Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 1168—1172

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Plastic containers and the whole-blood clotting test:glass remains the best option

Richard Stonea,∗, Jamie Seymourb, Oliver Marshall b

a Department of Emergency Medicine, Cairns Base Hospital, P.O. Box 902, Cairns, Queensland 4870, Australiab School of Tropical Biology, James Cook University, P.O. Box 6811, Cairns, Queensland 4870, Australia

Received 7 January 2006; received in revised form 31 January 2006; accepted 31 January 2006Available online 12 June 2006

KEYWORDSWhole-bloodcoagulation time;Whole-blood clottingtest;Snake bite;Glass;Plastic

Summary This is the first study to identify normal whole-blood clotting times in various plas-tic containers and to identify the effect of the addition of various concentrations of Pseudechisaustralis (Mulga snake) venom on the clotting time in glass and plastic. Polycarbonate was iden-tified as a potential alternative to glass as a testing container owing to a whole-blood clottingtime within acceptable limits for a bedside test (mean 29.5 min) and equivalent performanceto glass in the presence of P. australis venom. Other plastic containers (such as polypropyleneand polyethylene) were found to be unsuitable owing to very prolonged clotting times (>60 min)or impaired performance in the presence of venom. Overall, owing to the variation betweenthe performance of different plastics and the difficulty in differentiating between them, plastic

containers cannot be recommended as an alternative to glass when performing the whole-bloodclotting test for envenomed patients.

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. Introduction

oagulopathy is an important clinical manifestation ofnvenoming by many members of the viperid, elapid androtalid families of snakes. The incidence of snake bite-

elated coagulopathy and its impact on morbidity andortality is unknown, but in Africa and the Middle East

he Carpet Viper (Echis carinatus) probably bites and killsore people than any other snake in the world, and up

∗ Corresponding author. Present address: Department of Emer-ency Medicine, The Townsville Hospital, 100 Angus Smith Drive,ouglas, Queensland 4814, Australia. Tel.: +61 7 4796 1111;ax: +61 7 4796 2901.

E-mail address: richard stone@health.qld.gov.au (R. Stone).

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035-9203/$ — see front matter © 2006 Royal Society of Tropical Medicinoi:10.1016/j.trstmh.2006.01.012

l Medicine and Hygiene. Published by Elsevier Ltd. All rights

o 93% of patients envenomed have evidence of inco-gulable blood (Warrell et al., 1977). Across the world,p to 2.5 million envenomations occur each year, mainlyn warmer climates and where economic activities areargely agricultural (Chippaux, 1998). In Australia, snakeite is a much smaller but no less important healthssue. Coagulopathy is a feature of envenomation by allf the major Australian elapids except for the Deathdder (Acanthophis spp.) (Currie, 2000a; Dambisya et al.,995; Tibballs et al., 1991) and occurs in up to 50% ofhe 50—300 envenomed patients treated annually (Barrett

nd Little, 2003; Hughes, 2003; Sutherland and Leonard,995).

Objective evidence of coagulopathy is an important earlyndicator of envenomation and is usually evident 30—120 minfter the snake bite (Currie, 2000a). Although early systemic

e and Hygiene. Published by Elsevier Ltd. All rights reserved.

Whole-blood clotting test: the best option

clinical features (such as collapse, headache, nausea, vomit-ing and abdominal pain) may precede this, they are of a non-specific nature and other more specific objective clinicalsigns of envenomation such as neurotoxicity and myotoxicitytypically occur several hours after the onset of coagulopathy(Currie, 2000a). The whole-blood clotting test (WBCT) hasbeen identified as a valuable diagnostic test and an accurateindicator of coagulopathy in snake bite patients (Isbister andCurrie, 2003) and was originally described as a measure ofblood coagulability in patients envenomed by E. carinatus inNigeria (Warrell et al., 1977). In its most simple form, wholeblood is added to a clean, dry glass test tube and describedas either clotted or unclotted after 20 min (Warrell et al.,1977). This form of the test is sometimes referred to as theWBCT20. Clot formation can be timed more accurately todifferentiate between normal clotting (whole-blood clottingtime <10 min), mildly abnormal clotting (whole-blood clot-ting time 10—20 min) and severely abnormal clotting (whole-blood clotting time >20 min) (Isbister and Currie, 2003). The20-min threshold is based on original descriptions by Warrellet al. (1977) and is specific to glass as a testing container.There is no medical literature describing the WBCT using acontainer other than a glass test tube.

