Transfusion and Apheresis Science xxx (2014) xxx–xxx
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Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Serial assessment of biochemical changes in irradiated red blood cells Gopal Kumar Patidar a, Aparna Joshi a, Neelam Marwaha a,⇑, Rajendra Prasad b, Pankaj Malhotra c, Ratti Ram Sharma a, Hari Krishan Dhawan a a
Department of Transfusion Medicine, PGIMER, Chandigarh, India Department of Biochemistry, PGIMER, Chandigarh, India c Department of Internal Medicine, PGIMER, Chandigarh, India b
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 5 February 2014 Accepted 6 February 2014 Available online xxxx Keywords: Irradiation Biochemical parameters Potassium
a b s t r a c t Background: Transfusion associated graft vs host disease (TA-GVHD) is delayed effect of blood component therapy with a very high mortality rate. The use of irradiated blood components is the only proven method to prevent TA-GVHD in susceptible patients. Aim: Our study was designed to analyze the quality of irradiated PRBCs in terms of their biochemical parameters during a storage period up to 28 days post irradiation. Methods: A total of 80 PRBC units were analyzed, 40 units each stored in CPDA-1 and additive solution-SAGM. The units were evaluated serially for the following biochemical parameters, plasma/ supernatant potassium, sodium, pH, glucose, lactate, plasma/supernatant hemoglobin and red cell ATP. We further evaluated the differences in these parameters between units irradiated on day 1 and day 7 of storage and stored these units up to 28 days and 35 days respectively. Ten units in each group were used as control. The assessment was done at weekly intervals from the day of irradiation. Results: Within each group of red cells, there was a rise in mean concentration of plasma potassium (K+) from day 1 to last day of storage. There was a highly significant difference (P < 0.01) between irradiated and control units after first week of storage in both types of PRBCs. Irradiated CPDA-1 PRBC had significantly higher (K+) than irradiated SAGM PRBC. Intergroup comparison revealed significantly higher (P < 0.05) mean hemoglobin in irradiated CPDA-1 PRBC as compared to SAGM PRBC. The mean pH was significantly higher (P < 0.05) in irradiated CPDA-1 PRBC as compared to irradiated SAGM PRBC only on day 7 of storage. ATP levels significantly decreased in irradiated units as compared to control units. SAGM PRBCs had significantly higher (P < 0.05) mean ATP concentration than CPDA-1PRBCs. Conclusion: Our study demonstrates that SAGM-PRBCs show better stability after irradiation compared to CPDA-1 PRBCs. The limits of safety for CPDA-1 PRBCs appear to be two weeks after irradiation. SAGM-PRBCs on the other hand show acceptable limits of safety up to three weeks of irradiation. The shelf life of irradiated PRBCs may vary depending upon the storage solution and day of irradiation. Ó 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +91 9914209481. E-mail address: [email protected] (N. Marwaha). http://dx.doi.org/10.1016/j.transci.2014.02.002 1473-0502/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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G.K. Patidar et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
1. Introduction The use of modified blood components has increased to suit special requirements of immunosuppressed patients. These include leucoreduced and irradiated blood components. Established Indications of leukodepletions are to reduce frequency of recurrent febrile nonhemolytic transfusion reactions to RBCs, reduce rate of alloimmunization to leukocyte antigens for patients with malignant disease, and reduce risk of transmission of cytomegalovirus among persons at risk of infection by transfusion. Transfusion associated graft vs host disease (TA-GVHD) is an adverse effect with a very high mortality rate and this is not prevented by only leukodepletion. The use of irradiated blood components is the only proven method to prevent TA-GVHD in susceptible patients [1,2]. Irradiation eliminates the proliferative capacity of lymphocytes present in red cells, platelets and freshly collected plasma components, thereby inhibiting the production of cytokines which could act against patient’s tissues. Inhibiting lymphocyte proliferation does not only inhibit production of cytokines but also recognition of self and non-self (hence TA-GVHD). Groups at risk of GVHD include allogeneic/ autologous bone marrow transplant recipients, congenital immune deficiency syndrome patients, recipients of intrauterine transfusions and hematological malignancy patients. However, irradiation leads to certain biochemical changes in blood, reducing its shelf life from 35 to 28 days. These include increase in hemolysis, changes in supernatant potassium (K+), sodium (Na+), glucose, lactate, adenosine tri-phosphate (ATP) and decrease in pH. We studied serial changes in the biochemical parameters of irradiated and stored PRBCs preserved in CPDA-1 plasma and SAGM (sodium chloride, adenine, glucose and mannitol) to assess the feasibility of maintaining an inventory of these components. It is of great significance because
many healthcare facilities are not equipped for on-site irradiation. Thus, storage of pre-irradiated blood components could be considered in such centres, decreasing the time required for irradiation and transport of blood units as well as leading to better inventory management. 2. Materials and methods The prospective study was conducted in the Department of Transfusion Medicine, PGIMER, Chandigarh from April 2009 to April 2010 after obtaining permission from ethics committee of the institute. During this period, a total number of 80 units of PRBCs were studied; 40 PRBC-CPDA-1(packed red blood cell-citrate, phosphate, dextrose and adenine-1) and 40 PRBCSAGM(packed red blood cell-sodium chloride, adenine, glucose and mannitol). These were collected from healthy donors after screening and informed consent as per the criteria laid down in the Drugs and Cosmetics Act 1945 and Rules, Government of India. CPDA-1-PRBCs were prepared from 450 ml triple bags, the primary bag containing 63 ml CPDA-1 and SAGM-PRBC prepared from 450 ml quadruple bag which contain 63 ml CPD in primary bag and 100 ml SAGM in additional bag. Methods if preparation explain in Figs. 1 and 2. 2.1. Study design Two protocols were used for irradiation and storage as shown in Fig. 3. Details of study design are dictated in Fig. 3. As per AABB criteria the self life if irradiated PRBCs is 28 days. The PRBCs in the study groups were irradiated on day 1 and day7, and then assessed for biochemical changes up to day 28 and 35 respectively during the allowable self-life of 28 days in both groups. The CPDA-1 PRBCS were not leukoreduced. The SAGM-PRBCs were buffycoat
Fig. 1. Preparation of CPDA-1-PRBCs.
