- Email: [email protected]
Plastic containers and the whole-blood clotting test: glass remains the best option
Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 1168—1172
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
Plastic containers and the whole-blood clotting test: glass remains the best option Richard Stone a,∗, Jamie Seymour b, Oliver Marshall b a b
Department of Emergency Medicine, Cairns Base Hospital, P.O. Box 902, Cairns, Queensland 4870, Australia 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 2006 Available online 12 June 2006
KEYWORDS Whole-blood coagulation time; Whole-blood clotting test; Snake bite; Glass; Plastic
Summary This is the first study to identify normal whole-blood clotting times in various plastic containers and to identify the effect of the addition of various concentrations of Pseudechis australis (Mulga snake) venom on the clotting time in glass and plastic. Polycarbonate was identified as a potential alternative to glass as a testing container owing to a whole-blood clotting time within acceptable limits for a bedside test (mean 29.5 min) and equivalent performance to glass in the presence of P. australis venom. Other plastic containers (such as polypropylene and 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 between the 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-blood clotting test for envenomed patients. © 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Coagulopathy is an important clinical manifestation of envenoming by many members of the viperid, elapid and crotalid families of snakes. The incidence of snake biterelated coagulopathy and its impact on morbidity and mortality is unknown, but in Africa and the Middle East the Carpet Viper (Echis carinatus) probably bites and kills more people than any other snake in the world, and up
∗
Corresponding author. Present address: Department of Emergency Medicine, The Townsville Hospital, 100 Angus Smith Drive, Douglas, Queensland 4814, Australia. Tel.: +61 7 4796 1111; fax: +61 7 4796 2901. E-mail address: richard [email protected] (R. Stone).
to 93% of patients envenomed have evidence of incoagulable blood (Warrell et al., 1977). Across the world, up to 2.5 million envenomations occur each year, mainly in warmer climates and where economic activities are largely agricultural (Chippaux, 1998). In Australia, snake bite is a much smaller but no less important health issue. Coagulopathy is a feature of envenomation by all of the major Australian elapids except for the Death Adder (Acanthophis spp.) (Currie, 2000a; Dambisya et al., 1995; Tibballs et al., 1991) and occurs in up to 50% of the 50—300 envenomed patients treated annually (Barrett and Little, 2003; Hughes, 2003; Sutherland and Leonard, 1995). Objective evidence of coagulopathy is an important early indicator of envenomation and is usually evident 30—120 min after the snake bite (Currie, 2000a). Although early systemic
0035-9203/$ — see front matter © 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2006.01.012
Whole-blood clotting test: the best option clinical features (such as collapse, headache, nausea, vomiting and abdominal pain) may precede this, they are of a nonspecific nature and other more specific objective clinical signs of envenomation such as neurotoxicity and myotoxicity typically occur several hours after the onset of coagulopathy (Currie, 2000a). The whole-blood clotting test (WBCT) has been identified as a valuable diagnostic test and an accurate indicator of coagulopathy in snake bite patients (Isbister and Currie, 2003) and was originally described as a measure of blood coagulability in patients envenomed by E. carinatus in Nigeria (Warrell et al., 1977). In its most simple form, whole blood is added to a clean, dry glass test tube and described as either clotted or unclotted after 20 min (Warrell et al., 1977). This form of the test is sometimes referred to as the WBCT20. Clot formation can be timed more accurately to differentiate between normal clotting (whole-blood clotting time <10 min), mildly abnormal clotting (whole-blood clotting time 10—20 min) and severely abnormal clotting (wholeblood clotting time >20 min) (Isbister and Currie, 2003). The 20-min threshold is based on original descriptions by Warrell et al. (1977) and is specific to glass as a testing container. There is no medical literature describing the WBCT using a container 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 for a clean glass laboratory test tube renders the test useless if this basic requirement is not met. Modern health facilities (especially in Australia) have in many cases moved away from the use of glass products in favour of plastic alternatives both in laboratory and clinical settings. A possible alternative to glass is to modify the WBCT to use equivalent plastic containers; however, the effect and reliability of plastic used in this context has not been tested and there is considerable doubt as to its effectiveness. This study aims to identify the effectiveness of various plastic containers in performing the WBCT in envenomed snake bite patients.
2. Materials and methods 2.1. Ethics statement Ethical approval to perform this study was received from James Cook University, Cairns, Queensland, Australia (refer-
Table 1
1169 ence A10002). The free and informed consent of the experimental subjects was obtained.