The WBCT has been described as a simple bedside test(Currie, 2000b; Warrell et al., 1977) but the requirement fora clean glass laboratory test tube renders the test uselessif this basic requirement is not met. Modern health facili-ties (especially in Australia) have in many cases moved awayfrom the use of glass products in favour of plastic alter-natives both in laboratory and clinical settings. A possiblealternative to glass is to modify the WBCT to use equiva-lent plastic containers; however, the effect and reliabilityof plastic used in this context has not been tested and thereis considerable doubt as to its effectiveness. This study aimsto identify the effectiveness of various plastic containers inperforming the WBCT in envenomed snake bite patients.

2. Materials and methods

2.1. Ethics statement

Ethical approval to perform this study was received fromJames Cook University, Cairns, Queensland, Australia (refer-

Table 1 Container types

Container Container description

Polyethylene terephthalateand clot activator

‘Vacuette’ Z/serum separatoactivator blood collection tub

Polypropylene Sterile screw top plastic speccontainer

Polyethylene terephthalate Syringe

Polycarbonate Round base plastic test tube

Glass Round base glass test tube

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ence A10002). The free and informed consent of the exper-imental subjects was obtained.

2.2. Pilot study: whole-blood clotting times inglass and various plastic containers

A methodological study was performed to identify normalwhole-blood clotting times in glass and various plastic con-tainers. The container types are listed in Table 1. Containerswere identified on the basis of their ubiquitous availabil-ity in health facilities, low cost and ease of use. Blood wastaken from 16 healthy volunteer nursing staff. Subjects wereexcluded from the study if they were currently taking antico-agulant medication. A total of 10 ml of blood was collectedfrom each subject, with 2 ml of blood added to each ofthe five prepared replicate containers. Each replicate wasexamined at 5-min intervals until clotting had occurred. Clotformation was defined as ‘substantial’ clot within the sampleand was recorded as ‘yes’ or ‘no’. Agitation of the sampleduring and between readings was minimised to that neces-sary in order to determine whether substantial clot had orhad not formed. Data were analysed using AVOVA to deter-mine whether clotting time varied between subjects and todetermine mean whole-blood clotting times in glass and var-ious plastic containers for an unenvenomed subject. Resultsof the pilot study were used to determine the time thresh-old for clot examination as well as inclusion or exclusion ofparticular plastic types in subsequent stages of the study.

2.3. WBCT20 in glass containing various venomconcentrations

A blinded methodological study was performed to identifythe proportion of whole blood samples that had clotted at20 min in glass test tubes containing various concentrationsof Pseudechis australis (Mulga snake) venom. Venom wasextracted from a mature male P. australis as described byWillemse et al. (1979).

Twenty 4-ml glass test tubes were prepared with

lyophilised P. australis venom so that the addition of 2 mlof blood resulted in a blood sample venom concentrationof 0, 10, 100, 1000, 10 000 and 100 000 ng/ml, respec-tively. All venom concentrations were determined using

Volume Manufacturer

r; clote

8 ml Greiner Bio-One,Kremsmuenster, Austria

imen 70 ml Sarstedt Australia, Ingle Farm,South Australia, Australia

30 ml Terumo Corporation, MacquariePark, New South Wales,Australia

4 ml Abbott Diagnostics, NorthRyde, New South Wales,Australia

4 ml Sarstedt Australia, Ingle Farm,South Australia, Australia

R. Stone et al.

Figure 1 Whole-blood clotting test (WBCT) times in glass andvarious plastic containers with no added venom. PET, polyethy-lene terephthalate; PP, polypropylene; PC, polycarbonate. Errorbars represent 95% CI.

