Comparative sensitivity of commercially available aPTT reagents to mulga snake (Pseudechis australis) venom

Pathology (August 2014) 46(5), pp. 444–449

HAEMATOLOGY

Comparative sensitivity of commercially available aPTT reagents to mulga snake (Pseudechis australis) venom LISA F. LINCZ1,2, FIONA E. SCORGIE1, CHRISTOPHER I. JOHNSTON3,4, MARGARET O’LEARY5, RITAM PRASAD6, MICHAEL SELDON1, EMMANUEL FAVALORO7 AND GEOFFREY K. ISBISTER2,4,5 1Hunter Haematology Research Group, Calvary Mater Newcastle Hospital, Newcastle, NSW, 2Faculty of Health, University of Newcastle, NSW, 3School of Medicine Sydney, University of Notre Dame Australia, Darlinghurst, NSW, 4NSW Poisons Information Centre, Sydney Children’s Hospital Network, NSW, 5Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle Hospital, Newcastle, NSW, 6Haematology Unit, Royal Hobart Hospital, Hobart, Tas, and 7Diagnostic Haemostasis Laboratory, Haematology, ICPMR, Westmead

Hospital, Westmead, NSW, Australia

Summary This study aimed to determine the relative sensitivity of activated partial thromboplastin time (aPTT) reagents to the anticoagulant effects of phospholipases in mulga snake (Pseus) venom. Twenty-one haematology laboratories participating in the Royal College of Pathologists of Australasia Quality Assurance Programs were sent human plasma samples spiked with mulga venom (n ¼ 25 total results). Results for 17 patients with mulga snake envenoming were available through the Australian Snakebite Project. Only 12 of 25 venom spiked samples returned an abnormally prolonged aPTT. Tests performed with Dade Actin FS (n ¼ 7) did not identify any of the spiked samples as abnormal. Although clotting times were significantly prolonged using the lupus anticoagulant sensitive Actin FSL (n ¼ 5, p ¼ 0.043), only one was reported as abnormal. Only laboratories using TriniCLOT aPTT S (n ¼ 6), HemosIL APTT SP (n ¼ 2) and Stago PTT-A (n ¼ 1) consistently recorded the spiked sample as being above the upper normal reference interval. Abnormally prolonged aPTTs were recorded for four of eight patients whose tests were performed with Actin FSL, five of eight patients with TriniCLOT aPTT HS, and three of three patients using TriniCLOT aPTT S. We conclude that some reagents used for routine aPTT testing are relatively insensitive to the anticoagulant effects of mulga snake venom. Tests performed with these reagents should be interpreted with caution. Key words: Anticoagulant, aPTT, black snake, envenoming, lupus anticoagulant, mulga snake, phospholipase, phospholipid, Pseudechis australis. Received 8 October 2013, revised 6 February, accepted 11 February 2014

INTRODUCTION Australian black snake (Pseudechis species) venoms contain toxic phospholipases that have anticoagulant effects in vitro that prolong clotting times.1 Although the resulting coagulopathy in human envenoming is unlikely to be clinically significant, it is a useful early indicator of envenoming, allowing antivenom to be administered before more serious clinical sequelae develop, such as myotoxicity.2 Of equal importance is the potential to use the absence of an anticoagulant coagulopathy to exclude envenoming in suspected Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000120