2.2. Pilot study: whole-blood clotting times in glass and various plastic containers A methodological study was performed to identify normal whole-blood clotting times in glass and various plastic containers. The container types are listed in Table 1. Containers were identified on the basis of their ubiquitous availability in health facilities, low cost and ease of use. Blood was taken from 16 healthy volunteer nursing staff. Subjects were excluded from the study if they were currently taking anticoagulant medication. A total of 10 ml of blood was collected from each subject, with 2 ml of blood added to each of the five prepared replicate containers. Each replicate was examined at 5-min intervals until clotting had occurred. Clot formation was defined as ‘substantial’ clot within the sample and was recorded as ‘yes’ or ‘no’. Agitation of the sample during and between readings was minimised to that necessary in order to determine whether substantial clot had or had not formed. Data were analysed using AVOVA to determine whether clotting time varied between subjects and to determine mean whole-blood clotting times in glass and various plastic containers for an unenvenomed subject. Results of the pilot study were used to determine the time threshold for clot examination as well as inclusion or exclusion of particular plastic types in subsequent stages of the study.
2.3. WBCT20 in glass containing various venom concentrations A blinded methodological study was performed to identify the proportion of whole blood samples that had clotted at 20 min in glass test tubes containing various concentrations of Pseudechis australis (Mulga snake) venom. Venom was extracted from a mature male P. australis as described by Willemse et al. (1979). Twenty 4-ml glass test tubes were prepared with lyophilised P. australis venom so that the addition of 2 ml of blood resulted in a blood sample venom concentration of 0, 10, 100, 1000, 10 000 and 100 000 ng/ml, respectively. All venom concentrations were determined using
Container types
Container
Container description
Volume
Manufacturer
Polyethylene terephthalate and clot activator Polypropylene
8 ml
Polyethylene terephthalate
‘Vacuette’ Z/serum separator; clot activator blood collection tube Sterile screw top plastic specimen container Syringe
30 ml
Polycarbonate
Round base plastic test tube
4 ml
Glass
Round base glass test tube
4 ml
Greiner Bio-One, Kremsmuenster, Austria Sarstedt Australia, Ingle Farm, South Australia, Australia Terumo Corporation, Macquarie Park, New South Wales, Australia Abbott Diagnostics, North Ryde, New South Wales, Australia Sarstedt Australia, Ingle Farm, South Australia, Australia
70 ml
1170
R. Stone et al.
Bradford—Lowry analysis. A total of four replicates was used at each concentration and an additional 20 replicates were used as controls. Investigators were blinded to the concentration of venom present in each test tube. A total of 80 ml of blood was taken from a healthy volunteer author (J.S.), with 2 ml of blood added to each of the prepared glass test tubes and controls. Each of the samples was examined at 20 min. The percentage of samples clotted after 20 min was then determined and differences between the WBCT20 for different concentrations of venom were calculated using ANOVA.
2.4. WBCT results after 20 min (WBCT20) and 35 min (WBCT35) in containers containing various venom concentrations A blinded methodological study was performed to identify the proportion of WBCT20 and WBCT35 clotted samples in glass, polycarbonate and polyethylene terephthalate/clot activator containing various concentrations of P. australis venom. The methods used were identical to that for glass tubes with the exception that all samples were examined at 20 min and 35 min. WBCT35 was determined as a result of pilot study data for polycarbonate.
Figure 1 Whole-blood clotting test (WBCT) times in glass and various plastic containers with no added venom. PET, polyethylene terephthalate; PP, polypropylene; PC, polycarbonate. Error bars represent 95% CI.
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 and various plastic containers The whole-blood clotting time for blood with no added venom was significantly different between the five containers tested (F = 315.8, d.f. = 4 × 77, P < 0.05). Post-hoc analysis showed that there was no significant difference between whole-blood clotting time in glass and polyethylene/clot activator (mean 9.6 min) but that the clotting times for all other containers were significantly different. Wholeblood clotting time was greatest in polypropylene (mean 74.3 min) followed by polyethylene (mean 64 min) and polycarbonate (mean 29.5 min) (Figure 1). In view of excessive clotting times in polypropylene and plain polyethylene, these containers were excluded from further testing with venom.
