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Platelet function, but not thrombin generation, is impaired in acute normovolemic hemodilution (ANH) blood
Journal of Clinical Anesthesia 58 (2019) 39–43
Contents lists available at ScienceDirect
Journal of Clinical Anesthesia journal homepage: www.elsevier.com/locate/jclinane
Original contribution
Platelet function, but not thrombin generation, is impaired in acute normovolemic hemodilution (ANH) blood
T
Korrin J. Scott (MD)a, , J. William Shteamer (MD)a, Fania Szlam (MMSc)b, Roman M. Sniecinski (MD, MSc)a ⁎
a b
Emory University School of Medicine, Department of Anesthesiology, United States of America Emory University School of Medicine, United States of America
ABSTRACT
Study objective: We investigated the coagulation changes that might occur in acute normovolemic hemodilution (ANH) blood over several hours during cardiac surgery requiring cardiopulmonary bypass. Design: This study was a prospective observational study. Setting: This study took place at a university teaching hospital. Patients: This study included 26 patients, either ASA 3 or 4 and without known coagulation disorders, undergoing cardiac surgery. Patients were included if the use of cardiopulmonary bypass was expected to reach 2.5 h. Interventions: ANH blood was collected into CPDA-1 collection bags before systemic heparinization. Samples were taken directly from the bags at time of collection and reinfusion to assess changes in platelet and thrombin generation parameters. Measurements: Whole blood from citrated tubes was used immediately for rotational thromboelastometry and platelet aggregometry analyses. Thrombin generation was assessed using calibrated automated thrombography with platelet poor plasma. Main results: Despite no significant change in platelet count over the ANH storage period, there was significant degradation in platelet function as measured by thrombin receptor activating peptide stimulation on Mulltiplate™ analysis and maximum clot formation on ROTEM™ EXTEM. Notably, there was no change in the ability to generate thrombin. Conclusions: Little data exists regarding the quality of coagulation factors in autologous blood. Our study confirms ANH collection results in decreased platelet aggregation with TRAP stimulation; however, this is not appreciated with ADP stimulation. Thrombin generation capacity remains preserved.
1. Introduction Depending upon procedure type, between 30% and 80% of patients undergoing cardiac surgery receive allogeneic blood transfusions [1], which are associated with worse outcomes, even when given in small quantities [2,3]. Several cardiac surgery and anesthesiology societies have recommended intraoperative acute autologous normovolemic hemodilution (ANH) as a blood conservation technique, albeit a Class IIb one [4,5]. A recent meta-analysis including almost 30 randomized controlled trials concluded that ANH in cardiac surgery reduces allogeneic transfusions by about 1 unit of red blood cells (RBCs) [6]. Beyond reducing RBC transfusions, a large observational study including over 13,000 patients undergoing cardiac surgery concluded ANH use was also associated with fewer platelet and plasma transfusions [7]. This potential impact of ANH on the administration of non-RBC components has not been thoroughly investigated. Since ANH blood has not been exposed to the cardiopulmonary bypass circuit, fibrinogen and
platelet function should theoretically be preserved. Unfortunately, factors can be consumed when the coagulation system is not adequately suppressed in the collection bag, resulting in visible clot formation (see Fig. 1). The practice of ‘hemospasia,’ or sequestering blood from the cardiopulmonary bypass (CPB) circuit for later reinfusion, has been around for decades, but relies upon heparin as the anticoagulant [8]. Heparin for ANH has been shown to impair platelet function and coagulation as assessed by viscoelastic testing [9]. However, most clinicians utilize citrate phosphate dextrose (CPD) bags with or without the addition of adenine to collect the ANH blood. We performed the current study to investigate the coagulation changes that might occur in ANH blood in modern practice, particularly when stored for the several hours required during prolonged CPB cases. We hypothesized that there would be a decrease in the thrombin generation and platelet function in ANH blood between collection and reinfusion into the patient.
Corresponding author. E-mail addresses: [email protected] (K.J. Scott), [email protected] (J.W. Shteamer), [email protected] (F. Szlam), [email protected] (R.M. Sniecinski). ⁎
https://doi.org/10.1016/j.jclinane.2019.04.032 Received 15 January 2019; Received in revised form 9 April 2019; Accepted 26 April 2019 0952-8180/ © 2019 Elsevier Inc. All rights reserved.
Journal of Clinical Anesthesia 58 (2019) 39–43
K.J. Scott, et al.
were drawn just prior to reinfusion of the ANH blood after separation from CPB. Whole blood from citrated tubes was used immediately for rotational thromboelastometry and platelet aggregometry analyses. For thrombin generation measurements, samples were centrifuged for 20 min at 2000 ×g at room temperature to obtain platelet –poor plasma (PPP) and stored at −80 °C until time of batch analysis. 2.2. Standard hematologic testing Parameters of complete blood cell count (WBC, platelet count, hematocrit, hemoglobin) were measured from the samples in EDTA tubes using a Poch-100i analyzer (Sysmex America, Inc., Lincolnshire, IL). Fibrinogen concentrations were measured in thawed citrated plasma by the Clauss clotting method using STA Compact Analyzer and STA Fibrinogen reagents according to manufacturer's directions (Diagnostica Stago, Parsippany, NJ). Calibration was performed with STA-unicalibrator; ‘normal’ and ‘abnormal’ controls were tested prior to patients' plasma samples. 2.3. Rotational thromboelastometry measurements ROTEM™ device (Instrumentation Laboratory, Bedford, MA) was used for all of the viscoelastic testing measurements. EXTEM and FIBTEM testing were performed at 37 °C using 300 μl of the patient's whole blood according to manufacturer's directions. In EXTEM testing, blood is recalcified with 20 μl of start-tem reagent (0.2 M CaCl2) and clotting is activated via tissue factor (ex-tem reagent). The FIBTEM test is an EXTEM-based assay more specific for the fibrin part of the clot by inhibiting platelet contribution with cytochalasin D, albeit incompletely [10]. The following parameters of ROTEM were collected; a) Clotting time (CT; sec), which corresponds to initiation of clotting, b) clot formation time (CFT; sec), which corresponds to time from initiation of clotting until clot firmness of 20 mm is detected, c) angle (α; degrees), which reflects the rate of fibrin polymerization, and d) maximum clot firmness (MCF; mm), which reflects maximum amplitude of the tracing and the tensile strength of the clot.
