Advertisement

Transfusion ratios for postpartum hemodilutional coagulopathy: an in vitro thromboelastographic model

Published:November 21, 2013DOI:https://doi.org/10.1016/j.ajog.2013.11.029

      Objective

      A 1:1:1 ratio of packed red blood cells (PRBC), fresh frozen plasma (FFP), and platelets (PLT) has been advocated for trauma hemorrhage, but the effectiveness of this ratio for postpartum hemorrhage is unknown. We created an in vitro hemodilutional model to investigate this strategy.

      Study Design

      Blood from 20 parturients at term was diluted 50% with 0.9% normal saline. Diluted samples were reconstituted with 1:1 PRBC:FFP or 3:1 PRBC:FFP. In 10 samples, PLT were also added. Baseline, diluted, and reconstituted sample thromboelastographic values were compared.

      Results

      Maximum amplitude (MA) was lower compared to baseline values in both groups after 50% dilution with normal saline (P < .001) and remained lower than baseline despite reconstitution with 3:1:0 or 1:1:0 PRBC:FFP:PLT (P < .0001) or 3:1:1 PRBC:FFP:PLT (P < .01). MA approached baseline (P = not significant) in the samples with 1:1:1 PRBC:FFP:PLT.

      Conclusion

      The addition of PLT to 1:1 PRBC:FFP optimized MA in this in vitro hemodilutional model of postpartum hemorrhage.

      Key words

      For Editors’ Commentary, see Contents
      Postpartum hemorrhage (PPH) has been defined as blood loss >500 mL within 24 hours of vaginal delivery, >1000 mL after cesarean delivery,
      • Berg C.J.
      • Atrash H.K.
      • Koonin L.M.
      • Tucker M.
      Pregnancy-related mortality in the United States, 1987-1990.
      • McClure J.H.
      • Cooper G.M.
      • Clutton-Brock T.H.
      Center for Maternal and Child Enquiries
      Saving mothers' lives: reviewing maternal deaths to make motherhood safer: 2006-8: a review.
      change in hematocrit of >10%, or need for red blood cell transfusion.

      Arulkumaran S, Mavrides E, Penney GC, et al. Prevention and management of postpartum hemorrhage. Royal College of Obstetricians and Gynaecologists Green-top Guideline no. 52, May 2009. Minor revision Nov 2009 and April 2011.

      • Combs C.A.
      • Murphy E.L.
      • Laros Jr., R.K.
      Factors associated with postpartum hemorrhage with vaginal birth.
      • Combs C.A.
      • Murphy E.L.
      • Laros Jr., R.K.
      Factors associated with hemorrhage in cesarean deliveries.
      The incidence of PPH is increasing in many high-resource countries
      • Cameron C.A.
      • Roberts C.L.
      • Olive E.C.
      • Ford J.B.
      • Fischer W.E.
      Trends in postpartum hemorrhage.
      • Knight M.
      • Callaghan W.M.
      • Berg C.
      • et al.
      Trends in postpartum hemorrhage in high resource countries: a review and recommendations from the international postpartum hemorrhage collaborative group.
      • Bateman B.T.
      • Berman M.F.
      • Riley L.
      • Leffert L.
      The epidemiology of post partum hemorrhage in a large, nationwide sample of deliveries.
      ; PPH increased from 2.3-2.9% (26%) in the United States from 1994 through 2006.
      • Bateman B.T.
      • Berman M.F.
      • Riley L.
      • Leffert L.
      The epidemiology of post partum hemorrhage in a large, nationwide sample of deliveries.
      • Callaghan W.M.
      • Kuklina E.V.
      • Berg C.J.
      Trends in postpartum hemorrhage: United States, 1994-2006.
      Major obstetric hemorrhage, defined as blood loss of at least 2500 mL, transfusion of ≥5 U of blood, or transfusion of fresh frozen plasma (FFP), cryoprecipitate, or platelets (PLT), complicated 3.7 per 1000 births in the United Kingdom from 2005 through 2008.
      • Cantwell R.
      • Clutton-Brock T.
      • Cooper G.
      • Dawson A.
      • Drife J.
      • Garrod D.
      • et al.
      Saving mothers' lives: reviewing maternal deaths to make motherhood safer: 2006-2008. The eighth report of the confidential enquiries into maternal deaths in the United Kingdom.
      • Brace V.
      • Kernaghan D.
      • Penney G.
      Learning from adverse clinical outcomes: major obstetric hemorrhage in Scotland, 2003-05.
      PPH remains a common and increasing source of maternal morbidity and mortality worldwide, accounting for approximately one-quarter of maternal deaths per year.

      Department of Reproductive Health and Research, WHO. WHO recommendations for the prevention and treatment of postpartum hemorrhage. Geneva: Word Health Organization; 2012:3.

