Serial assessment of biochemical parameters of red cell preparations to evaluate safety for neonatal transfusions.Red blood cells (RBC RBC red blood cell.
RBC or rbc
red blood cell
n See red blood cell count.
red blood cells; red blood (cell) count (see blood count). ) are ideally suited to their primary function i.e., transport of oxygen from lungs to the tissues and carbon dioxide from the tissues to lungs. In neonates, RBC transfusion therapy is required in various clinical situations. During the first week of life, neonates experience a decline of RBCs caused by both physiologic factors and in sick premature infants due to sepsis, necrotizing enterocolitis or phlebotomy Phlebotomy Definition
Phlebotomy is the act of drawing or removing blood from the circulatory system through a cut (incision) or puncture in order to obtain a sample for analysis and diagnosis. blood losses. Apart from this, neonates may also need to undergo surgical procedures. In most of these situations neonates usually require repeated small volume transfusions (10-15 ml/kg body weight) (1) with RBCs suspended either in citrate phosphate dextrose dextrose: see glucose. adenine-1 (CPDA-1) solution at a haematocrit hematocrit, haematocrit
a centrifuge used for separating blood cells from the plasma.
See also: Blood and Blood Vessels
Noun 1. of approximately 70 per cent or in extended storage media (additive solution) at a haematocrit of approximately 60 per cent. Large volume transfusion (>25 ml/kg) are required in specific situations, e.g., exchange transfusion in hyperbilirubinaemia; exchange transfusion for sepsis, extracorporeal membrane oxygenation Extracorporeal Membrane Oxygenation Definition
Extracorporeal membrane oxygenation (ECMO) is a special procedure that uses an artificial heart-lung machine to take over the work of the lungs (and sometimes also the heart). (ECMO ECMO extracorporeal membrane oxygenation. ) and cardiac bypass surgery for congenital heart disease congenital heart disease, any defect in the heart present at birth. There is evidence that some congenital heart defects are inherited, but the cause of most cases is unknown. (2,3).
Many controversies exist regarding neonatal transfusion practice. Neonatologists often insist on transfusion of fresh RBC (<7 days old) because of various concerns regarding stored RBCs. There is an increase in extracellular potassium ([K.sup.+]), decrease in pH and 2,3 diphosphoglycerate (2,3 DPG DPG
diphosphoglycerate. ) in stored blood which is important for oxygen release in the tissues. There is also a possible risk of use of compounds like mannitol mannitol /man·ni·tol/ (man´i-tol) a sugar alcohol formed by reduction of mannose or fructose and widely distributed in plants and fungi; an osmotic diuretic used to prevent and treat acute renal failure, to promote excretion of toxic and glucose, in relatively large amounts, which are present in RBC additive solutions. However, when neonates require repeated transfusions, requisitions for fresh blood may lead to multiple allogeneic allogeneic /al·lo·ge·ne·ic/ (-je-ne´ik)
1. having cell types that are antigenically distinct.
2. in transplantation biology, denoting individuals (or tissues) that are of the same species but antigenically blood donor exposure and its consequent risks. Certain in vitro studies have revealed that alterations in various biochemical parameters on storage of whole blood/RBC do not significantly affect neonatal homeostasis homeostasis
Any self-regulating process by which a biological or mechanical system maintains stability while adjusting to changing conditions. Systems in dynamic equilibrium reach a balance in which internal change continuously compensates for external change in a feedback after small volume or top-up transfusions (4,5). These observations may help in reducing multiple donor exposures in neonates who require repeated small volume transfusions. One donor unit could be dedicated for one neonate neonate /neo·nate/ (ne´o-nat) newborn infant.
A neonatal infant.
a newborn animal. after aliquotting it into small volumes (6).
However, before changing neonatal transfusion practices in our scenario we need to study biochemical changes in various red cells preparation on storage. Hence this study was designed to evaluate the serial in vitro changes of the biochemical parameters in different RBC preparations during storage before considering their use for neonatal transfusion even after storage beyond one week.
