1

Intraoperative changes in platelet function in relation to moderate

haemorrhage

Authors: Jonathan J Hetherington, Aberdeen Royal Infirmary, NHS Grampian,

Isobel Ford, School of Medicine & Dentistry, University of Aberdeen

G Patrick Ashcroft, School of Medicine & Dentistry, University of Aberdeen

Jan O Jansen, Aberdeen Royal Infirmary, Aberdeen & Health Services

Research Unit, University of Aberdeen, Aberdeen, Scotland

Corresponding Author: Dr J Hetherington

Address: Anaesthetics Dept, Aberdeen Royal Infirmary, Aberdeen, Scotland AB25 2ZN

Email: jonathan.hetherington@nhs.net

2

AbstractBackground: Haemorrhage is associated with haemostatic dysfunction. Previous studies

have focused on coagulation factors, but platelet function plays an equally important role.

The time course of alterations in platelet function in relation to injurious stimuli is not known.

Aim: To evaluate short-term, intra-operative changes in platelet function, by whole blood

impedance aggregometry in patients undergoing hip arthroplasty. The primary outcome was

platelet aggregation in response to adenosine diphosphate (ADP) stimulation. We also

evaluated other agonists, and the feasibility of conducting platelet aggregometry

measurement in the operating theatre.

Methods: Patients undergoing primary cemented hip arthroplasty had six peri-operative

arterial blood samples analysed at pre-set stages of the operation, using the Multiplate®

Impedance Aggregometer. Four agonists were used: ADP, TRAP, Collagen and Arachidonic

Acid.

Results: There was a statistically significant change (p<0.05, ANOVA) in platelet response

to ADP over the course of the operations. The trend demonstrated an initial decrease in

responsiveness, followed by increased platelet responsiveness in the later stages. Other

agonists (TRAP, COL, ASPItest) demonstrated similar pattern of changes. Of 360 tests

conducted, 12 (3.3%) had to be re-run due to poor intra-assay variability. Satisfactory values

were obtained on the second attempt in all 12 samples.

Conclusion: Platelet function, as measured by impedance aggregometry, changes in response

to a surgical stimulus involving blood loss. The clinical significance of these changes, and the

potential of manipulating them for therapeutic purposes, remains to be elucidated.

3

Word count: 231 Keywords: Keywords: Haemorrhage, platelet aggregation, platelet function

tests, surgery

UKCRN Number: 13815

4

IntroductionChanges to the coagulability of blood in response to stimuli such as haemorrhage, trauma,

and surgery, are well recognised. Post-operatively, or following minor injury, patients are

more likely to develop thromboembolic complications, due to increased coagulability (1).

Conversely, haemorrhage-associated haemostatic dysfunction is now increasingly recognised.

(2,3)Error: Reference source not foundInitial efforts to elucidate the underlying mechanisms

for this change have focused on the roles of activated Protein C and hyperfibrinolysis (4,5).

Error: Reference source not foundPlatelet function has received less attention, although

recent studies have demonstrated an association between acute traumatic coagulopathy and

platelet aggregability, (6) and platelets are regarded as an integral part of the coagulation

system.

The assessment of platelet function is difficult, accounting for the paucity of studies in this

area. Flow cytometry and light transmission aggregometry are the techniques of choice in the

research laboratory, but require considerable operator training and experience. These methods

are also labour-intensive and time-consuming, making the rapid analysis of sequential

samples in a clinical or emergency setting difficult. Over the last decade, impedance

aggregometry has emerged as a more user-friendly alternative. Initially developed to

demonstrate platelet inhibition in cardiovascular patients receiving antiplatelet therapy, this

technique – whereby the rate of aggregation of platelets between electrodes is measured –

shows promise in the assessment of intrinsic platelet dysfunction.

Through the utilisation of a number of agonists to induce platelet aggregation an assessment

of platelet function can be made. Adenosine diphosphate (ADP) in particular has a substantial

contribution to the propagation of platelet activation at the site of injury (7) and the clinical

5

effectiveness of antiplatelet agents acting at the P2Y12 receptor in reducing thromboembolic

phenomena highlights the clinical importance of this pathway (8).

