Veterinary Clinical Pathology ISSN 0275-6382
ORIGINAL RESEARCH
Effects of four additive solutions on canine leukoreduced red cell concentrate quality during storage Luciana A. Lacerda1, Nicole R.C. Hlavac1, Silvia R. Terra2, Franciele P. Back1, K. Jane Wardrop3, lix H.D. Gonza lez1 Fe Departments of 1 Veterinary Clinical Pathology, and 2 Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; and Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA
3
Key Words Blood, dog, erythrocyte, filter, transfusion Correspondence Luciana de Almeida Lacerda, Av. Bento Goncßalves, 9090, B. Agronomia, LACVet, UFRGS, CEP 91540-000 Porto Alegre, RS, Brazil E-mail: [email protected] DOI:10.1111/vcp.12163
Background: Additive solutions (AS) and prestorage leukoreduction (LR) are important tools used to maintain erythrocyte viability during storage and avoid transfusion reactions in recipients, respectively. Objectives: The purpose of the study was to determine the efficacy of a WBC filter (Immugard IIIRC) and compare the effect of 4 AS (phosphateadenine-glucose-guanosine-gluconate-mannitol [PAGGGM], saline-adenine-glucose-mannitol [SAGM], Adsol, Optisol) on the in vitro quality of canine leukoreduced packed RBC units (pRBC) stored for 41 days. Methods: Five hundred milliliters of blood were collected from 8 healthy dogs each into 70 mL of citrate-phosphate-dextrose (CPD) solution, and were leukoreduced by a polyurethane filter. pRBC of each dog were divided equally into 4 bags containing a different AS. Bags were stored for 41 days at 4°C and evaluated every 10 days. Variables analyzed included pH, PCV, and% hemolysis, and lactate, glucose, potassium, sodium, ATP, and 2,3-diphosphoglycerate (2,3-DPG) concentrations. Results: The LR resulted in residual WBC counts comparable to human standards. During storage, pH, and glucose, 2,3-DPG, and ATP concentrations decreased, and hemolysis, and lactate, sodium, and potassium concentrations increased (P < .05). Significant differences between AS were seen in the glucose and sodium concentrations, due to the composition of AS. Also, the pH maintained by PAGGGM at day 21 was significantly higher than that seen with SAGM or Adsol. Conclusions: All AS used gave satisfactory results during the first 21 days of storage based on the degree of hemolysis, and on ATP and 2,3-DPG concentrations. When compared with day 1 values, significant changes were seen in these variables by day 31 with all AS.
Introduction When blood is stored for transfusion purposes, biochemical and morphologic changes, also known as the “storage lesion,” occur and can contribute to morbidity and mortality of critical patients.1–5 Over the last 4 decades, researchers have investigated various blood storage solutions and techniques that improve the quality of the stored blood and minimize the storage lesion in people and animals.6–16 Additive solutions (AS), which are protein-free solutions added to the pRBC following plasma removal, were developed to
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prolong the life of the RBC during storage; they continue to be improved to achieve better results.6–16 Leukoreduction (LR) has become another important technique to improve blood quality and to avoid adverse effects, such as inflammation, febrile and allergic responses, infectious diseases, and alloimmunization reactions in the recipient.17–21 The storage lesion of canine stored blood units is similar to that seen in human blood products.11 RBCrelated ATP and 2,3-diphosphoglycerate (2,3-DPG), and glucose concentrations decrease over time, while lactic and pyruvic acids accumulate, and pH decreases.
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The change in ATP and 2,3-DPG concentrations is extremely important, as without them the RBC cannot complete its in vivo functions properly.11–14 Recent human studies reported the maintenance of in vitro ATP and 2,3-DPG levels with a new additive solution, phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) (Sanquin Blood Supply Foundation, Amsterdam, the Netherlands). Compared with commercial solutions such as saline-adenineglucose-mannitol (SAGM) (Terumo Penpol Limited, Jawaharnagar, Gujarat, India), PAGGGM is more alkaline, and contains no chloride.15,16 LR and other prestorage techniques also can influence the RBC viability during storage, and may have an impact on the transfused patient.22–24 Studies on biochemical variables in stored LR pRBC are incomplete in the veterinary literature, with only changes in ATP concentrations previously documented.17 A significant reduction in markers of inflammation has also been noted in healthy dogs receiving autologous leukoreduced blood.19 In Brazil, SAGM is the AS that is commercially available, and its effects on canine RBC concentrates, also called packed RBC (pRBC), have not been previously reported. Effects of the new solution PAGGGM and the AS-5 solution Optisol (Terumo Corporation, Tokyo, Japan) have also not been documented in canine units. The purpose of our study was therefore to evaluate the efficacy of a prestorage LR filter and the effect of 4 AS on hematologic and biochemical variables of canine leukoreduced pRBC stored for 41 days. The formulations of all solutions used in this study are given in Table 1.
Materials and Methods Animal selection Eight large-breed dogs (body weight over 40 kg) with docile and calm temperament, including 4 sexually intact females and 4 sexually intact males, were selected with written consent from private owners. Results of a CBC, chemistry profile, infectious disease screen, and physical examination were normal for each dog before the procedure. All the animals were observed for at least 30 min and clinically reevaluated after the procedure to guarantee their safety before leaving the facility with the owner. This study was developed in compliance with the Standards of Animal Ethics and Welfare recommended by the National Council on the Control of Animal Experiments and was further approved by the Research Ethics Committee of Universidade Federal do Rio Grande do Sul (UFRGS) (n. 12482).
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Blood collection All dogs were physically restrained and not sedated for blood collection. Five hundred milliliters of blood were collected from each dog into a primary blood bag (triple bag system), containing 70 mL of CPD solution (Terumo Corporation) as the anticoagulant–preservative, and weighed before any other procedure. A small aliquot (approximately 5 mL) of blood was removed from the collection bag tubing using aseptic technique for prefiltration WBC and platelet count and PCV determination.
Filtration Bags containing the collected whole blood were cooled to 20–24°C using a closed system containing a buta-1,4-diol cooling plate (Compocool WB, Fresenius HemoCare, Bad Homburg, Germany). The bags were then attached to the filter system using sterile technique (Terumo Sterile Connecting Device – TSCD-II, Terumo Medical Corporation, Somerset, NJ, USA) and filtered through the LR filter into a secondary bag by gravity after breakage of an integral canula above the filter. The LR filter used in this study (Terumo Immugard III-RC, Terumo Corporation) is a third-generation polyurethane filter with a neutral charge, manufactured to remove both WBCs and platelets. The filtration time was determined from the moment blood started to flow through the tubing until the cessation of blood flow. The blood lost in the filter and tubing was determined by weighing the full secondary bag. The pre- and postfiltration weights were converted to milliliters by dividing the gram weight by 1.06.17 A second aliquot of the LR whole blood was taken using aseptic technique via sample tubing attached to the bag for postfiltration WBC, platelet count, and PCV determination.