Material & Methods
The study was conducted by the Department of Transfusion Medicine Postgraduate Institute of Medical Education & Research (PGIMER PGIMER Postgraduate Institute of Medical Education and Research (India) ),
Chandigarh, a tertiary care hospital, in collaboration with the Department of Biochemistry during the period from January 2007 to December 2007. Blood donors were screened as per regulations of Drugs and Cosmetics Rules, Govt. of India (7). Phlebotomy was performed after taking consent from donor. The study protocol was cleared by the Ethics committee of the Institute.
Twenty five units whole blood were collected in single plastic blood bags (J. Mitra Industries Pvt. Ltd. Haryana, India), 25 units of RBC suspended in citrate, phosphate, dextrose, adenine adenine (ăd`ənĭn, –nīn, –nēn), organic base of the purine family. Adenine combines with the sugar ribose to form adenosine, which in turn can be bonded with from one to three phosphoric acid units, yielding the three (CPDA-1) prepared in double blood bags (J. Mitra Industries Pvt. Ltd. Haryana, India) and 25 units of RBC suspended in additive solution composed of saline, adenine, glucose and mannitol (SAGM SAGM Saline, Adenine, Glucose, Mannitol
SAGM Separate Absorption Grading and Multiplication (Layers) ) were prepared in quadruple (Top and Top) bags (Teumo Penpol Ltd. Trivandrum, India) as per standard procedures (8).
Quality check as per Directorate General Health Services criteria (DGHS DGHS Director General of Health Services (India) ) (9). The initial quality of RBC preparations was assessed by utilizing the quality control parameters (visual examination of bag, volume and haematocrit) laid down by DGHS (Table I).
Biochemical parameters: Twenty five units each of whole blood, CPDA-1 RBC, SAGM RBC were tested serially for the following biochemical parameters: supernatant potassium ([K.sup.+]), pH, lactate Lactate
A salt or ester of lactic acid (CH3CHOHCOOH). In lactates, the acidic hydrogen of the carboxyl group has been replaced by a metal or an organic radical. Lactates are optically active, with a chiral center at carbon 2. , haemoglobin haemoglobin or US hemoglobin
a protein in red blood cells that carries oxygen from the lungs to the tissues [Greek haima blood + Latin globus ball]
Noun 1. , glucose and red cell 2,3 DPG up to 21 days of storage. Supernatant plasma potassium and plasma glucose level were estimated by automated analyzer (Hitachi 902, Roche, Germany). Supernatant plasma lactate was estimated by commercial kits (Randox Laboratories Limited, Ardmore, Diamond Road, UK), with the help of colorimeter A device that measures the red, green and blue values of color. See colorimetry and color calibration. Contrast with densitometer. . Plasma haemoglobin was determined by Hemcue AB (SE-26223, Angelholm, Sweden). Red cell 2,3 DPG was estimated by commercial kits (Roche Diagnostics, 38289, Manheim, Germany), with the help of UV spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. . Ten samples of cord blood red cells were also subjected to 2,3, DPG estimation. pH was measured by using digital pH meter (spectrophotometer, Indian Laboratory and Scientific Instrument Ltd., Chandigarh).
Plasma potassium ([K.sup.+]) and pH were estimated daily from day 1 till day 21. Estimation of glucose, lactate and plasma haemoglobin was performed at weekly intervals till three weeks. 2,3 DPG was estimated on day 1, 7, and 14 of storage.
Statistical analysis: ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there was applied for comparison of biochemical changes in the RBCs among intra- and inter-group blood bags. Independent T test was applied when significant difference was found. To assess the correlation between biochemical parameters of different red cell preparations during storage, Pearsons correlations coefficient (r) was applied. P<0.05 was considered statistically significant. All analyses were performed with the software package SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance. Version 13.0 for windows (USA).
