Publications by authors named "Soma Mohammed"

41 Publications

A multi-laboratory assessment of congenital thrombophilia assays performed on the ACL Top 50 family for harmonisation of thrombophilia testing in a large laboratory network.

Clin Chem Lab Med 2021 Jun 14. Epub 2021 Jun 14.

Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia.

Objectives: Thrombophilia testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new 'single platform' of hemostasis instrumentation. The study objective was to evaluate these instruments and manufacturer reagents for utility of congenital thrombophilia assays.

Methods: Comparative evaluations of various congenital thrombophilia assays (protein C [PC], protein S [PS], antithrombin [AT], activated protein C resistance [APCR]) using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing, predominantly STAGO, instrumentation and reagents. Verification of manufacturer assay normal reference ranges (NRRs).

Results: HemosIL PC and free PS assays showed good comparability with existing Stago methods (R>0.9) and could be considered as verified as fit for purpose. HemosIL AT showed high relative bias with samples from patients on direct anti-Xa agents, compromising utility. Manufacturer NRRs for PC, PS and AT were verified with minor variance. Given the interference with direct anti-Xa agents, an alternate assay (Hyphen) was evaluated for AT, and the NRR also verified. The HemosIL Factor V Leiden (APC Resistance V) evidenced relatively poor performance compared to existing assays, and could not be adopted for use in our network.

Conclusions: This evaluation of HemosIL reagents on ACL TOP 50 Family instruments identified overall acceptable performance of only two (PC, free PS) of four thrombophilia assays, requiring use of third-party reagents on ACL instruments for the other two assays (AT, APCR).
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http://dx.doi.org/10.1515/cclm-2021-0499DOI Listing
June 2021

Verification of the ACL Top 50 Family (350, 550, and 750) for Harmonization of Routine Coagulation Assays in a Large Network of 60 Laboratories.

Am J Clin Pathol 2021 Apr 23. Epub 2021 Apr 23.

Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, Australia.

Objectives: To verify a single platform of hemostasis instrumentation, the ACL TOP 50 Family, comprising 350, 550, and 750 instruments, across a large network of 60 laboratories.

Methods: Comparative evaluations of instrument classes (350 vs 550 and 750) were performed using a large battery of test samples for routine coagulation tests, comprising prothrombin time/international normalized ratio, activated partial thromboplastin time (APTT), thrombin time, fibrinogen and D-dimer, and using HemosIL reagents. Comparisons were also made against existing equipment (Diagnostica Stago Satellite, Compact, and STA-R Evolution) and existing reagents to satisfy national accreditation standards. Verification of manufacturer normal reference ranges (NRRs) and generation of an APTT heparin therapeutic range were undertaken.

Results: The three instrument types were verified as a single instrument class, which will permit standardization of methods and NRRs across all instruments (n = 75) to be deployed in 60 laboratories. In particular, ACL TOP 350 test result data were similar to ACL TOP 550 and 750 and showed no to limited bias. All manufacturer NRRs were verified with occasional minor variance.

Conclusions: This ACL TOP 50 Family (350, 550, and 750) verification will enable harmonization of routine coagulation across all laboratories in the largest public pathology network in Australia.
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http://dx.doi.org/10.1093/ajcp/aqab004DOI Listing
April 2021

Effect of sample heat inactivation on test levels of HIT-IgG detected by the ACL AcuStar.

Thromb Res 2021 04 18;200:12-15. Epub 2021 Jan 18.

Prince of Wales Hospital, NSW Health Pathology, Randwick, NSW, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.thromres.2021.01.004DOI Listing
April 2021

2B or not 2B? A diagnosis of von Willebrand disease a lifetime of 86 years in the making.

Blood Coagul Fibrinolysis 2021 Apr;32(3):229-233

NSW Health Pathology West, Institute of Clinical Pathology and Medical Research (ICPMR).

