Biomedical Techniques Blood Cell Counting using Haemocytometer## Biomedical Techniques Blood Cell Counting using Haemocytometer

The haemocytometer is a thick glass slide with a counting chamber in the middle. The counting chamber contains two grids with improved Neubaur rulings of 3 by 3 mm primary square. The primary square is further subdivide into 9 secondary squares, each 1 by 1 mm. The four corner squares are used for the white blood cell count which are further subdivided into 16 tertiary squares. The central secondary square is divided into 25 tertiary squares, each of which measure 0.2 by 0.2 mm, each single tertiary square is further divided into 16 smaller squares. The five black squares along with the shaded squares in the centre are used for platelet count, while the five black squares alone are used for red blood cells counting (Figure12.10).

Diluting fluid

The blood cells are diluted in specific diluting fluid to keep the cells intact. RBC diluting

IMPROVED NEUBAUER

DEPTH 0.1 mm 1/400 mm2

Co

1 m

RBC pipette Calibrated stem

1 unit

Bulb

100 units

101 units

1 unit 10 units

11 units

Bead (Red)

Calibrated stem Bulb Bead

Short stem

Stem

WBC pipette

fluid (Hayem’s) is isotonic with blood, hence haemolysis does not occur. The blood is diluted 1:200 times with RBC diluting fluid and the cells are counted under 45X objective of the microscope.

The diluting fluid used for WBC count is Turk’s solution which contains glacial acetic acid and Gentian violet. The glacial acetic acid lyses the red blood cells and the Gentian violet stains the nuclei of the leucocytes. The blood is diluted 1:20 times and the cell are counted under 10X objective of the microscope. The total number of cells counted is expressed in mm3.

Blood cell counting using hemocytometer

1. The blood is collected till the 0.5 graduation in the pipette.

2. The diluting fluid is taken till the graduations 11 and 101 of the WBC and RBC pipette respectively.

3. The blood is diluted and mixed well with the respective diluting fluid by rotating the pipette horizontally several times.

ver glass

Mounting support

Courting chambers Filling nobh

Overflow stem

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C pipette and WBC pipette

4. The cover slip is placed on top of the counting chamber.

5. The tip of the pipette is placed on the counting chamber and fluid discharged till it fills the chambers.

6. The cells are allowed to settle for several minutes and the ruled area is viewed under the microscope.

Preparation of Blood Smear The examination of peripheral dry blood smear is a very important laboratory test as it is possible to

• Estimate approximately the number of cellular components

• Study the morphology of these components

• Observe the presence of blood parasites

• Study the response of the body to various diseases

The methodology for the preparation of blood smear is as follows (Figure12.11)

1. Place a drop of blood on a clean glass slide about 1cm from one end

2. Using another glass slide placed at an angle of about 45o to the previous slide.

3. Spread the drop of blood quickly in one stroke as a thin film

4. Stain the film using Leishman’s stain 5. Allow the slide to dry and wash the

excess stain 6. Observe the slide under a light

microscope (Figure12.12).

A person suffering from severe fever, head ach and fatigue so he visits his doctor. What type of diagnosing procedure does the doctor recommended for him. Name the medical equipment is used for it?

1. Place a small drop of whole on a very clean slide. Hold a second slide at the angle shown.

3. Maintain firm contact with the bottom slide and push the top slide in one motion to produce the smear.

2. While maintaining contact with the bottom slide, pull the top slide back to contact the drop, which will spread by the capillary action.

Cell Differential Count

Neutrophil 50-70%

Eosinophil 1-4%

Basophil 0-1%

Monocyte 2-8%

Lymphocyte 20-40%

Figure 12.13 Differential Count of White blood cells

Differential Count

The differential WBC count is the method in which the numbers of different types of white blood cells present in the blood are counted by examining a well stained peripheral blood smear (prepared by the above method). The number of each type of white blood cell is then expressed as a percentage of the total number of cells counted (Figure12.13).