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Bradford—Lowry analysis. A total of four replicates was usedat each concentration and an additional 20 replicates wereused as controls. Investigators were blinded to the concen-tration of venom present in each test tube. A total of 80 ml ofblood was taken from a healthy volunteer author (J.S.), with2 ml of blood added to each of the prepared glass test tubesand controls. Each of the samples was examined at 20 min.The percentage of samples clotted after 20 min was thendetermined and differences between the WBCT20 for differ-ent concentrations of venom were calculated using ANOVA.

2.4. WBCT results after 20 min (WBCT20) and35 min (WBCT35) in containers containing variousvenom concentrations

A blinded methodological study was performed to identifythe proportion of WBCT20 and WBCT35 clotted samples inglass, polycarbonate and polyethylene terephthalate/clotactivator containing various concentrations of P. australisvenom. The methods used were identical to that for glasstubes with the exception that all samples were examined at20 min and 35 min. WBCT35 was determined as a result ofpilot study data for polycarbonate.

2.5. Statistical analysis

All data were analysed using SPSS, version 12.0.1 (SPSS Inc.,Chicago, IL, USA).

3. Results

3.1. Whole-blood clotting times in glass andvarious plastic containers

The whole-blood clotting time for blood with no addedvenom was significantly different between the five contain-ers tested (F = 315.8, d.f. = 4 × 77, P < 0.05). Post-hoc analy-sis showed that there was no significant difference betweenwhole-blood clotting time in glass and polyethylene/clotactivator (mean 9.6 min) but that the clotting times forall other containers were significantly different. Whole-blood clotting time was greatest in polypropylene (mean74.3 min) followed by polyethylene (mean 64 min) and poly-carbonate (mean 29.5 min) (Figure 1). In view of exces-sive clotting times in polypropylene and plain polyethylene,these containers were excluded from further testing withvenom.

3.2. WBCT20 in glass containing various venomconcentrations

WBCT20 in glass containing various concentrations of venomshowed that there was a significantly greater proportionof samples that clotted at 20 min for venom concentra-

tions of 0 ng/ml and 10 ng/ml compared with higher venomconcentrations (�2 = 36, d.f. = 3, P < 0.05). All samples withvenom concentrations of 0 ng/ml and 10 ng/ml were clottedat 20 min (Figure 2).

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igure 2 The effect of various concentrations of venom onhole-blood clotting test results after 20 min in glass.

.3. WBCT20 and WBCT35 in containers containingarious venom concentrations

ddition of venom to blood samples in polyethylene/clotctivator had no effect on WBCT20 results. All samples werelotted at 20 min regardless of venom concentration. In allamples where clotting occurred, there was either no dif-erence between the percentage clotted for either of thewo time intervals, or a higher percentage was clotted after5 min. For venom concentrations above 100 ng/ml, no clot-ing was seen in either the glass or the polycarbonate tubesFigure 3).

. Discussion

his is the first study to investigate the use of containersther than glass for performing the WBCT in the context of

Whole-blood clotting test: the best option

Figure 3 The effect of various concentrations of venom onwhole-blood clotting test results after 20 min (WBCT20) and

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paatmuopadoaWttour results, plastic is a theoretical rather than a practi-

35 min (WBCT35) in glass, polycarbonate, and polyethylene andclot activator. PET, polyethylene terephthalate; PC, polycarbon-ate.

snake bite. Results demonstrate that whole-blood clottingtimes are dependent on the specific plastic type used tomanufacture the container, with some plastics (polypropy-lene and plain polyethylene) shown to be unsuitable test-ing containers owing to their prolonged clotting times. Theability of polycarbonate containers to cause clotting withinan acceptable timeframe was unexpected; however, dis-tinguishing polycarbonate from other plastic containers isdifficult and for this reason there seems little advantage inchoosing polycarbonate over glass.