3.2. WBCT20 in glass containing various venom concentrations WBCT20 in glass containing various concentrations of venom showed that there was a significantly greater proportion of samples that clotted at 20 min for venom concentrations of 0 ng/ml and 10 ng/ml compared with higher venom concentrations (2 = 36, d.f. = 3, P < 0.05). All samples with venom concentrations of 0 ng/ml and 10 ng/ml were clotted at 20 min (Figure 2).
Figure 2 The effect of various concentrations of venom on whole-blood clotting test results after 20 min in glass.
3.3. WBCT20 and WBCT35 in containers containing various venom concentrations Addition of venom to blood samples in polyethylene/clot activator had no effect on WBCT20 results. All samples were clotted at 20 min regardless of venom concentration. In all samples where clotting occurred, there was either no difference between the percentage clotted for either of the two time intervals, or a higher percentage was clotted after 35 min. For venom concentrations above 100 ng/ml, no clotting was seen in either the glass or the polycarbonate tubes (Figure 3).
4. Discussion This is the first study to investigate the use of containers other 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 on whole-blood clotting test results after 20 min (WBCT20) and 35 min (WBCT35) in glass, polycarbonate, and polyethylene and clot activator. PET, polyethylene terephthalate; PC, polycarbonate.
snake bite. Results demonstrate that whole-blood clotting times are dependent on the specific plastic type used to manufacture the container, with some plastics (polypropylene and plain polyethylene) shown to be unsuitable testing containers owing to their prolonged clotting times. The ability of polycarbonate containers to cause clotting within an acceptable timeframe was unexpected; however, distinguishing polycarbonate from other plastic containers is difficult and for this reason there seems little advantage in choosing polycarbonate over glass. In the presence of blood containing P. australis venom, polycarbonate behaves in a similar way to glass. Polycarbonate correctly identified envenomed blood in this study if the threshold for identification of clot was increased to 35 min, and the WBCT35 in polycarbonate may be a possible alternative to the WBCT20 in glass. In contrast, polyethylene/clot activator (which behaves identically to glass in the absence of venom) causes clotted WBCT20 results at venom concentrations sufficient to cause unclotted WBCT20 results in glass. Glass causes clot formation by disruption of platelets and activation of clotting factors (Rapaport et al., 1954) therefore it is evident that clot activator (a micronised silica coating) is more effective at this process. Although it was not the aim of the study, our results illustrate some potential limitations of the WBCT20 in glass. Several investigators have found that an unclotted WBCT20 in glass is a sensitive and specific indicator of severe coagulopathy (Dambisya et al., 1995; Isbister and Currie, 2003; Tibballs et al., 1991). However, even if severely abnormal clotting is present (as defined by an unclotted WBCT20), clotting can occur in glass if the sample is left for more prolonged periods (in this case 35 min). This is an important finding and illustrates the potential for false-negative results if the WBCT is read beyond the 20-min time threshold. It is possible that this potential for error is specific to particular venom types and the different site of activity they have on the coagulation cascade. Pseudechis australis venom causes a mild anticoagulant effect via prevention of the generation of factor Xa and inhibition of platelet aggregation. Other venoms (including those produced by other Australian elapids and many viperids) primarily cause dis-
1171 seminated intravascular coagulation and this may result in incoagulable blood (regardless of the time at which the sample is read) secondary to afibrinogenaemia and depletion of other clotting factors (Sutherland and Tibballs, 2001). There are several limitations to this study that should be considered when interpreting the results. First, difficulties were identified in determining the point at which substantial clot had formed in the sample containers. This was particularly the case in large-volume containers where the clot was very mobile. Owing to the large number of samples, trained assistants were used to interpret the results and, despite their consistency of approach, measurement bias could have been introduced. Interobserver correlation in determining time to clot was not quantified. Second, container volumes were not standardised for the pilot study on the basis that any identified alternative container should be commonly available. It is assumed that identified differences in whole-blood clotting times are solely dependent on material type, but the impact of the volume of the container (or, more specifically, the surface area in contact with blood) is unknown. Third, the absence of a procoagulant effect of P. australis venom dictated its use in this study. Although this potentially influences extrapolation to other venom types, this is unlikely to be of clinical significance. Finally, focus on the WBCT20 rather than clotting time failed to allow more accurate differentiation between samples (particularly for polyethylene/clot activator), which may have indicated the need for a reduced time threshold for clot identification. Although unanswered questions remain, further investigation of the role of plastic containers in performing the WBCT seems unjustified in light of the conclusions of this study. The potential role of the WBCT in monitoring clinical response to antivenom is highlighted by the identified relationship between P. australis venom concentration and the proportion of clotted samples. Further study could investigate the nature of the correlation between venom concentration and clotting time for a range of venom types as well as the effect on clotting time of antivenom administration. Consideration needs to be given to in vivo testing in view of the technical difficulties of in vitro testing using procoagulant venoms. Despite its limitations, the WBCT in glass is a rapid test to perform compared with laboratory-based coagulation tests and is accurate in the context of snake bite. In small rural and remote health facilities without access to formal laboratory coagulation tests, the WBCT may be the only objective measure of envenoming. This was the basis for the initial use of the test and remains important to this day throughout the world. Early identification of coagulopathy allows prompt clinical decision-making, particularly with regard to administration of antivenom (if available), the need for high dependency or intensive care, and the need for transport or retrieval to a larger hospital depending on locally available facilities. Potential reasons for underutilisation of the WBCT include lack of knowledge of the use of the test in the context of snake bite and lack of access to glass test tubes in modern healthcare environments. On the basis of our results, plastic is a theoretical rather than a practical alternative to glass and efforts should be concentrated on educating clinicians and ensuring availability of glass test tubes rather than focusing on an alternative testing container.