Fig. 1. Photograph of a blood collection bag used during autologous normovolemic hemodilution (ANH). The arrow points toward a large clot that has formed.
2. Methods
2.4. Platelet aggregometry
2.1. Patient enrollment and sample collection
Whole blood aggregometry was evaluated using multiple electrode methodology using a Multiplate™ device (Diapharma Group Inc., West Chester, OH). The technique is based on change of resistance that is proportional to the amount of activated platelets sticking to sensor wires. This increase in resistance is recorded during the test. For each test, 300 μl of pre-warmed calcium chloride (CaCl2, 3 mM) – normal saline solution and 300 μl of citrated whole blood sample were added to the test cell. The CaCl2 present in the saline partially compensates for citrate calcium depletion by citrate, but not enough to start clotting, and it increases aggregation [11]. After 3 min of incubation at 37 °C, samples were activated with either adenosine diphosphate (ADP) or thrombin receptor activating peptide 6 (TRAP) according to the manufacturer's directions. These reagents were chosen as they have been shown to be the most clinically useful predictors of blood use in cardiac surgery patients [12]. After 6 min run time, platelet aggregations curves were assessed with the following Multiplate™ parameters; area under the curve (AUC, U), maximum aggregation (maximum amplitude of the curve, AU), and velocity (the maximum slope of the curve, AU/min) [13].
This study was approved by the Emory University Institutional Review Board. Male and female patients > 18 years old undergoing cardiac surgery at Emory University Hospital and expected to require the use of > 2.5 h of CPB were screened for participation. Exclusion criteria were emergency surgery, baseline hemoglobin < 11.0 g/dL, or patient refusal/inability to provide written informed consent. No patient was actively taking any oral anticoagulant medication, other than aspirin, within 5 days of the operation. On the day of surgery, the patient's hemoglobin was once again rechecked and confirmation that the patient could safely undergo ANH was obtained from the attending anesthesiologist. After induction of anesthesia and prior to any systemic heparin administration, ANH blood was collected into 450 ml collection bags containing citrate, phosphate, dextrose, and adenine (i.e. CPDA-1 bags) for temporary storage (Blood-Pack™, Fenwal Inc., Lake Zurich, IL). The volume collected was at the discretion of the attending anesthesiologist, as well as the amount of crystalloid fluid replacement. No colloids were used during the ANH collection phase. Collection bags were kept at room temperature in the operating room and gently agitated by hand at regular intervals per normal institutional practice. Samples were directly collected from the first bag of ANH into 10 ml buffered 3.2% sodium citrate and 2 ml spray-coated potassium EDTA (3.6 mg) B-D Vaccutainer tubes (Becton Dickinson, Franklin Lakes, NJ) at the following time points: 1) baseline samples were taken within 10 min of finishing collecting the first ANH bag, 2) reinfusion samples
2.5. Thrombin generation measurements Stored frozen plasma samples were thawed on water bath at 37 °C for 5–10 min. Thrombin generation (TG) measurements were performed at 37 °C using calibrated automated thrombinoscope methodology as first described by Hemker [14]. Briefly, 80 μl of thawed PPP was added to wells of 96 wells microtiter plate (Diagnostica Stago, 40
Journal of Clinical Anesthesia 58 (2019) 39–43
K.J. Scott, et al.
Parsippany, NJ) along with 20 μl of tissue factor reagent (final concentration of 5 pM TF, 4 μM phospholipids). Wells with 20 μl of thrombin calibrator instead of TF reagent were run in parallel. After 10 min incubation, TG was initiated by an automatic addition of FluCa reagent. All TG reagents were purchased from Diagnostica Stago (Parsippany, NJ). The progress of the TG reaction was monitored using Thrombinoscope™ software (Diagnostica Stago, Parsippany, NJ). The following TG parameters were measured; ETP (nM*min) - endogenous thrombin potential- area under thrombin curve, peak thrombin (nM)maximum thrombin concentration, lag time (min) – time between addition of trigger (TF) and initiation of TG, ttp (min) time to reach the peak of TG, rate of TG (nmol/min) velocity index.
Table 1 Clinical characteristics of patients.
2.6. Statistical analysis
Patient demographics and preoperative hematological laboratory values are provided. Continuous variables given as mean ± standard deviation with the exception of platelet count, CPB bags used for collection, and ANH volume, which is median (1st quartile, 3rd quartile). ANH = autologous normovolemic hemodilution, Hgb = hemoglobin, aPTT = activated partial thromboplastin time, ASA = aspirin.
Age (years) Females, number (%) Weight (Kg) Preoperative Hgb (g/dL) Preoperative platelets (1000/μl) Preoperative INR Preoperative aPTT (seconds) Patients on ASA, number (%) Baseline ACT (seconds) Volume of ANH blood removed from patients(ml) CPB bags collected (n) Time between ANH collection and reinfusion (minutes)
In a similar study using heparin as the anticoagulant, Gallandat et al. arrived at a sample size of 23 for detecting a 25% reduction in platelet aggregation as measured by Mutiplate™ AUC using α = 0.05 level with 80% power. We targeted the same sample size as Gallandt et al. of 26 subjects. In retrospect, the mean Mutiplate™ AUC for ADP stimulation using citrate as the anticoagulant has been reported as 68.6 ± 20.1 U in normal blood [15]. Using paired t-tests and an α = 0.01 and targeting a power of 90%, 24 samples would be required to detect a 25% decrease in this same parameter. Descriptive statistics were used for all variables. Their distribution and residuals were evaluated with Q-Q plots and the KolmogorovSmirnov test. All hematologic variables were found to be normally distributed with the exception of platelet count, fibrinogen level, EXT CFT, and EXT alpha angle. Paired t-tests were used to determine statistical significance of the difference for normally distributed parameters. Non-normally distributed parameters were evaluated with Wilcoxon signed rank test. Given the multiple parameters being compared, Bonferroni correction was used to determine statistical significance (α = 0.05/16 = 0.0031). Calculations for determining the difference in parameters between baseline and reinfusion were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). The correlation between time of storage and magnitude of change in parameters found to significantly differ between collection and reinfusion was assessed using regression plots and Pearson's correlation coefficient. The percentage change from collection to reinfusion was also calculated for those parameters found to be significantly different. These calculations were performed using SAS 9.4 (SAS Institute, Cary, NC).
