Comparative evaluation of the in-vitro viability of canine and human blood preserved in citrate phosphate dextrose adenine (CPDA)-1 anticoagulated blood bag

Blood banking is a pivotal medical logistic activity that tends to bring life-saving benefits of transfusion to the patients who need them by making whole blood or blood component available, safe, viable and effective (Hess, 2010). Blood transfusion are required by patients who have need for increasing their oxygen carrying capacity (Leo and Anneke, 2008; Daniel et al., 2011). Red blood cells transfer oxygen to all cells but with continuous exposure to the oxidative stress during storage, this function is altered. Hence, there is need to sustain the viability of stored blood in citrate phosphate dextrose adenine (CPDA)-1 anticoagulated blood bag (Somnath et al., 2010). Blood stored over time face changes in biophysical, biochemical and immunological specifications. These changes are generally called blood storage lesion (Daniel et al., 2011).
        
In as much as several studies have found that transfusions therapy using stored or older blood are associated with adverse clinical outcomes (Marik and Sibbald, 1993; Purdy et al., 1997), others have not recorded such associations (Klein, 2003; Adamson, 2008). The common practice in our locality by small animal veterinarians is to transfuse freshly collected blood with commercially available human blood bag. However, human hemotherapist use fresh blood or old/stored blood for transfusion purposes. Till date, no study has been able to evaluate the changes associated with canine blood preserved with commercially available human blood bag and stored for a period of tim.

Animals
 
Three (3) Nigerian indigenous apparently healthy adult dogs with an average mean weight of (11.5±0.38) kg aged between 2-4 years were used for this study. The dogs were procured from market in Nsukka Local Government Area and housed singly per each cage in Department of Veterinary Surgery dog kennel and acclimatized for 30 days before the commencement of the experiment. They were fed with commercial dog food (JO-JO® France) and water provided ad libitum. Within this period, they were confirmed free of blood and gastrointestinal parasites through blood smear examination and fecal examination, respectively. The dogs were vaccinated with antirabies, canine distemper, leptospirosis, hepatits, parvovirus and parainfluenza vaccines.
 
Human
 
Human donors were apparently healthy volunteer males who tested negative for sexually transmitted diseases and hemoparasites.

Ethics
 
Studies were performed in conformity with National Institute of Health (NIH) revised guidelines for laboratory animals care and use (NIH, 1985) and the University of Nigeria ethical codes and regulations for research.
 
Methodology
 
Two hundred and fifty millilitres (250mls) of blood each were drawn from two clinically healthy dogs and a volunteer human donor into humans (infant) CPDA-1 blood bags. The sample collection were done using standard aseptic procedures and within 20minutes. The samples were thoroughly but gently mixed with the anticoagulant immediately after filling the bags. Parameters assayed on days 0, 3, 7, 14 and 21 include packed cell volume (PCV), hemoglobin (hb) concentration and Red blood cell (RBC) count as described by Cheesbrough (2006). Mean corpuscular volume (MCV) was calculated as described by Schalm et al., (1975). Also erythrocyte catalase (CAT) was assayed as described by Sinha (1972), erythrocyte glutathione (GSH) as described by Moron et al., (1979) and plasma potassium ion (K⁺) was determined as described by Hillman and Beyer (1967).
 
Analysis
 
The calculated values were entered into excel sheet format for documentation and analysis. SPSS version 21 was used for statistical analysis. Mean, standard error of mean, confidence intervals were determined for each analysed variable. Data collected were statistically compared within group using one way analysis of variance (ANOVA). The least significant difference post hoc test was used to separate the variant means at p<0.05 as statistically significant. Using independent t-test, the hypothesis to determine significant variation between the canine and human stored blood were determined at p<0.05.

The main concern for stored blood cells is ex-vivo storage lesions that undermine red cell functions and consequently affect the metabolic status of the in-vivo milieu of the recipient (Strauss, 2006). In this study, we evaluated the duration of time during which canine and human blood would remain viable in-vitro in commercially available human CPDA-1 blood bag by monitoring some principal haematological and biochemical parameters. This was followed by comparing the degree of deterioration of the stored canine blood with that of human.
        
There was a significant (p<0.05) progressive rise in plasma potassium concentration of canine blood during storage in CPDA-1 blood bag while that of human bloods plasma potassium ion recorded a progressive significant (p<0.05) increase from day 0 to day 21 ps as seen in Table 2. The significant rise in supernatant potassium ion concentration (K⁺) in both canine and human blood was consistent with findings reported by Minetti et al., (2001) and Flat et al., (2014). Minetti et al., (2001) purported that it could be as a result of sodium potassium pump paralysis which possibly lead to precipitous increase in supernatant K⁺. Upon critical evaluation it was observed also that, the level of potassium in human blood was about five times that observed in canine blood at base line. The same observation was made by Moore et al., (1981) and Moroff et al., (1983). The rate of subsequent potassium efflux to extracellular compartment was higher in human than in canine blood. This finding may be because human blood had higher potassium concentration hence leaked rapidly into the plasma. As expected there was a significant positive correlation between plasma potassium and hemoglobin levels in both stored canine and human blood. This finding affirms with a recent research reported by Somnath et al., (2010) suggesting that there could be red cell membrane disruption probably due to peroxidation that has led to hemoglobin leakage as well as leakage of other ions (K⁺). Increasing hemoglobin concentration signifies deterioration because it has post transfusion perturbations and implications (Rubin et al., 2008). When compared, the percentage of haemoglobin leakage was higher in canine blood (0.40%) than in human (0.15%) where it was better preserved. This finding affirms to that reported by Sylvester and Jane (1998).
        