WBC and platelet counts Before filtration, WBCs and platelets were counted using an automated hematology analyzer (ABX Micros Abc Vet, Horiba Medical, ABX Diagnostics, Montpellier, France). The residual WBC and platelet counts were counted on a Nageotte hemocytometer (LOLaboroptik GmbH, Bad Homburg, Germany) using T€ urk’s solution (Newprov, Pinhais, PR, Brazil) and on a Neubauer Bright line hemocytometer (Boeco Germany, Hamburg, Germany) using 1% ammonium oxalate, respectively. The following formula was used to determine leukoreduction:
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Table 1. Composition of additive solutions used for storage of canine pRBC in the study. CPD Molecular
AS-1 (Adsol)
AS-5 (Optisol)
SAGM
PAGGGM
Concentration
Concentration
Concentration
Concentration
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
180.2 135.1 283.2 120
141.82 0 0 18.52
2555.56 0 0 222.22
122.09 2 0 0
2200 27 0 0
49.94 2.22 0 0
900.0 30.00 0 0
49.94 1.25 0 0
900 16.9 0 0
47.5 1.44 1.44 8
855 19.4 40.9 125
141.95
0
0
0
0
0
0
0
0
8
143
182.2 58.45 218.1 294.1 192.1
0 0 0 89.59 15.53
0 0 0 2634.92 298.41 5.71
41.16 153.98 0 0 0
750 900 0 0 0 3.88
28.81 150.04 0 0 0
525.00 877.00 0 0 0 2.33
28.81 150.04 0 0 0
525 877 0 0 0 2.32
55 0 40 0 0
1000 0 872 0 0 3.06
Weight Dextrose Adenine Guanosine Monobasic Sodium Phosphate (NaH2PO42H2O) Sodium phosphate Dibasic (Na2HPO42H2O) Mannitol Sodium Chloride Sodium Gluconate Sodium Citrate Citric Acid Total Mass (grams)
Concentration
PAGGGM indicates phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucose-mannitol.
% platelet and WBC depletion ðPrefiltration counts Postfiltration countsÞ 100 ¼ Prefiltration WBC or platelet counts
for 1 min every 10 days prior to the sampling procedure.25,26
In vitro analyses Component preparation Blood from the secondary bag (ie, LR whole blood) was processed into pRBC and plasma by centrifugation at 4,050g for 6 min at 4°C (Legend-RT plus, Sorvall, Thermo Scientific, Asheville, NC, USA).13,14 After centrifugation, plasma was expressed (Plasma Separation Stand, Terumo Medical Corporation) from the primary bag into a transfer bag, separated, and stored at 30°C for one year. Each pRBC specimen was split equally (approximately 55 mL per bag) into 4 smaller pediatric transfer bags (capacity of 100 mL) (JP Ind ustria Farmac^eutica S.A., Ribeir~ ao Preto, SP, Brazil), and 4 different additive solutions were added to the RBC concentrate (25 mL per bag), respecting the proportion of the standard method for a normal unit. The pRBC and additive solution transfer procedures were performed using sterile technique (Terumo Sterile Connecting Device – TSCD-II, Terumo Medical Corporation). The solutions used were SAGM, Adsol (Fenwal, Inc., Lake Zurich, IL, USA), Optisol, and PAGGGM (for detailed composition, see Table 1). Each smaller pRBC unit was stored at 4°C for 41 days in a standard blood bank refrigerator in a horizontal position; the pRBC units were manually gently mixed
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Blood samples were drawn with an 11-mL syringe from each unopened bag using sterile technique through a blood sampling port (JP Ind ustria Farmac^ eutica S.A.) and a 16-Gauge needle on days 1, 11, 21, 31, and 41 after collection. Each bag was manually mixed for 1 min before taking the sample. Samples were divided into 3 parts. One 2-mL aliquot was used for pH determination right after sampling by a pH benchtop meter (Thermo Scientific, Beverly, MA, USA). A second 3-mL aliquot was used for PCV and total HGB determination. The third 6-mL aliquot was used for the determination of % hemolysis, and supernatant lactate, glucose, potassium, sodium concentrations, RBC-ATP and 2,3-DPG concentrations. The PCV was measured manually by centrifugation for 5 min at 10,000g (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany). The total HGB concentration was measured by a cyanmethemoglobin method (Labtest Diagn ostica S.A., Lagoa Santa, MG, Brazil). The sample was then centrifuged at 2,000g for 10 min at 4°C (Legend-RT plus, Sorvall, Thermo Scientific). The supernatant was used for plasma HGB determination using a micromodification of the Drabkin hemoglobin assay.27 Hemolysis was expressed as a percentage of total HGB present in pRBC after
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correction for the PCV, as follows28,29: % hemolysis ¼ ð100 PCVÞ supernatant HGB (g/dL) total HGB (g/dL) The third part was centrifuged as described for HGB determination, and the supernatant was used to determine glucose, potassium, and sodium concentrations by a blood gas analyzer (I-Stat 1 and CG8+ cartridges, Abbott Point of Care Inc., Princeton, NJ, USA), and lactate concentrations by an enzymatic method (Labtest Diagn ostica S.A.). The RBCs were used to determine ATP and 2,3-DPG concentrations. Briefly, 1 mL of the pRBC of the sample was transferred to tubes containing 3 mL of ice-cold 8% trichloric acid (T6399, Sigma-Aldrich, St. Louis, MO, USA) and mixed thoroughly. The tubes were centrifuged as described previously, and supernatants were stored at 80°C until analysis. The ATP concentration of the acid-extracted RBC was determined by a luminescence ATP detection assay system (ATPLite, Perkin Elmer Inc., Waltham, MA, USA), and the 2,3-DPG concentration was determined by an enzymatic assay (Cat. No. 10 148 334 001, Roche Diagnostics GmbH, Mannheim, Germany), both using a microplate reader (Spectramax M5, Molecular Devices Inc., Sunnyvale, CA, USA).30 Both supernatant samples had to be neutralized before analysis due to the low pH of trichloric acid that could interfere with the 2,3-DPG and ATP method. Briefly, approximately 60 lL of 1 M sodium carbonate solution was added to 500 lL of supernatant of each sample to reach a neutral pH. At the end of 41 days of storage, the pRBC units were sent to a microbiology laboratory for aerobic and anaerobic blood culture.
Statistics Statistical analysis was performed using a statistical package software system (PASW Statistics 18, IBM Corporation, Somers, NY, USA). One-way ANOVA, followed by Bonferroni correction, and Tukey post hoc test were used to compare the solutions on each day of storage. A T-test was used to compare day 1 with the other days of each variable. The results were considered to be significant when P-values were < .05.
Results Efficacy of the WBC filter The filtration process took approximately 20 6 min, and the entire process (collection through storage)
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took approximately 6 h. Mean reductions of 99.8% and 95.9% were obtained for WBCs and platelets, respectively, with the filter used. The volume of blood lost to the filter was 46 mL. The residual WBC count mean was 0.48 0.09 9 106 per unit of canine whole blood. The mean RBC recovery was 98.14%.
Differences between additive solutions over time The PCV of all pRBC units complied with the American Association of Blood Banks standard for human RBC concentrates (≤ 80.0%). 18 Biochemical changes that occurred with storage are listed in Table 2 and shown in Figure 1. A statistically significant decrease (P < .05) in pH was observed in all solutions; the pH maintained by PAGGGM was significantly higher than that seen with storage in either SAGM or Adsol. A statistically significant increase (P < .05) in % hemolysis and in lactate concentration over time was observed in all solutions, while glucose concentration decreased statistically significantly (P < .05) over time in all solutions, with the exception of Adsol, which consistently maintained a significantly higher glucose concentration when compared with the other AS, due to its formulation (Figure 1). The ATP and 2,3-DPG concentrations were statistically significantly decreased (P < .05) in all solutions over time (Figure 1). The solution PAGGGM maintained the highest concentrations of ATP and 2,3-DPG at 21 days, but the change was not statistically significant. In contrast, sodium concentrations were statistically significantly increased (P < .05) over time in all solutions, except with PAGGGM. The sodium concentration in the PAGGGM stored product was significantly different from the concentration of all other solutions, at any time period. Potassium concentrations were statistically significantly increased (P < .05) over time in all solutions. Microbiologic cultures from all bags were negative.