57 ± 17 9 (35%) 87.6 ± 17.2 13.9 ± 1.4 196 ± 62 1.04 ± 0.10 32.8 ± 5.1 16 (62%) 105 ± 10 425 (350, 600) 1 (1, 2) 299 ± 94
statistically significant after adjusting for multiple comparisons. No parameters reflecting thrombin formation, as measured using TG testing or ROTEM® (i.e. clot time), significantly changed between collection and reinfusion of the ANH blood. When stimulated with ADP, no platelet aggregation parameters were found to significantly change either. Of the parameters that significantly changed between collection and reinfusion, the magnitude of change was not significantly correlated with time of storage (see Table 3). 4. Discussion It is widely accepted that ANH reduces the transfusion rate for allogenic RBCs, but its benefit with regard to hemostatic blood products remains unproven. Given that coagulopathy in cardiac surgery is often encountered secondary to exposure to the CPB circuit, the presumption is that blood not exposed to this insult maintains a more favorable coagulation profile. Reinfusion of this sequestered blood is postulated to improve hemostasis and further reduce the need for RBC transfusion. In a small clinical trial of patients undergoing cardiac surgery with CPB, however, Zisman et al. failed to demonstrate any benefit of ANH with respect to the administration of hemostatic blood products [16]. In fact, little data regarding the quality of coagulation factors in ANH blood exists. Our aim in this study was to look for possible degradation of coagulation parameters over time in the autologous blood under conditions that are considered standard practice for removal of ANH blood. Based upon our findings, platelet aggregation in response to some stimulants does seem to decrease in the collection bag. We found almost a 25% decrease in area under the curve using TRAP stimulation despite no appreciable decrease in platelet count. These findings are consistent with those of Gallandat et al. who reported decreased platelet aggregation in response to TRAP stimulation using heparin as the anticoagulant in ANH collection bags [9]. Unlike Gallandat et al., however, we did not appreciate a change in regard to ADP-induced aggregation. This may be due to the fact heparin has a different effect on Multiplate™ ADP results when compared to citrate [17]. Given that TRAP is a much stronger stimulant, this minor difference between anticoagulants was not likely as apparent. In an older study, Ramnarine et al. also found a decrease in platelet aggregation in CPDA bags with and without heparin [18]. However, they utilized collagen as an activator for platelet testing, as well as a gelatin solution for ANH replacement fluid, which is known to affect platelet aggregation [19]. In blood banking studies, platelet function is known to decrease with storage time [20]. Platelets obtained from whole blood in particular have been shown to have decreased responsiveness to thrombin over a period of 5 days [21]. While we saw a decrease in platelet aggregation from TRAP stimulation as well as with viscoelastic testing, it
3. Results Thirty-five patients scheduled to undergo cardiac surgery with prolonged CPB were screened and consented for the study between 12/ 29/15 and 3/5/18. Of these patients, 4 were deemed ineligible because of Hgb < 11.0 on initial OR blood gas, inadequate collected ANH volume (< 100 ml) in 2 patients, ANH blood being reinfused on CPB in 2 patients, and labs drawn into the wrong collection tubes on 1 patient. Twenty-six patients completed the protocol and were available for analysis. Their baseline demographics and standard preoperative labs are presented in Table 1. The measurements from the CPDA-1 bags at time of collection and at time of reinfusion, and their differences, are presented in Table 2. Platelet count itself did not significantly change, but Multiplate® parameters of aggregation, velocity, and AUC as stimulated by TRAP decreased by 24.9% (95% CI 12.3%, 37.4%), 17.5% (95% CI 4.1%, 31.0%), and 23.4% (95% CI 8.9%, 38.0%) respectively. Platelet-fibrinogen interaction as measured by EXTEM MCF and alpha similarly decreased by 9.8% (95% CI 5.0%, 14.4%) and 7.9% (95%CI 2.3%, 13.6%) respectively. Fibrinogen concentration and FIBTEM MCF showed very minor decreases over the storage time, but were not 41
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Table 2 Measured hemostatic variables of ANH Blood at Collection and Reinfusion. Parameter
Collection
Reinfusion
Difference
p
Platelet Count (x1000/μl) Fibrinogen Level (mg/dL) MP ADP AUC (units) MP ADP Aggregation (AU) MP ADP Velocity (AU/min) MP TRP AUC (units) MP TRP Aggregation (AU) MP TRP Vel (AU/min) TG TTP (min) TG PH (nM) TG ETP (nM*min) EXT CT (seconds) EXT CFT (seconds) EXT MCF (mm) EXT alpha (°) FIB MCF (mm)