      Transfusion recommendations for major hemorrhage, defined as >10 U of packed red blood cells (PRBC) in 24 hours, advocate more liberal use of FFP and PLT.
      • Fries D.
      • Innerhofer D.
      • Shobersberger W.
      Time for changing coagulation management in trauma-related massive bleeding.
      An analysis of 246 soldiers suffering mass casualties in Iraq revealed a significant decrease in mortality when large amounts of FFP were given,
      • Borgman M.A.
      • Spinella P.C.
      • Perkins J.G.
      • et al.
      The ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital.
      and transfusion of 1:1:1 PRBC:FFP:PLT for massive trauma resuscitation improved survival.
      • Holcomb J.B.
      • Zarzabal L.A.
      • Michalek J.E.
      • et al.
      Increased platelet: RBC ratios are associated with improved survival after massive transfusion.
      Because obstetric hemorrhage may be similar to trauma hemorrhage in regards to rapid, unanticipated blood loss and risk of hemodilutional coagulopathy, some centers have empirically implemented transfusion protocols for PPH that utilize a high FFP to PRBC ratio in addition to PLT transfusion.
      • Burtelow M.
      • Riley E.
      • Druzin M.
      • Fontaine M.
      • Viele M.
      • Goodnough L.T.
      How we treat: management of life-threatening primary postpartum hemorrhage with a standardized massive transfusion protocol.
      The use of point-of-care testing such as thromboelastography (TEG) for guiding hemostatic therapy during PPH has also been advocated.
      • Solomon C.
      • Collis R.E.
      • Collins P.W.
      Hemostatic monitoring during postpartum hemorrhage and implications for management.
      The effectiveness of a 1:1:1 ratio of PRBC:FFP:PLT for obstetric hemorrhage is unknown.
      We designed an in vitro model of obstetric hemodilutional coagulopathy using TEG to study the efficacy of a 1:1 PRBC:FFP transfusion ratio compared to the traditional 3:1 PRBC:FFP ratio. The effect of adding PLT in vitro to either 1:1 or 3:1 PRBC:FFP samples was also evaluated.

      Materials and Methods

      After institutional review board approval and written informed consent, 20 healthy parturients aged 18-40 years with uncomplicated pregnancies at term gestation (37-41 weeks) presenting in early labor were recruited. Exclusion criteria included a history of hypertension, preeclampsia, gestational diabetes, diabetes mellitus, preexisting coagulopathy, history of deep vein thrombosis or pulmonary embolism, or use of medications that enhanced or impaired coagulation. Women in active labor or who were receiving intravenous fluid, oxytocin, prostaglandin therapy, or epidural analgesia at the time of consent and blood draw were also excluded. A single blood sample was collected from participants within an hour of consent, at least 3 hours prior to delivery. No patients enrolled in this study experienced PPH coincidental to study participation.
      Two sets of 10 subjects were recruited to participate in this study (groups 1 and 2). Blood was obtained for complete blood cell count and TEG studies at the time of venipuncture and insertion of an 18-gauge intravenous catheter. The first 2 mL of blood was discarded to avoid tissue contamination, then venous blood was collected into 4 citrated Vacutainers (Becton Dickinson, Franklin Lakes, NJ), each with a maximum capacity of 2.7 mL of blood and containing 0.5 mL 0.109 molar, 3.2% sodium citrate. Citrated blood from each Vacutainer from a single patient was pooled to eliminate variability in citrate concentration between samples.
      Three physicians (M.K.F., N.S., and B.S.K.) trained to perform TEG processed all samples. Two Haemoscope dual-channel TEG analyzers (model 5000; Haemoscope Corp, Niles, IL) with 4 channels and disposable plastic cups and pins were used for this study. Analyzers were calibrated daily for quality control as per manufacturer guidelines. For analysis using TEG, 1 mL of whole blood was added to a vial of standardized kaolin for clot activation. After mixing by gentle inversion, 340 μL kaolin-activated whole blood was immediately added to a TEG analyzer cup prewarmed to 37°C and containing 20 μL of 0.2 mol/L calcium chloride for citrate reversal.
      TEG is a real-time monitor of whole blood coagulation that measures viscoelastic changes in the blood during normal and abnormal clot formation and fibrinolysis (Figure 1).
      • Srinivasa V.
      • Gilbertson L.I.
      • Bhavani-Shankar K.
      Thromboelastography: where is it and where is it heading?.
      TEG demonstrates initial fibrin formation, clot formation rate, clot strengthening, and eventual clot lysis (Figure 2). Standard TEG parameters were analyzed in terms of reaction (R) time (minutes), K time (minutes), α angle (degrees), and maximum amplitude (MA; mm). R time is the period of time from when blood is placed in the TEG until initial fibrin formation, detected as 2 mm in amplitude above baseline on the TEG tracing. The R time (normal range, 4–8 minutes) represents clotting factor function and is prolonged by anticoagulants and shortened in hypercoagulable conditions. The K time is measured from R time until a standardized level of clot firmness (20 mm amplitude on the TEG tracing; normal range, 1–4 minutes) is achieved, and represents speed of clot strengthening. K time is shortened by an increased fibrinogen level, to a lesser extent by PLT function, and is prolonged by anticoagulants. The α angle measures the slope of the TEG tracing from R time to K time and inversely correlates with K time, with a larger α angle reflecting enhanced fibrin deposition and strength (normal range, 47–74 degrees). The MA reflects overall clot strength determined by fibrin and PLT function, with a normal range of 55-73 mm amplitude on the TEG tracing.