Type 2B von Willebrand disease (2B VWD) is a rare, autosomal dominant bleeding disorder characterized by a hyperadhesive form of von Willebrand factor (VWF). 2B VWD expresses phenotypically as an enhanced ristocetin-induced platelet aggregation and usually also a discordance in VWF activity versus protein level, with loss of high molecular weight VWF and (mild) thrombocytopenia. While all cases of 2B VWD supposedly share these characteristics, there is significant heterogeneity in laboratory findings within this group of patients, which are largely dictated by the underlying genetic defect. We present a case of such a patient, expressing a clearly atypical VWF phenotype, but as still associated with enhanced ristocetin-induced platelet aggregation, thrombocytopenia, and a previously undescribed VWF variant (c.4130C>G; p.Ala1377Gly). The patient was misdiagnosed over his lifetime as idiotypic thrombocytopenia - a (mis)diagnosis that took a lifetime of 86 years to redress.
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http://dx.doi.org/10.1097/MBC.0000000000000994DOI Listing
April 2021

How we diagnose 2M von Willebrand disease (VWD): Use of a strategic algorithmic approach to distinguish 2M VWD from other VWD types.

Haemophilia 2021 Jan 20;27(1):137-148. Epub 2020 Nov 20.

Sydney Centres for Thrombosis and Haemostasis, Westmead Hospital, Westmead, NSW, Australia.

Introduction: von Willebrand disease (VWD) is the most common inherited bleeding disorder and caused by an absence, deficiency or defect in von Willebrand factor (VWF). VWD is currently classified into six different types: 1, 2A, 2B, 2N, 2M, 3. Notably, 2M VWD is more often misdiagnosed as 2A or type 1 VWD than properly identified as 2M VWD.

Aim: To describe an algorithmic approach to better ensure appropriate identification of 2M VWD, and reduce its misdiagnosis, as supported by sequential laboratory testing.

Methods: Comparative assessment of types 1, 2A, 2B and 2M VWD using various laboratory tests, including VWF antigen and several VWF activity assays, plus DDAVP challenge data, ristocetin-induced platelet agglutination (RIPA) data, multimer analysis and genetic testing.

Results: Types 1, 2A, 2B and 2M VWD give characteristic test patterns that can provisionally classify patients into particular VWD types. Notably, type 1 VWD shows low levels of VWF, but VWF functional concordance (VWF activity/Ag ratios >0.6), with both baseline assessment and post-DDAVP. Types 2A, 2B and 2M VWD show VWF functional discordance (low VWF activity/Ag ratio(s)) dependent on the defect, but type 2M separates from 2A/2B VWD based on specific test patterns, especially with collagen binding vs glycoprotein Ib binding assays. RIPA identifies 2B VWD. Multimers separate 2M from 2A/2B.

Conclusion: We provide strategies to improve correct diagnosis of VWD, especially focussed on 2M VWD, and which can be used by most diagnostic haemostasis laboratories, reserving genetic analysis (if required) for confirmation.
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http://dx.doi.org/10.1111/hae.14204DOI Listing
January 2021

A multicenter laboratory assessment of a new automated chemiluminescent assay for ADAMTS13 activity.

J Thromb Haemost 2021 02 21;19(2):417-428. Epub 2020 Nov 21.

Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia.

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal disorder caused by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency. Prompt identification/exclusion of TTP can thus be facilitated by rapid ADAMTS13 testing. The most commonly utilized (enzyme-linked immunosorbent assay [ELISA]-based) assay takes several hours to perform and so does not generally permit rapid testing.

Objectives: To evaluate the utility of a new automated test for ADAMTS13 activity, the HemosIL AcuStar ADAMTS13 Activity assay, based on chemiluminescence and able to be performed on an ACL AcuStar instrument within 33 minutes.

Patients/methods: This multicenter (n = 8) assessment included testing of more than 700 test samples, with similar numbers of prospective (n = 348) and retrospective (n = 385) samples. The main comparator was the Technozym ADAMTS13 Activity ELISA. We also assessed comparative performance for detection of ADAMTS13 inhibitors using a Bethesda assay.

