Measure The Resistance To Haemolysis Biology Essay

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According to Denise Harmening osmotic fragility test is used to measure the resistance to haemolysis when flow in increase dilutes saline solution. The sooner the haemolysis occur the greater the osmotic fragility. The resistance of the red blood cells depends on the surface to volme ratio and the functional state of the RBCs membrane. Cells with decreased surface:volume ratio (spherocytes) will lyse faster since the ability to hold water has being decreased due to the increase in the amount of haemoglobin present in the cell resulting in an increase in the osmotic fragility. Cells with increased surface:volume ratio will lyse slower since the ability to hold water has being increased due to their shape and size resulting in a decreased osmotic fragility.

When the red blood cell is placed in a hypotonic sodium chloride (NaCl) solution, the cell will take up fluid until an equilibrium is established. If the equilibrium established shifted, the cell membrane will become haemolysed. When red blood cell lyse it will result in the release of hemoglobin hence the degree of haemolysis can be measured using spectrophotometer. In the case of RBCs being placed in a hypertonic solution water will move out of the RBCs resulting in cell dehydration and shrinkage therefore no hemolysis. In isotonic solution there will be no net movement of water in or out of the cells therefore cell structure will be maintained.

Wright's Stain

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This is a Romanowsky metachromatic stain that facilitates the differentiation of blood cell types. This stain is used primarily to stain peripheral blood and bone marrow aspirates. It is made by mixing old or specialized treated methylene blue dye with eosin in a methanol diluent. It is a mixture two dyes, an acidic dye (Eosin) and a basic dye (New methylene blue). The Eosin stains the basic components of the cell in orange such as the haemoglobin, and cytoplasmic constituents. The new methylene blue stains the acidic components of the cell blue black such as the nuclei and granules. The dyes are mixed in a methanol diluent which fixes the cells and acts as a mordant keeping the cells in a life like state. A buffer is used in the staining process as the control system of the pH. The buffer of 6.8 is used to facilitate the cellular components picking up their respective dyes properly.

Venipuncture

Venipuncture is the procedure by which blood is taken from a vein for laboratory testing, different volume of blood is needed depending on the tests required. The blood is usually drawn from different areas on the body such as the vein on the inside of the elbow, back of the hand, or behind the knees, using a sterile needle and syringe or vacuum tube and needle collection system. The vacuum tube allows blood to be directly aspirated from the vein to the collection tube. During the venipuncture procedure there can be single or multiple draws of blood from the patient. Tubes are normally placed in a specific order to avoid any cross contamination of the collected samples and anticoagulants. Different tubes have different colour-coated stoppers which indicate the different additives specific for the different tests to be performed.

Method(s):

Osmotic Fragility

The dilutions as outlined in table 1 below were done using sample A (Hep).

Table 1 showing the different concentrations (%) and volume (ml) of NaCl along with the volume of blood (ml) required for the dilution.

TUBE

CONCENTRATION OF NaCl (%)

VOLUME OF BLOOD (ml)

VOLUME OF NaCl REQUIRED (ml)

1

0.90

0.05

5.0

2

0.75

0.05

5.0

3

0.65

0.05

5.0

4

0.60

0.05

5.0

5

0.55

0.05

5.0

6

0.50

0.05

5.0

7

0.45

0.05

5.0

8

0.40

0.05

5.0

9

0.35

0.05

5.0

10

0.30

0.05

5.0

11

0.20

0.05

5.0

12

0.10

0.05

5.0

The tubes were covered with parafilm and mixed well by inverting gently.

The tubes were allowed to stand for 30 minutes at RTP.

The tubes were gently re - mixed and centrifuged at low speed for five minutes.

Only the supernatant was transferred to a clean cuvette.

The absorbance values of the dilutions were read spectrophotometrically at 540 nm.

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The absorbance readings obtained were recorded and the percent hemolysis of each tube was calculated using this formula:

Absorbance of supernatants (tubes 1-12) x 100 = % Haemolysis Absorbance of Absorbance of supernatant in tube 12

A graph of the % haemolysis versus [NaCl] was constructed using a smooth curve for the results. This OF curve for sample A and Sample B was drawn alongside the curves of the reference ranges.

Wright's Stain

Using the EDTA Sample labelled B, a feathered edged smear was made and allowed to air dry.

A wright's stain was then applied to the smear using the following method:

The dried blood film was placed, smears side up on a leveled staining rack after allowed to dry.

The slide was flooded with Wright's stain and timed for exactly 4 minutes.

Without removing the stain buffers was added, drop wise to the slide so that a layer was formed on the slide but not spilled off. The two solutions were gently mixed by blowing back and forth over the surface of the slide. This was done until a metallic green sheen was seen at the top of the mixture when it was properly mixed.

The mixture was allowed to stay on the slide for exactly 10 minutes.

The slide was thoroughly rinsed with tap water.

Stain was removed from the back of the slide by wiping with paper towel.

The smear was allowed stand feather end up and air dry.

Venipuncture:

The patient was greeted and the procedure about to be undertaken explained to him/ her.

The arm was supported by a firm surface in an incline position

The equipment was assembled in preparation for the procedure such as the needle, barrel and the tubes. Additional tubes and spare alcohol pads and band aids were also placed within reach.

The tourniquet was applied firmly about the upper arm.

The median cubital vein or another suitable vein was located.

The site was then cleansed with 70% isopropyl alcohol by making outward concentric circles.