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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Fig. 2. Preparation of SAGM-PRBC.
Fig. 3. Study design.
depleted, hence only one log leukoreduced. The units were stored at 4 ± 2 °C in blood bank refrigerator from day 1 to day 35. The parameters of visual examination, volume and hematocrit were tested for quality control (as per national standards criteria) [3].
(25–26 °C). The aliquots of packed cells were used to prepare hemolysate, which was also stored at 70 °C for testing ATP.
2.3. Preparation of hemolysate [4] 2.2. Sampling procedure Sampling was done at weekly intervals. After a hard spin at 3000 rpm for 10 min, the supernatant was taken and 0.5 ml was stored in a labeled cryovial at 70 °C for lactate estimation. Another 0.5 ml was tested for Na+, K+ and glucose in the Department of Biochemistry, PGIMER, Chandigarh while 1 ml was tested for supernatant Hb and pH. The pH was measured at room temperature
Two ml of packed red cells were washed three times with normal saline. Equal quantity of distilled water was added and mixed thoroughly with the cells using vortex mixer for 5 min. Then equal volume of carbon-tetra chloride (Qualigens Fine Chemicals) was added and again mixed thoroughly using vortex mixer for 1 min. Tube was given hard spin at 2000 rpm for 20 min. The clear supernatant was collected in a cryovial and hemoglobin was measured using automated cell counter (SYSMEX KX-21,
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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G.K. Patidar et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
Kobe, Japan). The cryovial with the hemolysate was then stored at 70 °C till ATP estimation. The units were irradiated by gamma irradiation, using a Cesium137, self contained, turn-table, gamma cell irradiator (BIOBEAM 8000, STS, Braunschweig, Germany). The dose delivered to each bag was 25 Gray (Gy) requiring 9 min 55 s of exposure. Quality control of irradiated products was done using radiation exposure indicator strips (RADSURE, ISP Technologies Inc. – Wayne, NJ). Supernatant potassium, sodium and glucose levels were estimated by automated analyzer (Hitachi 902, Roche Diagnostics, Germany). Supernatant plasma lactate was estimated by commercial kits (CENTRONIC GmbH, Germany). Plasma supernatant hemoglobin was determined by HemoCue AB (Angelholm, Sweden). An estimation of supernatant pH of each PRBC unit was done by using the pH indicator solution (UNIVERSAL INDICATOR, GlaxoSmithKline Pharmaceuticals Limited, India). Red cell ATP concentration was determined using a commercial kit ATP Colorimetric Assay Kit (Bio Vision Inc, USA) as per manufacturer’s instructions [18]. All the parameters were estimated weekly from day 1 till day 28 in Group 1 bags and from day 7 till day 35 (28 days after irradiation) in Group 2 bags. 3. Statistical analysis In this study we compared the biochemical parameters between Non-IR and IR red cells of SAGM PRBC and CPDA-1 PRBC. This was normally distributed data. The observations recorded for all the RBC parameters were expressed in terms of the following descriptive statistical measures as mean, range and standard deviation. Independent ‘t’ test was applied where we found significant difference in our result. To assess the correlation between several biochemical parameters during red cells’ storage, Pearson’s correlation coefficient (r) was applied. A p value 25 ml/kg) such as for exchange transfusions [19,20].
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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Fig. 4. Correlations between various parameters.
Fig. 5. Increase in plasma potassium from day 1 to day 28 of storage in both groups of PRBCs.
Fig. 6. Decrease in pH from day 1 to day 28 of storage in both groups of PRBCs.
Fig. 7. Decrease in ATP from day 1 to day 28 of storage in both groups of PRBCs.
