This is an essay that I recently wrote for my General Biology class.
One of the main issues facing veterinary medicine today is a lack of efficient cryopreservation (the process of freezing and storing cells to maintain their activity) of erythrocytes, or red blood cells, of small companion animals, e.g. dogs and cats. A transfusion of erythrocyte concentration, as opposed to whole blood transfusion, contains a consistent amount of red blood cells with minimum levels of citric acid, white blood cells, platelets, and unwanted chemicals; and can potentially save the lives of small companion animals [1, 2].
This experiment was conducted to observe the effects of penetrating cryoprotectants, where small molecules are able to cross cell membranes to reduce ice growth and cell dehydration during freezing, and non-penetrating cryoprotectants, in which large molecules, e.g. polymers, are added to cryoprotectant solutions . A cryoprotectant is a substance used to protect cells or tissues from damage during freezing. This particular experiment was concerned with the capability of single-component and multi-component cryoprotectant solutions (using both penetrating and non-penetrating cryoprotectants), as well as pre-incubation and incubation time periods prior to the freezing of canine and feline erythrocytes.
There have been no previous studies of cryopreservation conducted on feline erythrocytes but there have been studies conducted on cryopreserving canine erythrocytes . Glycerol was used as a penetrating cryoprotectant for such studies, and has previously been proven to be effective for properly preserving canine erythrocytes with little damage after the red blood cells were thawed. However, the process is problematic and tedious, and may not be readily available at all times. If glycerol is not removed properly from the erythrocytes before transfusion during deglyceralization (removing glycerol from previously frozen red blood cells), hemolysis, the destruction of red blood cells, may occur. Non-penetrating cryoprotectants, such as PEO-1500 and hydroxethyl starch (HES) were also used in previous studies conducted on cryopreserving canine erythrocytes [5-9]. One advantage of using non-penetrating cryoprotectants over penetrating cryoprotectants is that the former eliminates “washing” or removing the cryoprotectant itself before it is available to use as a transfusion. Other methods that have been taken into consideration to cryopreserve erythrocytes of small companion animals include creating and utilizing a multi-component cryoprotectant. A multi-component cryoprotectant is acquired through various components of different cryoprotectants, including penetrating and non-penetrating cryoprotectants. The utilization of some multi-component cryoprotectants has been proven to be successful in the cryopreservation of erythrocytes of other animals, such as rats and humans [10-12].
Glycerol and ME2SO, both penetrating cryoprotectants, and hydroxethyl starch, a non-penetrating cryoprotectant, were used in the experiment. The conducted study found that glycerol was not as effective in cryopreserving erythrocytes of both species, despite previous studies demonstrating otherwise. ME2SO demonstrated a decreased level of hemolysis after the erythrocytes of both species were thawed but not as significant as expected. Hydroxethyl starch proved to be the most effective cryoprotectant for both species, having the lowest levels of hemolysis yet having the highest levels of osmotic fragility (or lowest levels of osmotic stability) of all cryoprotectants. Osmotic stability or fragility was tested for by placing the thawed erythrocytes, after being incubated in various cryoprotectants, into an isotonic sodium chloride solution and observing the levels of osmosis. The results obtained from the research and experiment was represented by the levels of hemolysis and osmotic stability of the thawed erythrocytes of both species. The experiment executed in a particular path has provided a means of utilization of the results to be further researched and experimented on in various veterinary medicine studies as well.
Overall, this particular scientific study was an example of an exceptional experiment yet there were a few aspects that could have been improved or clarified. The methods and the materials used in the study were adequate and proved to be extremely useful in achieving results. On the other hand, the sample size of retrieved erythrocytes could have been increased to better represent various results of more than just three, different cryoprotectant solutions. It would have also been quite interesting to read if not only the samples of erythrocytes were taken from healthy adult canines and felines but also from small companion animals who had hematological abnormalities, disorders, or diseases to fully comprehend the effects of cryopreservation and cryoprotectants on less than healthy companion animals. The central experimental control used in the majority of the scientific study was sufficient enough to fathom, although the control group used to study the osmotic stability of the erythrocytes was not as easy to perceive. However, the findings of the experiment were significant enough that the results may be used in the future in related veterinary scientific studies
For instance, every scientific experiment follows a specific set of guidelines encoded as the scientific method. Issues that can be resolved scientifically involve observation, questioning, hypothesizing, prediction, and experimentation. As a result, the conducted research and the obtained results from such experimentations can be further implemented in future scientific studies. It is this implied key component of using the scientific method that differentiates exceptional experiments from subpar experiments. A fantastic scientific study can be repeated by others who are interested in the subject using the exact same methods and materials presented in the original.
