Fresh Blood Units Contain Large Potent Platelets That Improve Hemostasis After Open Heart Operations

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1 Fresh Blood Units Contain Large Potent Platelets That Improve Hemostasis After Open Heart Operations Rephael Mohr, MD, Daniel A. Goor, MD, Alon Yellin, MD, Yaron Moshkovitz, MD, Ami Shinfeld, MD, and Uri Martinowitz, MD Departments of Cardiac Surgery, Thoracic Surgery, and Hematology, The Chaim Sheba Medical Center, Tel Hashomer, Israel Twenty units of fresh whole blood were separated into fresh packed red blood cells (PC) and platelet-rich plasma (PRP) and were transfused to 0 patients immediately after coronary bypass grafting. Patients were preoperatively randomized to receive either PRP (group A, 0 patients) or PC (group B, 0 patients). Platelet number in the PRP group was greater, but not significantly greater, than in the PC group (7.5 f 3 versus 5.9 f. X 10"; p = not significant). However, mean platelet volume in the PC group was significantly greater (.75 f 1.1 versus f 0.7 fl). Postoperatively, group A patients bled more than group B (5 f 1 versus 37 f 1 ml; p < 0.01) and received more red blood cell units (.7 f 1. versus 1. f 0.7 U; p < 0.05) and a larger number of blood products (5.9 f 3.7 versus. f 1. U; p < 0.05). Transfusion of PRP to group A increased platelet count from 1 f 0 to 1 f 110 X 109/L; however, platelet functions did not improve. Administration of PC to group B increased platelet count from 139 f to 15 f 3 x 109/L, improved platelet aggregation (with collagen from 33% f 0% to 53% f 3%, with epinephrine from 3% f % to 51% f 0%; p < 0.05), and corrected the prolonged bleeding time. The results suggest that the improved hemostasis observed after fresh whole blood administration is related to the large, potent platelets that remained in the PC and were not separated to the PRP during standard platelet concentrate preparation. (Ann Thoruc Surg 99;53:50) e [I, 1 have recently shown that when compared with platelet concentrates, fresh whole blood (FWB) has a superior effect on recovery of platelet function after open heart operations. We [3, 1 have also shown that the larger platelets, which are known to be more potent [511], play a major role in hemostasis after open heart procedures. If platelets are, indeed, the determinant of quality of hemostasis after open heart operations, why then are the platelets contained in FWB more effective than those of platelet concentrates? It was our assumption that the larger and more potent platelets are sedimented together with the packed cell fraction during the centrifugation for separation of the platelet concentrate. The present study was designed to evaluate this assumption and its clinical manifestations after open heart operations. Patients and Methods Forty patients (30 men and 10 women) who underwent coronary bypass grafting were randomized before operation into two groups. Group A (0 patients) received immediately after cardiopulmonary bypass platelet-rich plasma (PRP), which had been separated from FWB after donation on the morning of the operation and was kept at room temperature (0" to C) until transfusion. The Accepted for publication Sep 1, Address reprint requests to Dr Goor, Department of Cardiac Surgery, The Chaim Sheba Medical Center, Tel Hashomer 51, Israel. remaining 0 patients (group B) received fresh packed red blood cells (PC) after removal of PRP. Separation of FWB into PRP and PC was done by conventional centrifugation for 3 minutes at 1,70 g [1] (Sorvall RC-3 Dupont/Sorvall Inc, Newton, CT). Platelet count and volume were measured and compared in the PC and PRP. Platelet count was determined by electronically thresholding a selective size range of blood particles from to 0 pm3. The particles entering the sensing zone cause an impedance change between two silver-silver chloride electrodes that is proportionate to the size of the particle. The particles were counted according to the Coulter system [13]. Mean platelet volume (MPV) was derived by computing the arithmetic mean volume from the fitted platelet distribution curve. The result was expressed in femtoliters. The S-plus analyzer calculates plateletcrit by multiplying platelet count by MPV. Total platelet volume was calculated by multiplying plateletcrit by the volume of the respective blood component unit (PC or PRP). Each FWB unit was cross-matched and tested for hepatitis B surface antigen, antibody, and human immunodeficiency virus before administration. Whenever possible, potential donors were tested several days before donation. Patients who had been taking dipyridamole or aspirincontaining drugs during the weeks preceding their operation were excluded from the study. All patients were rewarmed to 35 C before discontinuation of cardiopulmonary bypass. Reversal of the heparin by The Society of Thoracic Surgeons /9/$5.00