Summary Medical instruments help in correct diagnosis and treatment to observing patients, examining organs, making measurements and administering medication. Today’s medical instruments are considerably more complicated and diverse, primarily because they incorporate electronic systems for sensing, transducing, manipulating, storing, and displaying data or information. The modern electronic stethoscope is a high precision instrument used to hear with clarity the internal sounds of the human body even in a noisy environment or through the heavy clothing of the patient. The sphygmomanometer is a device to diagnose pathological conditions such as hypertension and hypotension. A glucometer is a simple and portable medical device used to find out the approximate levels of blood glucose. Autoanalyser is a computer controlled device and used to quick estimation of various bio-chemical substances like glucose, urea, cholesterol, enzymes and other proteins present in body fluids.

Imaging instruments produce diagnostic images and help in the detection of the diseases or defects in an organ at a very early stage, when there are no clinical

manifestations or the disease is undetected by the conventional methods of investigations. Then proper therapeutic or surgical measures can be adopted. An EEG is a test to evaluate the electrical activities of the brain. X-Rays are a form of electromagnetic radiation to visualize the internal structures of a patient. Ultrasound waves are used to image the foetus at different stages of pregnancy used to hear foetal heart sound, blood flow, diagnose the damages in heart and diagnosis of tumours, gall stones, kidney stones, and obstructions in the genital tracts.

Evaluation 1. The instrument used to

measure blood pressure is a. Stethoscope b. Haemocytometer c. Sphygmomanometer d. Haemoglobinometer

2. Blood smear is used to study a. Total RBC b. Total WBC c. Haemoglobin d. Differential count of WBC

3. Diluting fluid used for total RBC count is a. Turk’s solution b. Toisson solution c. Hayem’s solution d. Folin phenol reagent

4. Normal diastolic blood pressure is a. 80 mmHg b. 100 mmHg c.120 mmHg d.140 mmHg

5. Foetus development can be observed using a. Ultra sonogram b. X-Ray c. ECG d. EEG

6. When electrical impulses in the heart are not generated properly _____ is used a. EEG b. Pace maker c. Auto analyser d. Glucometer

7. PET scan uses a. Radio isotopes b. UV rays c. Ultra sound d. IR waves

8. Write the normal values of total RBC and WBC.

9. What does a pace maker do? 10. What are the advantages of CT over

other imaging techniques? 11. Enumerate the uses of stethoscope. 12. Explain the working of MRI. 13. How does a normal EEG appear? 14. Write the clinical significance of ultra

sonogram. 15. Explain the principle involved in PET

scan.

Activity

What’s with all the Pressure? Students learn how to take blood pressure b then practicing on fellow classmates in sma of this activity is completed, the class brain might affect a person’s health.

Learning Objectives

◆ Describe the tools used to measure blood ◆ Measure a persons blood pressure using a ◆ Describe how blood pressure is measured

reading of systolic over diastolic pressure in

Measuring Blood Pressure Worksheet

1. Place the blood pressure cuff on the uppe clothing is very thick, you may need to a the bottom of the cuff to be just above th

2. Place the stethoscope so that it is between cuff. You want to place it just above the inside crook of the arm. This way, it is pla

3. Tighten the cuff around the patient’s arm an 4. Make sure the relief valve (the screw knob

cuff) is completely closed, and press the b reads 200

5. While listening through the stethoscope, s escapes at a slow but steady rate. You sho yet, and you should note that the needle o

6. Continue to release air and listen until you Mentally record the number the needle i number is the patient’s systolic pressure.

7. Continue to release air and listen, this time The number that the needle is on when the s

8. Record both numbers below. Release any and the stethoscope from the patient.

Record Results Student #1 Systolic Pressure………

y observing a teacher demonstration and ll groups. Once the hands-on component storms and discusses how blood pressure

pressure. ppropriate equipment. with a sphygmomanometer, which gives a units of mmHG (millimeters of mercury).

r arm of the student volunteer “patient” (if sk them to roll up their sleeve). You want eir elbow the patient’s skin and the blood pressure elbow, on the inside of the arm-on the ced over the brachial artery

d stethoscope so that is it snug, but not tight. attached to the bulb of the blood pressure ulb several times until the dial on the cuff

lowly turn the relief valve so that some air uld not hear any pulse in the stethoscope n the dial is slowly going down.

first hear a pulse through the stethoscope. s on when you first hear the sound. This

until the pulse sound goes completely away. ound stops is the patient’s diastolic pressure. residual air from the cuff, and remove it

Name……….. Diastolic Pressure……….

C on

ce pt

M ap