1172
5. Conclusions Three conclusions can be drawn from the results of this study. (1) Polypropylene and plain polyethylene are unsuitable testing containers for the WBCT owing to their prolonged whole-blood clotting times. Polyethylene/clot activator containers are unsuitable owing to clot formation in the presence of P. australis venom concentrations sufficient to cause unclotted WBCT20 in glass. (2) Although polycarbonate has been identified as a possible alternative WBCT testing container material, polycarbonate products are difficult to identify and are unlikely to be universally available in health facilities. Therefore, polycarbonate cannot be recommended as a viable alternative to glass. (3) The traditional WBCT20 in glass has the potential to give falsenegative results if clot identification occurs beyond 20 min. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements We thank the Cairns Tropical Zoo, Cairns, Queensland, Australia, for providing snake venom. We also thank Dr Peter Pereira and Theona Osborne of Cairns Base Hospital for their assistance in data collection.
References Barrett, R., Little, M., 2003. Five years of snake envenoming in far north Queensland. Emerg. Med. 15, 500—510.
R. Stone et al. Chippaux, J.P., 1998. Snake-bites: appraisal of the global situation. Bull. World Health Organ. 76, 515—524. Currie, B.J., 2000a. Snakebite in tropical Australia, Papua New Guinea and Irian Jaya. Emerg. Med. 12, 285—294. Currie, B., 2000b. Clinical toxicology: a tropical Australian perspective. Ther. Drug Monit. 22, 73—78. Dambisya, Y.M., Lee, T., Gopalakrishnakone, P., 1995. Anticoagulant effects of Pseudechis australis (Australian King Brown Snake) venom on human blood: a computerized thromboelastography study. Toxicon 33, 1378—1382. Hughes, A., 2003. Observation of snakebite victims: is twelve hours still necessary? Emerg. Med. 15, 511—517. Isbister, G.K., Currie, B.J., 2003. Suspected snakebite: one year prospective study of emergency department presentations. Emerg. Med. 15, 160—169. Rapaport, S., Aas, K., Owren, P.A., 1954. The effect of glass upon the activity of the various clotting factors. J. Clin. Invest. 34, 9—19. Sutherland, S., Leonard, R., 1995. Snakebite deaths in Australia 1992—1994 and a management update. Med. J. Aust. 163, 616—618. Sutherland, S., Tibballs, J., 2001. Australian animal toxins: the creatures, their toxins and care of the poisoned patient, second ed. Oxford University Press, Melbourne. Tibballs, J., Sutherland, S.K., Kerr, S., 1991. Studies on Australian snake venoms, Part II: the haematological effects of brown snake (Pseudonaja) species in the dog. Anaesth. Intensive Care 19, 338—342. Warrell, D.A., Davidson, N.McD., Greenwood, B.M., Ormerod, L.D., Pope, H.M., Watkins, B.J., Prentice, C.R., 1977. Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus) in Nigeria. Q. J. Med. 46, 33—62. Willemse, G.T., Hattingh, J., Karlsson, R.M., Levy, S., Parker, C., 1979. Changes in the composition and protein concentration of puff adder (Bitis arietans) venom due to frequent milking. Toxicon 17, 37—42.