2 Ann Thorac Surg 199;53:5M MOHRETAL 51 effect by protamine was monitored by the activated clotting time [1]. All pump blood was returned to the patient (through the aortic cannula or intravenously via infusion bags). The following postoperative hematological variables were studied in all patients: Bleeding time, platelet count, MPV, and platelet aggregation in response to collagen, adenosine diphosphate, and epinephrine. All assays were done after protamine adminiqtration and repeated after transfusion of PRP or PC tq groups A and B, respectively. Platelet aggregations were determined by the method of Born and Cross [15] on a PAP- aggregometer (BIO/ DATA Corporation, Hatboro, PA). The aggregation agents were adenosine diphosphate (final concentration, 3 pmol/l), collagen (Ethicon Inc, Somerville, PJ; final concentration, 0.0 mg/ml), epinephrine (1 Hg/mL), and ristocetin (1. mg/ml). All aggregations were performed on PRP with a platelet count higher than 190 x 109/L. Platelet-rich plasma was prepared by centrifuging citrated blood (3.%) at 00 g for 10 minutes. The normal slope of aggregation was 5 degrees or higher. Normal aggregation amplitude averaged 0% to 90%, and it was determined in our laboratory on 50 normal control subjects. The number of blood units transfused, the total number of blood products (red blood cells, plasma and platelet concentrates) transfused, and the total volume of chest tube drainage in the initial hours after patients arrived in the intensive care unit were recorded by the intensive care unit personnel for evaluation of clinical hemostasis. For calculation of blood components transfused to every patient, 1 U of blood was added to the final number of red blood cells transfused in group B, and 1 U of blood products to the final number of blood products in both groups to account for the PC and PRP transfused in the operating room to groups B and A, respectively. The policy of blood transfusion in the intensive care unit differed in the immediate and late postoperative periods. Most blood transfusions were given in the first postoperative hours. The indications for blood transfusion were hemodynamic variables, such as low systemic and left atrial pressures, and constricted periphery. The amount transfused depended strongly on the amount of blood lost through the chest tubes. Once the patient s condition was stabilized completely, the indication for further blood transfusion was a hemoglobin level lower than 10 g/100 ml. Platelet concentrate transfusions were administered only to patients with active bleeding if the platelet count was less than 100 x 109L All results were expressed as mean * standard deviation. Paired and nonpaired Student s t tests were used for statistical analysis. Results The two groups were similar in age, sex, bypass time, and lowest body temperature (Table 1). There were also no significant differences between the two groups in preoperative and postoperative bleeding time, platelet count, plateletcrit, and platelet function (Tables 1, ). The average platelet number in the whole blood units Table 1. Patient Characteristics Group A, PRP Group B, PC Variable (0 patients) (0 patients) Age (Y) Sex (WF) Redo CABG BSA (m ) Bypass time (min) Aortic cross-clamp time (min) Lowest body temp Preop PLT count ( x 109/L) Preop PCT Aggregation (%) Collagen ADP Epinephrine Ristocetin Preop bleeding time (min) 0 f f f t 1. 7 t t 0.09 f 5 7 t t zt 0. 