Discussion Biochemical changes occurring during storage of non-LR canine pRBC in specific AS or anticoagulantpreservatives have been previously reported, and pRBC stored in the AS Adsol and Nutricel (Pall Corporation, East Hills, NY, USA) have shown superior in vitro and in vivo results when compared with the anticoagulant-preservative citrate-phosphate-dextrose-adenine (CPDA-1) solution.12–14 However, some countries, such as Brazil, lack commercial access to the
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Table 2. Select variables determined on different days of storage of leukoreduced canine pRBC in 4 different additive solutions. Variable
Day 1
pH SAGM 7.27 (0.09) Adsol 7.28 (0.04) Optisol 7.30 (0.04) PAGGGM 7.22 (0.06) Hemolysis (%) SAGM 0.30 (0.09) Adsol 0.31 (0.08) Optisol 0.25 (0.04) PAGGGM 0.27 (0.05) Lactate (mmol/L) SAGM 6.06 (1.69) Adsol 6.22 (2.03) Optisol 6.26 (1.25) PAGGGM 6.50 (1.63) Glucose (mg/dL) SAGM 520.25a (43.93) Adsol 1176.22b (293.16) Optisol 532.38a (72.73) PAGGGM 491.29a (115.86) ATP (lmol/g Hb) SAGM 3.08 (0.84) Adsol 3.26 (0.84) Optisol 2.45 (1.06) PAGGGM 3.31 (1.55) 2.3-DPG (lmol/g Hb) SAGM 19.83 (2.38) Adsol 20.00 (4.20) Optisol 16.84 (4.46) PAGGGM 18.00 (4.06) Na+ (mmol/L) SAGM 144.63a (2.20) Adsol 143.13a (2.80) Optisol 145.38a (1.85) PAGGGM 115.00b (9.02) K+ (mmol/L) SAGM 3.13 (1.01) Adsol 3.04 (0.83) Optisol 3.16 (0.94) PAGGGM 2.79 (0.96)
Day 11
Day 21
Day 31
Day 41
7.33 (0.05) 7.31 (0.09) 7.34 (0.08) 7.39 (0.06)
7.08ª (0.06) 7.10ª (0.07) 7.15ª.b (0.06) 7.19b (0.06)
7.02 (0.07) 6.99 (0.09) 7.05 (0.05) 7.07 (0.08)
7.03a,b (0.07) 7.00a (0.09) 7.05a,b (0.08) 7.13b (0.08)
0.64 (0.21) 0.59 (0.18) 0.63 (0.23) 0.66 (0.19)
0.67 (0.24) 0.74 (0.35) 0.69 (0.23) 0.65 (0.27)
1.13 (0.51) 0.91 (0.36) 1.01 (0.38) 0.97 (0.39)
1.31 (0.56) 1.19 (0.46) 1.42 (0.67) 1.23 (0.61)
13.71 (2.22) 13.63 (1.28) 12.75 (1.66) 13.57 (2.48)
22.49 (3.39) 20.47 (2.20) 20.74 (1.75) 20.60 (2.35)
26.09 (3.55) 27.21 (4.12) 23.80 (2.85) 26.27 (3.67)
26.98 (2.14) 29.45 (5.08) 25.70 (1.68) 28.43 (6.57)
371.13a (111.14) 1454.67b (89.36) 397.00a (129.57) 350.86a (143.21)
441.75a (61.39) 1304.75b (109.26) 449.50a (33.20) 397.71a (26.74)
401.75a (42.90) 1264.92b (103.86) 416.38a (30.87) 331.86a (40.03)
372.50a (43.61) 1224.62b (92.23) 389.25a (32.41) 309.71a (36.53)
3.03 (1.30) 3.62 (2.07) 2.77 (1.45) 4.06 (2.28)
2.00 (0.57) 1.93 (0.51) 1.96 (0.45) 2.42 (0.93)
1.02 (0.65) 1.19 (0.62) 0.73 (0.50) 0.92 (0.76)
0.50 (0.41) 0.53 (0.50) 0.29 (0.35) 0.35 (0.30)
16.99 (4.96) 19.01 (2.94) 16.72 (4.26) 17.04 (4.67)
13.74 (4.19) 13.67 (4.66) 13.33 (3.49) 15.97 (4.66)
8.43 (1.52) 10.27 (3.06) 6.99 (2.36) 10.11 (3.93)
4.25 (2.21) 6.21 (2.83) 3.66 (1.66) 7.77 (4.05)
150.00b (0.53) 153.38a (1.19) 150.50b (0.53) 118.00c (1.15)
150.50a (0.76) 153.63b (0.74) 151.13b (0.83) 119.29c (1.80)
4.09 (0.35) 4.09 (0.38) 4.16 (0.47) 3.81 (0.35)
4.15 (0.36) 4.13 (0.42) 4.20 (0.48) 3.90 (0.37)
148.88a (1.55) 152.38b (1.92) 150.38a,b (1.92) 114.57c (4.31) 4.45 (0.73) 4.23 (0.61) 4.26 (0.68) 3.74 (0.48)
145.88a (10.87) 153.00a (1.07) 147.25a (12.98) 121.71b (12.11) 3.96 (0.37) 4.00 (0.39) 4.05 (0.50) 3.80 (0.33)
DPG indicates 2,3-diphosphoglycerate; PAGGGM, phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucose-mannitol. Data are means (SD). Superscript letters in the same column represent statistically significant differences (P < .05) between solutions at the same time.
above AS, prompting the current study using other, more available solutions. Only limited studies on storage of LR canine pRBC and its effects on the blood product quality and on the recipient have been reported, thus further emphasizing the need for this study. 17,19–21 Leukoreduction has been associated with reduced mortality in specific human patient populations, due to avoidance of some transfusion reactions and better quality of pRBC units.21,31,32 Many countries have
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adopted universal LR. LR filters have some disadvantages, such as higher cost; according to a recent study, each filter can add $20–$65 to the cost of a human pRBC unit depending on the country.33 The cost-effectiveness and the quality of leukoreduced blood products are a debated issue nowadays with many studies reporting that the higher costs can be justified by the reduction of hospital charges and shortened hospital length of stay.33,34 The use of LR filters can lead to 10% of RBCs lost due to trapping in the fil-
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Figure 1. Changes in pH, % hemolysis, and lactate. Glucose, ATP, 2,3-diphosphoglycerate (DPG), sodium, and potassium concentrations in leukoreduced canine pRBC stored in 4 different additive solutions. SAGM, Adsol, Optisol, PAGGGM. *Significantly different from the mean SD of the day 1 value. *P < .05. **P < .001. PAGGGM indicates phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucosemannitol.
