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Microparticle Release During Normal Cesarean Delivery

Hofer, Jennifer E., MD*; Scavone, Barbara M., MD*,†

doi: 10.1213/ANE.0000000000002290
Obstetric Anesthesiology: Brief Report
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Coagulation increases during pregnancy and peaks during parturition. We hypothesized that an increase in microparticle (MP) levels in plasma occurs around the time of placental separation and subsides over several hours. We performed a prospective observational pilot study to investigate plasma MP levels in healthy parturients immediately before and after cesarean delivery. The primary outcome was MP levels at postdelivery time points compared to baseline levels. Samples underwent flow cytometry and staining to determine MP levels. Placental-derived MPs were further characterized for the presence of procoagulant proteins. Placental-derived MPs increased immediately after delivery before returning to baseline in healthy parturients.

From the Departments of *Anesthesia and Critical Care

Obstetrics and Gynecology, the University of Chicago Medicine, Chicago, Illinois.

Published ahead of print August 4, 2017.

Accepted for publication May 15, 2017.

Funding: Departmental.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Jennifer E. Hofer, MD, Departments of Anesthesia and Critical Care, the University of Chicago Medicine, 5841 S Maryland Ave, MC 4028, Chicago, IL 60637. Address e-mail to jhofer@dacc.uchicago.edu.

Hypercoagulability peaks during parturition, independent of the mode of delivery.1 The increases in coagulation that accompany delivery may be partially mediated by circulating tissue factor (TF),2 but full characterization of the mediators of the increase in coagulation during normal delivery has yet to be undertaken.

Microparticles (MPs) are spherical cytoplasm-free parts of cell membranes that are released from cells during cell activation or apoptosis3 and may be physiologic or pathologic.4 Because MPs originate from cell membranes, they contain the same membrane proteins as their parent cells, which helps identify their origin (platelet, endothelium, white blood cell, placenta).5

Although treatment of PPH is improving, its exact pathogenesis is not known, and a better understanding of peripartum coagulation may permit better identification of those at risk for postpartum hemorrhage and allow for more targeted therapy. Specifically, the relationship between coagulability and MP levels surrounding normal delivery has not been characterized. We hypothesized that an increase in MP levels occurs around the time of delivery and subsides over several hours. To investigate this hypothesis, we performed a small prospective observational pilot study to investigate MP levels in healthy parturients immediately before and after scheduled cesarean delivery.

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METHODS

This study registered with the Clinical Trials Registry prior to patient enrollment and was approved by the Institutional Review Board at the University of Chicago. We obtained written informed consent from 10 healthy parturients with a singleton pregnancy, gestational age ≥36 weeks, who presented for scheduled cesarean delivery January 2014 through June 2015. Exclusion criteria included hypertensive diseases of pregnancy, diabetes mellitus, known placental abnormalities, and coagulation disorders. A 16-gauge intravenous line was placed for 5 blood draws: baseline antepartum on the day of delivery, immediately after delivery of the placenta, then 1, 4, and 24–36 hours postpartum. Intravenous fluids were stopped for several minutes prior to the laboratory draws, and the first 10 mL blood was wasted prior to each collection.

Blood was collected in a “yellow top” tube containing acid citrate dextrose and centrifuged at 2000g for 20 minutes to separate plasma. The plasma was centrifuged once again at 5000g for 10 minutes to generate platelet-poor plasma, which was aliquoted and stored at −80°C until assays were performed. Platelet-poor plasma samples were thawed and stained with a cocktail of antibodies that was first passed through a 0.1 μm pore–sized filter to remove any external source of small particles. Fluorescently coupled antibodies were used to identify MPs from various cell types including platelet- (coexpressing platelet glycoprotein 1b α chain [GP1bα] and platelet endothelial cell adhesion molecule 1 [PECAM-1]), endothelial- (expressing only PECAM-1), leukocyte- (expressing leukocyte common antigen-T200), and placental- (expressing placental alkaline phosphatase) derived. Antibody stains included anti-PLAP Purified (AbD Serotec; BioRad Laboratories, Inc, Hercules, CA) clone H17E2 custom conjugated to Pacific Blue (Life Technologies Pacific Blue Antibody Labeling Kit; Thermo Fisher Scientific, Inc, Waltham, MA) and used at 1 μg/mL (or 1 μL/test), CD31 BV605 (BD Biosciences, San Jose, CA) clone WM59 used at 3 μL/test, CD42b APC (BD Biosciences) clone HIP1 used at 10 μL/test, CD45 APC-Cy7 (BD Biosciences) clone 2D1 used at 3 μL/test, and CD142 PE (BD Biosciences) clone human tissue factor-1 (HTF-1) used at 10 μL/test. The antibody-stained samples were incubated at 4°C for 60 minutes and then washed once with 5 mL of wash buffer (phosphate buffered saline [PBS]/0.1% bovine serum albumin [BSA]). The samples were finally ultracentrifuged at 17,000g for 10 minutes to pellet the MPs, and the pellet was resuspended in exactly 100 µL wash buffer in preparation for acquisition. The samples were acquired on the ImageStreamX MkII (Millipore Sigma, Billerica, MA) image cytometer equipped with a ×60 objective lens, 4-laser lines (405, 488, 561, and 640 nm) capable of exciting the various fluorophores and emission filters to collect light emitting from each fluorophore independently. MPs were identified according to their size (>0.1 to <1 μm), and furthermore by their expression of surface markers yielding a fluorescence signal greater than negative controls. A gating strategy was created using the IDEAS 6.0 analysis software (Millipore Sigma) to identify each of the target phenotypes, and this gating method was applied to all samples equally.