5 f t t f t f f 7 73 f t t 0. ADP = adenosine diphosphate; BSA = body surface area; CABG = coronary artery bypass grafting; PC = fresh red blood cells; PLT = platelet; PRP = platelet-rich plasma. was x loll. The average number of platelets in the PRP was higher than in the PC (7.5 rtr 3 versus 5.9 t. x lo1, respectively). However, the difference did not reach statistical significance (Fig 1). Mean platelet volume in the PC fraction was significantly larger than in PRP (.75 f 1.1 versus * 0.7 fl; p < 0.001) (Figs 1, ). Moreover, in each individual unit, without exception, the MPV in the PC was larger than in the PRP (see Fig ). The calculated total platelet volume (plateletcrit x volume of fraction unit) was similar in the PC and PRP (53 * 19 versus 5.5 & 5 ml; p = not significant). Intravenous administration of PC improved platelet aggregation and corrected the prolonged bleeding time significantly better than PRP. Platelet counts increased similarly in both groups (group A, 0 * 9 x lo9; group B, 17 f X lo9) (see Table ). Twenty-four-hour blood loss was significantly lower in group B (37 rtr 1 versus 5 * 1; p < 0.01). The total volume of blood transfused and the amount of blood products transfused (ie, number of platelet concentrates, plasma, and red blood cell units) were significantly higher in group A (.7 rtr 1. versus 1. f 0.7 U and 5.9 * 3.7 versus. * 1. U, respectively; p < 0.05) (see Table ). Only 1 patient in group B required transfusion of platelet concentrates during the first postoperative hours compared with 5 patients in group A. The reduced exposure to donors of blood products in group B is mainly due to the lower number of platelet concentrates transfused (see Table ). One patient in group B had to be returned to the operating room because of excessive mediastinal bleeding (00 ml during the first hour in the intensive care unit); however, a surgical source of bleeding was found, and the patient was not excluded from the study.

3 5 MOHRETAL Ann Thorac Surg 199;53:50 Table. Effect of Fresh PRP versus Fresh PC Transfusion on Hemostasis After Cardiopulmonary Bypass Group A (PRP) Group B (PC) Post- Post- Post- Post- Variable operative transfusion operative transfusion PLT count (X 109/L) rt rt 15 f 3 PCT f f 0.0 Bleeding time (min) 7. rt f f 1.. f 1. Aggregation (%) Collagen ADP Epinephrine Ristocetin -hour blood loss (ml) RBC requirement (U) PLT concentrate transfusion (U) Total blood product consumption (U) 37 rt rt 3 5 * 5 f 7 0 rt 15 f 19 3 f f 51 0 * 0 7 f 1 73 rt 77 f 13 5 f 1b 37 f 1.7 f 1.= f 0.5 f. 5.9 f 3.7. rt 1. a p < 0.05, A versus B. p < 0.01, A versus B. ADP = adenosine diphosphate; PC = fresh red blood cells; PCT = plateletcrit; PLT = platelet; PRP = platelet-rich plasma; RBC = red blood cell. Comment The present study was designed in an attempt to solve the enigma why transfusion of fresh whole blood at the termination of cardiopulmonary bypass improves hemostasis better than transfusion of platelet concentrates [l,, 11. The key word for solving this enigma is large platelets. Large platelets are more potent and hemostatically superior to smaller platelets [ During preparation of platelet concentrates, fresh blood is separated into PC and PRP. The large platelets are sedimented during centrifugation together with the packed red blood cell fraction and are not included in the PRP, which is later used for preparation of platelet concentrates. One can arrive at this conclusion on the basis of the significantly higher MPV in the PC compared with PRP (see Fig ), particularly when comparing each of the 0 separated units, where MPV in the PC was larger than in PRP with no exception. The observation that large platelets are metabolically and functionally more active than the small platelets is not new. Large platelets have been found to contain more glycogen adenine nucleotides, orthophosphate, and reduced forms of gluthathione, nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phos- Fig 1. Comparison of platelet (PLT) number, mean platelet volume (MPV), and total platelet volume (TPV) in the fresh red blood cells and platelet-rich plasma. 10 L P C Q O o l 0 CELLS PLATELET-RICH PLASMA

