flat surface for 5 minutes.
  6. With the help of high power objective, count the total RBC cells in the area with RBC count.

Area of the grid where RBC are counted

Formula of Total RBC count 

Error of RBC Count:

  1. If the blood collection is done from the hemo-concentration area, the RBC count may be low.
  2. If the pipetting is not done properly, the RBC count may be high and low.
  3. If there is an error in the calculation, the RBC count can be more or less.
  4. RBC count may be high and low if the counting chamber is not charged properly.

WBC Count

Clinical Significance
  1.  The total WBC count is less than 4000/cumm then it is called Leukocytopenia.
  2. When the total WBC count is more than 11000/cumm, it is called Leukocytopenia.
  3. WBC count increases in conditions like bacterial infection , viral infection etc.
  4. WBC count is reduced in tha condition of Tuberculosis, Malaria, Anemia etc.
Principal 

Glacial Acetic Acid destroy RBC and Jensen Violet slightly stains WBC nucleus and dilute the blood specimen with WBC diluting fluid in 1:20 dilution and count WBC cells with the help of low power objective.

Normal Value :

Male/Female :-            4000 to 11000/cumm

Requirement 
  1. Microscope
  2. Neubauer Counting Chamber
  3. WBC pipette 
  4. WBC Diluting Fluid

Composition of WBC Diluting Fluid.

  1. Glacial Acetic Acid.       - 2ml
  2. 1% W/V Jensen Violet  -1ml
  3. Distill Water                  - 97ml
Store this solution in the Room Temperature.

Procedure :-

  1. The WBC takes blood up to 0.5 mark in the pipette and clean the extra blood on the side of the pipette with the help of cotton.
  2. Take the WBC Diluting Fluid up to 11 mark in the WBC pipette.
  3. Pipette mixes for 5 minutes.
  4. After 5 minutes, some drops are drained with a pipette and a small drop of fluid is filled to the counting Chamber.
  5. Place the counting chamber on the plane surface for 2 to 3 minutes.

Errors of WBC count:-

  1. If the blood collection is done from the Hemo- Concentration area , the return may be more or less.
  2. If pipetting is not done properly than the WBC count may be more and less 
  3. If there are errors in the counting the WBC count may be more and less.
  4. WBC count may be heigher or lower if the counting chamber is not charged properly.

Total platelets count 

Clinical Significance:-

  1. Total platelets count is used to investigate the diagnosis of bleeding disorder.
  2. Total platelets count in tha condition in thrombocytopenia, bleeding,  A plastic , Anemia , leukaemia hyper spleen etc.
Normal Value:-
 Male/female.         -   2.5 to 5 lac/cumm

Specimen :- 
Capillary blood or well mixed anticoagulant blood.

Notes :-  Use plastic syringe and plastic tubes for blood collection as the glass surface gets scratched. 

Requirement
  1. Microscope 
  2. Neubauer Counting Chamber
  3. RBC Pipette
  4. Platelets Diluting Fluid

Composition of platelets Diluting Fluid

  1. Hydrochloride               3g 
  2. Sodium Chloride.         10g
  3. Distill water.                  100 ml

This solution is filtered with filter paper and store in clean & dry plastic container at 2- 8 °c



 

Procedure :- 

  1. RBC take blood up to 0.5 marks in Pipette and clean the extra blood on the side of the Pipette with the help of cotton.
  2. Platelets take up to the 101 marks in the RBC Pipette to dilute fluid.
  3. Mixing blood and diluting fluid for 5 minutes.
  4. After 5 minutes a few drop are drained with a pipette and a small drop of fluid is filled to the counting chamber.
  5. Place the counting chamber in a moist filter paper in a petri-dish and leave it for 15 minutes.
  6. After 15 minutes count the total platelets in the RBC Count are in the counting chamber. 

Errors of Platelets Count:-

  • If the blood collection is done from the Hemo-Concentration area , the platelets count may be high or low.
  • If the pipette is not done properly the platelets count low or high.
  • Errors in the collection may result in a high or low platelets count.
  • Platelets Count may be high or low if counting chamber is not charged properly.

Total Absolute Eosinophil count.

Clinical Significance:-

1. Allergic Reaction
2. Eosinophil count increases in case of parasitic infection, Leukemia.

Principal:- 

The blood is diluted with a special types of diluting fluid that removes Red cells and eosinophil cell are stained is Red Color and these cells are counted by counting chamber with help of 40x objective.

Normal Value:- 

Male/Female                   40 to 440 cumm

Specimen:-  Well mixed Anticoagulant blood 

Requirement

  1. Microscope
  2. Neubauer Counting Chamber
  3. Test tube
  4. Petri Dish 
  5. Diluting Fluid

Composition of Diluting Fluid

  1. Yellow Eosin.          0.5gm
  2. 95% Phenol.            0.5 ml
  3. Formalin.                0.5ml
  4. Distill Water.         99ml
(Mix well and store Amber Colored Bottle)

Procedure:- 

  1. 0.36 ml of diluting fluid is taken in a test tube and 0.04ml of blood is added to it .
  2. Leave it for 10 minutes
  3. Mix well and change the counting chamber.
  4. Place the counting chamber for 2 to 3 minutes
  5. Counting the cells in all 9 squares of the chamber be reducing the light to low power objective and high power objective.

Errors of Absolute Eosinophil Count:-

  1. The quantity of blood in the test tube should not be more and less than 0.04ml.
  2. The quantity of diluting fluid should not increase or decrease.
  3. The test should be done within 30minutes after the dilution of blood.

Total Reticulocyte count

Clinical Significance:- 

  1. Reticulote count is peripheral blood, activity of bone marrow red cell formation provides information.
  2. An increase in the number of Reticulate indicates an increase in the activity of the bone marrow.
  3. Decrease in the number of Reticulate indicates a decrease in the bone marrow.
  4. This happen in the condition of A plastic Anemia.

Principal :-

The Supravital Stin method is used to count the Reticulocyte. When blood is mixed with a stain the stain differentiates into the living stage of the cell and the cell is precipitated by the RNA staining as a dark blue network. After this blood smear is made and its direct count is not possible. Therefore the Red cell is counted against the number and the Red cell is counted as percentage.

Normal Value:-
Male/Female.       -  0.2 to 2.5%

Specimen :- Well mixed Anticoagulant blood.

Notes :- Test should be done within 3 to 4 hours of blood collection.

Requirement:- 

  1. Microscope
  2. Glass slide
  3. Test tube
  4. Pasture Pipette
  5. Reagent (Supravital Stain)

Composition of Supravital Stain

  1. Brilliant Crystal Blue.        1gm
  2. Sodium Citrate.                    0.4gm
  3. Sodium Chloride.                0.85gm
  4. Distill Water.                        100ml
( First Sodium Citrate is dissolved with sodium chloride after that brilliant Crystal Blue is added and filter and store at 2 to 8C°)

Procedure:-

  1. Filter the 2ml stain
  2. In a test tube, two drop of blood and two drop of stain are added through different pipettes.
  3. Cover the tube with a cotton plug and keep it at 37C° for 30 minutes
  4. Air dry the smear.
  5. Observe with the help of low power objective and oil immersion objective 
  6. Reticulocyte cells are visible in voilet color .
  7. RBC cells are visible in pale blue color.
  8. Count Reticulocyte cells and RBC cells in about 15 fields.

Errors of Reticulocyte count

  1. Stain and blood should be pipette in repeatedly in quantity with the help of different pipettes.
  2. Stain and blood should be mixed well.
  3. Staining time should not be less than 30 minutes.