#

cases, since administration of antivenom carries an estimated 20% risk of a systemic hypersensitivity reaction.3,4 The early diagnosis of envenoming in patients with snakebite is based on identifying the major envenoming syndromes caused by Australian snakes, namely: coagulopathy (venom induced consumption coagulopathy or anticoagulant coagulopathy), myotoxicity, and neurotoxicity. In a recent review of 478 suspected snakebite patients recruited to the Australian Snakebite Project (ASP), the combined laboratory investigations of prothrombin time/international normalised ratio (PT/INR), activated partial thromboplastin time (aPTT) and creatine kinase (CK) level, in addition to neurological assessments, were able to identify severe envenoming in 96% of cases by 6 h and 99% by 12 h post-bite.2 In addition, the coagulation assays became abnormal very soon after the bite, unlike the rise in CK from myotoxicity that took up to 12 h to become abnormal. The aPTT in particular was the most valuable early laboratory indicator for envenoming in black snake (Pseudechis species) bites, becoming abnormal due to the anticoagulant coagulopathy within an hour of the bite. This suggests that the aPTT may be a useful early indicator of black snake envenoming, before there is any evidence of myotoxicity, allowing prompt use of antivenom. However, a study of red-bellied black snake (P. porphyriacus) envenoming found that the aPTT wasn’t always abnormal in envenomed patients, and rather than this being an absence of an anticoagulant coagulopathy, it appeared more likely to be a problem with the variability of the aPTT assay itself.5 A prolonged aPTT can be due to many other causes unrelated to envenoming, making it a useful screening test for various factor inhibitors and lupus anticoagulant, as well as for monitoring heparin therapy. As such, commercially available reagents used to measure aPTT are highly variable, being specifically formulated with different types and amounts of phospholipids and contact activators. In this regard, it has been well documented that the sensitivity of an aPTT reagent for lupus anticoagulant (an immunoglobulin that inhibits coagulation in vitro by interfering with protein-phospholipid complexes) is highly dependent on the phospholipid conditions within the assay.6–10 We hypothesised that a similar phenomenon could occur in the presence of black snake venom, where the anticoagulant effect is due to the presence of a phospholipase. If so, the diversity in aPTT reagents used by different

2014 Royal College of Pathologists of Australasia

Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.

47 (7.0%)

55 (8.1%) 9 (1.3%)

137 (20.3%)

220 (32.5%) Ellagic acid (1x10 M)

445

Silica Diagnostica Stago

TriniCLOT aPTT S HemosIL APTT SP

Stago PTT-A

*

Tcoag Ireland Instrumentation Laboratory

TriniCLOT aPTT HS

According to the RCPA QAP.13 Siemens Healthcare Diagnostics Products (Germany); Tcoag Ireland Ltd (Ireland); Instrumentation Laboratory (USA); Diagnostica Stago (France).

Cephalin from rabbit cerebral tissues 180

Micronised silica Silica Porcine and chicken Synthetic phospholipids 300 Refer to instrument

Micronised silica Porcine and chicken 300

Soy and rabbit brain


4

71 (10.5%) 180

Siemens Healthcare Diagnostics Products Siemens Healthcare Diagnostics Products Tcoag Ireland

‘determination of aPTT and other procedures requiring an aPTT reagent’ ‘aPTT reagent with increased sensitivity to lupus-like inhibitors’ ‘determination of aPTT; excellent sensitivity to factors II, V, VIII, IX, X, XI & XII’ ‘determination of aPTT’ ‘determination of aPTT as a general screening for evaluation of the intrinsic coagulation pathway and to monitor patients receiving heparin’ ‘determination of aPTT’

Recommended incubation time (seconds) Intended use/characteristics as indicated by manufacturer

Dade Actin FS

Samples for the in vitro survey were supplied to 21 New South Wales haematology diagnostic laboratories which were recruited through the Royal College of Pathologists Australasia Quality Assurance Program (RCPA QAP; n ¼ 25 total paired results). Laboratories were recruited with the aim of including as many different aPTT reagents as possible. Over a 2 week period in May 2012, each laboratory was sent two frozen de-identified plasma samples, shipped on dry ice, with instructions to perform a standard aPTT test within 2 h of thawing the samples. Sample 1 was reconstituted plasma spiked with 27 ng/mL P. australis venom, based on results from the in vitro dosing study and preliminary ex vivo patient samples. Sample 2 consisted of reconstituted control plasma. To ensure consistency, aliquots of the samples were tested in the primary investigator’s laboratory prior to dispatch, during the survey, and again after all laboratories had completed the survey. For each of the three time points, the thawed samples produced clotting times of 35, 38, 36 s (Sample 1) and 29, 29, 26 s (Sample 2) respectively, using Actin FSL; and 28, 28, 29 s (Sample 1)

Manufacturer

In vitro survey

aPTT reagent

Reconstituted normal human plasma was spiked with serial dilutions of P. australis venom and then analysed for aPTT on a Behring Coagulation System (BCS) analyser in the primary investigator’s laboratory using reagents and protocols from the manufacturer (Dade Behring, Siemens, Germany). Two aPTT assays were performed—one with Actin FS, and the other with Actin FSL—with the latter reported to have increased sensitivity to lupus-like inhibitors (Table 1). The aPTT times were recorded in seconds. Reference intervals were determined to be 24.6–33.4 s for Actin FS and 23.6–32.0 s for Actin FSL. Intra- and inter-assay variations, respectively, were calculated to be 1.5% and 11.5% for tests performed with Actin FS, and 2.4% and 5.1% for those done with Actin FSL.