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/trst
Plastic containers and the whole-blood clotting test: glass remains the best option Richard Stone a,∗, Jamie Seymour b, Oliver Marshall b a b
Department of Emergency Medicine, Cairns Base Hospital, P.O. Box 902, Cairns, Queensland 4870, Australia 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 2006 Available online 12 June 2006
KEYWORDS Whole-blood coagulation time; Whole-blood clotting test; Snake bite; Glass; Plastic
Summary This is the first study to identify normal whole-blood clotting times in various plastic containers and to identify the effect of the addition of various concentrations of Pseudechis australis (Mulga snake) venom on the clotting time in glass and plastic. Polycarbonate was identified as a potential alternative to glass as a testing container owing to a whole-blood clotting time within acceptable limits for a bedside test (mean 29.5 min) and equivalent performance to glass in the presence of P. australis venom. Other plastic containers (such as polypropylene and 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 between the 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-blood clotting test for envenomed patients. © 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Coagulopathy is an important clinical manifestation of envenoming by many members of the viperid, elapid and crotalid families of snakes. The incidence of snake biterelated coagulopathy and its impact on morbidity and mortality is unknown, but in Africa and the Middle East the Carpet Viper (Echis carinatus) probably bites and kills more people than any other snake in the world, and up
∗
Corresponding author. Present address: Department of Emergency Medicine, The Townsville Hospital, 100 Angus Smith Drive, Douglas, Queensland 4814, Australia. Tel.: +61 7 4796 1111; fax: +61 7 4796 2901. E-mail address: richard [email protected] (R. Stone).
to 93% of patients envenomed have evidence of incoagulable blood (Warrell et al., 1977). Across the world, up to 2.5 million envenomations occur each year, mainly in warmer climates and where economic activities are largely agricultural (Chippaux, 1998). In Australia, snake bite is a much smaller but no less important health issue. Coagulopathy is a feature of envenomation by all of the major Australian elapids except for the Death Adder (Acanthophis spp.) (Currie, 2000a; Dambisya et al., 1995; Tibballs et al., 1991) and occurs in up to 50% of the 50—300 envenomed patients treated annually (Barrett and Little, 2003; Hughes, 2003; Sutherland and Leonard, 1995). Objective evidence of coagulopathy is an important early indicator of envenomation and is usually evident 30—120 min after the snake bite (Currie, 2000a). Although early systemic
0035-9203/$ — see front matter © 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2006.01.012
Whole-blood clotting test: the best option clinical features (such as collapse, headache, nausea, vomiting and abdominal pain) may precede this, they are of a nonspecific nature and other more specific objective clinical signs of envenomation such as neurotoxicity and myotoxicity typically occur several hours after the onset of coagulopathy (Currie, 2000a). The whole-blood clotting test (WBCT) has been identified as a valuable diagnostic test and an accurate indicator of coagulopathy in snake bite patients (Isbister and Currie, 2003) and was originally described as a measure of blood coagulability in patients envenomed by E. carinatus in Nigeria (Warrell et al., 1977). In its most simple form, whole blood is added to a clean, dry glass test tube and described as either clotted or unclotted after 20 min (Warrell et al., 1977). This form of the test is sometimes referred to as the WBCT20. Clot formation can be timed more accurately to differentiate between normal clotting (whole-blood clotting time <10 min), mildly abnormal clotting (whole-blood clotting time 10—20 min) and severely abnormal clotting (wholeblood clotting time >20 min) (Isbister and Currie, 2003). The 20-min threshold is based on original descriptions by Warrell et al. (1977) and is specific to glass as a testing container. There is no medical literature describing the WBCT using a container 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 for a clean glass laboratory test tube renders the test useless if this basic requirement is not met. Modern health facilities (especially in Australia) have in many cases moved away from the use of glass products in favour of plastic alternatives both in laboratory and clinical settings. A possible alternative to glass is to modify the WBCT to use equivalent plastic containers; however, the effect and reliability of plastic used in this context has not been tested and there is considerable doubt as to its effectiveness. This study aims to identify the effectiveness of various plastic containers in performing the WBCT in envenomed snake bite patients.
2. Materials and methods 2.1. Ethics statement Ethical approval to perform this study was received from James Cook University, Cairns, Queensland, Australia (refer-
Table 1
1169 ence A10002). The free and informed consent of the experimental subjects was obtained.