      • Srinivasa V.
      • Gilbertson L.I.
      • Bhavani-Shankar K.
      Thromboelastography: where is it and where is it heading?.
      • Butwick A.
      • Ting V.
      • Ralls L.A.
      • Harter S.
      • Riley E.
      The association between thromboelastographic parameters and total estimated blood loss in patients undergoing elective cesarean delivery.
      Figure thumbnail gr1
      Figure 1Thromboelastography sample cup design
      Reprinted with permission from Wolters Kluwer Health.
      Pin attached to torsion wire is suspended in cup containing whole blood (0.36 mL); cup oscillates at rate of 4.45 degrees every 10 seconds (4°45'). As clot starts to form, torsion on pin is converted by electrical transducer to signal that is recorded by computer over time.
      Figure thumbnail gr2
      Figure 2Thromboelastography tracing parameters
      Reprinted with permission from Wolters Kluwer Health.
      Standard TEG parameters were analyzed, including reaction (R) time (minutes), K time (minutes; not shown), α angle (degrees), and maximum amplitude (MA; mm).
      TEG, thromboelastography.
      For each patient in group 1, 4 samples were created from the citrated pooled whole blood sample as follows: (1) control: 1 mL whole blood; (2) hemodiluted: 8 mL whole blood + 8 mL 0.9% normal saline; (3) 1:1:0 PRBC:FFP:PLT: reconstitution of diluted sample with PRBC, FFP, and PLT in a ratio of 1:1:0 (4 mL diluted blood + 2 mL PRBC + 2 mL FFP; no PLT added); and (4) 3:1:0 PRBC:FFP:PLT: reconstitution of diluted sample with PRBC, FFP, and PLT in a ratio of 3:1:0 (4 mL diluted blood + 3 mL PRBC + 1 mL FFP; no PLT added).
      In all, 1 mL of each of these samples was analyzed using TEG within 30 minutes of collection. A PLT count and hematocrit were measured from the control and reconstituted samples by our institution's hematology laboratory (Sysmex XE 5000; Sysmex Corp, Hyogo, Japan).
      The blood utilized for reconstitution was obtained from the institution's blood bank. The PRBC (type O, antibody negative) was stored at 4°C and used within 2 weeks of expiration. The FFP (type O) was stored at –20°C. The FFP was thawed and utilized within 2 hours for this study.
      In group 2, the same methodology was used to obtain the baseline, diluted, and reconstituted samples, with the addition of PLT to the reconstituted samples. PLT were added in a volume of 1.2 mL to the reconstituted samples. The amount of PLT added was predetermined to approximate a PLT count of 100,000/mm−3 based on pilot testing of PLT dilutions and resulting laboratory measurements. PLT aliquots were obtained by apheresis from healthy donors and were provided by our institution's blood bank. Reconstituted samples will be referred to as follows: group 1, PRBC:FFP:PLT = 1:1:0 and 3:1:0; group 2, PRBC:FFP:PLT = 1:1:1 and 3:1:1.
      Statistical analysis of TEG and laboratory data was performed using software (SAS, version 9.3; SAS Institute, Cary, NC). Repeated measures analysis of variance with a mixed model approach was used to analyze outcomes. Bonferroni adjusted pairwise comparisons (to control for familywise error rate) was performed to examine the effect of different tests on TEG parameters. P < .05 was used to indicate statistical significance. All analyses were 2-tailed.
      A power calculation based on the comparison of MA between the 2 groups for 1:1:0 and 3:1:0 was performed. The criterion for significance (α) was set at .05. The test was 2-tailed. With the proposed sample size of 10 and 10 for the 2 groups, the study would have a power of 99% to yield a statistically significant result. This computation assumes that the mean difference between MA is 12 mm and the common within-group SD is 2.85 mm. A 12-mm decrease in MA for the power calculation was based on a study in which this degree of reduction in MA was associated with significant bleeding after cardiac surgery.
      • Welsby I.J.
      • Jiao K.
      • Ortel T.L.
      • et al.
      The kaolin-activated Thromboelastograph predicts bleeding after cardiac surgery.
      Furthermore, a 12-mm decrease in MA (20-25% decrease) will result in MA values below normal range of MA in healthy pregnant subjects at term gestation (66.7–70.3 mm).
      • Karlsson O.
      • Sporrong T.
      • Hillarp A.
      • Jeppsson A.
      • Hellgren M.
      Prospective longitudinal study of thromboelastography and standard hemostatic laboratory tests in healthy women during normal pregnancy.
      A sample size of 10 per group was based on a previous in vitro hemodilutional rotational thromboelastometry (ROTEM) study in which 8 patient samples were used to demonstrate significantly impaired hemostasis after 60% dilution with normal saline 0.9%.
      • De Lorenzo C.
      • Calatzis A.
      • Welsch U.
      • Heindl B.
      Fibrinogen concentrate reverses dilutional coagulopathy induced in vitro by saline but not by hydroxyethyl starch 6%.