4 Ann Thorac Surg 199:53:5& MOHRETAL 53 Y J PLAI~LEFRICH PLASMA PACKED CELLS Fig. lndividual comparison of mean platelet volume (MPV) in the fresh red blood cells and platelet-rich plasma. phate [57]. They show greater rate of metabolism of protein [, 91, carbohydrate [7], and lipid metabolism [lo]. Large platelets aggregate better in response to adenosine diphosphate, epinephrine, thrombin, and collagen [5, 1 and respond to lower concentrations of adenosine diphosphate (171. They show greater adhesiveness to glass beads and collagen [1] and are less susceptible to inhibition by prostacyclin [ 111. Clinical observations also indicated the hemostatic superiority of large platelets [19]. In patients with severe thrombocytopenia, MPV was found more important than platelet count for prediction of bleeding tendency [0]. A unit of FWB contains both the small platelets that are separated to the platelet concentrate and the large and potent platelets that are lost from platelet concentrate during separation of blood to PC and PRP. Therefore there is no doubt that FWB is hemostatically superior to stored platelet concentrate. It must be emphasized that no attempt was made here to prove that fresh red blood cells restore hemostasis better than fresh platelet concentrate. The slightly lower postoperative bleeding and slightly higher need for blood transfusions in patients of group B, although statistically significant, does not bear real clinical significance. However, together with the better platelet aggregation and shorter bleeding time observed in this group, they serve as indicators of better hemostatic effect of PC compared with PRP. Those scientific observations of minor differences between the two groups are important, as they correspond to the "trapping" of the more potent large platelets within the fraction of the red blood cells. Because this study was not conducted in a double-blind fashion, a biased attitude to postoperative blood transfusions might be suspected, provoking distortion of the postoperative clinical picture. However, management of patients in the intensive care unit was conducted by the intensive care unit personnel, who were neither involved in nor aware of ongoing studies. The decision to transfuse homologous blood or blood components was based entirely on the hemodynamic situation of the patient and blood loss in the first to postoperative hours. In conclusion, this study demonstrates that the conventional blood bank method of blood separation is deficient in the sense that the larger and most potent platelets cannot be separated into the units of platelet concentrate. It should be noted that in searching for donors for FWB, donors who are blood-related to the patient should be avoided. This is because of the very rare but fatal risk of graft-versus-host reaction caused by the donated lymphocytes []. References 1. Mohr R, Martinowitz U, Lavee J, Amroch D. The hemostatic effect of transfusing fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg 19;9: Lavee J, Martinowitz U, Mohr R, et al. The effect of transfusion of fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg 199; Mohr R, Martinowitz U, Golan M, Lusky A. Platelet size and mass as an indicator for platelet transfusion after cardiopulmonary bypass. Circulation 195;7(Suppl 3):10.. Mohr R, Golan M, Martinowitz U, Rosner F. Effect of cardiac operation on platelets. J Thorac Cardiovasc Surg 19;9: Karpatkin S, Charmatz A. Heterogeneity of human platelets. I. Metabolic and kinetic evidence suggestive of young and old platelets. J Clin Invest 199;: Karpatkin S. Heterogeneity of human platelets. 11. Functional evidence suggestive of young and old platelets. J Clin Invest 199;: Hirsh J. Platelet age: its relationship to platelet size, function and metabolism. Br J Haematol 197;3: Steiner M, Boldini M. Protein synthesis in aging blood platelets. Blood 199;33:% Friedhoff A, Miller JC, Karpatkin S. Heterogeneity of human platelets. VII. Platelet monoamine oxidase activity in normals and patients with autoimmune thrombocytopenic purpura and reactive thrombocytosis: its relationship to platelet protein density. Blood 197;51: Leone G, Agostini A, DeCrescenzo A, Bizzi B. Platelet heterogeneity: relationship between buoyant density, size, lipid peroxidation and platelet age. Scand J Haematol 1979; 3: Jakubowski JA, Alder, Thompson CP, Valeri CR. Influence of platelet volume on the ability of prostacycline to inhibit platelet aggregation and the release reaction. J Lab Clin Med 195;105: Miller WV. Preparation of blood components. In: Technical manual of the American Association of Blood Banks. Philadelphia: J.B. Lippincott, 1977: Caulter Counter S plus I1 Field Manual I. Hialea, FL: Caulter Electronics Inc, Verska JJ. Control of heparinization by activated clotting time during bypass with improved postoperative hemostasis. Ann Thorac Surg 1977;: Born GVR, Cross MJ. The aggregation of blood platelets. J Phy siol 193; 1:

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