2.2. Pilot study: whole-blood clotting times in glass and various plastic containers A methodological study was performed to identify normal whole-blood clotting times in glass and various plastic containers. The container types are listed in Table 1. Containers were identified on the basis of their ubiquitous availability in health facilities, low cost and ease of use. Blood was taken from 16 healthy volunteer nursing staff. Subjects were excluded from the study if they were currently taking anticoagulant medication. A total of 10 ml of blood was collected from each subject, with 2 ml of blood added to each of the five prepared replicate containers. Each replicate was examined at 5-min intervals until clotting had occurred. Clot formation was defined as ‘substantial’ clot within the sample and was recorded as ‘yes’ or ‘no’. Agitation of the sample during and between readings was minimised to that necessary in order to determine whether substantial clot had or had not formed. Data were analysed using AVOVA to determine whether clotting time varied between subjects and to determine mean whole-blood clotting times in glass and various plastic containers for an unenvenomed subject. Results of the pilot study were used to determine the time threshold for clot examination as well as inclusion or exclusion of particular plastic types in subsequent stages of the study.
2.3. WBCT20 in glass containing various venom concentrations A blinded methodological study was performed to identify the proportion of whole blood samples that had clotted at 20 min in glass test tubes containing various concentrations of Pseudechis australis (Mulga snake) venom. Venom was extracted from a mature male P. australis as described by Willemse et al. (1979). Twenty 4-ml glass test tubes were prepared with lyophilised P. australis venom so that the addition of 2 ml of blood resulted in a blood sample venom concentration of 0, 10, 100, 1000, 10 000 and 100 000 ng/ml, respectively. All venom concentrations were determined using
Container types
Container
Container description
Volume
Manufacturer
Polyethylene terephthalate and clot activator Polypropylene
8 ml
Polyethylene terephthalate
‘Vacuette’ Z/serum separator; clot activator blood collection tube Sterile screw top plastic specimen container Syringe
30 ml
Polycarbonate
Round base plastic test tube
4 ml
Glass
Round base glass test tube
4 ml
Greiner Bio-One, Kremsmuenster, Austria Sarstedt Australia, Ingle Farm, South Australia, Australia Terumo Corporation, Macquarie Park, New South Wales, Australia Abbott Diagnostics, North Ryde, New South Wales, Australia Sarstedt Australia, Ingle Farm, South Australia, Australia
70 ml
1170
R. Stone et al.
Bradford—Lowry analysis. A total of four replicates was used at each concentration and an additional 20 replicates were used as controls. Investigators were blinded to the concentration of venom present in each test tube. A total of 80 ml of blood was taken from a healthy volunteer author (J.S.), with 2 ml of blood added to each of the prepared glass test tubes and controls. Each of the samples was examined at 20 min. The percentage of samples clotted after 20 min was then determined and differences between the WBCT20 for different concentrations of venom were calculated using ANOVA.
2.4. WBCT results after 20 min (WBCT20) and 35 min (WBCT35) in containers containing various venom concentrations A blinded methodological study was performed to identify the proportion of WBCT20 and WBCT35 clotted samples in glass, polycarbonate and polyethylene terephthalate/clot activator containing various concentrations of P. australis venom. The methods used were identical to that for glass tubes with the exception that all samples were examined at 20 min and 35 min. WBCT35 was determined as a result of pilot study data for polycarbonate.
Figure 1 Whole-blood clotting test (WBCT) times in glass and various plastic containers with no added venom. PET, polyethylene terephthalate; PP, polypropylene; PC, polycarbonate. Error bars represent 95% CI.
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 and various plastic containers The whole-blood clotting time for blood with no added venom was significantly different between the five containers tested (F = 315.8, d.f. = 4 × 77, P < 0.05). Post-hoc analysis showed that there was no significant difference between whole-blood clotting time in glass and polyethylene/clot activator (mean 9.6 min) but that the clotting times for all other containers were significantly different. Wholeblood clotting time was greatest in polypropylene (mean 74.3 min) followed by polyethylene (mean 64 min) and polycarbonate (mean 29.5 min) (Figure 1). In view of excessive clotting times in polypropylene and plain polyethylene, these containers were excluded from further testing with venom.