      Results

      Demographic and obstetric data are presented in Table 1. Results of TEG analysis are shown in Table 2 for group 1 and group 2.
      Table 1Maternal demographic and obstetric data
      Demographicn = 20
      Age, y32 (6)
      Weight, kg85 (17)
      Gestational age, wk39.3 (1.1)
      Parity0 (0–4)
      Ethnicity
       Non-Hispanic Caucasian12 (60)
       Hispanic6 (30)
       African American1 (5)
       Asian American1 (5)
      Age, weight, and gestational age are presented as mean (SD); parity is presented as median (range); ethnicity is presented as n (%).
      Farber. An in vitro thromboelastographic model for postpartum hemodilutional coagulopathy. Am J Obstet Gynecol 2014.
      Table 2Thromboelastographic and hematologic results
      GroupSample description
      Baseline50% dilution3:1:0
      3 packed red blood cells:1 packed red blood cells:0 platelets
      1:1:0
      1 packed red blood cells:1 fresh frozen plasma:1 platelets
      Group 1: no added platelets
       R time, min6.1 (1.2)5.9 (1.2)5.7 (1.5)5.1 (1.4)
      P < .01 compared to baseline
       K time, min1.4 (0.2)1.8 (0.3)2.9 (1.1)
      P < .001 compared to baseline
      2.4 (0.8)
      P < .001 compared to baseline
       α angle, degrees67.7 (6.9)64.3 (3.6)54.0 (8.9)
      P < .001 compared to baseline
      60.1 (7.3)
      P < .001 compared to baseline
       MA, mm68.7 (5.8)57.5 (4.2)
      P < .001 compared to baseline
      49.3 (5.0)
      P < .001 compared to baseline
      51.0 (4.2)
      P < .001 compared to baseline
       Hct, %34.5 (2.1)27.7 (3.1)
      P < .01 compared to baseline
      22.7 (2.4)
      P < .01 compared to baseline
       Plt, 1000 μL−1237 (60)61 (19)
      P < .001 compared to baseline
      68 (16)
      P < .001 compared to baseline
      Group 2: platelets added to 3:1 and 1:1 samples
       R time, min5.8 (1.3)6.0 (0.7)5.1 (1.0)4.8 (0.6)
      P < .01 compared to baseline
       K time, min1.4 (0.3)1.6 (0.3)1.2 (0.2)
      P < .001 compared to baseline
      P < .001 compared to values in group 1.
      1.1 (0.2)
      P < .001 compared to baseline
      P < .001 compared to values in group 1.
       α angle, degrees65.8 (8.2)66.7 (3.6)70.8 (4.1)
      P < .001 compared to values in group 1.
      72.6 (6.1)
      P < .001 compared to baseline
      P < .001 compared to values in group 1.
       MA, mm67.5 (4.8)59.3 (4.2)
      P < .01 compared to baseline
      61.2 (2.7)
      P < .01 compared to baseline
      P < .001 compared to values in group 1.
      63.2 (2.5)
      P < .001 compared to values in group 1.
       Hct, %34.5 (2.9)24.0 (1.8)
      P < .01 compared to baseline
      20.1 (2.6)
      P < .01 compared to baseline
       Plt, 1000 μL−1256 (65)189 (34)
      P < .001 compared to values in group 1.
      202 (52)
      P < .001 compared to values in group 1.
      Data presented as mean (SD).
      α angle, clot formation rate; Hct, hematocrit; K, clot formation; MA, maximum amplitude; Plt, platelet count; R, reaction.
      Farber. An in vitro thromboelastographic model for postpartum hemodilutional coagulopathy. Am J Obstet Gynecol 2014.
      a 3 packed red blood cells:1 packed red blood cells:0 platelets
      b 1 packed red blood cells:1 fresh frozen plasma:1 platelets
      c P < .01 compared to baseline
      d P < .001 compared to baseline
      e P < .001 compared to values in group 1.

      Baseline and hemodiluted sample comparison

      Fifty percent hemodilution of samples in groups 1 and 2 significantly decreased MA (reflecting a decrease in clot strength) compared to baseline MA (Table 2) (group 1: 68.7-57.5 mm; P < .0001; group 2: 67.5-59.3 mm; P = .0015). Baseline MA values in group 1 were not significantly different from baseline MA values in group 2, and diluted MA values in both groups were also similar (P = .541 and .363, respectively). Hemodilution had no effect on R time (time to initial clot formation), K time, or α angle (speed of clot formation). Of note, hematocrit and PLT count were not performed in hemodiluted samples for comparison to baseline values.

      Effect of reconstitution on R time: time to initial clot formation

      R time, time to initial clot formation, was not significantly longer in diluted samples compared to baseline samples in groups 1 or 2 (P = .691 and .372, respectively). R time decreased significantly in both samples reconstituted with 1:1:0 and 1:1:1 (P = .002, P = .002).

      Effect of reconstitution on K time and α angle: rate of clot formation

      K time, reflecting rate of clot formation, was prolonged in the samples reconstituted without PLT (1:1:0 and 3:1:0) compared to baseline samples. In contrast, K time decreased in the samples reconstituted with PLT (1:1:1, 3:1:1) and was significantly shorter compared to the respective reconstituted samples without PLT (1:1:0 and 3:1:0). This demonstrates a faster rate of clot formation in the samples reconstituted with PLT.
      In group 1, α angle in both 1:1:0 and 3:1:0 PRBC:FFP:PLT decreased further from diluted samples and was significantly lower than baseline α angle (P < .001 and P < .001, respectively). In group 2, reconstitution with both 1:1:1 and 3:1:1 PRBC:FFP:PLT increased the α angle (P = .003 and P = .030, respectively). Along with a decrease in K time, the increase in α angle is consistent with a faster rate of clot formation in the samples reconstituted with PLT.

      Effect of reconstitution on PLT and MA: clot strength

      In group 1, the PLT count was significantly lower in both the 1:1:0 and the 3:1:0 combinations compared to baseline PLT count (P < .001 and P < .001, respectively). In group 2, the PLT count increased toward baseline values in both reconstituted samples.
      In group 1, MA remained significantly lower than baseline MA despite reconstitution with either a 1:1:0 or 3:1:0 ratio (Table 2) (P < .0001 and P < .0001, respectively). MA was lower in the samples reconstituted with either 1:1:0 or 3:1:0 compared to diluted MA as well (P < .0001 and P < .0001, respectively). With the addition of PLT (group 2), MA increased significantly in the 1:1:1 samples compared to diluted samples (from 59.3-63.2 mm, Table 2) (P = .043), approaching baseline MA (P = not significant from baseline). In 3:1:1 samples, the increase in MA (from 59.3-61.2) is not statistically significant in comparison to diluted samples (P = .307) and MA in this group remained significantly lower than baseline MA (P < .0001).
      Although there was no significant difference between TEG variables in baseline or diluted samples in groups 1 and 2, the α angle and MA of group 2 samples reconstituted with 1:1:1 or 3:1:1 were significantly higher than corresponding values in group 1, 1:1:0 and 3:1:0 (P < .0025 and P < .001, respectively). In addition, K times in the reconstituted samples of group 2 were significantly lower than respective K times in group 1 (P = .0003).