Results: Overall, the chemiluminescent assay yielded similar results to the comparator ELISA, albeit with slight negative bias. ADAMTS13 inhibitor detection was also comparable, albeit with slight positive bias with the AcuStar assay. Assay precision was similar with both assays, and we also verified assay normal reference ranges.

Conclusions: The HemosIL AcuStar ADAMTS13 Activity assay provided results rapidly, which were largely comparable with the Technozym ADAMTS13 Activity ELISA assay, albeit lower on average. Conversely, inhibitor levels tended to be identified at a higher level on average. Thus, the HemosIL AcuStar ADAMTS13 Activity assay provides a fast and accurate means to quantitate plasma levels of ADAMTS13 for TTP/ADAMTS13 identification/exclusion, and potentially also for other applications.
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http://dx.doi.org/10.1111/jth.15157DOI Listing
February 2021

A multicentre assessment of contemporary laboratory assays for heparin induced thrombocytopenia.

Pathology 2021 Feb 5;53(2):247-256. Epub 2020 Oct 5.

NSW Health Pathology, NSW, Australia; Prince of Wales Hospital, Randwick, NSW, Australia.

Heparin induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy. In some patients, HIT causes platelet activation and thrombosis (sometimes abbreviated HITT), which leads to adverse clinical sequalae ('pathological HIT'). The likelihood of HIT is initially assessed clinically, typically using a scoring system, of which the 4T score is that most utilised. Subsequent laboratory testing to confirm or exclude HIT facilitates exclusion or diagnosis and management. The current investigation comprises a multicentre (n=9) assessment of contemporary laboratory testing for HIT, as performed over the past 1-3 years in each site and comprising testing of over 1200 samples. The primary laboratory test used by study participants (n=8) comprised a chemiluminescence procedure (HIT-IgG) performed on an AcuStar instrument. Additional immunological testing performed by study sites included lateral flow (STiC, Stago), enzyme linked immunosorbent assay (ELISA), Asserachrom (HPIA IgG), PaGIA (BioRad), plus functional assays, primarily serotonin release assay (SRA) or platelet aggregation methods. The chemiluminescence procedure yielded a highly sensitive screening method for identifying functional HIT, given high area under the curve (AUC, generally ≥0.9) in a receiver operator characteristic (ROC) analysis against SRA as gold standard. ELISA testing resulted in lower ROC AUC scores (<0.8) and higher levels of false positives. Although there is clear association with the likelihood of HIT, the 4T score had less utility than literature suggests, and was comparable to a previous study reported by some of the authors.
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http://dx.doi.org/10.1016/j.pathol.2020.07.012DOI Listing
February 2021

Plasma vs serum as test sample for the chemiluminescent AcuStar HemosIL HIT-IgG assay.

Int J Lab Hematol 2021 02 30;43(1):e41-e44. Epub 2020 Sep 30.

Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead, NSW, Australia.

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http://dx.doi.org/10.1111/ijlh.13353DOI Listing
February 2021

Sample stability for routine coagulation testing.

Thromb Res 2020 12 19;196:130-134. Epub 2020 Aug 19.

Section of Clinical Biochemistry, Department of Neuroscience, Biomedicine and Movement, University of Verona, Verona, Italy.

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http://dx.doi.org/10.1016/j.thromres.2020.08.025DOI Listing
December 2020

Comparative assessment of von Willebrand factor multimers vs activity for von Willebrand disease using modern contemporary methodologies.

Haemophilia 2020 May 11;26(3):503-512. Epub 2020 Mar 11.

Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia.

Introduction: Diagnosis of von Willebrand disease (VWD) is challenging due to heterogeneity of VWD and test limitations. Many von Willebrand factor (VWF) assays are utilized, including antigen (Ag), activity and multimer analysis. Activity assays include ristocetin cofactor using platelets (VWF:RCo) or other particles incorporating recombinant glycoprotein I ('VWF:GPIbR'), or other GPI binding assays using gain-of-function mutations ('VWF:GPIbM'), or collagen binding (VWF:CB).