ter, but prestorage LR also effectively removes WBCs and more than 90% of platelets before they can contribute to the storage lesion of RBCs or transfusion reactions mediated by platelets and RBCs.32,35 The LR filter used in this study showed a lower efficacy for WBC removal than that presented for
human RBC concentrates in other studies.31 The time of filtration was longer and volume of blood lost to the filter was lower when compared with one other study in dogs17, while a more recent study showed similar results.19 Although the filter efficacy was < 99.9%, the filtration process still met the AABB threshold of
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< 5 9 106 residual WBCs per unit, and the European requirements for WBC counts of < 1 9 106 WBCs per unit.18 Four different AS were used in this study. The original AS were formulated to dilute the RBC with saline prior to transfusion, and in addition supplied glucose and adenine to maintain energy metabolism. Later studies resulted in the addition of mannitol to decrease hemolysis of the stored cells. The newer AS PAGGGM contains guanosine for improved maintenance of ATP, 2,3 DPG, and phosphate concentrations, the latter of which may act as buffers or can be used in the formation of organic phosphates.15,36,37 All variables analyzed in all of the AS showed time-dependent changes. Hemolysis was higher than anticipated in the older units, as the limits of hemolysis for human RBC concentrates are 0.8% and 1.0% in Europe and the USA, respectively.18 The increased hemolysis seen in the bags stored for 31 and 41 days may be related to the smaller volume of pRBC remaining in the bags at these time points, as previously noted in human blood.37 Statistically significant differences between solutions at all time points were identified only for glucose (higher glucose concentrations in Adsol) and sodium (lower sodium concentrations in PAGGGM), both a result of the formulation of the AS themselves. The formulation of Adsol contains more than twice the amount of glucose (dextrose) compared with the other additives used in the study, contributing to the increased residual glucose concentration over time. High glucose levels can lead to an increase in insulin in the transfused individual38, which should be taken into consideration if a large volume of Adsol containing pRBC is needed and if the recipient suffers metabolic dysfunction.39 The lower sodium seen in PAGGGM is also a result of the formulation of this AS. The composition of PAGGGM was originally based on the hypothesis of Meryman and Hornblower that a chloride-free AS would aid in the maintenance of RBC-related 2,3 DPG and ATP concentrations throughout the storage period.40 Saline is thus replaced in this additive with a lower concentration of sodium as sodium gluconate used instead. It is unlikely that the low sodium concentration in PAGGGM would have any deleterious effect on the recipient. PAGGGM is also a more alkaline solution than other AS, with a higher pH, normally favoring formation of 2,3 DPG. In the present study, however, a statistically significant pH change was only seen at day 21, when compared with the other AS. In the current study, there were no statistically significant difference between the ATP and 2,3-DPG con-
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centrations, but ATP concentrations were transiently higher than those reported in previous studies on canine units11–14,17, which was different from a previous study on leukoreduced canine units.17 Recent studies have shown the influence of blood storage and concentrations of 2,3-DPG and ATP on the clinical outcome of patients who require blood transfusion.1–5 Maintenance of ATP and 2,3-DPG concentrations is a desirable characteristic, as ATP is needed to maintain RBC function and integrity; and 2,3-DPG improves the oxygen release to tissues.3–5 Low concentrations of ATP can lead to deformability and exposure of the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine, which are normally concentrated on the inner surface of the RBC membrane and are one of the signals for their removal from the circulation. People with ATP levels < 4 lmol per g HGB risk a higher incidence for PS exposure.15 The minimum recommended range of ATP concentration for stored pRBC is between 2.3 and 2.7 lmol/g HGB, which has been correlated with 75% of transfused RBCs remaining in circulation for 24 h posttransfusion in people.41 A significant association was found between RBC deformability and ATP concentrations < 2.5 lmol/g HGB.42 Concentrations of ATP < 0.75 lmol/g HGB are believed to be a limiting factor in RBC viability in dogs; therefore, storage of LR canine pRBC to day 41 would not be acceptable, as indicated in the present study.43 Concentrations of 2,3-DPG were > 11 lmol per g HGB until day 21 in all solutions, which is close to the values found in the blood of healthy and anemic dogs.44 PAGGGM and Adsol units appeared to have higher concentrations of 2,3-DPG at days 31 and 41 when compared with the other AS, but the differences were not statistically significant. The decrease in the concentrations of 2,3-DPG during storage was similar to that reported in previous human and canine studies for ADSOL and SAGM solutions11–14, but was lower than results previously reported for PAGGGM units in people.16 As 2,3-DPG binds to the b-subunits of HGB, shifting the oxygen dissociation curve to the right and allowing enhanced release of oxygen, its depletion during RBC storage is postulated to decrease microcirculatory oxygen release. Higher 2,3-DPG concentrations are a desirable characteristic as the de novo synthesis of 2,3DPG can take up to 24 h to reach normal levels and, although unlikely, it can affect critical patients with a precarious oxygen balance.14,45 Besides the in vitro biochemical alterations, RBC storage lesions can result in immunomodulatory and inflammatory consequences in transfusion recipients.19,21,22 The clinical impact of pRBC storage time is an important and controversial issue, and there are several large randomized human clinical
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trials currently underway that are investigating this issue.41 In the present study, satisfactory results were obtained with all AS during the first 21 days of storage when compared with other canine pRBC studies.12,13,17,19 Further evaluation of the AS used in this study is warranted, using in vivo analysis to determine posttransfusion stability and viability. Nonleukoreduced units could also be evaluated to further test these storage solutions in dogs.
Acknowledgment The authors would like to thank the Brazilian Funding Agency CNPq (EDITAL MCT/CNPQ N° 014/2008 – UNIVERSAL), the UFRGS’ department of Biochemistry (Dr. Fatima Guma), Dr. Korte and his colleagues from Sanquin Blood Supply Foundation (Amsterdam, NL), Dr. W Jean Dodds and her staff from Hemopet Blood Bank (California, US), Terumo Medical do Brasil LTDA for supplying equipment and solutions to the project, and Daniela Benzano for her help with statistical analysis. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
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8. Hess JR, Hill HR, Oliver CK, et al. Twelve-week RBC storage. Transfusion. 2003;43:867–872. 9. Kurup PA, Arun P, Gayathri NS, Dhanya CR, Indu AR. Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate. Vox Sang. 2003;85:253–261. 10. Hess JR. An update on solutions for red cell storage. Vox Sang. 2006;91:13–19, Review. 11. Price GS, Armstrong PJ, McLeod DA, Babineau CA, Metcalf MR, Sellett LC. Evaluation of citratephosphate-dextrose-adenine as a storage medium for packed canine erythrocytes. J Vet Intern Med. 1988;2:126–132. 12. Wardrop KJ, Owen TJ, Meyers KM. Evaluation of an additive solution for preservation of canine red blood cells. J Vet Intern Med. 1994;8:253–257. 13. Wardrop KJ, Young J, Wilson E. An in vitro evaluation of storage media for the preservation of canine packed red blood cells. Vet Clin Pathol. 1994;23:83–88. 14. Wardrop KJ, Tucker RL, Mugnai K. Evaluation of canine red blood cells stored in a saline, adenine, and glucose solution for 35 days. J Vet Intern Med. 1997;11:5–8. 15. de Korte D, Kleine M, Korsten HG, Verhoeven AJ. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage. Transfusion. 2008;48:1081–1089. 16. Burger P, Korsten H, De Korte D, Rombout E, Van Bruggen R, Verhoeven AJ. An improved red blood cell additive solution maintains 2,3-diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage. Transfusion. 2010;50:2386–2392.
