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.
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, change in hematocrit of >10%, or need for red blood cell transfusion. The incidence of PPH is increasing in many high-resource countries ; PPH increased from 2.3-2.9% (26%) in the United States from 1994 through 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. PPH remains a common and increasing source of maternal morbidity and mortality worldwide, accounting for approximately one-quarter of maternal deaths per year.
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. An analysis of 246 soldiers suffering mass casualties in Iraq revealed a significant decrease in mortality when large amounts of FFP were given, and transfusion of 1:1:1 PRBC:FFP:PLT for massive trauma resuscitation improved survival. 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. The use of point-of-care testing such as thromboelastography (TEG) for guiding hemostatic therapy during PPH has also been advocated. 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 ). 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.
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. 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). 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%.
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.
| Demographic | n = 20 |
|---|---|
| Age, y | 32 (6) |
| Weight, kg | 85 (17) |
| Gestational age, wk | 39.3 (1.1) |
| Parity | 0 (0–4) |
| Ethnicity | |
| Non-Hispanic Caucasian | 12 (60) |
| Hispanic | 6 (30) |
| African American | 1 (5) |
| Asian American | 1 (5) |
| Group | Sample description | |||
|---|---|---|---|---|
| Baseline | 50% dilution | 3:1:0 a | 1:1:0 b | |
| Group 1: no added platelets | ||||
| R time, min | 6.1 (1.2) | 5.9 (1.2) | 5.7 (1.5) | 5.1 (1.4) c |
| K time, min | 1.4 (0.2) | 1.8 (0.3) | 2.9 (1.1) d | 2.4 (0.8) d |
| α angle, degrees | 67.7 (6.9) | 64.3 (3.6) | 54.0 (8.9) d | 60.1 (7.3) d |
| MA, mm | 68.7 (5.8) | 57.5 (4.2) d | 49.3 (5.0) d | 51.0 (4.2) d |
| Hct, % | 34.5 (2.1) | – | 27.7 (3.1) c | 22.7 (2.4) c |
| Plt, 1000 μL −1 | 237 (60) | – | 61 (19) d | 68 (16) d |
| Group 2: platelets added to 3:1 and 1:1 samples | ||||
| R time, min | 5.8 (1.3) | 6.0 (0.7) | 5.1 (1.0) | 4.8 (0.6) c |
| K time, min | 1.4 (0.3) | 1.6 (0.3) | 1.2 (0.2) d,e | 1.1 (0.2) d,e |
| α angle, degrees | 65.8 (8.2) | 66.7 (3.6) | 70.8 (4.1) e | 72.6 (6.1) d,e |
| MA, mm | 67.5 (4.8) | 59.3 (4.2) c | 61.2 (2.7) c,e | 63.2 (2.5) e |
| Hct, % | 34.5 (2.9) | – | 24.0 (1.8) c | 20.1 (2.6) c |
| Plt, 1000 μL −1 | 256 (65) | – | 189 (34) e | 202 (52) e |
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
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).
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