Further, for equal days of storage after irradiation, increase in K+ levels was more significant in CPDA-1 PRBCs compared to SAGM-PRBCs (p < 0.001). This could be due to greater dilution of K+ in SAGM additive solution. Apart from visual assessment, plasma hemoglobin measurement in the supernatant from red cell units provides an objective measure of the extent of hemolysis during storage. The extent of hemolysis in blood components is an important indicator of cellular integrity and quality parameters. In CPDA-1 PRBCs Hb levels (Group 1) increased to more than double the initial value in one week and 6 times in four weeks after irradiation. Hb levels (Group 1) increased less exponentially to 1.5 times the initial value in one week and 3.5 times in four weeks after irradiation. Janatpour et al. [5] have reported the rise in Hb concentration in irradiated stored blood by day 28 of storage; a 5-fold increase in four weeks, Davey and colleagues [8] have reported in SAGM-PRBCs; 16-fold rise over 6 weeks similarly observed in Leitner et al. [10] study supernatant hemoglobin was rise up to 2.06 g/L after 35 days of storage. In our study, Hb levels in SAGM-PRBCs increased from mean of 0.37 g/L on day 1 to 3.4 g/L on day 28 and to 4.8 g/L on day 35 of storage. Hb levels (Group 1) increased to 3 times the initial value in one week to 9 times in four weeks. Hb levels (Group 2) increased to 1.5 times the initial value in one week to 4 times in four weeks. In our study, glucose levels in CPDA-1 PRBCs decreased from mean of 21.77 mmol/L on day 1 to 8.16 mmol/L on day 28 and to 6.73 mmol/L on day 35 of storage. By the last day of storage, glucose levels in both groups of irradiated units decreased to 1/3rd of initial values. In SAGM-PRBC glucose level decrease up to 18.12 mmol/L till 35 days of storage and it is comparable to study by Moroff et al. [6] (30.58 mmol/L on day 28), and Zimmermann and colleagues [11] (19.97 mmol/L on day 42). Further, for equal days of storage after irradiation, the glucose levels were almost double in SAGM-PRBCs compared to CPDA-1 PRBCs. This may be attributed to difference in composition of CPDA-1 anticoagulant (2 g dextrose in 63 ml, a part of which is removed while making plasma and platelet components from whole blood) and SAGM additive solution (2.2 g dextrose in 100 ml additive solution is added to the PRBCs). Lactate levels (Group 1) almost doubled the initial value in one week and increased to 3 times in four weeks after irradiation. Lactate levels (Group 2) increased less exponentially to 1.5 times the initial value in one week and 2 times in four weeks after irradiation. Lactate levels in both
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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Table 4 Comparison with AABB standards for biochemical parameters in non-leukoreduced red cells.
a
Days of storage
pH
ATP (% of initial value) (lmol/gm Hb)
Plasma K+ (mmol/L)a
CPDA-1 PRBC (AABB) SAGM PRBC (AABB) CPDA-1 PRBC (Group-1/Group-2)
35 42 7 14 21 28
6.71 6.6 7/6.6 6.6/6.5 6.5/6.5 6.5/6.3
45(±12) 60 88.7/86.9 (3.00/2.94) 76/72.4 (2.57/2.45) 66.2/63.3 (2.24/2.14) 60.7/55 (2.05/1.86)
78.5 50 42.8/69.9 73.3/88.3 90.2/103.2 103/113.7
SAGM-PRBC (Group-1/Group-2)
7 14 21 28
6.7/6.5 6.5/6.5 6.5/6.5 6.4/6.2
95.3/92.7 92.2/87.6 86.4/82.9 81.9/70.3
23/32.5 35/43.6 45.3/55.4 56.9/62.1
(3.31/3.22) (3.20/3.04) (3.00/2.88) (2.84/2.44)
Conversion of plasma potassium unit from meq/L to mmol/L with the help of formula 1 mmol = 1 meq No. of charges.
Table 5 Comparison of different studies with our study. Studies
Janatpour et al. [5] Moroff et al. [6]
Potassium Mean (mmol/L) (days)
Sodium Mean (mmol/L) (days)
Glucose Mean (mmol/L) (days)
Lactate Mean (mmol/L) (days)
pH Mean (days)
Supernatant Hb Mean (g/L) (days)
ATP Mean (lmol/ gm Hb) (days)
CPDA1 PRBC
SAGMPRBC
SAGM-PRBC
SAGM-PRBC
SAGM-PRBC
SAGMPRBC
CPDA1 PRBC
SAGMPRBC
SAGM-PRBC
111 (D-28) NA
NA
NA
NA
NA
NA
NA
NA
NA
30.58 (D-28)
28.41 (D-28)
NA
2.9 (D-28)
NA
NA
NA
6.4 (D28) NA
4.2 (D28) NA NA
NA
NA
NA
NA
NA
NA
NA
1.9 (D-42)
130 (D-28)
NA
NA
NA
6.23 (D-42) NA 2.06 (D-35) NA
NA
NA
NA
4.8 (D35)
2.8 (D-28)
Brugnara and Churchill [7] Davey and colleagues [8] Hillyer et al. [9]
NA
72.3 (D-28) 60–70 (D-41) 78.1 (D-42) NA
Leitner et al. [10]
NA
NA
95 (D-39)
NA
NA
6.56 (D28) NA
Zimmermann and colleagues [11] P. Agarwal et al. [14] Our Study
NA
NA
NA
19.97 (D-42)
NA
NA
NA
NA
NA
NA
NA
NA
NA
103 (D-28)
62.1 (D-35)
128.6 (D-35)
18.12(D-35)
24.30 (D-28)
6.35 (D28)
2.1(D21) 3.85 (D-28)
NA NA
NA
NA
4.7 (D-28)
NA – Not Analysed.