Placental-derived MPs were further characterized for the presence of TF, phosphatidylserine (PS), plasminogen activator inhibitor-1 (PAI-1), PAI-2, and soluble fms-like tyrosine kinase-1 (sFlt-1) using fluorescently coupled antibodies against each of these proteins. Samples were again stained and analyzed in a similar fashion as above.

Demographic data collected included age, height, weight, gravidity and parity, estimated gestational age, and presence of comorbidities. We also noted estimated blood loss and whether or not any blood products were transfused.

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Statistical Analysis

We choose to study 10 patients as a convenience sample since this was conceived of as a pilot project. A sample size of 10 achieves 92% power to detect a mean difference of 12,000 total MP level between baseline and postdelivery assuming a standard deviation of 100,000 with a significance level of .05. Demographic statistics were generated using mean and standard deviation for continuous variables, and frequency and percentiles for categorical variables.

Each measure at each time point was summarized by the use of median and quartiles. Using a nonparametric sign test, we tested if the median of the differences at each time point from baseline was zero. We used the Bonferroni correction to adjust for multiple comparisons so that the significance level was .01 (=.05/5). All analyses were performed with the use of Stata software, version 14.0 (StatCorp Medical, Jacksonville, FL).

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RESULTS

Written consent was obtained from 10 patients. Blood samples from 9 patients were collected. The 10th patient’s intravenous line would not return blood. Six patients had all 5 samples collected; the remaining patients had 4 samples collected (24- to 36-hour postpartum sample not collected). The mean patient age was 32 ± 5 years, and mean body mass index was 32 ± 6 kg/m2. One-third of patients were nulliparous; all patients were healthy and had gestational age >39 weeks. The mean estimated blood loss was 639 ± 150 mL. None of the patients hemorrhaged (ie, lost >1000 mL within 24 hours of cesarean delivery), and no one received a blood transfusion.

Table

Table

Figure

Figure

For each outcome measure, the median and quartiles are shown for each time point (Table). Significant changes in postdelivery MPs (total placenta TF-negative (−) MPs and total placental MPs) from baseline were found (P = .0078; Figure). There were no other differences in any median MP levels over time. The study was underpowered to detect significant changes in MP over time. A median of 56% (range, 33%–73%) of placental-derived MPs released immediately after delivery demonstrated at least one procoagulant protein (TF: 0% [range, 0%–1%], PS: 24% [range, 17%–40%], PAI-1: 18% [range, 15%–41%], PAI-2: 9% [range, 7%–14%], sFlt-1: 14% [range, 8%–25%]).

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DISCUSSION

We found plasma levels of placental-derived MPs peaked immediately following normal cesarean delivery and returned to baseline levels within 1 hour of delivery. The placental-derived MPs contained thromboplastic molecules PS, PAI-1, PAI-2, and sFlt that are known to stimulate coagulation.6,7 Coagulation activity reaches a peak shortly after delivery of the newborn and placenta,1 and we have determined that an acute rise in procoagulant-containing placental MPs occurs during this same time period. We hypothesize that at the time of placental separation, the placenta sheds MPs into the circulation, carrying the thromboplastins that are present on the parent cells, and that this contributes to the acceleration of coagulation activity observed immediately after parturition.8

Despite most of the literature on this topic being over a decade old, our results are similar to those of other more recent investigators. Trophoblastic MPs rose after placental separation in both normal and preeclamptic patients in a previous study, although the increase did not reach statistical significance.9 Others have demonstrated an increase in platelet-derived MPs after delivery among women who labored, but no increase in platelet- or trophoblast-derived MPs after cesarean delivery.10 We did not investigate laboring women. The placental-derived MPs that acutely increased at time of delivery were TF-negative in our study. Boer et al2 demonstrated a postdelivery rise in TF-dependent coagulation during and after delivery; however, those investigators measured soluble TF, not MP-bound TF. Our study indicates a source of TF other than placental-derived MPs. We observed that 24% of placental-derived MPs contained PS. PS bound to MPs may interact with non–MP-bound soluble TF, increasing the procoagulant profile of the MP.

Our study has limitations. Our sample size was small, and our results may not be generalizable to other populations. (Our patients did not labor and were largely obese.) We did not assess other coagulation parameters, and, therefore, we cannot state with certainty that the observed placental-derived MPs fueled a coagulation increase, despite the fact that procoagulants were bound to the MPs. Further investigation is needed to characterize MP release patterns in a larger patient population to determine if our results can be replicated in laboring patients, and to reveal the pattern of MP release in comorbid and pathologic states.

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DISCLOSURES

Name: Jennifer E. Hofer, MD.

Contribution: This author helped obtain consent from patients, collect blood samples, work with the statistician, actively find references, analyze the data, and write the manuscript.

Name: Barbara M. Scavone, MD.

Contribution: This author helped obtain consent from patients, collect blood samples, work with the statistician, actively find references, analyze the data, and write the manuscript.

This manuscript was handled by: Jill M. Mhyre, MD.

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REFERENCES

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