In the presence of blood containing P. australis venom,polycarbonate behaves in a similar way to glass. Polycar-bonate correctly identified envenomed blood in this studyif the threshold for identification of clot was increasedto 35 min, and the WBCT35 in polycarbonate may be apossible alternative to the WBCT20 in glass. In contrast,polyethylene/clot activator (which behaves identically toglass in the absence of venom) causes clotted WBCT20results at venom concentrations sufficient to cause unclot-ted WBCT20 results in glass. Glass causes clot formationby disruption of platelets and activation of clotting factors(Rapaport et al., 1954) therefore it is evident that clot acti-vator (a micronised silica coating) is more effective at thisprocess.

Although it was not the aim of the study, our results illus-trate some potential limitations of the WBCT20 in glass.Several investigators have found that an unclotted WBCT20in glass is a sensitive and specific indicator of severe coag-ulopathy (Dambisya et al., 1995; Isbister and Currie, 2003;Tibballs et al., 1991). However, even if severely abnormalclotting is present (as defined by an unclotted WBCT20),clotting can occur in glass if the sample is left for moreprolonged periods (in this case 35 min). This is an impor-tant finding and illustrates the potential for false-negativeresults if the WBCT is read beyond the 20-min time thresh-old. It is possible that this potential for error is specificto particular venom types and the different site of activitythey have on the coagulation cascade. Pseudechis australis

venom causes a mild anticoagulant effect via prevention ofthe generation of factor Xa and inhibition of platelet aggre-gation. Other venoms (including those produced by otherAustralian elapids and many viperids) primarily cause dis-

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eminated intravascular coagulation and this may result inncoagulable blood (regardless of the time at which the sam-le is read) secondary to afibrinogenaemia and depletion ofther clotting factors (Sutherland and Tibballs, 2001).

There are several limitations to this study that should beonsidered when interpreting the results. First, difficultiesere identified in determining the point at which substantiallot had formed in the sample containers. This was partic-larly the case in large-volume containers where the clotas very mobile. Owing to the large number of samples,

rained assistants were used to interpret the results and,espite their consistency of approach, measurement biasould have been introduced. Interobserver correlation inetermining time to clot was not quantified. Second, con-ainer volumes were not standardised for the pilot study onhe basis that any identified alternative container shoulde commonly available. It is assumed that identified differ-nces in whole-blood clotting times are solely dependent onaterial type, but the impact of the volume of the container

or, more specifically, the surface area in contact with blood)s unknown. Third, the absence of a procoagulant effect of P.ustralis venom dictated its use in this study. Although thisotentially influences extrapolation to other venom types,his is unlikely to be of clinical significance. Finally, focus onhe WBCT20 rather than clotting time failed to allow moreccurate differentiation between samples (particularly forolyethylene/clot activator), which may have indicated theeed for a reduced time threshold for clot identification.

Although unanswered questions remain, further investi-ation of the role of plastic containers in performing theBCT seems unjustified in light of the conclusions of this

tudy. The potential role of the WBCT in monitoring clinicalesponse to antivenom is highlighted by the identified rela-ionship between P. australis venom concentration and theroportion of clotted samples. Further study could investi-ate the nature of the correlation between venom concen-ration and clotting time for a range of venom types as wells the effect on clotting time of antivenom administration.onsideration needs to be given to in vivo testing in view ofhe technical difficulties of in vitro testing using procoagu-ant venoms.