In summary, there is evidence that platelet function is affected by tissue trauma and

haemorrhage (6,9), and that these changes are associated with poor outcome(9,10)(6). There

is also some evidence that platelet function may change earlier and more rapidly than was

previously assumed (11). However, for technical reasons these changes are difficult to

demonstrate.

We hypothesised that some of the changes in platelet function noted in injured patients may

also take place in response to planned surgery associated with blood loss, reflecting decreased

platelet responsiveness secondary to trauma and haemorrhage. The aim of this study was to

elucidate short-term changes in platelet function, as measured by stimulated impedance

aggregometry, in response to moderate blood loss, during the course of an elective operation.

6

MethodsThis was a prospective observational study. The study protocol was approved by the North of

Scotland Research Ethics Service (reference NS/12/0095). We identified patients scheduled

to undergo primary cemented hip arthroplasty at the pre-operative assessment clinic between

January 1st 2013 and July 31st 2013. Patients with a known platelet or coagulation disorder or

diabetes, and those taking warfarin were excluded. Study information was distributed to

patients in advance, and written consent obtained following a period of consideration of at

least seven days. Patients receiving antiplatelet drugs had their medication withheld as per the

routine practice of the operating surgeon (3-10 days prior to commencement of surgery).

On arrival in the anaesthetic room, baseline physiological parameters were recorded, and a

radial arterial cannula inserted under local anaesthetic, to facilitate intra-operative sampling.

Patients were then anaesthetised and underwent surgery. Blood samples were taken at six

time points during the perioperative period: Pre-operatively before administration of the

anaesthetic; on commencement of the skin incision; on removal of the femoral head; on

reaming of the femoral canal; on cementing of the femoral component of the prosthesis; and

following skin closure. Samples for full blood count and platelet function were drawn at each

time point via a Vacutainer system into tubes containing EDTA (3ml), citrate (2.7ml) and

hirudin (3ml) anticoagulants respectively. A single additional sample for measurement of

clottable fibrinogen was taken pre-operatively in the anaesthetic room. Platelet samples were

analysed immediately using the Multiplate® Impedance Aggregometer (Roche Diagnostics

International Ltd, Rotkreuz, Switzerland). 0.3 mL of whole blood was diluted in an equal

volume of warmed isotonic sodium chloride solution and incubated for 3 minutes at 37˚C

with continuous stirring by a magnetised chip in a customised test cell. Each cell contains two

sets of 3mm silver-coated copper wires, across which electrical resistance is measured.

7

Platelet adhesion to the electrodes is detected as increasing electrical impedance. Platelet

function was assessed using four agonists – ADPtest® (6.5M adenosine diphosphate, ADP),

TRAPtest® (32µM thrombin receptor activating peptide TRAP-6), COLtest® (3.2µg/mL

collagen), and ASPItest® (0.5mM Arachidonic Acid) (all final concentrations), all obtained

from Roche Diagnostics Ltd, Sussex, UK – in order to assess different pathways of platelet

activation. Agonist responses were reported as area under the aggregation curve in units

(AUC) during a 6-minute measurement period.

Full blood counts were analysed on a Siemens Advia 2120i by the hospital’s haematology

laboratory. Citrate samples taken pre-operatively were centrifuged at 2000 x g for 15 min on

completion of the operation, plasma separated and frozen at below -70oC, and batch-analysed

for clottable fibrinogen using a semi-automated Clauss method with reagents from Helena

Biosciences (Tyne And Wear, UK).

Estimated total blood loss was calculated using routine clinical methodology (swab weights,

suction loss and direct measurable losses). Data was collected on regular medication use,

drugs given intraoperatively, comorbidity and anaesthetic technique employed.

The primary outcome measure was ADP-induced platelet aggregation. Secondary outcomes

included aggregation in response to thrombin receptor activating peptide, arachidonic acid,

and collagen, as well as the feasibility of conducting repeated intraoperative impedance

aggregometry, measured in terms of the number of patients recruited (as a proportion of those

eligible), and the proportion in whom a complete set of results was obtained. Statistical

analyses were performed using SPSS Statistical Software Version 21 (IBM, Chicago, IL,

USA). Changes in platelet activity over time were assessed by analysis of variance using

repeated measures ANOVA. Descriptive statistics were performed on clinical, platelet and

8

biochemical parameters. Correlation between the primary measure of ADP-stimulated

platelet aggregation and blood loss was investigated using Spearman’s rank correlation.