the groups of irradiated PRBCs nearly doubled in one week and increased to almost 4 times in four weeks post irradiation. Thus, lactate levels in our study were 24.30 mmol/L on day 28 is comparable to earlier values by Moroff et al. [6] 28.41 mmol/L on day 28 in SAGM-PRBC. There was a small but significant (p < 0.05) difference between the lactate levels in CPDA-1 and SAGM-PRBCs on most days of storage, levels being higher in CPDA-1 PRBCs. We found that mean ATP value in CPDA1-PRBCs (Group 1) was 60% of initial value by day 28; and (Group 2) was 55% of day 1 value after 35 days of storage. Mean RBC ATP levels exceeding 40% of pre-storage values have been reported to correlate with satisfactory (>70%) in vivo recovery [12]. Leucoreduced PRBCs maintain a better quality after irradiation. Thus, our values (2.8 lmol/gm Hb on day 28) correspond with the findings of other studies Moroff et al. [6] (2.9 lmol/gm Hb on day 28), and Davey and colleagues [8] (1.9 lmol/gm Hb on day 42) and differ from the findings of Zimmermann et al. [11] (4.7 lmol/gm Hb
on day 28) because they did with leucoreduced red cells. Between irradiated CPDA-1 and SAGM-PRBCs, for similar days of storage, ATP levels were always higher in SAGMPRBCs (p < 0.001) on most days of storage. This variation could be due to higher values of dextrose in SAGM than CPDA-1 which helps in better RBC metabolism. Lactate and pyruvate production from glucose, as a consequence of red cell metabolism also leads to decrease in the pH of stored blood. In our study, pH levels in CPDA-1 PRBCs decreased from a mean of 7.15 on day 1 to 6.45 on day 28 and to 6.25 on day 35 of storage. pH levels in SAGM-PRBCs decreased from a mean of 7.1 on day 1 to 6.35 on day 28 and to 6.2 on day 35 of storage. Hillyer et al. [9] have shown a decrease in pH of irradiated AS-3 PRBCs to a mean of 6.56 on day 28 and 6.48 on day 42, while Moroff and colleagues [6] have reported pH value of 6.4 ± 0.1 on day 28 in AS-1 PRBCs. Lower values were observed in our study which may partly be related to the method of pH measurement in our study where the pH
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indicator solution was used and the reading was on a difference in scale of 0.5 units. A mathematical deduction of the pH curve of many samples of stored blood in various storage solutions revealed a lower limit of pH 6.2 below which RBCs had decreased ATP generation [21]. The biochemical alterations observed in our study on irradiated PRBCs were within recommended values as per international (AABB) [13] standards, except for concern regarding pH and K+. In Group-1 SAGM-PRBCs plasma K+ exceeded recommended levels after day 28 of storage while in Group-2 it exceeded after day 21. SAGM-PRBCs (irradiated day 7) exceeded recommended K+ levels after day 21 of storage and safe pH levels after day 28 of storage. The quality of stored irradiated PRBCs was better and longer preserved in SAGM additive solution than CPDA-1 PRBCs. Zimmermann and colleagues [11] have further showed that quality of irradiated PRBCs is even better in leucoreduced SAGM-PRBCs. Their study on units irradiated with 30 Gy on day 14 and stored till day 42 showed that PRBCs maintained their in vitro quality till 28 days post irradiation. 6. Conclusion Hence, we conclude that SAGM-PRBCs show better stability after irradiation compared to CPDA-1 PRBCs. The limits of recommended levels for CPDA-1 PRBCs appear to be two weeks after irradiation and thus the shelf life should be limited to this period. SAGM-PRBCs on the other hand show acceptable limits of safety up to three weeks of irradiation. The shelf life of irradiated red cell is different in various countries. The shelf life of irradiated PRBCs should be determined by taken into consideration the storage solution, day of irradiation after blood collection and leukoreduction. References [1] Anderson KC, Weinstein HJ. Transfusion-associated graft versus-host disease. N Engl J Med 1990;323:315–21. [2] Leitman SF. Use of blood cell irradiation in the prevention of posttransfusion graft-versus-host disease. Transfus Sci 1989;10: 2219–32. [3] Saran RK. Transfusion medicine – technical manual, vol. 32, 2nd ed. New Delhi, India: Directorate General of Health Services (DGHS), Ministry of Health and Family Welfare, Government of India; 2003. p. 352–3.
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[4] Lewis SM, Bain BJ, Bates I. Dacie and Lewis practical hematology. 10th ed. Elsevier; 2008. p. 281–2. [5] Janatpour K, Denning L, Nelson K, Betlach B, Mackenzie M, Holland P. Comparison of X-ray vs. gamma irradiation of CPDA-1 red cells. Vox Sang 2005;89:215–9. [6] Moroff G, Holme S, AuBuchon JP, Heaton WA, Sweeney JD, Friedman LI. Viability and in-vitro properties of AS-1 red cells after gamma irradiation. Transfusion 1999;39:128–34. [7] Brugnara C, Churchill WH. Effect of irradiation on red cell cation content and transport. Transfusion 1992;32:246–52. [8] Davey RJ, McCoy NC, Yu M, Sullivan JA, Spiegel DM, Leitman SF. The effect of pre-storage irradiation on post-transfusion red cell survival. Transfusion 1992;32:525–8. [9] Hillyer CD, Tiergerman KO, Berkman EM. Evaluation of the red cell storage lesion after irradiation in filtered packed red cell units. Transfusion 1991;31:497–9. [10] Leitner GC, Neuhauser M, Weigel G, Kurze S, Fischer MB, Höcker P. Altered intracellular purine nucleotides in gamma-irradiated red blood cell concentrates. Vox Sang 2001;81:113–8. [11] Zimmermann R, Wintzheimer S, Weisbach V, Strobel J, Zingsem J, Eckstein R. Influence of prestorage leucoreduction 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. [12] Peck CC, Moore GL, Bolin RB. Adenine in blood preservation. Crit Rev Clin Lab Sci 1981;13:173–212. [13] Brecher ME. Components from whole blood donations. American Association of Blood Banks. Technical Manual 16th ed.; 2008. p. 288–89. [14] Agarwal P, Ray VL, Choudhary N, Chaudhary