The most fundamental question concerning this specific experiment was in regards to what is the most effective cryoprotectant that can be applied to the cryopreservation of erythrocytes of small companion animals that can lead to a successful blood transfusion. Hence, various single-component and multi-component cryoprotectants were observed and tested in the experiment. A hypothesis and a prediction were not directly stated but rather implied by the various past studies and tests administered akin to the current experiment in proposition. As stated beforehand, based on previous studies and experiments, glycerol was proven to be an effective cryoprotectant in properly cryopreserving canine erythrocytes. The reader may come to the conclusion that the mention of a successful experiment of cryopreservation using glycerol may possibly lead to yet another successful experiment using the same glycerol as a cryoprotectant.
The samples of the erythrocytes were acquired from healthy, adult canines and felines. The majority of the study was organized mainly in Ukraine and Germany, and was attended to in accordance with standard ethical and moral regulations for animals used in experiments. The actual blood samples were collected from the brachial vein, located in the forelimb, through means of venipuncture (the puncture of a vein as part of a medical procedure). While canines did not require anesthesia, felines did; hence a sedative known as Sedazin was administered to the felines in care. Sedazin, according to Biowet Puławy in Poland, is a “Xylazine 2% solution with a sedative, analgesic, and myorelaxant effect used in cattle, horses, dogs, and cats” . Sedazin was executed subcutaneously (situated or applied under the skin) or intramuscularly (situated or taking place within, or administered into a muscle), using three milligrams per kilogram of weight as the ratio. Approximately, 20 milliliters of whole blood had been retrieved from canines and 7-10 milliliters had been attained from felines.
The sample size was roughly five blood samples for each of the both species. The control groups of the experiment were the erythrocyte concentrations of both species being presented to an isotonic saline solution comprised of sodium chloride, tris-hydrochloride, and a pH level of 7.4 at 10, 20, and 30 minutes without any additional interference. The levels of hemolysis of both feline and canine erythrocytes were measured, and it was thus concluded through the implication that the longer the red blood cells were incubated in the isotonic saline solution, the levels of hemolysis increased. The data of using single cryoprotectant solutions were presented by the levels of hemolysis of red blood cells as well, though after the erythrocytes had been frozen, thawed, and washed relying upon the pre-incubation periods of 10, 20, and 30 minutes with the cryoprotectant itself. The most effective cryoprotectant was determined to be a 17.5% solution of hydroxethyl starch. On the contrary, hydroxethyl starch had the highest levels of osmotic fragility compared to the other single component cryoprotectants.
The sample size in regards to the cryopreservation of feline and canine erythrocytes using multi-component cryoprotectant solutions was also five. The control groups are similar in nature to the one being applied in the central, original experiment, however, multi-component solutions were employed in lieu of the single component cryoprotectant solutions. The multi-component solutions were consisted of one penetrating cryoprotectant, either glycerol or ME2SO, and the non-penetrating cryoprotectant, hydroxethyl starch. The data of cryopreserving canine and feline erythrocytes were presented by the levels of hemolysis of red blood cells, after the erythrocytes had been frozen, thawed, and washed, relying upon the pre-incubation periods of 10, 20, and 30 minutes as well. The most effective multi-component cryoprotectant solution was a combination of glycerol and hydroxethyl starch, however, the effectiveness was not as significant as the hydroxethyl starch solution alone.
As mentioned beforehand, the 17.5% solution of hydroxethyl starch resulted in the highest levels of osmotic fragility for both feline and canine erythrocytes. Adding a penetrating cryoprotectant, such as ME2SO, to the hydroxethyl starch lowered the levels of osmotic fragility significantly, while it increased the levels of hemolysis of red blood cells concurrently. Osmotic fragility was tested for by placing the contents, the thawed erythrocytes, into an isotonic sodium chloride solution and observing the levels of osmotic stability. However, the majority of the experimental results differed significantly from that of the control group, which was placed into a 0.45% isotonic sodium chloride solution.
To conclude, the results obtained from both single component and multi-component cryoprotectant solutions used in cryopreserving erythrocytes of small companion animals proved that further implementation of research and experimentation is required to fully comprehend and achieve exceptional and capable solutions as to what is the most effective cryoprotectant that can accomplish the lowest levels of hemolysis of red blood cells as well as osmotic fragility. Overall, the scientific study in proposition was an example of an exceptional experiment for the methods and materials being utilized, the fundamental question that was kept in mind throughout the experiment, the observations and the experimentations conducted, the discussion of the results, and the indication that the particular experiment is repeatable. Therefore, this particular scientific study would be extremely advantageous for future scientific studies in veterinary medicine.
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