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20. Phelan HA, Gonzalez RP, Patel HD, et al. Prestorage leukoreduction ameliorates the effects of aging on banked blood. J Trauma. 2010;69:330–337. 21. Graf C, Raila J, Schweigert FJ, Kohn B. Effect of leukoreduction treatment on vascular endothelial growth
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33. AuBuchon JP, Custer B, Sher G. A comparison of health care and blood supply system structures. Vox Sang. 2011;100:22–35, Review
23. Ran Q, Hao P, Xiao Y, Zhao J, Ye X, Li Z. Effect of irradiation and/or leucocyte filtration on RBC storage lesions. PLoS ONE. 2011;6:e18328. 24. Zimmermann R, Wintzheimer S, Weisbach V, Strobel J, Zingsem J, Eckstein R. Influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (RBC) storage variables of RBCs in additive solution saline-adenine-glucose-mannitol. Transfusion. 2009;49:75–80. 25. Wood LA, Beutler E. The effect of periodic mixing on the preservation of 2,3-diphosphoglycerate (2,3-DPG) levels in stored blood. Blood. 1973;42:17–25. 26. H€ ogman CF, Eriksson L, Gong J, H€ ogman AB, Payrat JM. Shall red cell units stand upright, lie flat or be mixed during storage? In vitro studies of red cells collected in 0.5 CPD and stored in RAS2 (Erythrosol) Transfus Sci. 1995;16:193–199. 27. Moore GL, Ledford ME, Merydith A. A micromodification of the Drabkin hemoglobin assay for measuring plasma hemoglobin in the range of 5 to 2000 mg/dl. Biochem Med. 1981;26:167–173. 28. Sowemimo-Coker SO. Red blood cell hemolysis during processing. Transfus Med Rev. 2002;16:46–60. 29. Han V, Serrano K, Devine DV. A comparative study of common techniques used to measure haemolysis in stored red cell concentrates. Vox Sang. 2010;98: 116–123. 30. Niinist€ o K, Raekallio M, Sankari S. Storage of equine red blood cells as a concentrate. Vet J. 2008;176:227– 231. 31. Antonelou MH, Tzounakas VL, Velentzas AD, Stamoulis KE, Kriebardis AG, Papassideri IS. Effects of prestorage leukoreduction on stored red blood cells signaling: a time-course evaluation from shape to proteome. J Proteomics. 2012;76 Spec No.:220–238. 32. Lannan KL, Sahler J, Spinelli SL, Phipps RP, Blumberg N. Transfusion immunomodulation - the case for leukoreduced and (perhaps) washed transfusions. Blood Cells
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34. Shander A, Hofmann A, Gombotz H, Theusinger OM, Spahn DR. Estimating the cost of blood: past, present, and future directions. Best Pract Res Clin Anaesthesiol. 2007;21:271–289, Review. 35. Shapiro MJ. To filter blood or universal leukoreduction: what is the answer? Crit Care. 2004;8(Suppl 2):S27– S30. Epub 2004, Review. 36. Hogman CF, Hedlund K, Sahlestrom Y. Red cell preservation in protein-poor media III. Protection against in vitro hemolysis. Vox Sang. 1981;41:274–281. 37. H€ ogman CF. Liquid storage of human erythrocytes. In: Harris JR, ed. Blood Separation and Plasma Fractionation. New York: Wiley-Liss, Inc.; 1991:63–97. 38. Buchholz DH, Borgia JF, Ward M, Miripol JE, Simpson JM. Comparison of Adsol and CPDA-1 blood preservatives during simulated massive resuscitation after hemorrhage in swine. Transfusion. 1999;39:998–1004. 39. Goodstein MH, Locke RG, Wlodarczyk D, Goldsmith LS, Rubenstein SD, Herman JH. Comparison of two preservation solutions for erythrocyte transfusions in newborn infants. J Pediatr. 1993;123:783–788. 40. Meryman HT, Hornblower M. Manipulating red cell intr-and extracellular pH by washing. Vox Sang. 1991;60:99–104. 41. Grazzini G, Vaglio S. Red blood cell storage lesion and adverse clinical outcomes: post hoc ergo propter hoc? Blood Transfus. 2012;10(Suppl 2):s4–s6 42. Almizraq R, Tchir JD, Holovati JL, Acker JP. Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality. Transfusion. 2013;53:2258–2267. doi: 10.1111/trf.12080 43. Karger R, Lukow C, Kretschmer V. Deformability of red blood cells and correlation with ATP Content during Storage as WBC-Depleted Whole Blood. Transfus Med Hemother. 2012;39:277–282. 44. Smith JE. Storing and Preserving Erythrocytes. In: Apgar NS, Board PG, eds. Red Blood Cells of Domestic Animals. New York, NY: Elsevier; 1983:357–366. 45. Paltrinieri S, Comazzi S, Agnes F. Haematological parameters and altered erythrocyte metabolism in anaemic dogs. J Comp Pathol. 2000;122:25–34.
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ORIGINAL RESEARCH
Effects of four additive solutions on canine leukoreduced red cell concentrate quality during storage Luciana A. Lacerda1, Nicole R.C. Hlavac1, Silvia R. Terra2, Franciele P. Back1, K. Jane Wardrop3, lix H.D. Gonza lez1 Fe Departments of 1 Veterinary Clinical Pathology, and 2 Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; and Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA
3
Key Words Blood, dog, erythrocyte, filter, transfusion Correspondence Luciana de Almeida Lacerda, Av. Bento Goncßalves, 9090, B. Agronomia, LACVet, UFRGS, CEP 91540-000 Porto Alegre, RS, Brazil E-mail: [email protected] DOI:10.1111/vcp.12163
Background: Additive solutions (AS) and prestorage leukoreduction (LR) are important tools used to maintain erythrocyte viability during storage and avoid transfusion reactions in recipients, respectively. Objectives: The purpose of the study was to determine the efficacy of a WBC filter (Immugard IIIRC) and compare the effect of 4 AS (phosphateadenine-glucose-guanosine-gluconate-mannitol [PAGGGM], saline-adenine-glucose-mannitol [SAGM], Adsol, Optisol) on the in vitro quality of canine leukoreduced packed RBC units (pRBC) stored for 41 days. Methods: Five hundred milliliters of blood were collected from 8 healthy dogs each into 70 mL of citrate-phosphate-dextrose (CPD) solution, and were leukoreduced by a polyurethane filter. pRBC of each dog were divided equally into 4 bags containing a different AS. Bags were stored for 41 days at 4°C and evaluated every 10 days. Variables analyzed included pH, PCV, and% hemolysis, and lactate, glucose, potassium, sodium, ATP, and 2,3-diphosphoglycerate (2,3-DPG) concentrations. Results: The LR resulted in residual WBC counts comparable to human standards. During storage, pH, and glucose, 2,3-DPG, and ATP concentrations decreased, and hemolysis, and lactate, sodium, and potassium concentrations increased (P < .05). Significant differences between AS were seen in the glucose and sodium concentrations, due to the composition of AS. Also, the pH maintained by PAGGGM at day 21 was significantly higher than that seen with SAGM or Adsol. Conclusions: All AS used gave satisfactory results during the first 21 days of storage based on the degree of hemolysis, and on ATP and 2,3-DPG concentrations. When compared with day 1 values, significant changes were seen in these variables by day 31 with all AS.
Introduction When blood is stored for transfusion purposes, biochemical and morphologic changes, also known as the “storage lesion,” occur and can contribute to morbidity and mortality of critical patients.1–5 Over the last 4 decades, researchers have investigated various blood storage solutions and techniques that improve the quality of the stored blood and minimize the storage lesion in people and animals.6–16 Additive solutions (AS), which are protein-free solutions added to the pRBC following plasma removal, were developed to
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prolong the life of the RBC during storage; they continue to be improved to achieve better results.6–16 Leukoreduction (LR) has become another important technique to improve blood quality and to avoid adverse effects, such as inflammation, febrile and allergic responses, infectious diseases, and alloimmunization reactions in the recipient.17–21 The storage lesion of canine stored blood units is similar to that seen in human blood products.11 RBCrelated ATP and 2,3-diphosphoglycerate (2,3-DPG), and glucose concentrations decrease over time, while lactic and pyruvic acids accumulate, and pH decreases.
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The change in ATP and 2,3-DPG concentrations is extremely important, as without them the RBC cannot complete its in vivo functions properly.11–14 Recent human studies reported the maintenance of in vitro ATP and 2,3-DPG levels with a new additive solution, phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) (Sanquin Blood Supply Foundation, Amsterdam, the Netherlands). Compared with commercial solutions such as saline-adenineglucose-mannitol (SAGM) (Terumo Penpol Limited, Jawaharnagar, Gujarat, India), PAGGGM is more alkaline, and contains no chloride.15,16 LR and other prestorage techniques also can influence the RBC viability during storage, and may have an impact on the transfused patient.22–24 Studies on biochemical variables in stored LR pRBC are incomplete in the veterinary literature, with only changes in ATP concentrations previously documented.17 A significant reduction in markers of inflammation has also been noted in healthy dogs receiving autologous leukoreduced blood.19 In Brazil, SAGM is the AS that is commercially available, and its effects on canine RBC concentrates, also called packed RBC (pRBC), have not been previously reported. Effects of the new solution PAGGGM and the AS-5 solution Optisol (Terumo Corporation, Tokyo, Japan) have also not been documented in canine units. The purpose of our study was therefore to evaluate the efficacy of a prestorage LR filter and the effect of 4 AS on hematologic and biochemical variables of canine leukoreduced pRBC stored for 41 days. The formulations of all solutions used in this study are given in Table 1.