RK. Effect of prestorage gamma irradiation on red blood cells. Indian J Med Res 2005;122: 385–7. [15] Menitove JE. Standards for blood banks and transfusion services, 19th ed. Bethesda, MD: American Association of Blood Banks; 1999. [16] Chapman J, Fineey RD, Farman K, Kesley P, et al. Guidelines on gamma irradiation of blood components for the prevention of transfusion-associated-graft-versus-host disease. Transfus Med 1996;6:261–71. [17] Asai T, Inaba S, Ohto H, Osada K, Suzuki G, Takahashi K, et al. Guidelines for irradiation of blood and blood components to prevent post-transfusion graft vs host disease in Japan. Transfus Med 2000; 10:312–20. [18] Ambruso DR, Hawkins B, Johnson DL, Fritzberg AR, Klingensmith WC, McCabe ER. Measurement of adenosine triphosphate and 2,3diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy. Biochem Med Metab Biol 1986;35(3): 376–83. [19] Seghatchian MJ, Vickers M, Ip AH, Stivala JF, de Silva PM. The potassium haemoglobin and acid content of CPDA-1 whole blood, plasma reduced red cells and red cells suspended in SAG-M over 35 days. Transfus Med 1993;3(Suppl. 2):58–9. [20] Hall TL, Barnes A, Miller JR, Bethencourt DM, Nestor L. Neonatal mortality following transfusion of red cells with high plasma potassium levels. Transfusion 1993;33:606–9. [21] Valeri CR, Hirsch NM. Restoration in vivo of erythrocyte adenosine triphosphate, 2,3-diphosphoglycerate, potassium ion, and sodium ion concentrations following the transfusion of acidcitrate-dextrosestored human red blood cells. J Lab Clin Med 1969;73:722–33.
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Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Serial assessment of biochemical changes in irradiated red blood cells Gopal Kumar Patidar a, Aparna Joshi a, Neelam Marwaha a,⇑, Rajendra Prasad b, Pankaj Malhotra c, Ratti Ram Sharma a, Hari Krishan Dhawan a a
Department of Transfusion Medicine, PGIMER, Chandigarh, India Department of Biochemistry, PGIMER, Chandigarh, India c Department of Internal Medicine, PGIMER, Chandigarh, India b
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 5 February 2014 Accepted 6 February 2014 Available online xxxx Keywords: Irradiation Biochemical parameters Potassium
a b s t r a c t Background: Transfusion associated graft vs host disease (TA-GVHD) is delayed effect of blood component therapy with a very high mortality rate. The use of irradiated blood components is the only proven method to prevent TA-GVHD in susceptible patients. Aim: Our study was designed to analyze the quality of irradiated PRBCs in terms of their biochemical parameters during a storage period up to 28 days post irradiation. Methods: A total of 80 PRBC units were analyzed, 40 units each stored in CPDA-1 and additive solution-SAGM. The units were evaluated serially for the following biochemical parameters, plasma/ supernatant potassium, sodium, pH, glucose, lactate, plasma/supernatant hemoglobin and red cell ATP. We further evaluated the differences in these parameters between units irradiated on day 1 and day 7 of storage and stored these units up to 28 days and 35 days respectively. Ten units in each group were used as control. The assessment was done at weekly intervals from the day of irradiation. Results: Within each group of red cells, there was a rise in mean concentration of plasma potassium (K+) from day 1 to last day of storage. There was a highly significant difference (P < 0.01) between irradiated and control units after first week of storage in both types of PRBCs. Irradiated CPDA-1 PRBC had significantly higher (K+) than irradiated SAGM PRBC. Intergroup comparison revealed significantly higher (P < 0.05) mean hemoglobin in irradiated CPDA-1 PRBC as compared to SAGM PRBC. The mean pH was significantly higher (P < 0.05) in irradiated CPDA-1 PRBC as compared to irradiated SAGM PRBC only on day 7 of storage. ATP levels significantly decreased in irradiated units as compared to control units. SAGM PRBCs had significantly higher (P < 0.05) mean ATP concentration than CPDA-1PRBCs. Conclusion: Our study demonstrates that SAGM-PRBCs show better stability after irradiation compared to CPDA-1 PRBCs. The limits of safety for CPDA-1 PRBCs appear to be two weeks after irradiation. SAGM-PRBCs on the other hand show acceptable limits of safety up to three weeks of irradiation. The shelf life of irradiated PRBCs may vary depending upon the storage solution and day of irradiation. Ó 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +91 9914209481. E-mail address: [email protected] (N. Marwaha). http://dx.doi.org/10.1016/j.transci.2014.02.002 1473-0502/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
2
G.K. Patidar et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
1. Introduction The use of modified blood components has increased to suit special requirements of immunosuppressed patients. These include leucoreduced and irradiated blood components. Established Indications of leukodepletions are to reduce frequency of recurrent febrile nonhemolytic transfusion reactions to RBCs, reduce rate of alloimmunization to leukocyte antigens for patients with malignant disease, and reduce risk of transmission of cytomegalovirus among persons at risk of infection by transfusion. Transfusion associated graft vs host disease (TA-GVHD) is an adverse effect with a very high mortality rate and this is not prevented by only leukodepletion. The use of irradiated blood components is the only proven method to prevent TA-GVHD in susceptible patients [1,2]. Irradiation eliminates the proliferative capacity of lymphocytes present in red cells, platelets and freshly collected plasma components, thereby inhibiting the production of cytokines which could act against patient’s tissues. Inhibiting lymphocyte proliferation does not only inhibit production of cytokines but also recognition of self and non-self (hence TA-GVHD). Groups at risk of GVHD include allogeneic/ autologous bone marrow transplant recipients, congenital immune deficiency syndrome patients, recipients of intrauterine transfusions and hematological malignancy patients. However, irradiation leads to certain biochemical changes in blood, reducing its shelf life from 35 to 28 days. These include increase in hemolysis, changes in supernatant potassium (K+), sodium (Na+), glucose, lactate, adenosine tri-phosphate (ATP) and decrease in pH. We studied serial changes in the biochemical parameters of irradiated and stored PRBCs preserved in CPDA-1 plasma and SAGM (sodium chloride, adenine, glucose and mannitol) to assess the feasibility of maintaining an inventory of these components. It is of great significance because