The quality parameters such as blood volume and haematocrit of all the units included in the study conformed to the standards established by DGHS (9). There was a significant rise in [K.sup.+] concentration (P<0.001) from day 1 to day 21 of storage. CPDA-1 RBC had the highest significant mean plasma [K.sup.+] during storage (P<0.05). SAGM RBC had slightly higher mean plasma [K.sup.+] than whole blood from day 7 till 21. Mean pH fall was observed in each group of RBC and the pH change was also significant (P<0.001) during storage for each group of RBC. On day 1 CPDA-1 RBC had significantly lower pH than both whole blood and SAGM RBC (P<0.05). From day 3 to day 21, SAGM RBC had slightly higher mean pH value than CPDA-1 RBC though this difference was not statistically significant (Table II). Whole blood had significantly higher mean pH (P<0.005) than both SAGM RBC and CPDA-1 RBC. Fall in mean glucose concentration was noticed in each group RBC from day 1 to day 21 of storage and it was significant (P<0.005). SAGM RBC had significantly higher mean glucose concentration during storage than other two types of RBC preparations (P<0.005). Whole blood had higher mean glucose concentration than CPDA-1 RBC, but the difference was significant only from day 7 to 21 of storage. There was also significant rise (P<0.005) in mean lactate concentration during storage for each group of red cells. Mean lactate value was significantly higher in CPDA-1 RBC on day 7, 14 and 21 than the other red cells (P<0.05). However, mean lactate concentration was not significantly different between whole blood and SAGM RBC. Within each group of RBC fall in mean 2,3 DPG concentration from day 1 to 7 was significant (P<0.001). 2,3 DPG concentration was less than 1umol/g Hb in CPDA-1 RBC and SAGM RBC on day 14 of storage. Ten samples of cord blood were taken as negative control to estimate red cell 2,3 DPG values. 2,3 DPG was undetectable in all the cord blood samples tested.
Within each group of red cells rise in mean plasma haemoglobin concentration from day 1 to 21 was significant (P<0.005) (Table II). Mean plasma haemoglobin was significantly less (P<0.005) in whole blood as compared to CPDA-1 RBC and SAGM RBC during storage. However, the plasma haemoglobin level rose significantly in CPDA-1 RBC on last day of storage.
There was significant negative correlation between mean pH versus mean plasma lactate and plasma glucose versus plasma lactate in each group of red cells during storage. (r= -0.714, -0.732, -0.874, and -0.778, -0.869, -0.696 respectively for CPDA-1 RBC, SAGM RBC and whole blood respectively, P<0.005). Significant positive correlation existed between mean plasma K+ and plasma haemoglobin in all three types of red cells (r=0.726, 0.419, 0.605 for CPDA-1 RBC, SAGM RBC and whole blood respectively, P<0.005). Significant positive correlations were observed between mean pH and 2, 3 DPG (r=-0.623, 0.761 and 0.766 for CPDA-1 RBC, SAGM RBC and whole blood respectively). However, significant negative correlation were observed between lactate and 2,3 DPG concentration (r= -0.698, -0.716 and -0.764 for CPDA-1 RBC, SAGM RBC and whole blood respectively P<0.005) between plasma [K.sup.+] and 2,3 DPG (r= -0.694, -0.761, -0.751 respectively for CPDA-1 RBC, SAGM RBC and whole blood.
The main concerns for stored RBCs are ex vivo storage lesions that undermine red cell functions and may affect the metabolic status of the in vivo milieu of the neonatal recipients (10,11). Additional possible risks are also from the additives like glucose, mannitol which are present in red cells in large amount (12). However, when repeated top up transfusions are required, each fresh unit increases the donor exposure for neonate and its subsequent risks of developing transfusion transmitted diseases.
In the present study significant rise in supernatant [K.sup.+] was seen in the three RBC preparations on storage similar to other studies (12,13). In a study by Strauss (2), the supernatant plasma level after 42 days of RBC storage in additive solution rose to 50 meq/litre. However, the actual dose of bioavailable [K.sup.+] transfused (ionic [K.sup.+] in the volume of extracellular fluid) during small volume transfusion is very low. It has been estimated that the [K.sup.+] concentration of CPDA-1 RBC at haematocrit of 70 per cent at 35 days of storage (permitted shelf life) will be around 70-80 meq/litre. The transfusion dose in a neonate is 15 ml/kg and in a one kg neonate only 0.3 to 0.4 meq K+ will be infused. This dose is even smaller than the usual daily requirement of 2-3 meq/kg. However, this rationale will not apply to large volume transfusions (>25 ml/kg) such as for exchange transfusions. As expected, there was a significant positive correlation between plasma [K.sup.+] and haemoglobin levels in all the three RBC preparations and was in agreement with earlier studies (13,14). The plasma haemoglobin values were higher in CPDA-1 RBC as compared to leukoreduced SAGM RBC. Possible explanation for less hemolysis hemolysis (hĭmŏl`ĭsĭs), destruction of red blood cells in the bloodstream. Although new red blood cells, or erythrocytes, are continuously created and old ones destroyed, an excessive rate of destruction sometimes occurs. in SAGM RBC was due to presence of membrane stabilizers such as mannitol or citrate in the additive solutions (15,16). The degree of haemolysis Hae`mol´y`sis
n. 1. (Physiol.) Same as Hæmatolysis, Hæmatolytic.
the breaking down of erythrocytes with liberation of hemoglobin in the blood. was well below 0.8 per cent, the permissible value at the end of shelf life of all RBC preparations.