Materials and Methods Animal selection Eight large-breed dogs (body weight over 40 kg) with docile and calm temperament, including 4 sexually intact females and 4 sexually intact males, were selected with written consent from private owners. Results of a CBC, chemistry profile, infectious disease screen, and physical examination were normal for each dog before the procedure. All the animals were observed for at least 30 min and clinically reevaluated after the procedure to guarantee their safety before leaving the facility with the owner. This study was developed in compliance with the Standards of Animal Ethics and Welfare recommended by the National Council on the Control of Animal Experiments and was further approved by the Research Ethics Committee of Universidade Federal do Rio Grande do Sul (UFRGS) (n. 12482).
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Blood collection All dogs were physically restrained and not sedated for blood collection. Five hundred milliliters of blood were collected from each dog into a primary blood bag (triple bag system), containing 70 mL of CPD solution (Terumo Corporation) as the anticoagulant–preservative, and weighed before any other procedure. A small aliquot (approximately 5 mL) of blood was removed from the collection bag tubing using aseptic technique for prefiltration WBC and platelet count and PCV determination.
Filtration Bags containing the collected whole blood were cooled to 20–24°C using a closed system containing a buta-1,4-diol cooling plate (Compocool WB, Fresenius HemoCare, Bad Homburg, Germany). The bags were then attached to the filter system using sterile technique (Terumo Sterile Connecting Device – TSCD-II, Terumo Medical Corporation, Somerset, NJ, USA) and filtered through the LR filter into a secondary bag by gravity after breakage of an integral canula above the filter. The LR filter used in this study (Terumo Immugard III-RC, Terumo Corporation) is a third-generation polyurethane filter with a neutral charge, manufactured to remove both WBCs and platelets. The filtration time was determined from the moment blood started to flow through the tubing until the cessation of blood flow. The blood lost in the filter and tubing was determined by weighing the full secondary bag. The pre- and postfiltration weights were converted to milliliters by dividing the gram weight by 1.06.17 A second aliquot of the LR whole blood was taken using aseptic technique via sample tubing attached to the bag for postfiltration WBC, platelet count, and PCV determination.
WBC and platelet counts Before filtration, WBCs and platelets were counted using an automated hematology analyzer (ABX Micros Abc Vet, Horiba Medical, ABX Diagnostics, Montpellier, France). The residual WBC and platelet counts were counted on a Nageotte hemocytometer (LOLaboroptik GmbH, Bad Homburg, Germany) using T€ urk’s solution (Newprov, Pinhais, PR, Brazil) and on a Neubauer Bright line hemocytometer (Boeco Germany, Hamburg, Germany) using 1% ammonium oxalate, respectively. The following formula was used to determine leukoreduction:
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Table 1. Composition of additive solutions used for storage of canine pRBC in the study. CPD Molecular
AS-1 (Adsol)
AS-5 (Optisol)
SAGM
PAGGGM
Concentration
Concentration
Concentration
Concentration
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
(mmol/L)
mg/100 mL
180.2 135.1 283.2 120
141.82 0 0 18.52
2555.56 0 0 222.22
122.09 2 0 0
2200 27 0 0
49.94 2.22 0 0
900.0 30.00 0 0
49.94 1.25 0 0
900 16.9 0 0
47.5 1.44 1.44 8
855 19.4 40.9 125
141.95
0
0
0
0
0
0
0
0
8
143
182.2 58.45 218.1 294.1 192.1
0 0 0 89.59 15.53
0 0 0 2634.92 298.41 5.71
41.16 153.98 0 0 0
750 900 0 0 0 3.88
28.81 150.04 0 0 0
525.00 877.00 0 0 0 2.33
28.81 150.04 0 0 0
525 877 0 0 0 2.32
55 0 40 0 0
1000 0 872 0 0 3.06
Weight Dextrose Adenine Guanosine Monobasic Sodium Phosphate (NaH2PO42H2O) Sodium phosphate Dibasic (Na2HPO42H2O) Mannitol Sodium Chloride Sodium Gluconate Sodium Citrate Citric Acid Total Mass (grams)
Concentration
PAGGGM indicates phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucose-mannitol.
% platelet and WBC depletion ðPrefiltration counts Postfiltration countsÞ 100 ¼ Prefiltration WBC or platelet counts
for 1 min every 10 days prior to the sampling procedure.25,26
In vitro analyses Component preparation Blood from the secondary bag (ie, LR whole blood) was processed into pRBC and plasma by centrifugation at 4,050g for 6 min at 4°C (Legend-RT plus, Sorvall, Thermo Scientific, Asheville, NC, USA).13,14 After centrifugation, plasma was expressed (Plasma Separation Stand, Terumo Medical Corporation) from the primary bag into a transfer bag, separated, and stored at 30°C for one year. Each pRBC specimen was split equally (approximately 55 mL per bag) into 4 smaller pediatric transfer bags (capacity of 100 mL) (JP Ind ustria Farmac^eutica S.A., Ribeir~ ao Preto, SP, Brazil), and 4 different additive solutions were added to the RBC concentrate (25 mL per bag), respecting the proportion of the standard method for a normal unit. The pRBC and additive solution transfer procedures were performed using sterile technique (Terumo Sterile Connecting Device – TSCD-II, Terumo Medical Corporation). The solutions used were SAGM, Adsol (Fenwal, Inc., Lake Zurich, IL, USA), Optisol, and PAGGGM (for detailed composition, see Table 1). Each smaller pRBC unit was stored at 4°C for 41 days in a standard blood bank refrigerator in a horizontal position; the pRBC units were manually gently mixed
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Blood samples were drawn with an 11-mL syringe from each unopened bag using sterile technique through a blood sampling port (JP Ind ustria Farmac^ eutica S.A.) and a 16-Gauge needle on days 1, 11, 21, 31, and 41 after collection. Each bag was manually mixed for 1 min before taking the sample. Samples were divided into 3 parts. One 2-mL aliquot was used for pH determination right after sampling by a pH benchtop meter (Thermo Scientific, Beverly, MA, USA). A second 3-mL aliquot was used for PCV and total HGB determination. The third 6-mL aliquot was used for the determination of % hemolysis, and supernatant lactate, glucose, potassium, sodium concentrations, RBC-ATP and 2,3-DPG concentrations. The PCV was measured manually by centrifugation for 5 min at 10,000g (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany). The total HGB concentration was measured by a cyanmethemoglobin method (Labtest Diagn ostica S.A., Lagoa Santa, MG, Brazil). The sample was then centrifuged at 2,000g for 10 min at 4°C (Legend-RT plus, Sorvall, Thermo Scientific). The supernatant was used for plasma HGB determination using a micromodification of the Drabkin hemoglobin assay.27 Hemolysis was expressed as a percentage of total HGB present in pRBC after
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correction for the PCV, as follows28,29: % hemolysis ¼ ð100 PCVÞ supernatant HGB (g/dL) total HGB (g/dL) The third part was centrifuged as described for HGB determination, and the supernatant was used to determine glucose, potassium, and sodium concentrations by a blood gas analyzer (I-Stat 1 and CG8+ cartridges, Abbott Point of Care Inc., Princeton, NJ, USA), and lactate concentrations by an enzymatic method (Labtest Diagn ostica S.A.). The RBCs were used to determine ATP and 2,3-DPG concentrations. Briefly, 1 mL of the pRBC of the sample was transferred to tubes containing 3 mL of ice-cold 8% trichloric acid (T6399, Sigma-Aldrich, St. Louis, MO, USA) and mixed thoroughly. The tubes were centrifuged as described previously, and supernatants were stored at 80°C until analysis. The ATP concentration of the acid-extracted RBC was determined by a luminescence ATP detection assay system (ATPLite, Perkin Elmer Inc., Waltham, MA, USA), and the 2,3-DPG concentration was determined by an enzymatic assay (Cat. No. 10 148 334 001, Roche Diagnostics GmbH, Mannheim, Germany), both using a microplate reader (Spectramax M5, Molecular Devices Inc., Sunnyvale, CA, USA).30 Both supernatant samples had to be neutralized before analysis due to the low pH of trichloric acid that could interfere with the 2,3-DPG and ATP method. Briefly, approximately 60 lL of 1 M sodium carbonate solution was added to 500 lL of supernatant of each sample to reach a neutral pH. At the end of 41 days of storage, the pRBC units were sent to a microbiology laboratory for aerobic and anaerobic blood culture.