many healthcare facilities are not equipped for on-site irradiation. Thus, storage of pre-irradiated blood components could be considered in such centres, decreasing the time required for irradiation and transport of blood units as well as leading to better inventory management. 2. Materials and methods The prospective study was conducted in the Department of Transfusion Medicine, PGIMER, Chandigarh from April 2009 to April 2010 after obtaining permission from ethics committee of the institute. During this period, a total number of 80 units of PRBCs were studied; 40 PRBC-CPDA-1(packed red blood cell-citrate, phosphate, dextrose and adenine-1) and 40 PRBCSAGM(packed red blood cell-sodium chloride, adenine, glucose and mannitol). These were collected from healthy donors after screening and informed consent as per the criteria laid down in the Drugs and Cosmetics Act 1945 and Rules, Government of India. CPDA-1-PRBCs were prepared from 450 ml triple bags, the primary bag containing 63 ml CPDA-1 and SAGM-PRBC prepared from 450 ml quadruple bag which contain 63 ml CPD in primary bag and 100 ml SAGM in additional bag. Methods if preparation explain in Figs. 1 and 2. 2.1. Study design Two protocols were used for irradiation and storage as shown in Fig. 3. Details of study design are dictated in Fig. 3. As per AABB criteria the self life if irradiated PRBCs is 28 days. The PRBCs in the study groups were irradiated on day 1 and day7, and then assessed for biochemical changes up to day 28 and 35 respectively during the allowable self-life of 28 days in both groups. The CPDA-1 PRBCS were not leukoreduced. The SAGM-PRBCs were buffycoat
Fig. 1. Preparation of CPDA-1-PRBCs.
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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Fig. 2. Preparation of SAGM-PRBC.
Fig. 3. Study design.
depleted, hence only one log leukoreduced. The units were stored at 4 ± 2 °C in blood bank refrigerator from day 1 to day 35. The parameters of visual examination, volume and hematocrit were tested for quality control (as per national standards criteria) [3].
(25–26 °C). The aliquots of packed cells were used to prepare hemolysate, which was also stored at 70 °C for testing ATP.
2.3. Preparation of hemolysate [4] 2.2. Sampling procedure Sampling was done at weekly intervals. After a hard spin at 3000 rpm for 10 min, the supernatant was taken and 0.5 ml was stored in a labeled cryovial at 70 °C for lactate estimation. Another 0.5 ml was tested for Na+, K+ and glucose in the Department of Biochemistry, PGIMER, Chandigarh while 1 ml was tested for supernatant Hb and pH. The pH was measured at room temperature
Two ml of packed red cells were washed three times with normal saline. Equal quantity of distilled water was added and mixed thoroughly with the cells using vortex mixer for 5 min. Then equal volume of carbon-tetra chloride (Qualigens Fine Chemicals) was added and again mixed thoroughly using vortex mixer for 1 min. Tube was given hard spin at 2000 rpm for 20 min. The clear supernatant was collected in a cryovial and hemoglobin was measured using automated cell counter (SYSMEX KX-21,
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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G.K. Patidar et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
Kobe, Japan). The cryovial with the hemolysate was then stored at 70 °C till ATP estimation. The units were irradiated by gamma irradiation, using a Cesium137, self contained, turn-table, gamma cell irradiator (BIOBEAM 8000, STS, Braunschweig, Germany). The dose delivered to each bag was 25 Gray (Gy) requiring 9 min 55 s of exposure. Quality control of irradiated products was done using radiation exposure indicator strips (RADSURE, ISP Technologies Inc. – Wayne, NJ). Supernatant potassium, sodium and glucose levels were estimated by automated analyzer (Hitachi 902, Roche Diagnostics, Germany). Supernatant plasma lactate was estimated by commercial kits (CENTRONIC GmbH, Germany). Plasma supernatant hemoglobin was determined by HemoCue AB (Angelholm, Sweden). An estimation of supernatant pH of each PRBC unit was done by using the pH indicator solution (UNIVERSAL INDICATOR, GlaxoSmithKline Pharmaceuticals Limited, India). Red cell ATP concentration was determined using a commercial kit ATP Colorimetric Assay Kit (Bio Vision Inc, USA) as per manufacturer’s instructions [18]. All the parameters were estimated weekly from day 1 till day 28 in Group 1 bags and from day 7 till day 35 (28 days after irradiation) in Group 2 bags. 3. Statistical analysis In this study we compared the biochemical parameters between Non-IR and IR red cells of SAGM PRBC and CPDA-1 PRBC. This was normally distributed data. The observations recorded for all the RBC parameters were expressed in terms of the following descriptive statistical measures as mean, range and standard deviation. Independent ‘t’ test was applied where we found significant difference in our result. To assess the correlation between several biochemical parameters during red cells’ storage, Pearson’s correlation coefficient (r) was applied. A p value 25 ml/kg) such as for exchange transfusions [19,20].