      Comment

      Emerging transfusion protocols for massive hemorrhage after trauma recommend 1:1:1 PRBC:FFP:PLT.
      • Fries D.
      • Innerhofer D.
      • Shobersberger W.
      Time for changing coagulation management in trauma-related massive bleeding.
      • Borgman M.A.
      • Spinella P.C.
      • Perkins J.G.
      • et al.
      The ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital.
      • Holcomb J.B.
      • Zarzabal L.A.
      • Michalek J.E.
      • et al.
      Increased platelet: RBC ratios are associated with improved survival after massive transfusion.
      Patients who received higher ratios of PLT:PRBC after trauma and massive hemorrhage had decreased blood loss, improved survival, and decreased ventilator days.
      • Holcomb J.B.
      • Zarzabal L.A.
      • Michalek J.E.
      • et al.
      Increased platelet: RBC ratios are associated with improved survival after massive transfusion.
      The optimal ratio of blood components for massive PPH has not been determined. The obstetric patient is profoundly different from a nonpregnant patient; marked plasma volume expansion with a lesser increase in hemoglobin leads to a hemodilutional anemia at baseline. Incidental thrombocytopenia occurs in 7.6% of women in the third trimester of gestation and is attributed to expansion of maternal plasma volume as well as PLT consumption in the uteroplacental unit.
      • Burrows R.F.
      • Kelton J.G.
      Thrombocytopenia at delivery: a prospective survey of 6715 deliveries.
      Many procoagulant factors increase in pregnancy, while anticoagulant factors such as proteins C and S decrease. Hypofibrinolysis occurs due to increased plasminogen activator inhibitor. Based on these differences, it is prudent to determine whether PRBC:FFP:PLT ratios optimal for trauma hemorrhage are applicable to the management of PPH. Our study suggests that the coagulation defects created by the hemodilution of blood from pregnant patients at term can best be corrected toward baseline values with PRBC and FFP in the ratio of 1:1 and the addition of PLT.
      Hemodilution 50% by volume with crystalloid solution significantly decreased MA, reflecting decreased clot strength determined largely by PLT and to a lesser extent by fibrinogen activity. None of the other TEG parameters analyzed, including R time (time to initial clot formation; determined largely by procoagulant factor activity), K time, or α angle (rate of clot strengthening, both determined largely by fibrinogen effectiveness) were altered by hemodilution in this model, suggesting that 50% crystalloid dilution of blood from pregnant patients at term gestation does not decrease clotting factors or fibrinogen levels sufficiently enough to alter these TEG variables. This is in contrast to previous in vitro models unrelated to pregnancy where fibrinogen was the first factor to become critically low.
      • De Lorenzo C.
      • Calatzis A.
      • Welsch U.
      • Heindl B.
      Fibrinogen concentrate reverses dilutional coagulopathy induced in vitro by saline but not by hydroxyethyl starch 6%.
      • Fries D.
      • Innerhofer P.
      • Reif C.
      • et al.
      The effect of fibrinogen substitution on reversal of dilutional coagulopathy: an in vitro model.
      • Haas T.
      • Fries D.
      • Velik-Salchner C.
      • Reif C.
      • Klingler A.
      • Innerhofer P.
      The in vitro effects of fibrinogen concentrate, factor XIII and fresh frozen plasma on impaired clot formation after 60% dilution.
      The difference may be due to pregnancy-induced increases in the baseline fibrinogen levels (100%) and other clotting factors (20-100%) in this study compared to studies in nonpregnant patients.
      • Hui C.
      • Lili M.
      • Libin C.
      • et al.
      Changes in coagulation and hemodynamics during pregnancy: a prospective longitudinal study of 58 cases.
      • Stirling Y.
      • Woolf L.
      • North W.R.
      • Seghatchian M.J.
      • Meade T.W.
      Hemostasis in normal pregnancy.
      Fibrinogen is a prerequisite for clot formation and is the first hemostatic component that declines following trauma and hemodilution in nonpregnant subjects.
      • Hiipala S.T.
      • Myllyla G.J.
      • Vahtera E.M.
      Hemostatic factors and replacement of major blood loss with plasma-poor red cell concentrates.
      • Singbartl K.
      • Innerhofer P.
      • Radvan J.
      • et al.
      Hemostasis and hemodilution: a quantitative mathematical guide for clinical practice.
      A decrease in MA with no impact on α angle or K time suggests that pregnant women may be more vulnerable to decreased PLT concentration than from decreased clotting factors and fibrinogen when hemodilution occurs. This inference is further strengthened by the TEG results in reconstituted samples in our study. There was no significant improvement in MA after reconstitution with 3:1:0 or 1:1:0 PRBC:FFP:PLT; the efficacy of the 1:1 PRBC:FFP ratio occurred only after PLT were added. This in vitro model suggests that PLT contribution may be as vital as liberal plasma for restoring clot strength after hemodilution during PPH.
      A significantly higher α angle and shorter K time in the 1:1:1 PRBC:FFP:PLT samples was observed, representing enhanced kinetics of clot formation. Alpha angle and K time are reciprocally related and reflect fibrinogen-PLT interaction during clot formation. Alpha angle is an indirect measure of fibrinogen, and faster clot strengthening kinetics mediated by enhanced fibrinogen and PLT interactions are reflected by a larger α angle and a shorter K time. The most superior clot strengthening kinetics in this in vitro study, reflected by a larger α angle and shorter K time, were seen in the reconstituted samples with 1:1:1 PRBC:FFP:PLT. The addition of PLT improved clot strength (MA) in both 1:1:1 and 3:1:1 samples (61.2 vs 63.2), a between-group difference that is likely to have no clinical relevance. However, the addition of PLT in the 1:1:1 samples also enhanced the rate of clot strengthening, yielding a shorter K time and larger α angle. Taken together, the TEG results of 1:1 samples with PLT reflect superior global coagulation compared to other samples.
      To our knowledge, this is the first study to evaluate the effects of transfusion ratios using an in vitro obstetric hemodilutional model. The findings of our study suggest superiority of a 1:1 ratio of PRBC: FFP over a 3:1 PRBC:FFP ratio and the potential benefit of PLT in this setting. In our in vitro model, a PLT count of 50,000/mm−3 was found to be inadequate despite a 1:1 PRBC:FFP ratio of blood products added to the diluted sample. The significance of PLT in PPH has been suggested in vivo: women with a PLT count of <100,000/mm3 or fibrinogen concentration of <2.9 g/L−1 during labor had an increased incidence of PPH (odds ratio, 19.7).