Statistics Statistical analysis was performed using a statistical package software system (PASW Statistics 18, IBM Corporation, Somers, NY, USA). One-way ANOVA, followed by Bonferroni correction, and Tukey post hoc test were used to compare the solutions on each day of storage. A T-test was used to compare day 1 with the other days of each variable. The results were considered to be significant when P-values were < .05.
Results Efficacy of the WBC filter The filtration process took approximately 20 6 min, and the entire process (collection through storage)
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took approximately 6 h. Mean reductions of 99.8% and 95.9% were obtained for WBCs and platelets, respectively, with the filter used. The volume of blood lost to the filter was 46 mL. The residual WBC count mean was 0.48 0.09 9 106 per unit of canine whole blood. The mean RBC recovery was 98.14%.
Differences between additive solutions over time The PCV of all pRBC units complied with the American Association of Blood Banks standard for human RBC concentrates (≤ 80.0%). 18 Biochemical changes that occurred with storage are listed in Table 2 and shown in Figure 1. A statistically significant decrease (P < .05) in pH was observed in all solutions; the pH maintained by PAGGGM was significantly higher than that seen with storage in either SAGM or Adsol. A statistically significant increase (P < .05) in % hemolysis and in lactate concentration over time was observed in all solutions, while glucose concentration decreased statistically significantly (P < .05) over time in all solutions, with the exception of Adsol, which consistently maintained a significantly higher glucose concentration when compared with the other AS, due to its formulation (Figure 1). The ATP and 2,3-DPG concentrations were statistically significantly decreased (P < .05) in all solutions over time (Figure 1). The solution PAGGGM maintained the highest concentrations of ATP and 2,3-DPG at 21 days, but the change was not statistically significant. In contrast, sodium concentrations were statistically significantly increased (P < .05) over time in all solutions, except with PAGGGM. The sodium concentration in the PAGGGM stored product was significantly different from the concentration of all other solutions, at any time period. Potassium concentrations were statistically significantly increased (P < .05) over time in all solutions. Microbiologic cultures from all bags were negative.
Discussion Biochemical changes occurring during storage of non-LR canine pRBC in specific AS or anticoagulantpreservatives have been previously reported, and pRBC stored in the AS Adsol and Nutricel (Pall Corporation, East Hills, NY, USA) have shown superior in vitro and in vivo results when compared with the anticoagulant-preservative citrate-phosphate-dextrose-adenine (CPDA-1) solution.12–14 However, some countries, such as Brazil, lack commercial access to the
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Table 2. Select variables determined on different days of storage of leukoreduced canine pRBC in 4 different additive solutions. Variable
Day 1
pH SAGM 7.27 (0.09) Adsol 7.28 (0.04) Optisol 7.30 (0.04) PAGGGM 7.22 (0.06) Hemolysis (%) SAGM 0.30 (0.09) Adsol 0.31 (0.08) Optisol 0.25 (0.04) PAGGGM 0.27 (0.05) Lactate (mmol/L) SAGM 6.06 (1.69) Adsol 6.22 (2.03) Optisol 6.26 (1.25) PAGGGM 6.50 (1.63) Glucose (mg/dL) SAGM 520.25a (43.93) Adsol 1176.22b (293.16) Optisol 532.38a (72.73) PAGGGM 491.29a (115.86) ATP (lmol/g Hb) SAGM 3.08 (0.84) Adsol 3.26 (0.84) Optisol 2.45 (1.06) PAGGGM 3.31 (1.55) 2.3-DPG (lmol/g Hb) SAGM 19.83 (2.38) Adsol 20.00 (4.20) Optisol 16.84 (4.46) PAGGGM 18.00 (4.06) Na+ (mmol/L) SAGM 144.63a (2.20) Adsol 143.13a (2.80) Optisol 145.38a (1.85) PAGGGM 115.00b (9.02) K+ (mmol/L) SAGM 3.13 (1.01) Adsol 3.04 (0.83) Optisol 3.16 (0.94) PAGGGM 2.79 (0.96)
Day 11
Day 21
Day 31
Day 41
7.33 (0.05) 7.31 (0.09) 7.34 (0.08) 7.39 (0.06)
7.08ª (0.06) 7.10ª (0.07) 7.15ª.b (0.06) 7.19b (0.06)
7.02 (0.07) 6.99 (0.09) 7.05 (0.05) 7.07 (0.08)
7.03a,b (0.07) 7.00a (0.09) 7.05a,b (0.08) 7.13b (0.08)
0.64 (0.21) 0.59 (0.18) 0.63 (0.23) 0.66 (0.19)
0.67 (0.24) 0.74 (0.35) 0.69 (0.23) 0.65 (0.27)
1.13 (0.51) 0.91 (0.36) 1.01 (0.38) 0.97 (0.39)
1.31 (0.56) 1.19 (0.46) 1.42 (0.67) 1.23 (0.61)
13.71 (2.22) 13.63 (1.28) 12.75 (1.66) 13.57 (2.48)
22.49 (3.39) 20.47 (2.20) 20.74 (1.75) 20.60 (2.35)
26.09 (3.55) 27.21 (4.12) 23.80 (2.85) 26.27 (3.67)
26.98 (2.14) 29.45 (5.08) 25.70 (1.68) 28.43 (6.57)
371.13a (111.14) 1454.67b (89.36) 397.00a (129.57) 350.86a (143.21)
441.75a (61.39) 1304.75b (109.26) 449.50a (33.20) 397.71a (26.74)
401.75a (42.90) 1264.92b (103.86) 416.38a (30.87) 331.86a (40.03)
372.50a (43.61) 1224.62b (92.23) 389.25a (32.41) 309.71a (36.53)
3.03 (1.30) 3.62 (2.07) 2.77 (1.45) 4.06 (2.28)
2.00 (0.57) 1.93 (0.51) 1.96 (0.45) 2.42 (0.93)
1.02 (0.65) 1.19 (0.62) 0.73 (0.50) 0.92 (0.76)
0.50 (0.41) 0.53 (0.50) 0.29 (0.35) 0.35 (0.30)
16.99 (4.96) 19.01 (2.94) 16.72 (4.26) 17.04 (4.67)
13.74 (4.19) 13.67 (4.66) 13.33 (3.49) 15.97 (4.66)
8.43 (1.52) 10.27 (3.06) 6.99 (2.36) 10.11 (3.93)
4.25 (2.21) 6.21 (2.83) 3.66 (1.66) 7.77 (4.05)
150.00b (0.53) 153.38a (1.19) 150.50b (0.53) 118.00c (1.15)
150.50a (0.76) 153.63b (0.74) 151.13b (0.83) 119.29c (1.80)
4.09 (0.35) 4.09 (0.38) 4.16 (0.47) 3.81 (0.35)
4.15 (0.36) 4.13 (0.42) 4.20 (0.48) 3.90 (0.37)
148.88a (1.55) 152.38b (1.92) 150.38a,b (1.92) 114.57c (4.31) 4.45 (0.73) 4.23 (0.61) 4.26 (0.68) 3.74 (0.48)
145.88a (10.87) 153.00a (1.07) 147.25a (12.98) 121.71b (12.11) 3.96 (0.37) 4.00 (0.39) 4.05 (0.50) 3.80 (0.33)
DPG indicates 2,3-diphosphoglycerate; PAGGGM, phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucose-mannitol. Data are means (SD). Superscript letters in the same column represent statistically significant differences (P < .05) between solutions at the same time.