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Fig. 4. Correlations between various parameters.
Fig. 5. Increase in plasma potassium from day 1 to day 28 of storage in both groups of PRBCs.
Fig. 6. Decrease in pH from day 1 to day 28 of storage in both groups of PRBCs.
Fig. 7. Decrease in ATP from day 1 to day 28 of storage in both groups of PRBCs.
Further, for equal days of storage after irradiation, increase in K+ levels was more significant in CPDA-1 PRBCs compared to SAGM-PRBCs (p < 0.001). This could be due to greater dilution of K+ in SAGM additive solution. Apart from visual assessment, plasma hemoglobin measurement in the supernatant from red cell units provides an objective measure of the extent of hemolysis during storage. The extent of hemolysis in blood components is an important indicator of cellular integrity and quality parameters. In CPDA-1 PRBCs Hb levels (Group 1) increased to more than double the initial value in one week and 6 times in four weeks after irradiation. Hb levels (Group 1) increased less exponentially to 1.5 times the initial value in one week and 3.5 times in four weeks after irradiation. Janatpour et al. [5] have reported the rise in Hb concentration in irradiated stored blood by day 28 of storage; a 5-fold increase in four weeks, Davey and colleagues [8] have reported in SAGM-PRBCs; 16-fold rise over 6 weeks similarly observed in Leitner et al. [10] study supernatant hemoglobin was rise up to 2.06 g/L after 35 days of storage. In our study, Hb levels in SAGM-PRBCs increased from mean of 0.37 g/L on day 1 to 3.4 g/L on day 28 and to 4.8 g/L on day 35 of storage. Hb levels (Group 1) increased to 3 times the initial value in one week to 9 times in four weeks. Hb levels (Group 2) increased to 1.5 times the initial value in one week to 4 times in four weeks. In our study, glucose levels in CPDA-1 PRBCs decreased from mean of 21.77 mmol/L on day 1 to 8.16 mmol/L on day 28 and to 6.73 mmol/L on day 35 of storage. By the last day of storage, glucose levels in both groups of irradiated units decreased to 1/3rd of initial values. In SAGM-PRBC glucose level decrease up to 18.12 mmol/L till 35 days of storage and it is comparable to study by Moroff et al. [6] (30.58 mmol/L on day 28), and Zimmermann and colleagues [11] (19.97 mmol/L on day 42). Further, for equal days of storage after irradiation, the glucose levels were almost double in SAGM-PRBCs compared to CPDA-1 PRBCs. This may be attributed to difference in composition of CPDA-1 anticoagulant (2 g dextrose in 63 ml, a part of which is removed while making plasma and platelet components from whole blood) and SAGM additive solution (2.2 g dextrose in 100 ml additive solution is added to the PRBCs). Lactate levels (Group 1) almost doubled the initial value in one week and increased to 3 times in four weeks after irradiation. Lactate levels (Group 2) increased less exponentially to 1.5 times the initial value in one week and 2 times in four weeks after irradiation. Lactate levels in both
Please cite this article in press as: Patidar GK et al. Serial assessment of biochemical changes in irradiated red blood cells. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.02.002
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G.K. Patidar et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
Table 4 Comparison with AABB standards for biochemical parameters in non-leukoreduced red cells.
a
Days of storage
pH
ATP (% of initial value) (lmol/gm Hb)
Plasma K+ (mmol/L)a
CPDA-1 PRBC (AABB) SAGM PRBC (AABB) CPDA-1 PRBC (Group-1/Group-2)
35 42 7 14 21 28
6.71 6.6 7/6.6 6.6/6.5 6.5/6.5 6.5/6.3
45(±12) 60 88.7/86.9 (3.00/2.94) 76/72.4 (2.57/2.45) 66.2/63.3 (2.24/2.14) 60.7/55 (2.05/1.86)
78.5 50 42.8/69.9 73.3/88.3 90.2/103.2 103/113.7
SAGM-PRBC (Group-1/Group-2)
7 14 21 28
6.7/6.5 6.5/6.5 6.5/6.5 6.4/6.2
95.3/92.7 92.2/87.6 86.4/82.9 81.9/70.3
23/32.5 35/43.6 45.3/55.4 56.9/62.1
(3.31/3.22) (3.20/3.04) (3.00/2.88) (2.84/2.44)
Conversion of plasma potassium unit from meq/L to mmol/L with the help of formula 1 mmol = 1 meq No. of charges.