Manufacturer defined characteristics of aPTT reagents from this study and their relative usage in Australian laboratories

In vitro dosing study

Table 1

Pseudechis australis venom, originating from Eyre Peninsula and from Alice Springs, was purchased from Venom Supplies (Tanunda, South Australia) and prepared in a 1:1 mixture as a 4 mg/mL stock in 50% glycerol and stored at
208C. Dilutions were prepared immediately before use, and analysed with minimal delay. In vitro studies were performed with normal human plasma (Dade Ci-Trol Coagulation Control Level 1; Siemens Healthcare Diagnostics Products, Germany) reconstituted in sterile water as per the manufacturer’s instructions. Reagents for aPTT assays included Dade Actin FS/FSL Activated PTT reagents (Siemens Healthcare Diagnostics Products), TriniCLOT aPTT HS/S (Tcoag Ireland Ltd, Ireland), HemosIL APTT SP (Instrumentation Laboratory, USA), and Stago PTT-A, (Diagnostica Stago, France). Polyclonal monospecific IgY antibodies raised against P. australis venom were purchased from GenWay Biotech (USA). IgY was biotinylated using EZ-link sulfo-NHSLC-Biotin (Pierce #21335; Thermo Fisher Scientific, USA) as per manufacturer’s instructions. Bovine serum albumin (BSA) and tetramethylbenzidine (TMB) were purchased from Sigma (USA). Streptavidin-conjugated horseradish peroxidase was purchased from Millipore Chemicon (USA). Enzyme immunoassays were performed using Greiner microlon high binding 96 well plates which were read at 450 nm on a BioTek ELx808 microplate reader (BioTek, USA) .

Source of phospholipids

Materials

Soy

The Australian Snakebite Project (ASP) is approved by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research, for the period 02/04/2004–31/12/2020.

Dade Actin FSL

MATERIALS AND METHODS

180

Activator (concentration)

diagnostic laboratories could create variable sensitivity to the presence of black snake venom, raising the possibility of false negative results in patients with snake envenoming. Therefore, we undertook an in vitro survey of local diagnostic laboratories using various aPTT reagents to test their ability to detect a clinically relevant amount of mulga snake (Pseudechis australis) venom in blinded samples of human plasma. These results were then compared with ex vivo laboratory results available from 17 patients with confirmed mulga snake envenoming.

Ellagic acid (1x10
4M)

Used by number of laboratories* (%)

APTT SENSITIVITY TO MULGA SNAKE VENOM

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446

LINCZ et al.

Pathology (2014), 46(5), August

a lupus anticoagulant sensitive reagent, Actin FSL, obtained from the same manufacturer, produced longer clotting times at low venom concentrations, and these exceeded the upper limits of the normal clotting reference interval time range at 16 ng/mL. These results were maintained after a single freeze thaw of the reconstituted spiked samples with average variations of 3.2% for Actin FS and 2.3% for Actin FSL (data not shown). We chose 27 ng/mL, a clinically relevant venom concentration12 that produced aPTT results just outside of the upper reference interval in these dosing studies with Actin FSL, to test the sensitivity of other commonly used aPTT reagents.

and 29, 28, 29 s (Sample 2), respectively, with Actin FS. Individual laboratory results, reagents and instrument details were recorded on a standardised sheet and sent back to the primary investigator. All laboratories completed the survey within 2 weeks of receiving their samples. Ex vivo study Samples and laboratory investigations for the ex vivo study were obtained from patients with mulga snakebites recruited to the Australian Snakebite Project, an ongoing national multicentre prospective study of snakebite including over 120 hospitals.3,5,11 Approval has been obtained from all Human Research Ethics Committees covering the institutions involved, and all participants provided written informed consent to participate in the study. All patients recruited to ASP have demographic, clinical, laboratory and treatment information recorded on a standardised clinical research form, as well as serum samples collected for measurement of venom concentrations. For the present study, additional information was obtained on the aPTT reagent and analyser used to test plasma samples from each patient with possible mulga snakebite, identified as previously described.12 In brief, possible cases of mulga snake envenoming were identified based on either a positive snake venom detection kit for mulga/black snake venom, expert identification of the snake, or clinical effects consistent with mulga snake envenoming. Mulga snake venom was detected pre-antivenom using a venom specific immunoassay on plates coated with anti-P.australis IgY, and detected with a biotinylated antibody and streptavidin horseradish peroxidase as previously described.12 The limit of detection for this assay is 0.3 ng/mL and it is equally effective using plasma or serum (M. O’Leary, unpublished data).