Despite its limitations, the WBCT in glass is a rapid test toerform compared with laboratory-based coagulation testsnd is accurate in the context of snake bite. In small ruralnd remote health facilities without access to formal labora-ory coagulation tests, the WBCT may be the only objectiveeasure of envenoming. This was the basis for the initial

se of the test and remains important to this day through-ut the world. Early identification of coagulopathy allowsrompt clinical decision-making, particularly with regard todministration of antivenom (if available), the need for highependency or intensive care, and the need for transportr retrieval to a larger hospital depending on locally avail-ble facilities. Potential reasons for underutilisation of theBCT include lack of knowledge of the use of the test in

he context of snake bite and lack of access to glass testubes in modern healthcare environments. On the basis of

al alternative to glass and efforts should be concentratedn educating clinicians and ensuring availability of glassest tubes rather than focusing on an alternative testingontainer.

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. Conclusions

hree conclusions can be drawn from the results of thistudy. (1) Polypropylene and plain polyethylene are unsuit-ble testing containers for the WBCT owing to their pro-onged whole-blood clotting times. Polyethylene/clot acti-ator containers are unsuitable owing to clot formation inhe presence of P. australis venom concentrations sufficiento cause unclotted WBCT20 in glass. (2) Although polycar-onate has been identified as a possible alternative WBCTesting container material, polycarbonate products are dif-cult to identify and are unlikely to be universally avail-ble in health facilities. Therefore, polycarbonate cannote recommended as a viable alternative to glass. (3) Theraditional WBCT20 in glass has the potential to give false-egative results if clot identification occurs beyond 20 min.

onflicts of interest statementhe authors have no conflicts of interest concerning the workeported in this paper.

cknowledgements

e thank the Cairns Tropical Zoo, Cairns, Queensland, Aus-ralia, for providing snake venom. We also thank Dr Peterereira and Theona Osborne of Cairns Base Hospital for theirssistance in data collection.

eferences

arrett, R., Little, M., 2003. Five years of snake envenoming in farnorth Queensland. Emerg. Med. 15, 500—510.

W

R. Stone et al.

hippaux, J.P., 1998. Snake-bites: appraisal of the global situation.Bull. World Health Organ. 76, 515—524.

urrie, B.J., 2000a. Snakebite in tropical Australia, Papua NewGuinea and Irian Jaya. Emerg. Med. 12, 285—294.

urrie, B., 2000b. Clinical toxicology: a tropical Australian perspec-tive. Ther. Drug Monit. 22, 73—78.

ambisya, Y.M., Lee, T., Gopalakrishnakone, P., 1995. Anticoagulanteffects of Pseudechis australis (Australian King Brown Snake)venom on human blood: a computerized thromboelastographystudy. Toxicon 33, 1378—1382.

ughes, A., 2003. Observation of snakebite victims: is twelve hoursstill necessary? Emerg. Med. 15, 511—517.

sbister, G.K., Currie, B.J., 2003. Suspected snakebite: one yearprospective study of emergency department presentations.Emerg. Med. 15, 160—169.

apaport, S., Aas, K., Owren, P.A., 1954. The effect of glass uponthe activity of the various clotting factors. J. Clin. Invest. 34,9—19.

utherland, S., Leonard, R., 1995. Snakebite deaths in Australia1992—1994 and a management update. Med. J. Aust. 163,616—618.

utherland, S., Tibballs, J., 2001. Australian animal toxins: the crea-tures, their toxins and care of the poisoned patient, second ed.Oxford University Press, Melbourne.

ibballs, J., Sutherland, S.K., Kerr, S., 1991. Studies on Australiansnake venoms, Part II: the haematological effects of brown snake(Pseudonaja) species in the dog. Anaesth. Intensive Care 19,338—342.

arrell, D.A., Davidson, N.McD., Greenwood, B.M., Ormerod, L.D.,Pope, H.M., Watkins, B.J., Prentice, C.R., 1977. Poisoning bybites of the saw-scaled or carpet viper (Echis carinatus) in Nige-

ria. Q. J. Med. 46, 33—62.

illemse, G.T., Hattingh, J., Karlsson, R.M., Levy, S., Parker, C.,1979. Changes in the composition and protein concentration ofpuff adder (Bitis arietans) venom due to frequent milking. Tox-icon 17, 37—42.