As this was a pilot study, we did not perform a sample size calculation. However previous

work by our group showed a statistically significant difference (p<0.05) in pre vs. post-op

platelet activation measures in 10 hip surgery patients (% platelets expressing P-Selectin: pre

1.0 (0.29-3.47); immediately post-op 0.53 (0.13-2.27); median (IQR)). We therefore believe

a group of 20 patients will be adequate to detect a significant difference and to provide data

to inform further studies.

9

ResultsRecruitment

In all, 53 eligible patients were identified at the pre-assessment clinic. The recruitment

process can be seen in the flowchart in figure 1.

Demographics and medication

The baseline characteristics of the study population are shown in table 1. The median age was

69 years (range 56-78 years), with nine males and six females. Four patients had previously

received anti-platelet therapy with aspirin, which had been discontinued at 3, 5, 7 and 10 days

before surgery. None had been taking thienopyridines.

All patients were undergoing hip replacement for osteoarthritis of the hip. One patient had

concurrent rheumatoid arthritis, which was documented to be well controlled on

methotrexate.

The first patient received their operation in March 2013 with the final study patient

completing surgery in late August 2013. All patients received spinal anaesthesia, but one

required conversion to a general anaesthetic prior to commencement of surgery. Median

operation time was 2h1min (range 1h15min to 2h49min) with a median blood loss of 555

(range 200-1000) mL. Tranexamic acid was used intra-operatively in 11 patients and was

administered at a median of 20 min before the completion of the operation (range 8 min post-

operatively to 87 min prior to completion of operation).

Feasibility of platelet testing

Each patient had 24 Multiplate® assays performed in total. Of the 360 tests, 12 (3.3%) were

re-run due to poor intra-assay variability (difference from mean values >15%, as calculated

by Multiplate® software) and satisfactory values were obtained in all on the second

10

measurement with a median variability of 7 AUC (range 5-28). Median time to commencing

assay analysis from sampling was 5 min (range 1-31min), the longest times being as a result

of two patients’ operations occurring simultaneously (all other patients’ analyses were

completed within 13min of sampling).

Platelet Responsiveness

The results of platelet aggregation and blood counts are shown in tables 2 and 3. There was a

statistically significant change in platelet responsiveness to ADP over time (p=0.012,

repeated measures ANOVA) (figure 1). There was a trend towards increased responsiveness

to ADP at the two final time points: cementing of the femoral head and immediately after

skin closure, when compared with the preoperative baseline. Platelet aggregation with the

other agonists, TRAP, collagen and arachidonic acid, followed a similar pattern (figure 2 a-

d).

The platelet count and haemoglobin fell over the course of the operation and white cell count

increased (see Table 3). Median plasma fibrinogen pre-operatively was 3.81 (Range 3.08 –

5.52) g/L.

There was a statistically significant inverse correlation between platelet responsiveness to

ADP at the pre-operative baseline and total blood loss during the operation (r=-0.624 p=0.04,

figure 3).

11

DiscussionThe results of this study demonstrate a statistically significant change in platelet aggregation

during elective primary cemented hip arthroplasty. Although statistical analysis was only

performed on one pathway of platelet function (ADP) – as a result of small sample size and

concerns over multiple testing – platelet aggregation by all four agonists followed a similar

pattern, showing alterations in platelet responsiveness across multiple pathways at the same

time points trending towards increased platelet responsiveness towards the end of surgery.

These findings are at variance with those reported in patients suffering from acute traumatic

coagulopathy (9,12), but this difference may be accounted for by the magnitude and type of

the insult. The maximal blood loss in the present study was only 1000ml, and given the

nature of the surgery, this deficit is unlikely to have been associated with hypoperfusion,

which is recognised as a key driver of haemorrhage-related coagulopathy. The changes in

platelet responsiveness may be in keeping with a hypercoagulable state, representing an

initial and effective adaptation to trauma, which has previously been demonstrated in less

severely injured(2) and postoperative patients (13).