142 {100, 157} 223 {173, 285} 11.0 ± 7.9 24.8 ± 14.0 4.7 ± 2.9 39.5 ± 18.1 65.9 ± 25.9 11.0 ± 4.9 6.5 ± 2.2 206.7 ± 73.7 1325.8 ± 262.3 87.6 ± 35.9 113 {88.5, 141.5} 61.2 ± 6.2 67.5 {65.0, 72.5} 13.5 ± 5.6
135 {91, 153} 222 {147, 274} 10.1 ± 6.3 22.6 ± 11.9 3.9 ± 1.3 30.4 ± 18.4 48.7 ± 25.4 8.9 ± 4.8 6.7 ± 2.5 201.5 ± 61.8 1320.2 ± 223.5 93.5 ± 32.3 137.5 {105.5, 166.8} 55.3 ± 8.8 63.0 {58.0, 70.0} 11.8 ± 6.0
3 {−5, 23} 7 {−4, 15} 0.9 (−2.2, 4.1) 2.2 (−3.2, 7.5) 0.8 (−0.7, 2.2) 9.1 (2.7, 15.5) 17.2 (7.0, 27.4) 2.1 (0.3, 3.9) −0.2 (−1.1, 0.7) 5.1 (−15.2, 25.4) 5.6 (−62.1, 73.4) −5.9, (−22.7, 10.9) −16.0 {−50.8, 10.3} 5.9 (2.2, 9.7) 3.5 {−0.3, 8.5} 1.7 (−0.1, 3.6)
0.0976 0.0215 0.4183 0.2711 0.1584 0.0006 < 0.0001 0.0027 0.6694 0.6075 0.8652 0.3378 0.0088 0.0002 0.0010 0.0147
Parameter results presented as mean ± standard deviation for normally distributed parameters and median {1st quartile, 2nd quartile} for non-normally distributed parameters (platelet count, fibrinogen level, EXT CFT, EXT alpha). Difference is Baseline minus Reinfusion, with results presented as mean (99% CI) for normally distributed parameters and median {1st quartile, 3rd quartile} for non-normally distributed. Paired t-tests used to determine statistical significance of the difference except for non-normally distributed parameters which were evaluated with Wilcoxon signed rank test. Bolded parameters indicate the difference was significant after Bonferroni correction for multiple comparisons (0.05/16 = 0.0031). MP = multiplate, ADP = adenosine stimulation, TRP = thrombin receptor peptide stimulation, AUC = area under curve given in arbitrary units, AU = aggregation units, Vel = velocity, TG = thrombin generation test, TTP = time to peak, PH = peak height given in nanomoles of thrombin (nM), ETP = endogenous thrombin potential (nM*min), EXT = ROTEM® Extem test, CT = clot time, CFT = clot formation time, MCF = maximum clot formation, FIB = ROTEM® Fibtem test.
was not found to be significant after adjusting for multiple comparisons. It seems likely this was a chance finding given the fibrinogen concentration did not change significantly. We did not find any decrement in the ability of ANH blood to generate thrombin. This was consistent when assessed by calibrated automated thrombography and point of care viscoelastic testing. In a 2003 study by Flom-Halvorsen, et al., sequestered heparinized blood (i.e. ‘hemospasia’) had no activation of thrombin after 1 h of storage as measured by thrombin-antithrombin complexes or prothrombin 1.2 [23]. Our data would indicate that collection of ANH blood into CPDA bags preserves coagulation factors at least as well, making it unlikely that activation of the coagulation cascade is the primary reason for the observed decrease in platelet response to stimulation. Our study has the limitation of not comparing the ANH blood to that of the patient at time of reinfusion. This would help answer the question of whether ANH blood offered any clinically relevant hemostatic advantages. It is likely that stimulation of platelets in vitro may not reflect their action once transfused to the patient. There are multiple confounders that happen on CPB, however, and we were focused solely on changes occurring within the collection bag. In conclusion, we examined changes in thrombin generating ability and platelet aggregation that occur in CPDA collection bags, commonly used in the contemporary practice of ANH. We found no evidence of decreased ability of ANH blood to generate thrombin after an average of almost 5 h in storage. While there was an appreciable decrease in platelet aggregation from TRAP stimulation, we did not find this with ADP stimulation. The clinical relevance of this remains to be clarified, but further research into platelet preservation seems warranted.
Table 3 Correlation of magnitude of change with time of ANH storage. Hematologic variable
Correlation of change with storage time (95% CI)
P
MP TRP AUC MP TRP Aggregation MP TRP Velocity EXTEM MCF EXTEM alpha
−0.1145 −0.1491 −0.1579 −0.2675 −0.1912
0.5738 0.4624 0.4359 0.1800 0.3427
(−0.4805, (−0.5072, (−0.5138, (−0.5933, (−0.5389,
0.2856) 0.2529) 0.24438) 0.1338) 0.2115)
Of the parameters that were found to significantly change in the ANH bag (Table 2), the correlation between magnitude of change and time in the bag was investigated. Correlation presented as Pearson correlation coefficient (95% Confidence Intervals). Calculations based upon Fischer's Z transformation. Hypothesis test is rho = 0. MP = Multiplate™, TRP = thrombin receptor peptide stimulation, AUC = area under curve. EXTEM = ROTEM™ measurement using tissue factor as an activator, MCF = maximum clot firmness, alpha = angle between center line and tangential to the curve at 2 mm amplitude.