Glucose is the main source of energy for red cell metabolism via glycolytic pathway. In blood bags the glucose concentration is limited and as glucose is utilized, there is a concomitant ATP ATP: see adenosine triphosphate.
in full adenosine triphosphate
Organic compound, substrate in many enzyme-catalyzed reactions (see catalysis) in the cells of animals, plants, and microorganisms. (adenosine triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. ) depletion and decrease in red cell viability. We observed a fall in glucose on storage in all the three RBC preparations, but SAGM RBC had significantly higher glucose concentration than whole blood. The highest glucose concentration in SAGM RBC was due to additional 900 mg dextrose present in 100 ml of additive solution. This helps to prolong the shelf life of RBCs by ATP generation through glycolytic pathway (17).
Lactate, the end product of anaerobic anaerobic /an·aer·o·bic/ (an?ah-ro´bik)
1. lacking molecular oxygen.
2. growing, living, or occurring in the absence of molecular oxygen; pertaining to an anaerobe. metabolism of red cells increased during storage. The glucose utilization and lactate production were negatively correlated in all the red cell preparations with CPDA-1 RBC having highest lactate concentration on day 21. Possible explanation may be due to less quantity of adenine and other nutrients present in CPDA-1 RBCs for ATP generation as compared to SAGM RBC or whole blood. pH is an important marker of RBC metabolism during storage which slows as pH falls. 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 (18). Though ATP measurement was not done in the present study, by day 21, CPDA-1 RBC, SAGM RBC and whole blood mean pH was above the lower limit of pH threshold of 6.2, thus ATP generation would most likely be persisting in all the red cell preparations.
In the present study a significant decline was found in 2,3 DPG levels in all the red cells by 2 wk of storage with a significant positive correlation with pH. Beutler (19,20) also found 2,3 DPG to be totally depleted from RBCs by 21 days of storage. Although the levels decline during storage, they increase rapidly after transfusion in the recipient. This regeneration of 2,3 DPG is also supported by a study which observed that red cells stored for 3 wk were as efficacious as erythrocytes of 3.5 h of storage in reversing neurocognitive deficit of acute anemia (21). The p50 of transfused adult RBCs increases rapidly as compared to low p50 values of preterm infant's endogenous red cells due to high foetal foe·tal
adj. Chiefly British
Variant of fetal.
Adj. 1. foetal - of or relating to a fetus; "fetal development"
fetal Hb concentration. Moreover, cord blood has undetectable 2,3 DPG levels (similar result was observed by us on cord blood sample) which along with high foetal Hb concentration cause difficulty in offloading oxygen to the tissues. Thus, transfusing the stored adult RBCs to preterm infants still has an advantage over endogenously produced infant's own RBCs.
In conculsion, results from the present study indicate and substantiate the fact that biochemical alterations do occur in stored red cells. The three red cell preparations tested revealed these changes within acceptable limits of safety till 21 days of storage. Though, whole blood had least biochemical alterations followed by SAGM RBC, but for transfusing the same amount of blood (15 ml/kg) for correcting anaemia, CPDA-1 or SAGM RBC are preferred over whole blood because of higher post-transfusion haemoglobin increment by RBCs as compared to whole blood. Additional benefits like pre-storage leucoreduction and better inventory management are possible with RBC preparations and various adverse effects of fresh whole blood, both immunological and cytomegalovirus transmission can be minimized. Though, CPDA-1 RBCs had highest degree of alterations, these changes need to be considered in light of their effect on neonatal top-up transfusions. A pilot in vivo study needs to be carried out to confirm the post-transfusion safety in neonates. A double blind multi-center randomized control trial is ongoing out in Canada Out In Canada is a travel magazine focused on gay and lesbian also known as LGBT tourism, exclusively within Canada. The magazine is printed twice yearly, and is distributed free in gay villages across North America. to study the effectiveness of age of red cells (stored vs. fresh) in neonates requiring at least one transfusion (22) with the aim to determine if RBCs stored for 7 days or less (fresh RBCs) compared to the practice of using dedicated single units of donated RBCs decrease major nosocomial infections and organ dysfunction in neonates.