The small number of patients included in this pilot precludes a statistical analysis of the

temporal changes in aggregability. The initial apparent decrease may relate to the

administration of anaesthesia, instrumentation, and perhaps a neuroendocrine response. All

patients received spinal anaesthesia, had venous and arterial cannulae inserted, and received

sedative medication in preparation for the operative period; with the exception of one patient

who required conversion to GA following an ineffective spinal. The administration of spinal

anaesthesia is known to cause haemodynamic effects which may influence platelet function

(14). The effect of sedative anaesthetic agents on platelet function is unclear. Anaesthetic

doses of propofol have been reported to have either no effect (15) or to decrease platelet

12

aggregation (16), but at considerably higher doses than those administered in the present

study.

The trend in platelet aggregation response in the later stages of the operations, towards

increased aggregability, could be related to increasing systemic inflammation, reflected in an

apparent increase in white cell count over the course of the operation, perhaps exacerbated by

the cementing of the femoral components. Microcirculatory changes in response to cementing

are well recognised, but previous work showed no significant difference in activation of the

coagulation cascade between cemented and uncemented hemi-arthroplasty (17). More

recently, platelet activation in response to reaming/cementing of intramedullary nails was

found in animal studies (18).

Pre-operative plasma fibrinogen levels in patients in the present study were noted to be at the

upper end of normal range, perhaps representing the underlying inflammatory osteoarthritic

process (19), although the significance of this is uncertain. The widespread effects of

inflammatory conditions on Mean Platelet Volume (MPV) and platelet activation have been

recently reported (20). In long term conditions (e.g. MI and stroke), thrombopoiesis

determines long term changes of MPV, yet the activation of the sympathetic system and

release of platelets from the spleen may underlie rapid shifts in MPV values in response to

stress(20). This rapid increase of MPV due to the release of large platelets from the spleen is

more likely in the presence of an underlying prothrombotic condition (21) and this may have

been apparent in our patient population.

The effect of platelet count is said to be minimal within the normal range but a few

investigators have found a positive correlation with impedance aggregometry (22). Thus a

fall in platelet count during surgery would if anything be expected to influence a downward

trend in aggregation results, whereas we found the opposite.

13

Interestingly, our results indicated a statistically significant inverse correlation between

platelet responsiveness to ADP at the pre-operative baseline and total blood loss during the

operation (Fig 3). There was no significant correlation between blood loss and post-operative

platelet aggregability. Some previous studies, conducted in cardiac surgery patients, have also

reported an association between pre-operative ADP-test and blood loss or requirement for

platelet concentrates (22-24). Most of the patients in the previous studies were taking aspirin

(23) or thienopyridines (22) at the time of the study, or had stopped in the few days

previously, which would influence the correlation between platelet aggregation and blood

loss. The current study is the first to show such a correlation in non-cardiac patients. The

patients in the current study were non-coagulopathic and importantly, those who were taking

aspirin had stopped taking it at least 3 days previously, and 2 for at least 7 days. The clinical

significance of this association is unclear but the finding merits further investigation.

Intraoperative platelet function testing was feasible, although requiring a dedicated member

of staff. Data were obtained for 15 out of the 17 patients who gave consent and who

underwent the planned surgery. Difficulty of arterial access or change in surgical planning

prevented data collection in two subjects. Valid results were obtained for all 15 patients for

whom data were collected, with a very small requirement for repeat testing. On average,

platelet function measurement was completed within 11 minutes of sampling. Jambor et al

(25) showed that valid results are obtained between 0 and 60 minutes after sampling, with no

significant change to aggregation over these times. For the purpose of this study, the

equipment was located in the operating theatre, with no discernible impact on the conduct of

the operation, demonstrating the potential for the implementation of the device in this

environment. Although the investigation of patients undergoing elective surgery is more

straightforward, because the injurious stimulus is similar in type and magnitude, and the

14

degree of blood loss more predictable, the use of the analyser in patients with more severe

haemorrhage, analogous to the use of a thromboelastograph, is feasible.

This study has limitations, the most obvious being the sample size. The study recruited 28.3%

of patients identified as eligible for the study. It is likely that patients’ refusal to consent to

take part was related to the prospect of additional invasive monitoring in the conduct of the

protocol, or patients reluctant to expose themselves to perceived additional risk/pain

associated with the insertion of an arterial line. However, in order to facilitate repeat testing

within a short time period, the insertion an arterial cannula was essential in the conduct of the

study. Most previous studies have presented data from venous samples, but work by Kafian et

al revealed similar results are obtained when sampling from both arterial and venous points

(26).