With the bevel of the needle up, the needle was inserted smoothly and quickly into the vein and the evacuated tube engaged.

The EDTA tube was drawn first then the HEPARIN(green top) tube

Once the blood was flowing in the tube and the final tube was filled, the tourniquet was removed.

When the last tube was filled and it was removed from the tube holder and the needle was removed.The patient was told to apply pressure and keep the arm straight.

The needle was properly disposed of.

The specimens were immediately labelled and the specimen that required mixing was done by inverting the sample six to eight times for proper mixing of the sample.

Results:

Table 2.2 Showing the Red Blood Cells morphology of sample A and Sample B

Sample A

Sample B

3+ spherocytes

Normochromic

1+Tear drop cell

2+ spherocytes

2+ Schistocytes

2+Eliptocytes

1+ tear drop cell

Table 2.4 showing results for Absorbance readings of Sample A at 540 nm

Tube #

Absorbance

% Haemolysis

1

0

0

2

0

0

3

0

0

4

0.002

0.9

5

0.003

2.4

6

0.004

0.3

7

0.005

0.6

8

0.034

0.9

9

0.144

10.9

10

0.432

9.7

11

0.808

88.8

12

0.885

100

Table 2.4 showing results for Absorbance readings of Sample B at 540 nm

Tube #

Absorbance

% Haemolysis

1

0

0

2

0

0

3

0

0

4

0.002

0

5

0.003

0

6

0.004

0

7

0.005

20.0

8

0.034

43.4

9

0.144

84.8

10

0.432

83.9

11

0.808

90.6

12

0.885

100

Calculations(Examples of Sample A):

Tube 1: 0.000 x 100 = 0%

0.893

Tube 6: 0.005 x 100 = 0.6%

0.893

Tube 12: 0.893 x 100 = 100%

Calculations(Examples of Sample B):

Tube 1: 0.000 x 100 = 0%

1.257

Tube 6: 0.000 x 100 = 0%

1.257

Tube 12: 1.257 x 100 = 100%

1.257

Discussion

In Sample B RBCs Morphology showed normochromic red blood cell, however, there were spherocytes elipocyes and tear drop cells present. These cells have a increase surface to volume ratio meaning that there is more space in the cell. The presence of these cells would result in a slight shift to the left of the curve which is seen on the graph hence the graph corroborate with the RBC morphology of the smear.

Conclusion:

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The findings on both smears for samples A and B corroborated with the osmotic fragility curves obtained for both samples. The fixed sickle cell and target cells seen on the smear for sample B contributed to the left shifted curve for that sample. The Stomatocytes and target cells seen on sample A contributed to the relatively shift of the curve to the left obtained for sample B.

Post Lab Questions

Why is an absorbance of 540 nm used in this test?

When RBCs are lysed, haemoglobin is released and measuring the haemoglobin determines the degree of lysis. The haemolysis is will then is used because it is the optimal wavelength at which haemoglobin is measured. This wavelength is complimentary to the colour of the supernatant of the cell suspension

Briefly comment on the lysis pattern seen for Sample A (Hep). Does the RBC morphology of the smear you made corroborate the osmotic fragility results obtained? Support your answer.

In Sample A RBC Morphology there were normochromic however, there were stomatocytes and occasional target cells. These cells have a increase surface to volume ratio meaning that there is more space in the cell. The presence of these cells would result in a shift to the left of the normal curve. From the graph there was a shift to the left which corroborate with the RBC morphology of the smear.

Briefly comment on the lysis pattern seen for Sample B. Does the RBC morphology of Sample B corroborate the osmotic fragility results obtained? Support your answer.

On the basis of the results obtained it can be said that the RBCs morphology corroborated with the OF curve. The RBCs morphology of sample B showed Hypochromic cell an extent of piokilocytosis in the form polychromatic macrocytes, schistocytes, elliptocytes, target Cells and fixed sickle cell. The OF curve for sample B shift to the left when compared to the normal curve. This is because of the extent of piokilocytosis occurring in the sample as mentioned above. These types of cells possess an increased surface to volume ratio and therefore have the ability to take in a greater volume of water than normal RBC before lysis occurs. There is an increased resistance to lysis than normal RBCs and are therefore able to sustain a greater level of hypotonicity than normal RBCs.

Explain the mechanism by which the RBC population of Sample B yields the results documented. In your explanation give two (2) examples of RBC poikilocytosis that can cause the osmotic fragility pattern seen for B.

Target cells are cells than have a larger surface area: volume ratio, so it will have the capacity to hold more water and the red blood cell will take a longer time to lyse which explain the shift to the left of the OF curve. Fixed sickle cell are more resistant to lysing because of its ability to accommodate more water so this will also cause a shift of the OF curve to the left.

State the expected effect that a homogenous population of spherocytes will have on the osmotic fragility curve.

The osmotic fragility curve will shift to the right due to the equal population of spherocytes and normal red blood cell on the smear.

For the stated results in 5; explain the mechanism by which it occurs.

Spherocytes are RBCs without a central area of pallor. Because of this abnormality, spherocytes are said to have a decreased surface to volume ratio when compared to normal RBCs. This decreased surface:volume ratio means that there is not much space within the cell. This decrease in space results in the decreased ability of the spherocytes to take in a great volume of water so cells will become swollen and burst at a faster rate. Therefore in hypertonic solution lysis of sperocytes occurs because osmotic fragility increases and resistance to lysis decreases