Received June 15, 2009
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Of or relating to pediatrics. transfusion practice. Technical manual, 15th ed. Bethesda, Maryland, USA: American Association of Blood Banks; 2005. p. 55777.
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(7.) Malik V. Drugs and Cosmetic Act, 1940, 16th ed. New Delhi: Eastern Book Company; 2003. p. 279-303.
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or red blood cell or red blood corpuscle
Blood cell that carries oxygen from the lungs to the body tissues. adenosine triphosphate, 2,3-diphosphoglycerate, potassium ion, and sodium ion concentrations following the transfusion of acid-citrate-dextrose-stored human red blood cells. J Lab Clin Med 1969; 73 : 722-33.
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1. the act or process of adding oxygen.
2. the result of having oxygen added. deficits in humans. Anesthesiology 2006; 104 : 911-20.
(22.) Fergusson D, Hutton B, Hogan DL, LeBel L, Blajchman MA, Ford JC, et al. The age of red blood cells in premature infants (ARIPI) randomized controlled trial A randomized controlled trial (RCT) is a scientific procedure most commonly used in testing medicines or medical procedures. RCTs are considered the most reliable form of scientific evidence because it eliminates all forms of spurious causality. : study design. Transfus Med Rev 2009; 23 : 55-61.
Reprint requests: Dr Neelam Marwaha, Professor & Head, Department of Transfusion Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh 160 012, India e-mail: email@example.com
Somnath Mukherjee, Neelam Marwaha, Rajendra Prasad *, Ratti Ram Sharma & Be enu Thakral
Departments of Transfusion Medicine, * Biochemistry, Postgraduate Institute of Medical Education & Research,
Table I. Results of quality control parameters in different red cell preparations Parameters DGHS criteria * Volume (ml) Haematocrit (%) CPDA-1 RBC 280 [+ or -] 40 70 [+ or -] 5 SAGM RBC 350 [+ or -] 20 60 [+ or -] 5 Whole blood 350 [+ or -] 10% 40 [+ or -] 5 Parameters Present study Volume (ml) Haematocrit (%) (mean [+ or -] SD) (mean [+ or -] SD) CPDA-1 RBC 281.8 [+ or -] 21.1 69.6 [+ or -] 3.9 SAGM RBC 347 [+ or -] 12.2 60.4 [+ or -] 2.9 Whole blood 351.2 [+ or -] 8.3% 42.8 [+ or -] 4.1 * Directorate General Health Services, Transfusion Medicine Technical Manual 20039 Table II. Results of serially assessed biochemical parameters (mean [+ or -] SD) in each red cell preparations CPDA-1 RBC (n=25) Parameter Days of storage 1 7 Mean pH 7.10 [+ or -] 0.1 6.7 [+ or -] 0.1 (room temp.) Mean plasma [K.sup.