Despite these limitations, this study adds to the literature supporting the utilisation of platelet

function testing in the clinical environment. The use of the Multiplate® system within the

operating theatre is feasible and allows multiple rapid measures of platelet function to be

collected.

To our knowledge, this is the first study to demonstrate dynamic changes in platelet function,

over a short time period, in response to a surgical stimulus. The clinical significance of these

changes, and the potential of manipulating them for therapeutic purposes, remains to be

elucidated, although implications for the peri-operative management of patients in response

to aberrant platelet function may be substantial and should form the basis of further study, in

both elective and emergency surgery.

ACKNOWLEDGEMENTS

15

The authors are extremely grateful for the expert assistance and contributions of Dr Neil Scott

(Medical Statistician, University of Aberdeen), Win Culley (Research Nurse, Woodend

Hospital), Dr Karen Cranfield (Consultant Anaesthetist), and Ms Sharon Wood, (Research

Technician, Rowett Institute of Nutrition and Health) for fibrinogen measurement.

DECLARATIONS OF INTEREST

None

FUNDING

The study was funded by TENOVUS Scotland (G12/14). Jan Jansen is in receipt of salary

support through the NHS Research Scotland (NRS) fellowship scheme.

DISCLAIMER

The Health Services Research Unit receives funding from the Chief Scientist Office of the

Scottish Government Health and Social Care Directorates.  The opinions expressed in this

article are those of the authors alone.

AUTHORS CONTRIBUTION

J.H. J.J.: Hypothesis and study design; J.H.: patient recruitment, data collection and analysis,

wrote first draft of paper; J.J., I.F., G.PA.: Data interpretation, co-writing and revision of

manuscript

16

References:

(1) Bombeli T, Spahn DR. Updates in perioperative coagulation: physiology and management of thromboembolism and haemorrhage. British Journal of Anaesthesia 2004 August 01;93(2):275-287.

(2) Brohi K, Singh J, Heron M, Coats T. Acute traumatic coagulopathy. J Trauma 2003 Jun;54(6):1127-1130.

(3) MacLeod JB, Lynn M, McKenney MG, Cohn SM, Murtha M. Early coagulopathy predicts mortality in trauma. J Trauma 2003 Jul;55(1):39-44.

(4) Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie RC, Pittet JF. Acute traumatic coagulopathy: initiated by hypoperfusion: modulated through the protein C pathway? Ann Surg 2007 May;245(5):812-818.

(5) Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, et al. Acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. J Trauma 2008 May;64(5):1211-7; discussion 1217.

(6) Hess JR, Brohi K, Dutton RP, Hauser CJ, Holcomb JB, Kluger Y, et al. The coagulopathy of trauma: a review of mechanisms. J Trauma 2008 Oct;65(4):748-754.

(7) Woulfe D, Yang J, Brass L. ADP and platelets: the end of the beginning. J Clin Invest 2001 Jun;107(12):1503-1505.

(8) Yusuf S, Bijsterveld N, Moons A. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation: the Clopidogrel in Unstable Angina to Prevent recurrent Events Trial Investigators. N Engl J Med 2001;345(7):494-502.

(9) Solomon C, Traintinger S, Ziegler B, Hanke A, Rahe-Meyer N, Voelckel W, et al. Platelet function following trauma. A multiple electrode aggregometry study. Thromb Haemost 2011 Aug;106(2):322-330.

(10) Kutcher ME, Redick BJ, McCreery RC, Crane IM, Greenberg MD, Cachola LM, et al. Characterization of platelet dysfunction after trauma. J Trauma Acute Care Surg 2012 Jul;73(1):13-19.

(11) Jansen JO, Luke D, Davies E, Spencer P, Kirkman E, Midwinter MJ. Temporal changes in ROTEM(R)-measured coagulability of citrated blood samples from coagulopathic trauma patients. Injury 2013 Jan;44(1):36-39.

(12) Platelet function in trauma measured by multiple electrode aggregometry. ISTH; 2011.