The concentration of stored canine RBC catalase showed progressive decrease during storage such that the mean value of catalase on day 0 was significantly (p<0.05) higher than values obtained on day 3, 7, 14 and 21 post storage in CPDA-1 blood. The mean RBC-GSH concen- tration of stored canine blood progressively decreased during storage such that GSH concentration on day 0 was significantly (p<0.05) highest and values obtained on day 14 and 21 were significantly (p<0.05) the lowest (Table 1). Human erythrocyte catalase concentration in this study on day 0 was significantly (p< 0.05) higher than that of day 3, 7, 14 and 21. But the values recorded on days 7, 14 and 21 showed no significant (p>0.05) difference among themselves but were significantly (p< 0.05) lower than those of days 0 and 3 as seen in Table 2. Erythrocyte GSH concentration of human stored blood recorded on day 3 showed no significant (p>0.05) changes with that obtained on days 0 and 7. However, the value recorded on day 7 showed a significant (p< 0.05) difference when compared with the value recorded on day 0. The values of RBC-GSH on days 14 and 21 showed no significant (p>0.05) difference between themselves but were significantly (p< 0.05) lower when compared with the other experimental days. From Table 4, the percentage change of the RBC-CAT of human stored blood was higher than that of canine on days 3 and 7 but by day 14 and 21, the reverse was the case. However, percentage change in the erythrocyte GSH of humans was higher than that of canine on day 3 but by days 7, 14 and 21 that of canine was higher than humans. Red blood cell catalase and GSH levels which indicate erythrocyte antioxidant capacity decreased progressively during storage. This finding may suggest an increase in oxidative stress.  Another observation made was that by day 21 of storage, the percentage decrease in both CAT, and GSH was higher in canine blood (90.8% and 91.35% respectively) than in human blood (78.7% and 74% respectively). Catalase and GSH represents a fraction of enzymatic and non–enzymatic erythrocyte antioxidants which neutralizes both endogenous and exogenous reactive oxygen species (hydrogen peroxide, superoxides) to which RBCs are constantly exposed to (Almac and Ince, 2007). Therefore a major contributing factor to the decreasing life span of stored blood could be due to the decrease in the antioxidant defence system or an increase in oxidative stress (Huyut, 2016). Erythrocytes are rich in unconjugated polyunsaturated fatty acids which are susceptible to hydrogen atom abstraction (Stocks and Dormandy, 1971). Oxygenation of these fatty acids decreases erythrocyte antioxidant defence system such that continuous oxidation would lead to progressive exhaustion of the in-vitro antioxidants thus justifying the reason for the observed decrease in GSH and catalase levels during storage in both stored canine and human blood. Nevertheless, when compared the rate of deterioration was higher in stored canine blood (CAT-12.4% to 90.8%, GSH-13.95 to 91.35%) than in stored human blood (CAT-42.7% to 78.7%, GSH- 20.03% to 74%) by days 14 and 21. This may provide evidence that the oxidative stress that happens in canine blood preserved with commercially available human blood bag was more than that of human.
 

 

 

        
Hematologic data generated from the PCV and RBC count was used to calculate the MCV value. Table (3) compared the mean corpuscular volume (MCV) of canine and human blood stored under the same medium and environment. The values recorded on days 0, 3 and 14 showed no significant (p>0.05) difference. But on days 7 and 14, there was a significant (p<0.05) change between the two. The mean MCV of stored canine blood was significantly (p<0.05) lower than that recorded from that of human stored blood on day 7 but on day 14, the reverse was the case. The mean MCV value was significantly higher in dogs than in human blood by day 21 of storage. This finding has also been reported by Karama, (2008) and Somnath et al., (2010) where they suggested that increasing MCV indicates progressive increase in erythrocyte size due to influx and accumulation of extracellular sodium ion in the cytosol. This is also a pointer that canine blood degenerated faster than human blood.
 

               
In conclusion, this study have shown that canine blood can be preserved in CPDA-1 blood bag for 21 days and still remain viable for transfusion. It was also established that a negative relationship exists between storage time and erythrocyte viability cum function in both human and canine blood. However based on results generated from this study, it has been established that canine blood when stored in CPDA-1 blood bag deteriorates faster than human blood. It could be suggested that the reason for a faster deterioration of canine blood can be that the storage media used was specified for humans that preserved their parameters better. Although there was marked biochemical and hematological changes in stored canine blood, 21-day-old unit may be of accepTable quality for blood transfusion. We therefore recommend that further studies be done with canine blood bags to investigate their deterioratory propensities.