      • Simon L.
      • Santi T.M.
      • Sacquin P.
      • Hamza J.
      Pre-anesthetic assessment of coagulation abnormalities in obstetric patients: usefulness, timing, and clinical implications.
      Although our study cannot define the minimum PLT count needed for optimal coagulation, the range of PLT counts in the 1:1:1 PRBC:FFP:PLT ranged from 112,000–255,000/mm−3. Further evaluation of PLT count and qualitative analysis of clot strength using TEG during in vivo PPH may define the optimal threshold for PLT transfusion in PPH. A retrospective evaluation of severe PPH utilizing high FFP:PRBC transfusion ratios showed lower odds for requiring interventional procedures compared to higher PRBC:FFP ratios.
      • Pasquier P.
      • Gayat E.
      • Rackelboom T.
      • et al.
      An observational study of the fresh frozen plasma: red blood cell ratio in postpartum hemorrhage.
      The impact that PLT may have had on the study results cannot be defined due to the retrospective nature of the study and changing transfusion protocols over the study duration.
      • Pasquier P.
      • Gayat E.
      • Rackelboom T.
      • et al.
      An observational study of the fresh frozen plasma: red blood cell ratio in postpartum hemorrhage.
      We utilized TEG in our study, although investigators have historically relied on prothrombin time, activated partial thromboplastin time, PLT count, and fibrinogen levels to guide transfusion therapy. Unlike TEG, prothrombin time, activated partial thromboplastin time, PLT count, and fibrinogen levels are static tests of coagulation that do not reveal the qualitative effect that PLT have on global hemostasis. TEG and ROTEM can facilitate resuscitation in trauma patients by providing a global, dynamic assessment of coagulation during severe hemorrhage,
      • Cushing M.
      • Shaz B.H.
      Blood transfusion in trauma patients: unresolved questions.
      and are increasingly advocated for the identification and management of hypocoagulability and hyperfibrinolysis that can occur in major trauma.
      • Johansson P.I.
      • Stensballe J.
      • Vindelov N.
      • Perner A.
      • Espersen K.
      Thomboelastography and thromboelastometry in assessing coagulopathy in trauma.
      • Kashuk J.L.
      • Moore E.E.
      • Wohlauer M.
      • et al.
      Initial experiences with point-of-care rapid thrombelastography for management of life-threatening post-injury.
      • Johansson P.I.
      • Ostrowski S.R.
      • Secher N.H.
      Management of major blood loss: an update.
      TEG and ROTEM point-of-care tests offer the potential of rapid diagnosis and correction of coagulopathic bleeding during PPH.
      • Solomon C.
      • Collis R.E.
      • Collins P.W.
      Hemostatic monitoring during postpartum hemorrhage and implications for management.
      Based on the finding that early fibrinolysis in the setting of PPH is associated with subsequent severe hemorrhage,
      • Charbit B.
      • Mandelbrot L.
      • Samain E.
      • et al.
      PPH Study Group
      The decrease of fibrinogen is an early predictor of the severity of postpartum hemorrhage.
      a randomized trial using fibrinogen concentrate and hemostatic response using TEG monitoring during PPH is currently underway.
      • Wikkelsoe A.J.
      • Afshari A.
      • Stensballe J.
      • et al.
      The FIB-PPH trial: fibrinogen concentrate as initial treatment for postpartum hemorrhage: study protocol for a randomized controlled trial.
      Standard reference ranges for TEG at term gestation and a transfusion algorithm that incorporates TEG values to guide transfusion has been published,
      • Hill J.S.
      • Devenie G.
      • Powell M.
      Point-of-care testing of coagulation and fibrinolytic status during postpartum hemorrhage: developing a thromboelastography®-guided transfusion algorithm.
      further facilitating the use of TEG for PPH management.
      A major limitation of this study is that it is an in vitro model. The purpose of our design was to mimic the known hypercoagulable state of pregnancy and the early stages of PPH when crystalloid resuscitation begins and blood product transfusion is anticipated. We recognize that this model cannot replicate temperature and pH change, hypocalcemia, and other humoral or systemic effects that occur during PPH in vivo. In addition, it is not possible to recreate the capillary endothelium in vitro, thus its effect on coagulation cannot be evaluated by TEG. Nevertheless, TEG has been successfully used to study the effects of amniotic fluid, magnesium, oxytocin, and estrogen on coagulation in the pregnant population.
      • Harnett M.J.
      • Hepner D.L.
      • Datta S.
      • Kodali B.S.
      Effect of amniotic fluid on coagulation and platelet function in pregnancy: an evaluation using thromboelastography.
      • Harnett M.J.
      • Datta S.
      • Bhavani-Shankar K.
      The effect of magnesium on coagulation in parturients with preeclampsia.
      • Butwick A.
      • Harter S.
      An in vitro investigation of the coagulation effects of exogenous oxytocin using thromboelastography in healthy parturients.
      • Harnett M.J.
      • Bhavani-Shankar K.
      • Datta S.
      • Tsen L.C.
      In vitro fertilization-induced alterations in coagulation and fibrinolysis as measured by thromboelastography.
      Further in vivo studies to determine the role of PLT in PPH are needed.
      In conclusion, our in vitro model of hemodilution at term gestation suggests that a 1:1 ratio of PRBC:FFP provides optimal clot strength in the presence of PLT. Prospective randomized trials are needed to analyze the benefit of a 1:1:1 ratio of PRBC:FFP:PLT for resuscitation of massive PPH, and to define the optimal PLT count in this setting. The transfusion of blood components is not without risk; high ratios of FFP and PLT to PRBC do not necessarily lower mortality in all transfused patients.
      • Sambasivan C.N.
      • Kunio N.R.
      • Nair P.V.
      • et al.
      Trauma Outcomes Group
      High ratios of plasma and platelets to packed red blood cells do not affect mortality in nonmassively transfused patients.
      The use of point-of-care hemostatic testing such as TEG to guide transfusion for obstetric hemorrhage may optimize the ratio of products administered.