17

(13) Payen JF, Baruch M, Horvilleur E, Richard M, Gariod T, Polack B. Changes in specific markers of haemostasis during reduction mammoplasty. Br J Anaesth 1998 Apr;80(4):464-466.

(14) Jackson SP, Nesbitt WS, Westein E. Dynamics of platelet thrombus formation. J Thromb Haemost 2009 Jul;7 Suppl 1:17-20.

(15) Dordoni PL, Frassanito L, Bruno MF, Proietti R, de Cristofaro R, Ciabattoni G, et al. In vivo and in vitro effects of different anaesthetics on platelet function. Br J Haematol 2004 Apr;125(1):79-82.

(16) De La Cruz JP, Paez MV, Carmona JA, De La Cuesta FS. Antiplatelet effect of the anaesthetic drug propofol: influence of red blood cells and leucocytes. Br J Pharmacol 1999 Dec;128(7):1538-1544.

(17) Bredbacka S, Andreen M, Blomback M, Wykman A. Activation of cascade systems by hip arthroplasty. No difference between fixation with and without cement. Acta Orthop Scand 1987 Jun;58(3):231-235.

(18) Blankstein M, Byrick RJ, Nakane M, Bang AK, Freedman J, Garvey MB, et al. A preliminary study of platelet activation after embolization of marrow contents. J Orthop Trauma 2012 Nov;26(11):e214-20.

(19) Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: latest findings and interpretations. Ther Adv Musculoskelet Dis 2013 Apr;5(2):77-94.

(20) Gasparyan AY, Ayvazyan L, Mikhailidis DP, Kitas GD. Mean platelet volume: a link between thrombosis and inflammation? Curr Pharm Des 2011;17(1):47-58.

(21) Yilmaz MB, Saricam E, Biyikoglu SF, Guray Y, Guray U, Sasmaz H, et al. Mean platelet volume and exercise stress test. J Thromb Thrombolysis 2004 Apr;17(2):115-120.

(22) Ranucci M, Baryshnikova E, Soro G, Ballotta A, De Benedetti D, Conti D, et al. Multiple electrode whole-blood aggregometry and bleeding in cardiac surgery patients receiving thienopyridines. Ann Thorac Surg 2011 Jan;91(1):123-129.

(23) Emeklibas N, Kammerer I, Bach J, Sack FU, Hellstern P. Preoperative hemostasis and its association with bleeding and blood component transfusion requirements in cardiopulmonary bypass surgery. Transfusion 2013 Jun;53(6):1226-1234.

(24) Rahe-Meyer N, Winterhalter M, Boden A, Froemke C, Piepenbrock S, Calatzis A, et al. Platelet concentrates transfusion in cardiac surgery and platelet function assessment by multiple electrode aggregometry. Acta Anaesthesiol Scand 2009 Feb;53(2):168-175.

(25) Jambor C, Weber CF, Gerhardt K, Dietrich W, Spannagl M, Heindl B, et al. Whole blood multiple electrode aggregometry is a reliable point-of-care test of aspirin-induced platelet dysfunction. Anesth Analg 2009 Jul;109(1):25-31.

18

(26) Kafian S, Mobarrez F, Kalani M, Wallen H, Samad BA. Comparison of venous and arterial blood sampling for the assessment of platelet aggregation with whole blood impedance aggregometry. Scand J Clin Lab Invest 2011 Dec;71(8):637-640.

19

Legends

Fig 1 Study Recruitment Process.

Fig 2. Platelet aggregation in whole blood by MEA (multi-electrode aggregometry) using Multiplate ® analyser, with four agonists. n=15 patients at 6 time points before and during hip replacement operation: Sample 1. Pre-operatively before anaesthetic; 2. On commencement of the skin incision; 3. At removal of the femoral head; 4. Reaming of the femoral canal; 5. Cementing of the femoral component of the prosthesis; and 6. Immediately following skin closure. Results presented as AUC (area under the aggregation curve), means with standard deviation.Panel

(a) ADP-test (Adenosine diphosphate 6.5M), ANOVA p=0.012(b) ASPI-test (0.5mM arachidonic acid)(c) TRAP-test (32M thrombin receptor activating peptide)(d) Coll-test (3.2g/mL collagen)

