Aim:To demonstrate quality assurance techniques in performing cell washing. Principle:Cell washing is a common procedure performed in the laboratory. This is a series of careful steps taken to wash red blood cells normally three times intermittently with centrifugation and decanting (Harmening 2012).
The procedure serves to remove unbound immunoglobulins, Wharton’s jelly (from cord blood), hemolysed cells and also fibrin. The principle of the centrifuge entails centrifugal filtering of components due to centrifugal force applied and also centrifugal sedimentation which allows the separation of these components based on their weight and density due to the gravitational pull (Marieb 2009).
In washing red blood cells, quality assurance is ensured so that testing such as cross matching in blood transfusion can be accurately undertaken. The centrifuge used in this exercise was the fixed angle centrifuge. According to Lawrence Kaplan (2003) these rotors are so named due to the fact that the samples are kept at a fixed angle, 25 to 52 degrees of vertical axis of rotation. As the sample rotates, small particles are forced to the sides and bottom on the tube then settles to the bottom due to gravity as rotation ceases. These centrifuges are used when rapid separation of small particles is required. Materials:
CHEMICAL REAGENTS| APPARATUS & EQUIPTMENT|
WaterBlood samples in purple top tube0.85% salineNaCl| Centrifuge5ml Test tubestest tube rackParafilmDisposable and automatic pipette|
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Procedure:Reagent Make-Up and Standard Preparation
* 500ml of 0.85% saline was made by dissolving 4.5g of NaCl in 200ml of distilled water then adding up the solution in a measuring cylinder with distilled water until 500ml was obtained. * All the necessary equipments were obtained, checked and test tubes labeled 2%, 5% and sample. * To make the 2% standard 80 micro liter of blood was carefully pipetted into a clean test tube labeled 2% using an automated pipette with range 1-100 micro liter. * Pipetting was done by securing a tip to the automatic pipette without touching it, pressing the plunger to the first stop, submerging the tip vertically below the surface of the blood, slowly releasing the plunger, gently gliding the tip against the tube while coming out then vertically releasing the blood into the appropriate tube by pressing the plunger to the first then second stop. * 3920 micro liter of 0.85% saline was then pipetted into the same test tube using an automated pipette with range 500-1000 micro liter. * A piece of parafilm was used to cover the tube then it was inverted and color observed. * To make up the 5% standard 200 micro liter of blood was carefully pipetted into a clean test tube labeled 5% using an automated pipette with appropriate range.
* Apparatus, equipment and whole blood labeled and collected according to proper phlebotomy procedures was obtained. * Using a disposable pipette 2 drops of the blood sample from the purple top tube was dropped in a clean 5ml test tube labeled ‘sample’ * Using a wash bottle, 0.85% saline was forcefully added to the center of the sample in the tube until a cherry red color was observed. * The parafilm was used to cover the suspension and a second 5ml tube was filled with equal amount of water and also covered with a parafilm. * The 2 were set at opposite sides from each other in the centrifuge and centrifuged for 1 minute at high speed. * When the centrifuge had ended and stopped, the test tubes were retrieved after which decanting (removal of all the supernatant in a quick smooth motion) was done immediately. The water was discarded. * Steps 3 to 6 were repeated twice.
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* The final pellet was re-suspended until a cherry red color (similar to 2-5% standards) was obtained. Results:
It was observed that the more the sample was washed, the clearer the supernatant appeared and the smaller the pellet became. The final pellet was re-suspended until a cherry red color was observed. The colors and volume of the 2% and 5% standards were also observed and compared to that of the sample since the aim was to achieve a 2-5% suspension. The 2% standard was of a paler red than the 5%. The final suspension of the sample was observed to be between the two standards, however, closer to the color of the 5% standard after further comparison while the volume was equal to that of the 5% standard. Discussion:
In cell washing 0.85% NaCl was used to wash the cell each time. This solution was made prior to the lab exercise, and not overnight or days ahead, to ensure that the concentration remained the same as required and no evaporation could take place to alter / increase the such, as this would have adverse effects on the cells. This concentration was chosen since it was isotonic to the concentration of the red blood cells. Since both solutions were isotonic no particle movement occurred and the cells were not altered in any way. After cell washing centrifugation took place for 1 minute at high speed each time. This allowed for adequate separation of the red blood cells (pellet) of the blood from the plasma with unbound immunoglobulins, hemolysed cells and fibrin (all in supernatant).
The decanting step which always followed is described as a swift yet careful movement to dispense the supernatant from the pellet. This step was repeated twice to ensure that the cells were being washed as clean as possible, it also accounted for the fact that decanting may not always be done to remove all the supernatant.
The standards created (2% and 5% solution) were used to compare the accuracy of the washed cells determined by the color viewed since the aim was to have 2-5% cell suspension. Any color achieved between the two would be acceptably, however the 5% standard was a perfect example of the cherry red color to be attained. The cherry red color indicated that the cells were now clean and at an ideal concentration for further procedures. The volume of the final suspension was equal to that of the 5% standard. Conclusion: The lab exercise was a success since quality assurance techniques were used in the cell washing procedure and a 2-5% suspension was attained. Quality assurance steps included freshly prepared saline, 2% and 5% standards, accuracy in calculation and measurement of fluid as well as clean equipment and work field. The final suspension was near in both color and volume to the 5% standard prepared.
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Harmening D. 2012. Modern Blood Banking & Transfusion Practices. 6th Ed. Philadelphia: FA Davis. p 26. Kaplan LA. Pesce, Armadeo J. 2010. Clinical Chemistry Theory Analysis and Correlation. 5th Ed. Missouri: Mosby Inc. p 23-24. Marieb EN. 2009. Human Anatony and Physiology. Redwood City, Calf: Benjamin Cummings Pub. Co. p 301.