was not correlated with storage time. This could be due to the relatively short storage time of ANH blood compared to allogeneic platelets from the blood bank. Additionally, it is possible that the volume in the collection bag, and therefore the ratio of the anticoagulant to whole blood, is more important than storage time. The collection of ANH blood was done in a pragmatic fashion; clinicians are not likely to precisely weigh the collection bags while it is being filled and this was not done during this study. It is quite probable that the citrate concentration varied among the ANH units tested, which may have affected the absolute value of the various assays. Unfortunately, our precision in measuring the volume of ANH blood collected was not high enough to assess this relationship, which represents a limitation of this study. Despite this potential inter-subject variance, the citrate concentration was not altered between collection and reinfusion, allowing us to compare changes that occurred over time in any single bag. Like platelet count, fibrinogen concentration remained relatively constant from ANH collection to reinfusion. Although EXTEM alpha angle decreased slightly, this parameter is also influenced by platelets [22], and its change is probably more of a reflection of their decreased aggregation. ROTEM FIBTEM MCF did slightly decrease, although this
Disclosures Dr. Roman Sniecinski has received research funding from Grifols. All other authors have no disclosures. Funding The work for this study was funded by an internal Emory Department of Anesthesiology Team Based Science grant. 42
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Contents lists available at ScienceDirect
Journal of Clinical Anesthesia journal homepage: www.elsevier.com/locate/jclinane
Original contribution
Platelet function, but not thrombin generation, is impaired in acute normovolemic hemodilution (ANH) blood
T
Korrin J. Scott (MD)a, , J. William Shteamer (MD)a, Fania Szlam (MMSc)b, Roman M. Sniecinski (MD, MSc)a ⁎
a b
Emory University School of Medicine, Department of Anesthesiology, United States of America Emory University School of Medicine, United States of America
ABSTRACT
Study objective: We investigated the coagulation changes that might occur in acute normovolemic hemodilution (ANH) blood over several hours during cardiac surgery requiring cardiopulmonary bypass. Design: This study was a prospective observational study. Setting: This study took place at a university teaching hospital. Patients: This study included 26 patients, either ASA 3 or 4 and without known coagulation disorders, undergoing cardiac surgery. Patients were included if the use of cardiopulmonary bypass was expected to reach 2.5 h. Interventions: ANH blood was collected into CPDA-1 collection bags before systemic heparinization. Samples were taken directly from the bags at time of collection and reinfusion to assess changes in platelet and thrombin generation parameters. Measurements: Whole blood from citrated tubes was used immediately for rotational thromboelastometry and platelet aggregometry analyses. Thrombin generation was assessed using calibrated automated thrombography with platelet poor plasma. Main results: Despite no significant change in platelet count over the ANH storage period, there was significant degradation in platelet function as measured by thrombin receptor activating peptide stimulation on Mulltiplate™ analysis and maximum clot formation on ROTEM™ EXTEM. Notably, there was no change in the ability to generate thrombin. Conclusions: Little data exists regarding the quality of coagulation factors in autologous blood. Our study confirms ANH collection results in decreased platelet aggregation with TRAP stimulation; however, this is not appreciated with ADP stimulation. Thrombin generation capacity remains preserved.
1. Introduction Depending upon procedure type, between 30% and 80% of patients undergoing cardiac surgery receive allogeneic blood transfusions [1], which are associated with worse outcomes, even when given in small quantities [2,3]. Several cardiac surgery and anesthesiology societies have recommended intraoperative acute autologous normovolemic hemodilution (ANH) as a blood conservation technique, albeit a Class IIb one [4,5]. A recent meta-analysis including almost 30 randomized controlled trials concluded that ANH in cardiac surgery reduces allogeneic transfusions by about 1 unit of red blood cells (RBCs) [6]. Beyond reducing RBC transfusions, a large observational study including over 13,000 patients undergoing cardiac surgery concluded ANH use was also associated with fewer platelet and plasma transfusions [7]. This potential impact of ANH on the administration of non-RBC components has not been thoroughly investigated. Since ANH blood has not been exposed to the cardiopulmonary bypass circuit, fibrinogen and
platelet function should theoretically be preserved. Unfortunately, factors can be consumed when the coagulation system is not adequately suppressed in the collection bag, resulting in visible clot formation (see Fig. 1). The practice of ‘hemospasia,’ or sequestering blood from the cardiopulmonary bypass (CPB) circuit for later reinfusion, has been around for decades, but relies upon heparin as the anticoagulant [8]. Heparin for ANH has been shown to impair platelet function and coagulation as assessed by viscoelastic testing [9]. However, most clinicians utilize citrate phosphate dextrose (CPD) bags with or without the addition of adenine to collect the ANH blood. We performed the current study to investigate the coagulation changes that might occur in ANH blood in modern practice, particularly when stored for the several hours required during prolonged CPB cases. We hypothesized that there would be a decrease in the thrombin generation and platelet function in ANH blood between collection and reinfusion into the patient.
Corresponding author. E-mail addresses: [email protected] (K.J. Scott), [email protected] (J.W. Shteamer), [email protected] (F. Szlam), [email protected] (R.M. Sniecinski). ⁎
https://doi.org/10.1016/j.jclinane.2019.04.032 Received 15 January 2019; Received in revised form 9 April 2019; Accepted 26 April 2019 0952-8180/ © 2019 Elsevier Inc. All rights reserved.
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were drawn just prior to reinfusion of the ANH blood after separation from CPB. Whole blood from citrated tubes was used immediately for rotational thromboelastometry and platelet aggregometry analyses. For thrombin generation measurements, samples were centrifuged for 20 min at 2000 ×g at room temperature to obtain platelet –poor plasma (PPP) and stored at −80 °C until time of batch analysis. 2.2. Standard hematologic testing Parameters of complete blood cell count (WBC, platelet count, hematocrit, hemoglobin) were measured from the samples in EDTA tubes using a Poch-100i analyzer (Sysmex America, Inc., Lincolnshire, IL). Fibrinogen concentrations were measured in thawed citrated plasma by the Clauss clotting method using STA Compact Analyzer and STA Fibrinogen reagents according to manufacturer's directions (Diagnostica Stago, Parsippany, NJ). Calibration was performed with STA-unicalibrator; ‘normal’ and ‘abnormal’ controls were tested prior to patients' plasma samples. 2.3. Rotational thromboelastometry measurements ROTEM™ device (Instrumentation Laboratory, Bedford, MA) was used for all of the viscoelastic testing measurements. EXTEM and FIBTEM testing were performed at 37 °C using 300 μl of the patient's whole blood according to manufacturer's directions. In EXTEM testing, blood is recalcified with 20 μl of start-tem reagent (0.2 M CaCl2) and clotting is activated via tissue factor (ex-tem reagent). The FIBTEM test is an EXTEM-based assay more specific for the fibrin part of the clot by inhibiting platelet contribution with cytochalasin D, albeit incompletely [10]. The following parameters of ROTEM were collected; a) Clotting time (CT; sec), which corresponds to initiation of clotting, b) clot formation time (CFT; sec), which corresponds to time from initiation of clotting until clot firmness of 20 mm is detected, c) angle (α; degrees), which reflects the rate of fibrin polymerization, and d) maximum clot firmness (MCF; mm), which reflects maximum amplitude of the tracing and the tensile strength of the clot.