In vitro survey: spiked plasma samples Results of 25 paired tests (control and venom spiked plasma samples) from 21 different NSW diagnostic laboratories are illustrated in Fig. 2. There were six different aPTT reagents employed, the majority of which were manufactured by either Siemens or Tcoag (a subsidiary of Stago). There was also a wide range in the types of instruments used by the various laboratories. The Stago analysers were most common, with nine STA-R/STA-R Evolutions and four STA Compacts, while ten tests were performed on Dade Behring Sysmex instruments ranging from the CA 500 to the CS-2100i. There was also one Trinity Biotech Coag-A-Mate XM and one Beckman Coulter ACL TOP 500 in the laboratories surveyed. The normal reference intervals also varied widely, between 20 and 42 s, and were largely independent of the instruments. Overall, the survey reported an average clotting time of 29.4 s for the control sample and 35.2 s for the mulga snake venom spiked sample, with a mean difference of 5.8 s between the samples. All but five (20%) of paired tests correctly identified the sample spiked with mulga snake venom as having a longer clotting time. Individually, there were two of 25 (8%) of controls and 12 of 25 (48%) of mulga samples reported as abnormal. As expected, tests performed with the relatively lupus anticoagulant insensitive Dade Actin FS did not show any significant difference in clotting times between the paired samples (n ¼ 7, p ¼ 0.062), and did not identify any of the spiked samples as abnormal. For the reportedly higher lupus

Statistical analysis Data are presented as mean  standard deviation or individual results and normal reference intervals where appropriate. Differences between aPTT test results were analysed using Wilcoxon matched pairs test and correlations were assessed by Spearman rank order.

RESULTS In vitro dosing study An in vitro dosing study was performed to determine the relative ability of aPTT reagents with high versus low lupus anticoagulant sensitivities to detect a clinically relevant concentration of mulga snake venom in human plasma. Figure 1 shows that aPTT testing with a lupus anticoagulant insensitive reagent, Actin FS, did not produce a prolonged clotting time until the venom concentration was 250 ng/mL, and was not recorded as abnormal until 500 ng/mL. In contrast, testing with 100

Actin FS

Reference interval

90

Actin FSL

Reference interval

80

aPTT (sec)

70 60 50 40 30 20 10 0 0

2

4

8

16

31

63

125

250

500

1000

2000

Mulga venom (ng/ml) in control plasma Fig. 1 aPTT results from in vitro dosing study using reagents with high versus low lupus anticoagulant sensitivities on plasma spiked with various concentrations of mulga snake venom. Actin FSL, high lupus anticoagulant sensitivity; Actin FS, low lupus anticoagulant sensitivity. Data represents mean and standard deviation of three independent experiments. Solid lines indicate reference interval for Actin FSL (23.6–32.0 s); dashed lines indicates reference interval for Actin FS (24.6–33.4 s).

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APTT SENSITIVITY TO MULGA SNAKE VENOM aPTT (sec)

Reagent/ Lab ID

Instrument

447

aPTT [reference interval] and sample results (O,X) for each laboratory and instrument (sec)

reference interval

'O' control sample

'X' spiked sample

'X-O' absolute difference

20-30

26.7

26.9

0.2

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

>45

Dade Actin FS 1

Stago

STA Compact

OX

2

Stago

STA-R evolution

28-38

30.7

30.8

0.1

3

Stago

STA-R evolution

26-35

30.3

29.3

1.0

1

Dade Behring

CA1500

20-30

25.7

24.5

1.2

4

Dade Behring

CA1500

20-32

26.9

25.8

1.1

X

5

Dade Behring

CA 7000

20-32

26.7

25.6

1.1

X

6

Dade Behring

CS-2100i

20-32

32.2

25.7

6.5

(p-value)

(0.062)