3.2. WBCT20 in glass containing various venom concentrations WBCT20 in glass containing various concentrations of venom showed that there was a significantly greater proportion of samples that clotted at 20 min for venom concentrations of 0 ng/ml and 10 ng/ml compared with higher venom concentrations (2 = 36, d.f. = 3, P < 0.05). All samples with venom concentrations of 0 ng/ml and 10 ng/ml were clotted at 20 min (Figure 2).
Figure 2 The effect of various concentrations of venom on whole-blood clotting test results after 20 min in glass.
3.3. WBCT20 and WBCT35 in containers containing various venom concentrations Addition of venom to blood samples in polyethylene/clot activator had no effect on WBCT20 results. All samples were clotted at 20 min regardless of venom concentration. In all samples where clotting occurred, there was either no difference between the percentage clotted for either of the two time intervals, or a higher percentage was clotted after 35 min. For venom concentrations above 100 ng/ml, no clotting was seen in either the glass or the polycarbonate tubes (Figure 3).
4. Discussion This is the first study to investigate the use of containers other 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 on whole-blood clotting test results after 20 min (WBCT20) and 35 min (WBCT35) in glass, polycarbonate, and polyethylene and clot activator. PET, polyethylene terephthalate; PC, polycarbonate.
snake bite. Results demonstrate that whole-blood clotting times are dependent on the specific plastic type used to manufacture the container, with some plastics (polypropylene and plain polyethylene) shown to be unsuitable testing containers owing to their prolonged clotting times. The ability of polycarbonate containers to cause clotting within an acceptable timeframe was unexpected; however, distinguishing polycarbonate from other plastic containers is difficult and for this reason there seems little advantage in choosing polycarbonate over glass. In the presence of blood containing P. australis venom, polycarbonate behaves in a similar way to glass. Polycarbonate correctly identified envenomed blood in this study if the threshold for identification of clot was increased to 35 min, and the WBCT35 in polycarbonate may be a possible alternative to the WBCT20 in glass. In contrast, polyethylene/clot activator (which behaves identically to glass in the absence of venom) causes clotted WBCT20 results at venom concentrations sufficient to cause unclotted WBCT20 results in glass. Glass causes clot formation by disruption of platelets and activation of clotting factors (Rapaport et al., 1954) therefore it is evident that clot activator (a micronised silica coating) is more effective at this process. Although it was not the aim of the study, our results illustrate some potential limitations of the WBCT20 in glass. Several investigators have found that an unclotted WBCT20 in glass is a sensitive and specific indicator of severe coagulopathy (Dambisya et al., 1995; Isbister and Currie, 2003; Tibballs et al., 1991). However, even if severely abnormal clotting is present (as defined by an unclotted WBCT20), clotting can occur in glass if the sample is left for more prolonged periods (in this case 35 min). This is an important finding and illustrates the potential for false-negative results if the WBCT is read beyond the 20-min time threshold. It is possible that this potential for error is specific to particular venom types and the different site of activity they have on the coagulation cascade. Pseudechis australis venom causes a mild anticoagulant effect via prevention of the generation of factor Xa and inhibition of platelet aggregation. Other venoms (including those produced by other Australian elapids and many viperids) primarily cause dis-
1171 seminated intravascular coagulation and this may result in incoagulable blood (regardless of the time at which the sample is read) secondary to afibrinogenaemia and depletion of other clotting factors (Sutherland and Tibballs, 2001). There are several limitations to this study that should be considered when interpreting the results. First, difficulties were identified in determining the point at which substantial clot had formed in the sample containers. This was particularly the case in large-volume containers where the clot was very mobile. Owing to the large number of samples, trained assistants were used to interpret the results and, despite their consistency of approach, measurement bias could have been introduced. Interobserver correlation in determining time to clot was not quantified. Second, container volumes were not standardised for the pilot study on the basis that any identified alternative container should be commonly available. It is assumed that identified differences in whole-blood clotting times are solely dependent on material type, but the impact of the volume of the container (or, more specifically, the surface area in contact with blood) is unknown. Third, the absence of a procoagulant effect of P. australis venom dictated its use in this study. Although this potentially influences extrapolation to other venom types, this is unlikely to be of clinical significance. Finally, focus on the WBCT20 rather than clotting time failed to allow more accurate differentiation between samples (particularly for polyethylene/clot activator), which may have indicated the need for a reduced time threshold for clot identification. Although unanswered questions remain, further investigation of the role of plastic containers in performing the WBCT seems unjustified in light of the conclusions of this study. The potential role of the WBCT in monitoring clinical response to antivenom is highlighted by the identified relationship between P. australis venom concentration and the proportion of clotted samples. Further study could investigate the nature of the correlation between venom concentration and clotting time for a range of venom types as well as the effect on clotting time of antivenom administration. Consideration needs to be given to in vivo testing in view of the technical difficulties of in vitro testing using procoagulant venoms. Despite its limitations, the WBCT in glass is a rapid test to perform compared with laboratory-based coagulation tests and is accurate in the context of snake bite. In small rural and remote health facilities without access to formal laboratory coagulation tests, the WBCT may be the only objective measure of envenoming. This was the basis for the initial use of the test and remains important to this day throughout the world. Early identification of coagulopathy allows prompt clinical decision-making, particularly with regard to administration of antivenom (if available), the need for high dependency or intensive care, and the need for transport or retrieval to a larger hospital depending on locally available facilities. Potential reasons for underutilisation of the WBCT include lack of knowledge of the use of the test in the context of snake bite and lack of access to glass test tubes in modern healthcare environments. On the basis of our results, plastic is a theoretical rather than a practical alternative to glass and efforts should be concentrated on educating clinicians and ensuring availability of glass test tubes rather than focusing on an alternative testing container.