      References

        • Berg C.J.
        • Atrash H.K.
        • Koonin L.M.
        • Tucker M.
        Pregnancy-related mortality in the United States, 1987-1990.
        Obstet Gynecol. 1996; 88: 161-167
        • McClure J.H.
        • Cooper G.M.
        • Clutton-Brock T.H.
        • Center for Maternal and Child Enquiries
        Saving mothers' lives: reviewing maternal deaths to make motherhood safer: 2006-8: a review.
        Br J Anaesth. 2011; 107: 127-132
      1. Arulkumaran S, Mavrides E, Penney GC, et al. Prevention and management of postpartum hemorrhage. Royal College of Obstetricians and Gynaecologists Green-top Guideline no. 52, May 2009. Minor revision Nov 2009 and April 2011.

        • Combs C.A.
        • Murphy E.L.
        • Laros Jr., R.K.
        Factors associated with postpartum hemorrhage with vaginal birth.
        Obstet Gynecol. 1991; 77: 69-76
        • Combs C.A.
        • Murphy E.L.
        • Laros Jr., R.K.
        Factors associated with hemorrhage in cesarean deliveries.
        Obstet Gynecol. 1991; 77: 77-82
        • Cameron C.A.
        • Roberts C.L.
        • Olive E.C.
        • Ford J.B.
        • Fischer W.E.
        Trends in postpartum hemorrhage.
        Aust N Z J Public Health. 2006; 30: 151-156
        • Knight M.
        • Callaghan W.M.
        • Berg C.
        • et al.
        Trends in postpartum hemorrhage in high resource countries: a review and recommendations from the international postpartum hemorrhage collaborative group.
        BMC Pregnancy Childbirth. 2009; 9: 55
        • Bateman B.T.
        • Berman M.F.
        • Riley L.
        • Leffert L.
        The epidemiology of post partum hemorrhage in a large, nationwide sample of deliveries.
        Anesth Analg. 2010; 110: 1368-1373
        • Callaghan W.M.
        • Kuklina E.V.
        • Berg C.J.
        Trends in postpartum hemorrhage: United States, 1994-2006.
        Am J Obstet Gynecol. 2010; 202: 353-355
        • Cantwell R.
        • Clutton-Brock T.
        • Cooper G.
        • Dawson A.
        • Drife J.
        • Garrod D.
        • et al.
        Saving mothers' lives: reviewing maternal deaths to make motherhood safer: 2006-2008. The eighth report of the confidential enquiries into maternal deaths in the United Kingdom.
        BJOG. 2011; 118: 1-203
        • Brace V.
        • Kernaghan D.
        • Penney G.
        Learning from adverse clinical outcomes: major obstetric hemorrhage in Scotland, 2003-05.
        BJOG. 2007; 114: 1388-1396
      2. Department of Reproductive Health and Research, WHO. WHO recommendations for the prevention and treatment of postpartum hemorrhage. Geneva: Word Health Organization; 2012:3.