above AS, prompting the current study using other, more available solutions. Only limited studies on storage of LR canine pRBC and its effects on the blood product quality and on the recipient have been reported, thus further emphasizing the need for this study. 17,19–21 Leukoreduction has been associated with reduced mortality in specific human patient populations, due to avoidance of some transfusion reactions and better quality of pRBC units.21,31,32 Many countries have
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adopted universal LR. LR filters have some disadvantages, such as higher cost; according to a recent study, each filter can add $20–$65 to the cost of a human pRBC unit depending on the country.33 The cost-effectiveness and the quality of leukoreduced blood products are a debated issue nowadays with many studies reporting that the higher costs can be justified by the reduction of hospital charges and shortened hospital length of stay.33,34 The use of LR filters can lead to 10% of RBCs lost due to trapping in the fil-
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Figure 1. Changes in pH, % hemolysis, and lactate. Glucose, ATP, 2,3-diphosphoglycerate (DPG), sodium, and potassium concentrations in leukoreduced canine pRBC stored in 4 different additive solutions. SAGM, Adsol, Optisol, PAGGGM. *Significantly different from the mean SD of the day 1 value. *P < .05. **P < .001. PAGGGM indicates phosphate-adenine-glucose-guanosine-gluconate-mannitol; SAGM, saline-adenine-glucosemannitol.
ter, but prestorage LR also effectively removes WBCs and more than 90% of platelets before they can contribute to the storage lesion of RBCs or transfusion reactions mediated by platelets and RBCs.32,35 The LR filter used in this study showed a lower efficacy for WBC removal than that presented for
human RBC concentrates in other studies.31 The time of filtration was longer and volume of blood lost to the filter was lower when compared with one other study in dogs17, while a more recent study showed similar results.19 Although the filter efficacy was < 99.9%, the filtration process still met the AABB threshold of
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< 5 9 106 residual WBCs per unit, and the European requirements for WBC counts of < 1 9 106 WBCs per unit.18 Four different AS were used in this study. The original AS were formulated to dilute the RBC with saline prior to transfusion, and in addition supplied glucose and adenine to maintain energy metabolism. Later studies resulted in the addition of mannitol to decrease hemolysis of the stored cells. The newer AS PAGGGM contains guanosine for improved maintenance of ATP, 2,3 DPG, and phosphate concentrations, the latter of which may act as buffers or can be used in the formation of organic phosphates.15,36,37 All variables analyzed in all of the AS showed time-dependent changes. Hemolysis was higher than anticipated in the older units, as the limits of hemolysis for human RBC concentrates are 0.8% and 1.0% in Europe and the USA, respectively.18 The increased hemolysis seen in the bags stored for 31 and 41 days may be related to the smaller volume of pRBC remaining in the bags at these time points, as previously noted in human blood.37 Statistically significant differences between solutions at all time points were identified only for glucose (higher glucose concentrations in Adsol) and sodium (lower sodium concentrations in PAGGGM), both a result of the formulation of the AS themselves. The formulation of Adsol contains more than twice the amount of glucose (dextrose) compared with the other additives used in the study, contributing to the increased residual glucose concentration over time. High glucose levels can lead to an increase in insulin in the transfused individual38, which should be taken into consideration if a large volume of Adsol containing pRBC is needed and if the recipient suffers metabolic dysfunction.39 The lower sodium seen in PAGGGM is also a result of the formulation of this AS. The composition of PAGGGM was originally based on the hypothesis of Meryman and Hornblower that a chloride-free AS would aid in the maintenance of RBC-related 2,3 DPG and ATP concentrations throughout the storage period.40 Saline is thus replaced in this additive with a lower concentration of sodium as sodium gluconate used instead. It is unlikely that the low sodium concentration in PAGGGM would have any deleterious effect on the recipient. PAGGGM is also a more alkaline solution than other AS, with a higher pH, normally favoring formation of 2,3 DPG. In the present study, however, a statistically significant pH change was only seen at day 21, when compared with the other AS. In the current study, there were no statistically significant difference between the ATP and 2,3-DPG con-
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centrations, but ATP concentrations were transiently higher than those reported in previous studies on canine units11–14,17, which was different from a previous study on leukoreduced canine units.17 Recent studies have shown the influence of blood storage and concentrations of 2,3-DPG and ATP on the clinical outcome of patients who require blood transfusion.1–5 Maintenance of ATP and 2,3-DPG concentrations is a desirable characteristic, as ATP is needed to maintain RBC function and integrity; and 2,3-DPG improves the oxygen release to tissues.3–5 Low concentrations of ATP can lead to deformability and exposure of the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine, which are normally concentrated on the inner surface of the RBC membrane and are one of the signals for their removal from the circulation. People with ATP levels < 4 lmol per g HGB risk a higher incidence for PS exposure.15 The minimum recommended range of ATP concentration for stored pRBC is between 2.3 and 2.7 lmol/g HGB, which has been correlated with 75% of transfused RBCs remaining in circulation for 24 h posttransfusion in people.41 A significant association was found between RBC deformability and ATP concentrations < 2.5 lmol/g HGB.42 Concentrations of ATP < 0.75 lmol/g HGB are believed to be a limiting factor in RBC viability in dogs; therefore, storage of LR canine pRBC to day 41 would not be acceptable, as indicated in the present study.43 Concentrations of 2,3-DPG were > 11 lmol per g HGB until day 21 in all solutions, which is close to the values found in the blood of healthy and anemic dogs.44 PAGGGM and Adsol units appeared to have higher concentrations of 2,3-DPG at days 31 and 41 when compared with the other AS, but the differences were not statistically significant. The decrease in the concentrations of 2,3-DPG during storage was similar to that reported in previous human and canine studies for ADSOL and SAGM solutions11–14, but was lower than results previously reported for PAGGGM units in people.16 As 2,3-DPG binds to the b-subunits of HGB, shifting the oxygen dissociation curve to the right and allowing enhanced release of oxygen, its depletion during RBC storage is postulated to decrease microcirculatory oxygen release. Higher 2,3-DPG concentrations are a desirable characteristic as the de novo synthesis of 2,3DPG can take up to 24 h to reach normal levels and, although unlikely, it can affect critical patients with a precarious oxygen balance.14,45 Besides the in vitro biochemical alterations, RBC storage lesions can result in immunomodulatory and inflammatory consequences in transfusion recipients.19,21,22 The clinical impact of pRBC storage time is an important and controversial issue, and there are several large randomized human clinical
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trials currently underway that are investigating this issue.41 In the present study, satisfactory results were obtained with all AS during the first 21 days of storage when compared with other canine pRBC studies.12,13,17,19 Further evaluation of the AS used in this study is warranted, using in vivo analysis to determine posttransfusion stability and viability. Nonleukoreduced units could also be evaluated to further test these storage solutions in dogs.
Acknowledgment The authors would like to thank the Brazilian Funding Agency CNPq (EDITAL MCT/CNPQ N° 014/2008 – UNIVERSAL), the UFRGS’ department of Biochemistry (Dr. Fatima Guma), Dr. Korte and his colleagues from Sanquin Blood Supply Foundation (Amsterdam, NL), Dr. W Jean Dodds and her staff from Hemopet Blood Bank (California, US), Terumo Medical do Brasil LTDA for supplying equipment and solutions to the project, and Daniela Benzano for her help with statistical analysis. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
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