Table 5 Comparison of different studies with our study. Studies
Janatpour et al. [5] Moroff et al. [6]
Potassium Mean (mmol/L) (days)
Sodium Mean (mmol/L) (days)
Glucose Mean (mmol/L) (days)
Lactate Mean (mmol/L) (days)
pH Mean (days)
Supernatant Hb Mean (g/L) (days)
ATP Mean (lmol/ gm Hb) (days)
CPDA1 PRBC
SAGMPRBC
SAGM-PRBC
SAGM-PRBC
SAGM-PRBC
SAGMPRBC
CPDA1 PRBC
SAGMPRBC
SAGM-PRBC
111 (D-28) NA
NA
NA
NA
NA
NA
NA
NA
NA
30.58 (D-28)
28.41 (D-28)
NA
2.9 (D-28)
NA
NA
NA
6.4 (D28) NA
4.2 (D28) NA NA
NA
NA
NA
NA
NA
NA
NA
1.9 (D-42)
130 (D-28)
NA
NA
NA
6.23 (D-42) NA 2.06 (D-35) NA
NA
NA
NA
4.8 (D35)
2.8 (D-28)
Brugnara and Churchill [7] Davey and colleagues [8] Hillyer et al. [9]
NA
72.3 (D-28) 60–70 (D-41) 78.1 (D-42) NA
Leitner et al. [10]
NA
NA
95 (D-39)
NA
NA
6.56 (D28) NA
Zimmermann and colleagues [11] P. Agarwal et al. [14] Our Study
NA
NA
NA
19.97 (D-42)
NA
NA
NA
NA
NA
NA
NA
NA
NA
103 (D-28)
62.1 (D-35)
128.6 (D-35)
18.12(D-35)
24.30 (D-28)
6.35 (D28)
2.1(D21) 3.85 (D-28)
NA NA
NA
NA
4.7 (D-28)
NA – Not Analysed.
the groups of irradiated PRBCs nearly doubled in one week and increased to almost 4 times in four weeks post irradiation. Thus, lactate levels in our study were 24.30 mmol/L on day 28 is comparable to earlier values by Moroff et al. [6] 28.41 mmol/L on day 28 in SAGM-PRBC. There was a small but significant (p < 0.05) difference between the lactate levels in CPDA-1 and SAGM-PRBCs on most days of storage, levels being higher in CPDA-1 PRBCs. We found that mean ATP value in CPDA1-PRBCs (Group 1) was 60% of initial value by day 28; and (Group 2) was 55% of day 1 value after 35 days of storage. Mean RBC ATP levels exceeding 40% of pre-storage values have been reported to correlate with satisfactory (>70%) in vivo recovery [12]. Leucoreduced PRBCs maintain a better quality after irradiation. Thus, our values (2.8 lmol/gm Hb on day 28) correspond with the findings of other studies Moroff et al. [6] (2.9 lmol/gm Hb on day 28), and Davey and colleagues [8] (1.9 lmol/gm Hb on day 42) and differ from the findings of Zimmermann et al. [11] (4.7 lmol/gm Hb
on day 28) because they did with leucoreduced red cells. Between irradiated CPDA-1 and SAGM-PRBCs, for similar days of storage, ATP levels were always higher in SAGMPRBCs (p < 0.001) on most days of storage. This variation could be due to higher values of dextrose in SAGM than CPDA-1 which helps in better RBC metabolism. Lactate and pyruvate production from glucose, as a consequence of red cell metabolism also leads to decrease in the pH of stored blood. In our study, pH levels in CPDA-1 PRBCs decreased from a mean of 7.15 on day 1 to 6.45 on day 28 and to 6.25 on day 35 of storage. pH levels in SAGM-PRBCs decreased from a mean of 7.1 on day 1 to 6.35 on day 28 and to 6.2 on day 35 of storage. Hillyer et al. [9] have shown a decrease in pH of irradiated AS-3 PRBCs to a mean of 6.56 on day 28 and 6.48 on day 42, while Moroff and colleagues [6] have reported pH value of 6.4 ± 0.1 on day 28 in AS-1 PRBCs. Lower values were observed in our study which may partly be related to the method of pH measurement in our study where the pH
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indicator solution was used and the reading was on a difference in scale of 0.5 units. A mathematical deduction of the pH curve of many samples of stored blood in various storage solutions revealed a lower limit of pH 6.2 below which RBCs had decreased ATP generation [21]. The biochemical alterations observed in our study on irradiated PRBCs were within recommended values as per international (AABB) [13] standards, except for concern regarding pH and K+. In Group-1 SAGM-PRBCs plasma K+ exceeded recommended levels after day 28 of storage while in Group-2 it exceeded after day 21. SAGM-PRBCs (irradiated day 7) exceeded recommended K+ levels after day 21 of storage and safe pH levels after day 28 of storage. The quality of stored irradiated PRBCs was better and longer preserved in SAGM additive solution than CPDA-1 PRBCs. Zimmermann and colleagues [11] have further showed that quality of irradiated PRBCs is even better in leucoreduced SAGM-PRBCs. Their study on units irradiated with 30 Gy on day 14 and stored till day 42 showed that PRBCs maintained their in vitro quality till 28 days post irradiation. 6. Conclusion Hence, we conclude that SAGM-PRBCs show better stability after irradiation compared to CPDA-1 PRBCs. The limits of recommended levels for CPDA-1 PRBCs appear to be two weeks after irradiation and thus the shelf life should be limited to this period. SAGM-PRBCs on the other hand show acceptable limits of safety up to three weeks of irradiation. The shelf life of irradiated red cell is different in various countries. The shelf life of irradiated PRBCs should be determined by taken into consideration the storage solution, day of irradiation after blood collection and leukoreduction. References [1] Anderson KC, Weinstein HJ. Transfusion-associated graft versus-host disease. N Engl J Med 1990;323:315–21. [2] Leitman SF. Use of blood cell irradiation in the prevention of posttransfusion graft-versus-host disease. Transfus Sci 1989;10: 2219–32. [3] Saran RK. Transfusion medicine – technical manual, vol. 32, 2nd ed. New Delhi, India: Directorate General of Health Services (DGHS), Ministry of Health and Family Welfare, Government of India; 2003. p. 352–3.
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