+] 6.4 [+ or -] 1 15.8 [+ or -] 2.7 (mEq/l) Mean 2,3 DPG 11.5 [+ or -] 6.2 4.1 [+ or -] 1.6 [micro]mol/g Hb (100%) (36%) * (% of initial) Mean plasma 51.6 [+ or -] 16.7 106.4 [+ or -] 17.1 lactate (+) (mg/dl) Mean plasma 350.3 [+ or -] 62.6 252.1 [+ or -] 46.4 glucose (mg/dl) * Mean plasma 39.7 [+ or -] 13.1 85.9 [+ or -] 20.1 haemoglobin (mg/dl) (##) CPDA-1 RBC (n=25) Parameter Days of storage 14 21 Mean pH 6.5 [+ or -] 0.1 6.3 [+ or -] 0.1 * (room temp.) Mean plasma [K.sup.+] 24.3 [+ or -] 5.9 37.8 [+ or -] 5.7 * (mEq/l) Mean 2,3 DPG 0.4 [+ or -] 0.2 N D [micro]mol/g Hb (3.5%) ** (% of initial) Mean plasma 127.4 [+ or -] 14.1 141.9 [+ or -] 16.6 lactate (+) (mg/dl) Mean plasma 173.4 [+ or -] 48.1 116.2 [+ or -] 33.1 glucose (mg/dl) * Mean plasma 124.4 [+ or -] 18 142.6 [+ or -] 14.5 haemoglobin (mg/dl) (##) SAGM RBC (n=25) Parameter Days of storage 1 7 Mean pH 7.2 [+ or -] 0.1 6.8 [+ or -] 0.1 (room temp.) Mean plasma [K.sup.+] 4.01 [+ or -] 0.8 9.1 [+ or -] 1.6 (mEq/l) Mean 2,3 DPG 10.2 [+ or -] 3.9 4.5 [+ or -] 0.2 [micro]mol/g Hb (100%) (44%) * (% of initial) Mean plasma 47.3 [+ or -] 10.9 95.6 [+ or -] 17.3 lactate (+) (mg/dl) Mean plasma 426.3 [+ or -] 50 358.4 [+ or -] 40.2 glucose (mg/dl) * Mean plasma 34.5 [+ or -] 13.2 77.9 [+ or -] 22 haemoglobin (mg/dl) (##) SAGM RBC (n=25) Parameter Days of storage 14 21 Mean pH 6.6 [+ or -] 0.1 6.4 [+ or -] 0.1 * (room temp.) Mean plasma [K.sup.+] 17.3 [+ or -] 3.5 25.2 [+ or -] 4.5 * (mEq/l) Mean 2,3 DPG 0.7 [+ or -] 0.2 ND [micro]mol/g Hb (6.8%) ** (% of initial) Mean plasma 109.3 [+ or -] 21.9 128.5 [+ or -] 10.8 lactate (+) (mg/dl) Mean plasma 310.1 [+ or -] 43.5 252.7 [+ or -] 40.3 glucose (mg/dl) * Mean plasma 104.9 [+ or -] 26.1 128.8 [+ or -] 19.8 haemoglobin (mg/dl) (##) Whole blood (n=25) Parameter Days of storage 1 7 Mean pH 7.3 [+ or -] 0.1 6.9 [+ or -] 0.1 (room temp.) Mean plasma [K.sup.+] 4.23 [+ or -] 0.7 9.2 [+ or -] 1.7 (mEq/l) Mean 2,3 DPG 14.1 * [+ or -] 4.4 6.1 [+ or -] 2.3 [micro]mol/g Hb (100%) (43.4%) * (% of initial) Mean plasma 44.1 [+ or -] 14.2 88.4 [+ or -] 12.8 lactate (+) (mg/dl) Mean plasma 372.1 [+ or -] 52.7 292.8 [+ or -] 51.1 glucose (mg/dl) * Mean plasma 28.1 [+ or -] 12.6 57.5 [+ or -] 24.9 haemoglobin (mg/dl) (##) Whole blood (n=25) Parameter Days of storage 14 21 Mean pH 6.7 [+ or -] 0.1 6.5 [+ or -] 0.1 * (room temp.) Mean plasma [K.sup.+] 15.7 [+ or -] 3.3 22.8 [+ or -] 5.2 * (mEq/l) Mean 2,3 DPG 2.2 [+ or -] 0.9 ND [micro]mol/g Hb (15.5%) ** (% of initial) Mean plasma 114.1 [+ or -] 16.1 130.9 [+ or -] 14.6 lactate (+) (mg/dl) Mean plasma 228.2 [+ or -] 59.4 159.7 [+ or -] 60.6 glucose (mg/dl) * Mean plasma 83.8 [+ or -] 24.6 113.7 [+ or -] 23.3 haemoglobin (mg/dl) (##) ND, not done. * P <0.001 compared to day 1; ** P <0.001 compared to day 7; mean value from day 1 to 7 P<0.05, from day 7 to 14 and 14 to 21 P<0.001 for all RBC; (#) mean value from day 1 to 21 P <0.005 for all RBC; (##) mean value from day 1 to 21 P<0.005.