Mean difference

OX X X

O

O O O X

O

1.6

Dade Actin FSL 7

Trinity Biotech USA Coag-A -Mate XM 20-35

8

Dade Behring

CA 500

26-36

21.9 25.2

30.2 32.7

8.3 7.5

9

Dade Behring

CA 560

24-37

27.9

32.8

4.9

10

Dade Behring

CA 560

25-35

26.8

34.4

7.6

11

Dade Behring

CA 1500

24-34

26.4

34.1

7.7

(p-value)

(0.043)

Mean difference

X

O O

X O

X

O

X

O

X

7.2

TriniCLOT aPTT HS 12

Stago

STA Compact

25-36

29.4

36.7

7.3

13

Stago

STA-R Evolution

25-36

30.8

39.5

8.7

14

Dade Behring

CA 560

24-40

28.8

34.3

5.5

O

15

Dade Behring

CA 560

24-40

28.9

35.7

6.8

O

(p-value)

(0.067)

Mean difference

O

X O

X X X

7.1

TriniCLOT aPTT S 16

Stago

STA Compact

30.4

36.6

6.2

17

Stago

STA-R #1

25-37

32.2

37.4

5.2

O

17

Stago

STA-R #2

25-37

32.0

37.5

5.5

O

18

Stago

STA-R Evolution

25-37

31.5

38.1

6.6

19

Stago

STA-R Evolution

25-37

32.1

37.1

5.0

2

Stago

STA-R Evolution

25-37

31.6

37.9

6.3

(p-value)

25-35

Mean difference

O

X X X

O

X O

X

O

X

5.8

(0.028)

HemosIL APTT SP 2

Stago

STA-R Evolution

27-38

30.5

50.1

20

Beckman Coulter

ACL TOP 500

23-38

36.0

59

Mean difference

O

19.6

X

23.0

O

X

11.9

Stago PTT-A 21

Stago

STA Compact

28-42

33.9

47.9

29.8

35.2

Mean difference Total means

14.0

X

O

17.1 5.8

Fig. 2 aPTT results on paired blinded samples from 21 NSW laboratories. Bars indicate normal reference intervals. O, normal control plasma; X, normal control plasma spiked with 27 ng/mL mulga venom.

PTT-A (n ¼ 1) consistently recorded the spiked sample as being outside of the upper reference interval.

anticoagulant sensitive Actin FSL, the clotting times for the spiked samples were significantly prolonged by an average of 7.2 s (n ¼ 5, p ¼ 0.043), but only one was reported as abnormal. The TriniCLOT aPTT HS reagent produced a similar difference of 7.1 s between the samples, but this was not statistically significant (n ¼ 4, p ¼ 0.067) and only two were reported as abnormal. The aPTT S reagent from the same manufacturer had a smaller difference of 5.8 s between the paired sample clotting times, but this was statistically significant (n ¼ 6, p ¼ 0.028), and all six venom spiked samples were outside of the upper reference interval. In conclusion, only laboratories using TriniCLOT aPTT S (n ¼ 6), HemosIL APTT SP (n ¼ 2) and Stago aPTT (sec) Reagent/ patient ID Dade Actin FSL 1 Dade Behring 2 Dade Behring 3 Dade Behring 4 Dade Behring 5 Dade Behring 6 Dade Behring 7 Dade Behring 8 Dade Behring

Instrument

Reference interval

Pre antivenom

CA 500 CA 560 CA 560 CA 560 CA 560 CA 560 CA 1500 CA 1500

23-35 22-33 23-33 23-33 23-33 23-33 21-36 25-36

28.0 32.6 27.5 38.8 46.2 94.0 81.0 27.0

mean TriniCLOT aPTT HS 9 Stago 10 Stago 11 Stago 12 Stago 13 Dade Behring 14 Dade Behring 15 Dade Behring

Total mean

aPTT [reference interval] and pre-antivenom patient sample results (x) for each patient (sec)

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

>45

X X X X X X X X

46.9 Start-4 Start-4 Start-R evolution Start-R evolution CA 560 CA 560 CA 1500

27-43 27-43 26-40 25-36 25-38 26-41 24-39

mean TriniCLOT aPTT S 16 Stago 17 Dade Behring Mean

Ex vivo study: mulga snake envenoming Pre-antivenom aPTT test results from 17 confirmed cases of mulga snake envenoming are illustrated in Fig. 3. Once again, reagents from Siemens and Tcoag predominated, but the range of products and instruments was more restricted due to the smaller number of centres to which these patients presented (n ¼ 12). The only Stago instruments represented were the STA-R evolution (n ¼ 3 tests) and Start-4 (n ¼ 2), with the

82.2 37.9 29.3 82.0 26.0 95.0 93.0

X X X X X X X

63.6 Start-R evolution CA 500

24-38 25-37

42.0 82.0 62.0

X X

55.6

Fig. 3 aPTT results from 17 patients with mulga snake envenoming. Bars indicate normal reference intervals. X, patient plasma aPTT result pre-antivenom administration.