Fig. 1. Photograph of a blood collection bag used during autologous normovolemic hemodilution (ANH). The arrow points toward a large clot that has formed.
2. Methods
2.4. Platelet aggregometry
2.1. Patient enrollment and sample collection
Whole blood aggregometry was evaluated using multiple electrode methodology using a Multiplate™ device (Diapharma Group Inc., West Chester, OH). The technique is based on change of resistance that is proportional to the amount of activated platelets sticking to sensor wires. This increase in resistance is recorded during the test. For each test, 300 μl of pre-warmed calcium chloride (CaCl2, 3 mM) – normal saline solution and 300 μl of citrated whole blood sample were added to the test cell. The CaCl2 present in the saline partially compensates for citrate calcium depletion by citrate, but not enough to start clotting, and it increases aggregation [11]. After 3 min of incubation at 37 °C, samples were activated with either adenosine diphosphate (ADP) or thrombin receptor activating peptide 6 (TRAP) according to the manufacturer's directions. These reagents were chosen as they have been shown to be the most clinically useful predictors of blood use in cardiac surgery patients [12]. After 6 min run time, platelet aggregations curves were assessed with the following Multiplate™ parameters; area under the curve (AUC, U), maximum aggregation (maximum amplitude of the curve, AU), and velocity (the maximum slope of the curve, AU/min) [13].
This study was approved by the Emory University Institutional Review Board. Male and female patients > 18 years old undergoing cardiac surgery at Emory University Hospital and expected to require the use of > 2.5 h of CPB were screened for participation. Exclusion criteria were emergency surgery, baseline hemoglobin < 11.0 g/dL, or patient refusal/inability to provide written informed consent. No patient was actively taking any oral anticoagulant medication, other than aspirin, within 5 days of the operation. On the day of surgery, the patient's hemoglobin was once again rechecked and confirmation that the patient could safely undergo ANH was obtained from the attending anesthesiologist. After induction of anesthesia and prior to any systemic heparin administration, ANH blood was collected into 450 ml collection bags containing citrate, phosphate, dextrose, and adenine (i.e. CPDA-1 bags) for temporary storage (Blood-Pack™, Fenwal Inc., Lake Zurich, IL). The volume collected was at the discretion of the attending anesthesiologist, as well as the amount of crystalloid fluid replacement. No colloids were used during the ANH collection phase. Collection bags were kept at room temperature in the operating room and gently agitated by hand at regular intervals per normal institutional practice. Samples were directly collected from the first bag of ANH into 10 ml buffered 3.2% sodium citrate and 2 ml spray-coated potassium EDTA (3.6 mg) B-D Vaccutainer tubes (Becton Dickinson, Franklin Lakes, NJ) at the following time points: 1) baseline samples were taken within 10 min of finishing collecting the first ANH bag, 2) reinfusion samples
2.5. Thrombin generation measurements Stored frozen plasma samples were thawed on water bath at 37 °C for 5–10 min. Thrombin generation (TG) measurements were performed at 37 °C using calibrated automated thrombinoscope methodology as first described by Hemker [14]. Briefly, 80 μl of thawed PPP was added to wells of 96 wells microtiter plate (Diagnostica Stago, 40
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Parsippany, NJ) along with 20 μl of tissue factor reagent (final concentration of 5 pM TF, 4 μM phospholipids). Wells with 20 μl of thrombin calibrator instead of TF reagent were run in parallel. After 10 min incubation, TG was initiated by an automatic addition of FluCa reagent. All TG reagents were purchased from Diagnostica Stago (Parsippany, NJ). The progress of the TG reaction was monitored using Thrombinoscope™ software (Diagnostica Stago, Parsippany, NJ). The following TG parameters were measured; ETP (nM*min) - endogenous thrombin potential- area under thrombin curve, peak thrombin (nM)maximum thrombin concentration, lag time (min) – time between addition of trigger (TF) and initiation of TG, ttp (min) time to reach the peak of TG, rate of TG (nmol/min) velocity index.
Table 1 Clinical characteristics of patients.
2.6. Statistical analysis
Patient demographics and preoperative hematological laboratory values are provided. Continuous variables given as mean ± standard deviation with the exception of platelet count, CPB bags used for collection, and ANH volume, which is median (1st quartile, 3rd quartile). ANH = autologous normovolemic hemodilution, Hgb = hemoglobin, aPTT = activated partial thromboplastin time, ASA = aspirin.
Age (years) Females, number (%) Weight (Kg) Preoperative Hgb (g/dL) Preoperative platelets (1000/μl) Preoperative INR Preoperative aPTT (seconds) Patients on ASA, number (%) Baseline ACT (seconds) Volume of ANH blood removed from patients(ml) CPB bags collected (n) Time between ANH collection and reinfusion (minutes)
In a similar study using heparin as the anticoagulant, Gallandat et al. arrived at a sample size of 23 for detecting a 25% reduction in platelet aggregation as measured by Mutiplate™ AUC using α = 0.05 level with 80% power. We targeted the same sample size as Gallandt et al. of 26 subjects. In retrospect, the mean Mutiplate™ AUC for ADP stimulation using citrate as the anticoagulant has been reported as 68.6 ± 20.1 U in normal blood [15]. Using paired t-tests and an α = 0.01 and targeting a power of 90%, 24 samples would be required to detect a 25% decrease in this same parameter. Descriptive statistics were used for all variables. Their distribution and residuals were evaluated with Q-Q plots and the KolmogorovSmirnov test. All hematologic variables were found to be normally distributed with the exception of platelet count, fibrinogen level, EXT CFT, and EXT alpha angle. Paired t-tests were used to determine statistical significance of the difference for normally distributed parameters. Non-normally distributed parameters were evaluated with Wilcoxon signed rank test. Given the multiple parameters being compared, Bonferroni correction was used to determine statistical significance (α = 0.05/16 = 0.0031). Calculations for determining the difference in parameters between baseline and reinfusion were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA). The correlation between time of storage and magnitude of change in parameters found to significantly differ between collection and reinfusion was assessed using regression plots and Pearson's correlation coefficient. The percentage change from collection to reinfusion was also calculated for those parameters found to be significantly different. These calculations were performed using SAS 9.4 (SAS Institute, Cary, NC).
