Investigating the Resistance of Erythrocytes to Haemolysis

Aim

• To fix accurate consecutive dilutions from a standard stock solution of Na chloride
• To derive expertness in utilizing an automatic pipette to present really little volumes of liquids
• To read, grok and use the instructions on how to utilize a spectrophotometer and hemacytometer
• To number and detect ruddy blood cells and other blood atoms
• To build haemolysis curves for poulet and fish ruddy blood cells and compare the differences

Introduction

Osmosis is the inactive motion of H2O through a semipermeable membrane from a comparatively low osmotic force per unit area to a high osmotic force per unit area to accomplish an equilibrium province.

A 0.8 % NaCl solution is said to beisotonic.When blood cells reside in an isosmotic medium, the intracellular and extracellular fluids are in osmotic equilibrium across the cell membrane, and there is no net inflow or outflow of H2O. However, when they are placed in a sufficiently-strong hypotonic fluid, there is an uncontrolled net inward osmosis, ensuing in the uninterrupted puffiness of the cells until the cells burst unfastened, or lysed.Hemolysisis the lysis of ruddy blood cells. The usage of haemolysis curve as a mention is a practical application since it can find the points of initial and 100 % hemolysis, and supply a better comparing among different species i.e towards understanding the animate beings red blood cells osmotic breakability.

Red blood cells, besides known as red blood cell can change in term of size, figure, form, lifetime, agreement and coloring material of cells harmonizing to the species. Animals like fish, birds, reptilians and amphibious vehicles have egg-shaped nucleated red blood cells go arounding in their peripheral blood. ( Carla Simone Seibert, 2001 )

The osmotic breakability trial ( OFT ) is used to mensurate erythrocyte opposition to hemolysis while being exposed to changing degrees of dilution of a saline solution. ( Usman Khalid, 2012 ) The breakability of red blood cells to osmotic hemolysis has been investigated in assorted carnal species ( Perk et al. , 1964 ; Soliman & A ; Amrousi, 1966 ; Jain, 1973 ; J. O. Oyewale and L. A. Durotoye, 1988 ) . In this experiment we studied the ruddy blood cells breakability of fish ( aquatic ) and chicken ( land ) in saline solutions under assortment concentration ranges.

Equipment and Chemicals

1. Phosphate buffer
2. 1.0 N HCl
3. 10 % Na chloride stock solution
4. Heparin tubings for blood aggregation, Pateur pipettes, 2 volumetric flasks ( IL ) and 13 volumetric flasks of 100mL, 4 glass pipettes of 10mL, trial tubings ( 15mL ) = 26 and cuvettes = 26
5. Automatic pipettes ( 100µL ) , extractor, spectrophotometer

Method

1. A scope of buffered saline solution was prepared with the undermentioned concentrations: 0.10 % , 0.20 % , 0.25 % , 0.30 % , 0.35 % , 0.40 % , 0.45 % , 0.50 % , 0.55 % , 0.60 % , 0.70 % , 0.80 % NaCl from the stock solution of 10 % NaCl. 100mL volumetric flask was used.
2. 5mL of each NaCl solution was transferred utilizing a glass pipette into a separate trial tubing.
3. Fish blood was collected by break uping the caudal peduncle of the fish utilizing a crisp knife and have oning cut protective baseball mitts. The blood was collected into heparinized tubings. To forestall blood coagulum, the blood was dripped onto the underside of the tubing where Lipo-Hepin is located. The poulet blood has been prepared earlier.
4. 0.025 milliliter ( 25µL ) blood sample is pipetted into each trial tubing ( 12 tubings for each blood type ) that we have prepared. The blood was so assorted with saline solution really gently by inverting the trial tubing three times. ( Shaking the trial tubing was non allowed as it might interrupt the ruddy blood cells )
5. The trial tubing were left at room temperature for 40 proceedingss before centrifugating them for 10 proceedingss.
6. The supernatant of the haemoglobin solution is carefully removed utilizing a Pasteur pipette into a cuvette. The optical density is measured at 540nm in a spectrophotometer. The 0.8 % saline was used as a space to set the readings.

Consequence

Table 1.0 . The sum of optical density value obtained and calculated haemolysis ( % ) of poulet and fish under buffered saline solution of NaCl concentration ( % ).