1172
5. Conclusions Three conclusions can be drawn from the results of this study. (1) Polypropylene and plain polyethylene are unsuitable testing containers for the WBCT owing to their prolonged whole-blood clotting times. Polyethylene/clot activator containers are unsuitable owing to clot formation in the presence of P. australis venom concentrations sufficient to cause unclotted WBCT20 in glass. (2) Although polycarbonate has been identified as a possible alternative WBCT testing container material, polycarbonate products are difficult to identify and are unlikely to be universally available in health facilities. Therefore, polycarbonate cannot be recommended as a viable alternative to glass. (3) The traditional WBCT20 in glass has the potential to give falsenegative results if clot identification occurs beyond 20 min. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements We thank the Cairns Tropical Zoo, Cairns, Queensland, Australia, for providing snake venom. We also thank Dr Peter Pereira and Theona Osborne of Cairns Base Hospital for their assistance in data collection.
References Barrett, R., Little, M., 2003. Five years of snake envenoming in far north Queensland. Emerg. Med. 15, 500—510.
R. Stone et al. Chippaux, J.P., 1998. Snake-bites: appraisal of the global situation. Bull. World Health Organ. 76, 515—524. Currie, B.J., 2000a. Snakebite in tropical Australia, Papua New Guinea and Irian Jaya. Emerg. Med. 12, 285—294. Currie, B., 2000b. Clinical toxicology: a tropical Australian perspective. Ther. Drug Monit. 22, 73—78. Dambisya, Y.M., Lee, T., Gopalakrishnakone, P., 1995. Anticoagulant effects of Pseudechis australis (Australian King Brown Snake) venom on human blood: a computerized thromboelastography study. Toxicon 33, 1378—1382. Hughes, A., 2003. Observation of snakebite victims: is twelve hours still necessary? Emerg. Med. 15, 511—517. Isbister, G.K., Currie, B.J., 2003. Suspected snakebite: one year prospective study of emergency department presentations. Emerg. Med. 15, 160—169. Rapaport, S., Aas, K., Owren, P.A., 1954. The effect of glass upon the activity of the various clotting factors. J. Clin. Invest. 34, 9—19. Sutherland, S., Leonard, R., 1995. Snakebite deaths in Australia 1992—1994 and a management update. Med. J. Aust. 163, 616—618. Sutherland, S., Tibballs, J., 2001. Australian animal toxins: the creatures, their toxins and care of the poisoned patient, second ed. Oxford University Press, Melbourne. Tibballs, J., Sutherland, S.K., Kerr, S., 1991. Studies on Australian snake venoms, Part II: the haematological effects of brown snake (Pseudonaja) species in the dog. Anaesth. Intensive Care 19, 338—342. Warrell, D.A., Davidson, N.McD., Greenwood, B.M., Ormerod, L.D., Pope, H.M., Watkins, B.J., Prentice, C.R., 1977. Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus) in Nigeria. Q. J. Med. 46, 33—62. Willemse, G.T., Hattingh, J., Karlsson, R.M., Levy, S., Parker, C., 1979. Changes in the composition and protein concentration of puff adder (Bitis arietans) venom due to frequent milking. Toxicon 17, 37—42.