57 ± 17 9 (35%) 87.6 ± 17.2 13.9 ± 1.4 196 ± 62 1.04 ± 0.10 32.8 ± 5.1 16 (62%) 105 ± 10 425 (350, 600) 1 (1, 2) 299 ± 94
statistically significant after adjusting for multiple comparisons. No parameters reflecting thrombin formation, as measured using TG testing or ROTEM® (i.e. clot time), significantly changed between collection and reinfusion of the ANH blood. When stimulated with ADP, no platelet aggregation parameters were found to significantly change either. Of the parameters that significantly changed between collection and reinfusion, the magnitude of change was not significantly correlated with time of storage (see Table 3). 4. Discussion It is widely accepted that ANH reduces the transfusion rate for allogenic RBCs, but its benefit with regard to hemostatic blood products remains unproven. Given that coagulopathy in cardiac surgery is often encountered secondary to exposure to the CPB circuit, the presumption is that blood not exposed to this insult maintains a more favorable coagulation profile. Reinfusion of this sequestered blood is postulated to improve hemostasis and further reduce the need for RBC transfusion. In a small clinical trial of patients undergoing cardiac surgery with CPB, however, Zisman et al. failed to demonstrate any benefit of ANH with respect to the administration of hemostatic blood products [16]. In fact, little data regarding the quality of coagulation factors in ANH blood exists. Our aim in this study was to look for possible degradation of coagulation parameters over time in the autologous blood under conditions that are considered standard practice for removal of ANH blood. Based upon our findings, platelet aggregation in response to some stimulants does seem to decrease in the collection bag. We found almost a 25% decrease in area under the curve using TRAP stimulation despite no appreciable decrease in platelet count. These findings are consistent with those of Gallandat et al. who reported decreased platelet aggregation in response to TRAP stimulation using heparin as the anticoagulant in ANH collection bags [9]. Unlike Gallandat et al., however, we did not appreciate a change in regard to ADP-induced aggregation. This may be due to the fact heparin has a different effect on Multiplate™ ADP results when compared to citrate [17]. Given that TRAP is a much stronger stimulant, this minor difference between anticoagulants was not likely as apparent. In an older study, Ramnarine et al. also found a decrease in platelet aggregation in CPDA bags with and without heparin [18]. However, they utilized collagen as an activator for platelet testing, as well as a gelatin solution for ANH replacement fluid, which is known to affect platelet aggregation [19]. In blood banking studies, platelet function is known to decrease with storage time [20]. Platelets obtained from whole blood in particular have been shown to have decreased responsiveness to thrombin over a period of 5 days [21]. While we saw a decrease in platelet aggregation from TRAP stimulation as well as with viscoelastic testing, it
3. Results Thirty-five patients scheduled to undergo cardiac surgery with prolonged CPB were screened and consented for the study between 12/ 29/15 and 3/5/18. Of these patients, 4 were deemed ineligible because of Hgb < 11.0 on initial OR blood gas, inadequate collected ANH volume (< 100 ml) in 2 patients, ANH blood being reinfused on CPB in 2 patients, and labs drawn into the wrong collection tubes on 1 patient. Twenty-six patients completed the protocol and were available for analysis. Their baseline demographics and standard preoperative labs are presented in Table 1. The measurements from the CPDA-1 bags at time of collection and at time of reinfusion, and their differences, are presented in Table 2. Platelet count itself did not significantly change, but Multiplate® parameters of aggregation, velocity, and AUC as stimulated by TRAP decreased by 24.9% (95% CI 12.3%, 37.4%), 17.5% (95% CI 4.1%, 31.0%), and 23.4% (95% CI 8.9%, 38.0%) respectively. Platelet-fibrinogen interaction as measured by EXTEM MCF and alpha similarly decreased by 9.8% (95% CI 5.0%, 14.4%) and 7.9% (95%CI 2.3%, 13.6%) respectively. Fibrinogen concentration and FIBTEM MCF showed very minor decreases over the storage time, but were not 41
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Table 2 Measured hemostatic variables of ANH Blood at Collection and Reinfusion. Parameter
Collection
Reinfusion
Difference
p
Platelet Count (x1000/μl) Fibrinogen Level (mg/dL) MP ADP AUC (units) MP ADP Aggregation (AU) MP ADP Velocity (AU/min) MP TRP AUC (units) MP TRP Aggregation (AU) MP TRP Vel (AU/min) TG TTP (min) TG PH (nM) TG ETP (nM*min) EXT CT (seconds) EXT CFT (seconds) EXT MCF (mm) EXT alpha (°) FIB MCF (mm)
142 {100, 157} 223 {173, 285} 11.0 ± 7.9 24.8 ± 14.0 4.7 ± 2.9 39.5 ± 18.1 65.9 ± 25.9 11.0 ± 4.9 6.5 ± 2.2 206.7 ± 73.7 1325.8 ± 262.3 87.6 ± 35.9 113 {88.5, 141.5} 61.2 ± 6.2 67.5 {65.0, 72.5} 13.5 ± 5.6
135 {91, 153} 222 {147, 274} 10.1 ± 6.3 22.6 ± 11.9 3.9 ± 1.3 30.4 ± 18.4 48.7 ± 25.4 8.9 ± 4.8 6.7 ± 2.5 201.5 ± 61.8 1320.2 ± 223.5 93.5 ± 32.3 137.5 {105.5, 166.8} 55.3 ± 8.8 63.0 {58.0, 70.0} 11.8 ± 6.0
3 {−5, 23} 7 {−4, 15} 0.9 (−2.2, 4.1) 2.2 (−3.2, 7.5) 0.8 (−0.7, 2.2) 9.1 (2.7, 15.5) 17.2 (7.0, 27.4) 2.1 (0.3, 3.9) −0.2 (−1.1, 0.7) 5.1 (−15.2, 25.4) 5.6 (−62.1, 73.4) −5.9, (−22.7, 10.9) −16.0 {−50.8, 10.3} 5.9 (2.2, 9.7) 3.5 {−0.3, 8.5} 1.7 (−0.1, 3.6)
0.0976 0.0215 0.4183 0.2711 0.1584 0.0006 < 0.0001 0.0027 0.6694 0.6075 0.8652 0.3378 0.0088 0.0002 0.0010 0.0147