The value of optical density between the fish and poulet ruddy blood cells are compared in Table 1. A important difference in tendency between the two species is noted, where the optical density value of fish declined before it peaks up at NaCl concentration of 0.35 % , and so undergoes a steady lessening. Chicken in the other manus undergoes a little addition and reaches the extremum at 0.2 % NaCl concentration before bit by bit diminutions.

% Hemolysis is calculated by utilizing the undermentioned method:

From the deliberate value of % haemolysis obtained, a graph of haemolysis curve of the poulet and fish ruddy blood cells can be plotted.

Figure 1.0. Hemolysis curve of poulet and fish ruddy blood cells

From the experiment conducted, both trying informations of poulet and fish should be able to obtain a theoretical sigmoidal form haemolysis curve. However, the failure of both carnal informations to follow the theoretical S-shaped graph could be due to the inaccurate application of the usual saline-solution technique used in adult male ) to the measuring of osmotic breakability of the red blood cells of birds ( Parpart, Lorenz, Parpart, Gregg & A ; Chase, 1947 ; G. Viscor, 1982 ) . The average corpuscular breakability of both animate beings comparison from the graph obtained ( Fig. 1 ) showed that the poulet red blood cell undergoes 50 % haemolysis at 0.25 % NaCl concentration, while fish experienced partial haemolysis at a higher NaCl concentration that is 0.4 % . This indicates that the poulet had a lower red blood cell osmotic breakability ( higher osmotic opposition ) compared to the fish. The ground is that when both ruddy blood cells were introduced to a more hypotonic solution, the poulet experienced partial haemolysis at a lower saline concentration.

However, both showed merely a little addition in % haemolysis from the isosmotic solution between 0.8 % to 0.3 % for poulet and 0.8 % to 0.45 % for fish before both readings rise aggressively, responding to the hypotonic environment and undergo lysis.

Discussion

The ground why both graph doesn’t represent a sigmoid form could be due to the technique used. G. Viscor proposed that the technique used to compare osmotic breakability in adult male is non applicable to chicken as he found out that haemolysis of the poulet red blood cell was non complete and that portion of the haemoglobin remained attached to the broken red blood cells in the most hypotonic solutions ( 100 % haemolysis ) . The values obtained did non ever represent entire haemolysis, even though the per centum of haemolysis was calculated in relation to the highest value obtained with the spectrophotometer. Hence, this can lend to error in finding the osmotic breakability from the haemolysis curve.

When comparing both species opposition towards osmotic breakability, it is best to utilize the mean corpuscular breakability ( MCF ) , which represents the saline concentration at which 50 % haemolysis is produced. ( Fourie, 1977 ) From ( Fig. 1 ) above we can see that both fish and poulets have different curves to compare with, hence, partial haemolysis comparing is used.

When ruddy blood cells were bathed in hypotonic environment ( e.g. & lt ; 0.8 % NaCl or distilled H2O ) , net motion of solution into the cells took topographic point. The cells lyses as the unity of their membranes were disrupted.

Hemolysis completed in a higher saline concentration indicated a higher osmotic breakability of red blood cells. Fish blood hemolysed at a higher NaCl concentration compared to the poulet blood suggested that the fish has a greater osmotic breakability due to several factors that may hold influenced the osmotic ordinances in animate beings.

Chicken ruddy blood cells had higher ability to defy emphasis caused by the hypotonic environment before the cells undergo entire haemolysis. Hence, the poulet ruddy blood cell is more osmotic opposition than the fish.

Blood osmotic breakability in animate being is influenced by several extrinsic and intrinsic factors. Extrinsic factors such as pH, temperature, osmolality and blood storage affect the osmotic breakability of red blood cells ( Dacie and Lewis, 1995 ; Lewis and Ferguson, 1966 ; Oyewale et al. , 1991 ; Oyewale, 1994 ) . Structural features and the snap of the membrane of red blood cell besides may be an of import determiner in osmotic opposition ( G. Viscor, 1982 ) . Nucleated ruddy blood cells have lower osmotic breakability compared to the un-nucleated ruddy blood cells. However in this experiment, both fish and poulet has a nucleated ruddy blood cell. As add-on, life span and size of red blood cell could play an of import function of ruddy blood cells osmotic breakability. ( Kevin J. Aldrich, 2006 )

Equipment:

1. Hemacytometer- blood numeration chamber
2. Manual hand-held counter
3. Micropipette and pipette tips
4. Pphosphate buffer saline ( PBS ) solution- dissolve one tablet in 100mL distilled H2O
5. Light microscope

Method:

1. 10µL blood with 990µL PBS solution was diluted in a trial tubing ( 1:100 dilution )
2. The solution was assorted gently by inverting the trial tubing 3 times
3. The hemacytometer and screen glass faux pas was cleaned carefully. The screen faux pas was so placed on top of the numeration chamber. Each hemacytometer has 2 numbering Chamberss on opposite sides.
4. The infinite of the numbering chamber was filled with the diluted blood by touching the tip of the pipette gently against the screen faux pas at a 45 grade angle. Capillary action so sucked in the diluted blood from the micropipette. The numbering chamber was carefully filled so that it didn’t get over flooded. 10µL of the diluted blood sample was used.
5. The hemacytometer was placed under a light microscope and was allowed to settle for several seconds. Using 40x magnification ( 4mm aim and x10 ocular ) , the aim was focused on the cardinal square millimetre of the numeration chamber and the cells were counted in 5 squares ( 1/25mm²each ) , the four corner squares and the cardinal one of the ruled country.

Calculation

Entire figure of ruddy blood cells per millimeter³inpoulet

Entire figure of ruddy blood cells per millimeter³infish

Discussion

From the computation, there is a important difference between the figure of ruddy blood cells per millimeter³between the fish and poulet, where the poulet has a higher figure of ruddy blood cells per millimeter³compared to the fish.

Red blood cells and white blood cells are two critical constituents of blood but execute different functions in the organic structures. Red blood cells primary map is to transport O from the lungs to the tissues around the organic structure. They besides carry waste C dioxide from the tissues to the lungs, where it can be exhaled out.

Leukocytes function as a organic structure defense mechanism mechanism as they ingest pathogens and destruct them. Approximately, they besides produce antibodies to develop unsusceptibility against infections. Some are even phagocytic. There are 5 types of leucocytes with distinguishable map ; neutrophils, eosinophils, lymph cells, monocytes, basophils.

Neutrophils are the most active leucocytes in the initial phases of redness. In terrible infections, the neutrophils destroy bacteriums or little atoms, particularly the coccus signifiers, by phagocytosis.

Eosinophils helps on detoxification. They accumulate at the site of antigen-antibody reactions and inactivate histamine or histamine-like toxic stuffs.

Lymphocytes function in the immune response of the host where they are involved in production of antibody. Their phagocytic ability appears to be limited to ultramicroscopic atoms such as viruses.

Monocytes are macrophages and their particular enzyme systems helps in remotion of foreign big atoms from the organic structure.

Basophils react to injury by let go ofing histamine which initiates the inflammatory reaction by doing dilation and increased permeablenes of blood vass.

Red blood cells ( red blood cells ) differ from white blood cells ( leucocytes ) in many facets, such as structural features, lifetime, production and volume.

Red blood cells have biconcave phonograph record shaped, and is an anucleate with size about runing from 6-8 ?m. Relatively, white blood cells are irregular in form and has nucleus.

Red blood cells have a life span of 120 years, while white blood cells have a shorter lifetime from 4-30 yearss depending on the organic structure.

Red blood cells are produced at the ruddy bone marrow, while white blood cells are produced in lymph nodes and lien.

Even their % content in blood is besides different. Red blood cells make up 36-50 % of our blood ( depending on sex, height & A ; weight ) , while white blood cells are merely near to 1 % of the blood content.

Decision

Different species has different red blood cells osmotic breakability and blood count.

Osmotic breakability can be influenced by several factors i.e pH, temperature, structural features and the snap of the membrane of red blood cell.

There is no widely accepted set of normal consequences because different research labs use different testing process and this can well impact the consequences.