        • Fries D.
        • Innerhofer D.
        • Shobersberger W.
        Time for changing coagulation management in trauma-related massive bleeding.
        Curr Opin Anesthesiol. 2009; 22 (267-7)
        • Borgman M.A.
        • Spinella P.C.
        • Perkins J.G.
        • et al.
        The ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital.
        J Trauma. 2007; 63: 805-813
        • Holcomb J.B.
        • Zarzabal L.A.
        • Michalek J.E.
        • et al.
        Increased platelet: RBC ratios are associated with improved survival after massive transfusion.
        J Trauma. 2011; 71: S318-S328
        • Burtelow M.
        • Riley E.
        • Druzin M.
        • Fontaine M.
        • Viele M.
        • Goodnough L.T.
        How we treat: management of life-threatening primary postpartum hemorrhage with a standardized massive transfusion protocol.
        Transfusion. 2007; 47: 1564-1572
        • Solomon C.
        • Collis R.E.
        • Collins P.W.
        Hemostatic monitoring during postpartum hemorrhage and implications for management.
        Br J Anaesth. 2012; 109: 851-863
        • Srinivasa V.
        • Gilbertson L.I.
        • Bhavani-Shankar K.
        Thromboelastography: where is it and where is it heading?.
        Int Anesthesiol Clin. 2001; 39: 35-49
        • Butwick A.
        • Ting V.
        • Ralls L.A.
        • Harter S.
        • Riley E.
        The association between thromboelastographic parameters and total estimated blood loss in patients undergoing elective cesarean delivery.
        Anesth Analg. 2011; 112: 1041-1047
        • Welsby I.J.
        • Jiao K.
        • Ortel T.L.
        • et al.
        The kaolin-activated Thromboelastograph predicts bleeding after cardiac surgery.
        J Cardiothorac Vasc Anesth. 2006; 20: 531-535
        • Karlsson O.
        • Sporrong T.
        • Hillarp A.
        • Jeppsson A.
        • Hellgren M.
        Prospective longitudinal study of thromboelastography and standard hemostatic laboratory tests in healthy women during normal pregnancy.
        Anesth Analg. 2012; 115: 890-898
        • De Lorenzo C.
        • Calatzis A.
        • Welsch U.
        • Heindl B.
        Fibrinogen concentrate reverses dilutional coagulopathy induced in vitro by saline but not by hydroxyethyl starch 6%.
        Anesth Analg. 2006; 102: 1194-1200
        • Burrows R.F.
        • Kelton J.G.
        Thrombocytopenia at delivery: a prospective survey of 6715 deliveries.
        Am J Obstet Gynecol. 1990; 162: 731-734
        • Fries D.
        • Innerhofer P.
        • Reif C.
        • et al.
        The effect of fibrinogen substitution on reversal of dilutional coagulopathy: an in vitro model.
        Anesth Analg. 2006; 102: 347-351
        • Haas T.
        • Fries D.
        • Velik-Salchner C.
        • Reif C.
        • Klingler A.
        • Innerhofer P.
        The in vitro effects of fibrinogen concentrate, factor XIII and fresh frozen plasma on impaired clot formation after 60% dilution.
        Anesth Analg. 2008; 106: 1360-1365
        • Hui C.
        • Lili M.
        • Libin C.
        • et al.
        Changes in coagulation and hemodynamics during pregnancy: a prospective longitudinal study of 58 cases.
        Arch Gynecol Obstet. 2012; 285: 1231-1236
        • Stirling Y.
        • Woolf L.
        • North W.R.
        • Seghatchian M.J.
        • Meade T.W.
        Hemostasis in normal pregnancy.
        Thromb Haemost. 1984; 52: 176-182
        • Hiipala S.T.
        • Myllyla G.J.
        • Vahtera E.M.
        Hemostatic factors and replacement of major blood loss with plasma-poor red cell concentrates.
        Anesth Analg. 1995; 81: 360-365
        • Singbartl K.
        • Innerhofer P.
        • Radvan J.
        • et al.
        Hemostasis and hemodilution: a quantitative mathematical guide for clinical practice.
        Anesth Analg. 2003; 96: 929-935
        • Simon L.
        • Santi T.M.
        • Sacquin P.
        • Hamza J.
        Pre-anesthetic assessment of coagulation abnormalities in obstetric patients: usefulness, timing, and clinical implications.
        Br J Anaesth. 1997; 78: 678-683
        • Pasquier P.
        • Gayat E.
        • Rackelboom T.
        • et al.
        An observational study of the fresh frozen plasma: red blood cell ratio in postpartum hemorrhage.
        Anesth Analg. 2013; 116: 155-161
        • Cushing M.
        • Shaz B.H.
        Blood transfusion in trauma patients: unresolved questions.
        Minerva Anestesiol. 2011; 77: 349-359
        • Johansson P.I.
        • Stensballe J.
        • Vindelov N.
        • Perner A.
        • Espersen K.
        Thomboelastography and thromboelastometry in assessing coagulopathy in trauma.
        Scand J Trauma Resusc Emerg Med. 2009; 17: 45-52
        • Kashuk J.L.
        • Moore E.E.
        • Wohlauer M.
        • et al.
        Initial experiences with point-of-care rapid thrombelastography for management of life-threatening post-injury.
        Transfusion. 2012; 52: 23-33
        • Johansson P.I.
        • Ostrowski S.R.
        • Secher N.H.
        Management of major blood loss: an update.
        Acta Anaesthesiol Scand. 2010; 54: 1039-1049
        • Charbit B.
        • Mandelbrot L.
        • Samain E.
        • et al.
        • PPH Study Group
        The decrease of fibrinogen is an early predictor of the severity of postpartum hemorrhage.
        J Thromb Haemost. 2007; 5: 266-273
        • Wikkelsoe A.J.
        • Afshari A.
        • Stensballe J.
        • et al.
        The FIB-PPH trial: fibrinogen concentrate as initial treatment for postpartum hemorrhage: study protocol for a randomized controlled trial.
        Trials. 2012; 13: 110
        • Hill J.S.
        • Devenie G.
        • Powell M.
        Point-of-care testing of coagulation and fibrinolytic status during postpartum hemorrhage: developing a thromboelastography®-guided transfusion algorithm.
        Anaesth Intensive Care. 2012; 40: 1007-1015
        • Harnett M.J.
        • Hepner D.L.
        • Datta S.
        • Kodali B.S.
        Effect of amniotic fluid on coagulation and platelet function in pregnancy: an evaluation using thromboelastography.
        Anaesthesia. 2005; 60: 1068-1072
        • Harnett M.J.
        • Datta S.
        • Bhavani-Shankar K.
        The effect of magnesium on coagulation in parturients with preeclampsia.
        Anesth Analg. 2001; 92: 1257-1260
        • Butwick A.
        • Harter S.
        An in vitro investigation of the coagulation effects of exogenous oxytocin using thromboelastography in healthy parturients.
        Anesth Analg. 2011; 113: 323-326
        • Harnett M.J.
        • Bhavani-Shankar K.
        • Datta S.
        • Tsen L.C.
        In vitro fertilization-induced alterations in coagulation and fibrinolysis as measured by thromboelastography.
        Anesth Analg. 2002; 95: 1063-1066
        • Sambasivan C.N.
        • Kunio N.R.
        • Nair P.V.
        • et al.
        • Trauma Outcomes Group
        High ratios of plasma and platelets to packed red blood cells do not affect mortality in nonmassively transfused patients.
        J Trauma. 2011; 71: S329-S336