Parameter results presented as mean ± standard deviation for normally distributed parameters and median {1st quartile, 2nd quartile} for non-normally distributed parameters (platelet count, fibrinogen level, EXT CFT, EXT alpha). Difference is Baseline minus Reinfusion, with results presented as mean (99% CI) for normally distributed parameters and median {1st quartile, 3rd quartile} for non-normally distributed. Paired t-tests used to determine statistical significance of the difference except for non-normally distributed parameters which were evaluated with Wilcoxon signed rank test. Bolded parameters indicate the difference was significant after Bonferroni correction for multiple comparisons (0.05/16 = 0.0031). MP = multiplate, ADP = adenosine stimulation, TRP = thrombin receptor peptide stimulation, AUC = area under curve given in arbitrary units, AU = aggregation units, Vel = velocity, TG = thrombin generation test, TTP = time to peak, PH = peak height given in nanomoles of thrombin (nM), ETP = endogenous thrombin potential (nM*min), EXT = ROTEM® Extem test, CT = clot time, CFT = clot formation time, MCF = maximum clot formation, FIB = ROTEM® Fibtem test.
was not found to be significant after adjusting for multiple comparisons. It seems likely this was a chance finding given the fibrinogen concentration did not change significantly. We did not find any decrement in the ability of ANH blood to generate thrombin. This was consistent when assessed by calibrated automated thrombography and point of care viscoelastic testing. In a 2003 study by Flom-Halvorsen, et al., sequestered heparinized blood (i.e. ‘hemospasia’) had no activation of thrombin after 1 h of storage as measured by thrombin-antithrombin complexes or prothrombin 1.2 [23]. Our data would indicate that collection of ANH blood into CPDA bags preserves coagulation factors at least as well, making it unlikely that activation of the coagulation cascade is the primary reason for the observed decrease in platelet response to stimulation. Our study has the limitation of not comparing the ANH blood to that of the patient at time of reinfusion. This would help answer the question of whether ANH blood offered any clinically relevant hemostatic advantages. It is likely that stimulation of platelets in vitro may not reflect their action once transfused to the patient. There are multiple confounders that happen on CPB, however, and we were focused solely on changes occurring within the collection bag. In conclusion, we examined changes in thrombin generating ability and platelet aggregation that occur in CPDA collection bags, commonly used in the contemporary practice of ANH. We found no evidence of decreased ability of ANH blood to generate thrombin after an average of almost 5 h in storage. While there was an appreciable decrease in platelet aggregation from TRAP stimulation, we did not find this with ADP stimulation. The clinical relevance of this remains to be clarified, but further research into platelet preservation seems warranted.
Table 3 Correlation of magnitude of change with time of ANH storage. Hematologic variable
Correlation of change with storage time (95% CI)
P
MP TRP AUC MP TRP Aggregation MP TRP Velocity EXTEM MCF EXTEM alpha
−0.1145 −0.1491 −0.1579 −0.2675 −0.1912
0.5738 0.4624 0.4359 0.1800 0.3427
(−0.4805, (−0.5072, (−0.5138, (−0.5933, (−0.5389,
0.2856) 0.2529) 0.24438) 0.1338) 0.2115)
Of the parameters that were found to significantly change in the ANH bag (Table 2), the correlation between magnitude of change and time in the bag was investigated. Correlation presented as Pearson correlation coefficient (95% Confidence Intervals). Calculations based upon Fischer's Z transformation. Hypothesis test is rho = 0. MP = Multiplate™, TRP = thrombin receptor peptide stimulation, AUC = area under curve. EXTEM = ROTEM™ measurement using tissue factor as an activator, MCF = maximum clot firmness, alpha = angle between center line and tangential to the curve at 2 mm amplitude.
was not correlated with storage time. This could be due to the relatively short storage time of ANH blood compared to allogeneic platelets from the blood bank. Additionally, it is possible that the volume in the collection bag, and therefore the ratio of the anticoagulant to whole blood, is more important than storage time. The collection of ANH blood was done in a pragmatic fashion; clinicians are not likely to precisely weigh the collection bags while it is being filled and this was not done during this study. It is quite probable that the citrate concentration varied among the ANH units tested, which may have affected the absolute value of the various assays. Unfortunately, our precision in measuring the volume of ANH blood collected was not high enough to assess this relationship, which represents a limitation of this study. Despite this potential inter-subject variance, the citrate concentration was not altered between collection and reinfusion, allowing us to compare changes that occurred over time in any single bag. Like platelet count, fibrinogen concentration remained relatively constant from ANH collection to reinfusion. Although EXTEM alpha angle decreased slightly, this parameter is also influenced by platelets [22], and its change is probably more of a reflection of their decreased aggregation. ROTEM FIBTEM MCF did slightly decrease, although this
Disclosures Dr. Roman Sniecinski has received research funding from Grifols. All other authors have no disclosures. Funding The work for this study was funded by an internal Emory Department of Anesthesiology Team Based Science grant. 42
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