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LINCZ et al.

Pathology (2014), 46(5), August

remaining 12 tests performed on Dade Behring Sysmex machines ranging from the CA 500 to the CA 1500. Normal reference ranges varied between 21 and 43 s. Abnormally prolonged aPTTs were recorded for four of eight (50%) patients whose tests were performed with Actin FSL, four of seven (57%) with TriniCLOT aPTT HS, and two of two (100%) using Triniclot aPTT S. Serum venom concentrations were available for 16 of the patients and ranged from undetectable to 624 ng/mL (median 18 ng/mL). There was a strong positive correlation between venom concentration and clotting times (Rho ¼ 0.822, p < 0.0001, Fig. 4), with all samples from patients with venom concentrations 12.8 ng/mL having an abnormally prolonged aPTT.

DISCUSSION The aPTT is a useful test that has been adapted for numerous purposes, from a general coagulation function test to a more specialised screen for lupus anticoagulant antibodies. The aPTT reagent itself is also used for coagulation factor assays. Among the 676 Australian diagnostic haematology laboratories registered with the RCPA QAP, there are a reported 16 different commercially available aPTT reagents in use, with those encountered in the present survey accounting for 80% of laboratories (Table 1).13 The most commonly used Dade Actin FSL (32.5% of Australian laboratories) was only able to detect mulga snake toxin in one of five spiked samples and four of eight patient plasma samples. The even less sensitive Dade Actin FS, used in 10% of Australian laboratories, only produced abnormal aPTT results in the presence of very high concentrations (>500 ng/mL) of venom. In theory, it would have only detected envenoming in one of 16 of the patients analysed here. Thus, over 40% of diagnostic haematology laboratories in Australia are not sufficiently equipped to reliably detect systemic black snake envenoming by routine aPTT. The anticoagulant properties of black snake (Pseudechis species) venoms are attributable to the activity of phospholipase (PLA2) toxins.1,14 These toxins are also responsible for systemic myotoxicity or rhabdomyolysis, an uncommon but delayed effect of envenoming by mainly Australian black snakes and tiger snakes (Notechis species), but also taipans (Oxyuranus scutellatus), and certain sea snakes.2 Systemic Serum venom concentration (ng/ml)

1000

100

10

1

0.1 0

20

30

40

50

60

aPTT(sec) Fig. 4 Correlation between patient serum venom concentration and aPTT results. *, results within normal reference interval; * results outside of normal reference interval.

myotoxicity can be complicated by acute kidney injury and hyperkalaemia.15 Because the PLA2 s have both anticoagulant and myotoxic activity, the aPTT can be used as a surrogate marker of envenoming, allowing antivenom to be given early. Early antivenom therapy (within 2–3 h of the bite) has recently been shown to be associated with reduced or absent myotoxicity in mulga snake envenoming, even in patients with very high venom concentrations. This is in contrast to antivenom administered between 3–6 h post-bite, which was not able to prevent development of myotoxicity.12 A study in red-bellied black snake similarly showed that early use of antivenom (< 6 h) was associated with a complete absence of myotoxicity, compared to an occurrence in 20% of cases given antivenom after 6 h or not at all.5 Therefore, an abnormal aPTT recorded soon after the bite would expedite the early administration of antivenom. However, this requires the aPTT to be sensitive to the anticoagulant coagulopathy. Presumably, the phospholipases in black snake venoms would affect an aPTT reaction in much the same way as a lupus anticoagulant, and as such the results would depend on the source and concentration of phospholipid6–10 as well as the activator used in the assay.9,10,16 Although most of these factors are specified by the manufacturers of aPTT reagents (refer to Table 1) the exact concentration of phospholipid in these and other commercial mixtures used for aPTT is generally not revealed, making it impossible to know how these substances compare. In addition, manufacturers provide little information on the differences that exist between their own products. Much of what is known about the relative sensitivity of such reagents to various clotting abnormalities has been determined from independent large scale surveys or individual laboratory testing employing standardised plasma samples.7,10,17,18 When results from these studies are compared to the present findings, it appears that the ability of some aPTT reagents to detect lupus anticoagulant can also predict their sensitivities to mulga venom; i.e., Dade Actin FS
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APTT SENSITIVITY TO MULGA SNAKE VENOM