Fig 3: Correlation of ADP-stimulated platelet aggregation (Multiplate ADP-test) at pre-operative time point with total blood loss. n= 12 patients for whom blood loss data was available. Spearman’s Rank correlation coefficient r=-0.624, p=0.04

1

FIGURE 1: Study recruitment process

2 Patients

1 No arterial access, 1 change of surgical plan

15 Patients

Data collected

14 Patients

3 Unavailable, 11 surgery dates unsuitable for testing

17 Patients

Formally consented

22 Patients

Declined Study involvement

51 Patients

Identified

31 Patients

Initial consent given

15 Patients

Final Study Group

1

Legends

Fig 1 Study Recruitment Process.

Fig 2. Platelet aggregation in whole blood by MEA (multi-electrode aggregometry) using Multiplate ® analyser, with four agonists. n=15 patients at 6 time points before and during hip replacement operation: Sample 1. Pre-operatively before anaesthetic; 2. On commencement of the skin incision; 3. At removal of the femoral head; 4. Reaming of the femoral canal; 5. Cementing of the femoral component of the prosthesis; and 6. Immediately following skin closure. Results presented as AUC (area under the aggregation curve), means with standard deviation.Panel

(e) ADP-test (Adenosine diphosphate 6.5M), ANOVA p=0.012

(f) ASPI-test (0.5mM arachidonic acid)(g) TRAP-test (32M thrombin receptor activating peptide)(h) Coll-test (3.2g/mL collagen)

Fig 3: Correlation of ADP-stimulated platelet aggregation (Multiplate ADP-test) at pre-operative time point with total blood loss. n= 12 patients for whom blood loss data was available. Spearman’s Rank correlation coefficient r=-0.624, p=0.04

2

3

4

TABLE 1: Baseline characteristics of study population

Age, median (range) 69 (56-78)

Gender, male: female, n 9:6

ASA grade, n

ASA 1 1

ASA 2 12

ASA 3 2

Antiplatelet drug use, n 4

Pre-operative vital signs, median (range)

Systolic blood pressure (mmHg) 150 (109-199)

Heart rate (bpm) 82 (62-97)

Temperature (°C) 37.2 (36.7-37.6)

O2 saturation 97 (92-100)

Type of anaesthetic, n

Spinal 14

Spinal + general 1

Tranexamic acid use, n 11

Laterality, left: right, n 6:9

Blood loss, ml, median (range) 555 (200-1000)ASA – American Society of Anaethesiologists grade

5

TABLE 2: Platelet aggregation, as area under curve (AUC) mean and standard deviation, by

time point

Time Point Agonist

ADP TRAP COL ASPI

Pre-op 80 (17.06) 152 (23.48) 73 (16.29) 82 (26.37)

Knife to skin 76 (21.62) 136 (26.23) 69 (18.64) 72 (33.44)

Femoral Head Removal 78 (24.76) 143 (23.71) 72 (17.05) 73 (34.74)

Femoral Reaming 79 (21.20) 141 (25.13) 75 (20.80) 72 (36.16)

Femoral Cementing 80 (25.22) 152 (48.60) 79 (22.07) 75 (41.72)

Post-op 90 (24.02) 179 (36.18) 91 (29.01) 87 (42.33)

[Mean (SD)]

ADP – Adenosine Diphosphate; TRAP – Thrombin Receptor Activating Peptide; COL – Collagen; ASPI – Arachidonic acid

6

TABLE 3: Haematological parameters, mean and standard deviation, by time point

Time Point Hb (g/l) Plts (x109/l) WCC(x109/l)

Pre-op 139.7 (10.54) 283 (71.6) 6.3 (1.23)

Commencement 131.7 (10.77) 264 (67.5) 4.9 (1.28)

Femoral Head Removal 129.3 (10.43) 264 (59.1) 4.8 (1.11)

Femoral Reaming 126.1 (11.41) 260 (60.0) 5.0 (1.71)

Femoral Cementing 123.6 (12.88) 255 (63.3) 5.7 (2.23)

Post-op 121.3 (13.52) 255 (51.7) 7.5 (2.60)

Mean (SD)

Hb – Haemoglobin; Plts –Platelet count; WCC – White Cell Count