Mentions

1. A Eljack ( n.d ) . Leukocyte Function and Clinical Interpretation. Retrieved from hypertext transfer protocol: //compepid.tuskegee.edu/syllabi/pathobiology/pathology/clinpath/chapter4.html on 23rd March 2014
2. Arthur K. Parpart, Philip B. Lorenz, Ethel R. Parpart, John R. Gregg, and Aurin M. Chase ( 1946 ) . The osmotic opposition ( breakability ) of human ruddy cells. Retrieved from hypertext transfer protocol: //www.ncbi.nlm.nih.gov/pmc/articles/PMC439273/pdf/jcinvest00384-0046.pdf on 23rd March 2014
3. Carla Simone Seibert, Elvira Maria Guerra-Shinohara, Elianora Gomes de Carvalho1 and Elineide Eugenio Marques ( 2001 ) . Red blood cell parametric quantities and osmotic breakability curve of Colossoma macropomum ( Pisces, Osteichthyes, Mileinae ) in imprisonment. Maringa , 23 ( 2 ) ,515-520. Retrieved from hypertext transfer protocol: //www.periodicos.uem.br/ojs/index.php/ActaSciBiolSci/article/viewFile/2709/2028 on 23rd March 2014
4. Dacie, J. V. , S. M. Lewis ( 1995 ) : Practical Haematology, 8th erectile dysfunction. Churchill Livingstone, New York. pp. 216-220.
5. Erich Rosenberger ( 2007 Dec, 27 ) . The Functions of Red Blood Cells. Retrieved from hypertext transfer protocol: //www.sciences360.com/index.php/the-functions-of-red-blood-cells-22247/ on 23rd March 2014
6. Fourie, F. R. ( 1977 ) . Effectss of decoagulants on the haematocrit, osmolarity and pH of avian blood. Poultry Science 56, 1842-1846.
7. G. Viscor and J. Palomeque ( 1982 ) . Method for finding the osmotic breakability curves of red blood cells in birds. Laboratory Animals 16, 48-50.
8. J. O. Oyewale and L. A. Durotoye ( 1988 ) . Osmotic breakability of red blood cells of two strains of domestic poultry in the warm humid Torrid Zones. Lab Animal. 22: 250-254. Retrieved from hypertext transfer protocol: //lan.sagepub.com/content/22/3/250.full.pdf on 24^ThursdayMarch 2014
9. Jain NC ( 1973 ) . Osmotic breakability of red blood cells of Canis familiariss and cats in wellness and in certain haematologic upsets. Cornell Veterinarian: 63, 411-423
10. Kevin J. A. , David K. S. , Lynette M. S. , Greg S. ( 2006 ) Comparison of erythrocyte osmotic breakability among amphibious vehicles, reptilians, birds and mammals. Minutess of the Kansas Academy of Science, 109 ( 3 ) :149-158. Retrieved from hypertext transfer protocol: //www.bioone.org/doi/pdf/10.1660/0022-8443 % 282006 % 29109 % 5B149 % 3ACOEOFA % 5D2.0.CO % 3B2 on 23rd March 2014
11. Lewis, J. H. , E. E. Ferguson ( 1966 ) : Osmotic breakability of premammalian red blood cells. Comp.
12. Biochem. Physiol. 18, 589-595
13. Oyewale, J. O. , A. A. Sanni, H. A. Ajibade ( 1991 ) : Effectss of temperature, pH, and blood  storage on osmotic breakability of duck red blood cells. J. Vet. Med. A. 38, 261-264
14. Parpart, A. K. , Lorenz, P. B. , Parpart, E. R. , Gregg, J. P. & A ; Chase, A. M. ( 1947 ) . The osmotic opposition ( breakability ) of human ruddy cells. Journal of Clinical Investigation: 26, 636-640.
15. Perk K, Frei, YF & A ; Herz A ( 1964 ) Osmotic breakability of ruddy blood cells of immature and mature domestic and laboratory animate beings. American Journal of Veterinary Research: 25, 1241-1248
16. S. M. Durham ( n.d ) . Red blood cells. Retrieved from hypertext transfer protocol: //loudoun.nvc.edu/vetonline/vet131/erythrocytes.htm on 23^rdMarch 2014
17. Soliman MK & A ; Amrousi SE ( 1966 ) . Erythrocyte breakability of healthy poultry, Canis familiaris, sheep, cowss, American bison, Equus caballus and camel blood. Veterinary Record: 78, 429-430
18. Stephen Gallik ( 2011 ) . Cell Biology: Osmosis. Retrieved from hypertext transfer protocol: //cellbiologyolm.stevegallik.org/node/64 on 23rd March 2014
19. Usman Khalid ( 2012 Dec, 3 ) . Osmotic Fragility of Erythrocytes. Retrieved from hypertext transfer protocol: //emedicine.medscape.com/article/2085814-overview # aw2aab6b2 on 23rd March 2014