even simply compare results for patients being tested at two hospitals in the public and/or private sectors. The INR was developed to standardise PT results because of similar problems. However the aPTT predicament lies more in the fact that the reagent is used for multiple different tests. Thus smaller laboratories may favour a single reagent with broader uses, whereas larger well-staffed hospital haemostasis laboratories may stock several different aPTT reagents for use in more specialised assays. Recent recommendations for appropriate aPTT reagent selection advocate the use of a reagent with low sensitivity to lupus anticoagulant for routine aPTT testing.19 This is consistent with the International Society on Thrombosis and Haemostasis (ISTH) guidelines for lupus anticoagulant detection which state that such testing should be limited to those patients who have a significant probability of having the antiphospholipid syndrome, and then screening should consist of two tests, one of which is an aPTT performed with a low phospholipid containing reagent.16 Although the present study is not comprehensive, it suggests that in some hospitals, a lupus anticoagulant screen might be more useful than a routine aPTT when black snakebite is suspected and specific protocols should be established for appropriate coagulation testing in the event of possible envenoming. This may require timely transfer of samples to another nearby laboratory equipped with an aPTT reagent that is more sensitive to anticoagulant toxins, or perhaps development of a simple screen and confirm kit akin to the dilute Russell’s viper venom test for detection of lupus anticoagulants, whereby an anticoagulant toxin sensitive reagent, in combination with normal and abnormal control plasmas, could be used to calculate a ratio that provides an indication of the extent of envenoming. Acknowledgements: The authors would like to thank and acknowledge the invaluable contribution of the RCPA QAP Haematology and RCPA QAP staff (in particular Roslyn Bonar) as well as all RCPA QAP survey participants: Ronald Cheung, Douglass Hanly Moir Pathology, Macquarie Park; Don Clausen/Bob Cushan, Pathology North, Pathology New England, Tamworth; Emmanuel Favaloro, ICPMR, Westmead Hospital, Westmead; Stephanie Gay, Healthscope Pathology, Wagga Wagga; Rosalie Gemmell, SEALS, St George Hospital, Kogarah; Anita Ghevondian, Pathology North, Royal North Shore Hospital, St Leonards; Leanne Hall, Douglass Hanly Moir Pathology, Gateshead; Sayed Hamdam, ICPMR, Blacktown Hospital, Blacktown; Barry Jones/Robyn Green, Pathology North, Lismore; Sue Kehrer/ Vicki Ware, Sullivan Nicolaides Pathology, Coffs Harbour; Geoffrey Kershaw, Royal Prince Alfred Hospital, Camperdown; Jennine Lim, Sullivan Nicolaides Pathology, St Vincent’s Hospital, Lismore; Gabriella Manea, Nepean Hospital Haematology Dept, Kingswood; Beatrice Mui, Haematology Diagnostic Pathology Unit, Concord Hospital, Concord West; Rebecca Perry, SWPS, Cootamundra Hospital, Cootamundra; Michael Ryan, SEALS South, Wollongong Hospital, Wollongong; Alison Saul, Kempsey Hospital Pathology, Kempsey; Ray Thomas, Laverty Pathology, Barrack Heights; Lynn Worsley, Drs Barratt & Smith, Penrith; Geordie Zaunders, HAPS, Calvary Mater Newcastle, Waratah; Diane Zebeljan, SWAPS, Liverpool Hospital, Liverpool.

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Conflicts of interest and sources of funding: This study was supported in part by a National Health and Medical Research Council (NHMRC) Project Grant (ID490405). GKI is supported by a NHMRC Clinical Career Development Award (ID605817). The authors state that there are no conflicts of interest to disclose. Address for correspondence: Dr L. F. Lincz, Hunter Haematology Research Group, Calvary Mater Newcastle Hospital, Waratah, NSW 2298, Australia. E-mail: [email protected]

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