Making extrapolations from human studies to what happens in animals has been a misleading diagnostic tool for Veterinarians. Whole blood stability following storage is defined as the capability of blood to retain the initial value of a measured quantity for a defined period within specific limits when preserved under defined conditions. The results of the studied parameters showed an ongoing biomechanical metabolism following storage. Morphometric studies of red blood cells have been the subject of recent several studies in various animal species
(Adili et al., 2016) together with morphologic alterations
(Ibrahim et al., 2016) and vitality information
(Cunbo et al., 2017) for diagnostic purposes. Morphometric changes is based on linear measurements of erythrocyte sizes (diameter, surface and circumference)
(Adili et al., 2016).
The statistical significance of the experimental data obtained from the erythrocyte parameters (diameter, circumference and surface) was indicated in Table 1. The erythrocyte diameter, circumference and surface findings on day 0 ps showed no significant (p>0.05) variation when compared with the values obtained on days 7 and 28 ps but significantly (p<0.05) smaller to that of day 35. The findings on days 14 and 21 were significantly (p<0.05) smaller to that of the base value with no significant (p>0.05) variation between them. This could be due to compromised membrane integrity leading to loss of fluids. But by day 35, there could be influx of fluids that inadvertently interfere and induce some cytoplasmic and morphometric changes and on the extreme provoke degranulation of red cells (Adili and Melizi, 2014).
During storage, red blood cells undergo marked morphological changes (Ibrahim, 2008). Findings revealed about 25-50% spherocytes with 64-73% dicosyte by day 14 ps and about 87-100% crenocytes by day 28 ps. Our findings was in line with the work of
Ibrahim et al., (2016) on human pRBC where the reversible spherocytes were seen by day 14 ps, whereas this study showed about 87-100% irreversible crenocytes by day 28, Ibrahim’s work showed a 100% crenocytes by day 42 ps. This faster degeneration of canine stored blood could be attributed to progressive increase in erythrocyte size due to influx and accumulation of extracellular sodium ion in the cytosol as previously described by
Okereke et al., (2020) and
Udegbunam et al., (2020). Discoid shape of red blood cell influences its resistance to membrane distortions owing to a special organization of the membrane-skeleton system. This particular proûle maximizes the surface to volume ratio and thus may expedite diffusion and exchange. It is seen that during storage, red blood cells gradually loses the normal morphology of discoid shape and progress to spherocytes, with decreased surface area to volume ratio
(Cluitmans et al., 2012) as a result of increased reactive oxygen species (ROS) and decreased antioxidant defence mechanism leading to oxidation and degradation of these proteins. These ROS has known cytotoxic effects (Hess, 2010) and can cross-link erythrocyte membrane phospholipids and proteins
(Hoehn et al., 2015). As membrane proteins are lost coupled with increasing ROS, ion pumps begin to fail causing the starving RBC’s to swell as a result of influx of fluids. The erythrocytes will then take on a more spherical or hemispherical appearance shifting into numerous non-deformable protrusions of cellular membrane sticking out in all directions (Hess, 2010). These changes could be due to the continued biochemical, biomechanical and haematological alterations in the stored blood over time.
Red cell vitality was done in this study with trypan blue which in biosciences, it is used as a vital stain to selectively colour dead tissues or cells blue
(Cunbo et al., 2017). Trypan blue stain for erythrocytes can also be termed dye exclusion method. Here, live cells with intact membranes are not coloured because they are selective to compounds that pass through them. In membrane compromised or dead cells, the trypan blue passes the membrane with the cell appearing blue colour under the microscope
(Klaus et al., 2005). This work revealed blue staining erythrocytes by day 21 ps. Post storage days 0, 7 and 14 revealed live cells (as seen in Plates 1, 2 and 3 respectively). However, by day 21 post storage day, there were clusters of blue stained cells indicating presence of deformed/ dead red cells (Plate 4). This shows that with vital staining technique, compromised cells or dead cells was evident by day 21 with greater percentage by day 28 ps and day 35 ps as seen in Plates 5, 6, 7. In as much as no documentation in literature has used this technique to actually study morphological changes associated with SCB in CPDA-1, we suggest that this could form a baseline study and the cause be attributed to the oxidative changes and distortion of membrane skeleton system of the erythrocytes (Uzoigwe, 2006).
Anesthesiologists and surgeons often estimate intra-operative blood loss by simple observation, which can offer wide-ranging values. This often leads to poor management with the transfusion of blood, blood products and fluids in the perioperative patient. Intra operative blood loss was estimated using the gravimetric method as against the Spectrophotometric method which though was more reliable but clinically impractical
(Dominic et al., 2015). Under estimation of blood loss can lead to unnecessary transfusion practices. On the other hand,
Udegbunam et al., (2009) documented the need for anaesthesiologist and surgeons to consider the anaesthetic protocol used with respect to the nature of surgery been done. They argued that in surgical procedures such as splenectomy, some anaesthetic agents such as diazepam, ketamine and midazolam have the high tendency of causing increase in splenic volume where the blood cells are sequestrated in the spleen. Also, the use of barbiturates such as pentobarbitone, sodium amytal, pentothal have all also shown to cause increase in splenic size following administration
(Hausner et al., 1938). Though no report in literature exist on the effects of atropine and xylazine on the splenic size following administration, ketamine is known to have a vasorelaxant effect which could cause an increase in splenic size of dogs following administration
(Udegbunam et al., 2009). Findings shown in Table 3, however confirms the effect of ketamine administration on splenic size and validates the need for anaesthesiologist and surgeons to routinely estimate the volume of blood lost during each procedure.
Haldane et al., (2004), proposed that the goal for blood transfusion would be to achieve 25-30 per cent hematocrit in recipient dogs. Based on the information from Haldane and other researchers on haematocrit selection and the knowledge that no literature documented the clinical trial on the use of very old blood for transfusion therapy in order to achieve desired haematocrit, this work was designed to clinically validate the claims of Haldane and other researchers. This study also tried to reveal that following the alterations associated with stored blood, if prolonged stored blood when used for transfusion will achieve the desired haematocrit as compared with when fresh blood is used. The findings of this study showed that 4 day old blood was able to achieve 15% increase (from 31% to 46%) in haematocrit even when the desired haematocrit was 39% with 120 ml of blood. The use of 14 day old blood was also able to achieve a 7% increase (30% to 37%) (Table 2) in haematocrit which agreed with the desired haematocrit. The use of 21 day old blood recorded a significant increase from the haematocrit 24 hours post transfusion (31.7%) without a non-significant percentage desired haematocrit increase (33.7%) while 28 day old blood usage caused significant decrease of haematocrit 24 hours post transfusion (27% to 25.3%) and desired haematocrit (29%) (Table 2). This could be agreed to the progressive irreversible membrane cytoskeleton damage resulting to cell apoptosis (Kristensen and Feldman, 1995).