Aim: To comparatively evaluate modern contemporary VWF activity assays vs VWF multimer analysis using modern contemporary methods.

Materials And Methods: Several VWF activity assays (VWF:RCo, VWF:GPIbR, VWF:GPIbM, VWF:CB) assessed (typically as a ratio against VWF:Ag) against a new semi-automated procedure for different types of VWD (1, 3, 2A, 2B, 2M), plus control material (n = 580). The evaluation also focussed on relative loss of high and very high molecular weight multimers (HMWM and VHMWM) by densitometric scanning.

Results: All evaluated VWF activity/Ag ratios showed high correlation to the presence/absence of HMWM and VHMWM, although VWF:CB/Ag and VWF:GPIbR/Ag ratios using an automated chemiluminescence method yielded highest correlation coefficients (r = .909 and .874, respectively, for HMWM). Use of the investigative procedure (VHMWM) identified fewer false positives for 'loss' in type 1 VWD.

Conclusions: This comparative investigation identified that new automated chemiluminescence VWF activity assays best identified relative loss or presence of HMWM and VHMWM according to activity to Ag ratios and an alternative investigative method for identifying VHMWM in multimer testing for a new commercial multimer method may lead to fewer false identifications of HMW loss in type 1 VWD.
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http://dx.doi.org/10.1111/hae.13957DOI Listing
May 2020

Laboratory testing for activated protein C resistance: rivaroxaban induced interference and a comparative evaluation of andexanet alfa and DOAC Stop to neutralise interference.

Clin Chem Lab Med 2020 07;58(8):1322-1331

Western Australian Centre for Thrombosis and Haemostasis (WACTH), Murdoch University, Perth, WA, Australia.

Background Investigation of hemostasis is problematic when patients are on anticoagulant therapy. Rivaroxaban especially causes substantial interference, extending many clot-based tests, thereby leading to false positive or negative events. In particular, rivaroxaban affects some assays for activated protein C resistance (APCR). Methods We assessed, in an international setting, cross laboratory (n = 31) testing using four samples to evaluate rivaroxaban induced interference in APCR testing, and whether this interference could be neutralised. The samples comprised: (A) pool of normal plasma (APCR-negative control); (B) this normal pool spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (potential 'false' positive APCR event sample); (C) the rivaroxaban sample subsequently treated with a commercial direct oral anticoagulant 'DOAC-neutraliser' (DOAC Stop), or (D) treated with andexanet alfa (200 μg/mL). Testing was performed blind to sample type. Results The rivaroxaban-spiked sample generated false positive APCR results for some, but unexpectedly not most APCR-tests. The sample treated with DOAC Stop evidenced a correction in the rivaroxaban-affected APCR assays, and did not otherwise adversely affect the rivaroxaban 'unaffected' APCR assays. The andexanet alfa-treated sample did not evidence correction of the false positive APCR, and instead unexpectedly exacerbated false positive APCR status with many tests. Conclusions DOAC Stop was able to neutralise any APCR interference induced by rivaroxaban. In contrast, andexanet alfa did not negate such interference, and instead unexpectedly created more false-positive APCR events.
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http://dx.doi.org/10.1515/cclm-2019-1160DOI Listing
July 2020

Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects.

Haemophilia 2020 Mar 21;26(2):354-362. Epub 2020 Jan 21.

Western Australian Centre for Thrombosis and Haemostasis (WACTH), Murdoch University, Perth, WA, Australia.

Introduction: Investigation of factors (F) VIII and IX is common, with testing important for diagnosis or exclusion of haemophilia A or B, associated acquired conditions and factor inhibitors. Rivaroxaban, a common direct anti-Xa agent, causes significant interference in clotting assays, including substantial false reduction of factor levels.

Aim: To assess whether rivaroxaban-induced interference of FVIII and FIX testing could be neutralized.

Materials And Methods: An international, cross-laboratory exercise for FVIII (n = 84) and FIX (n = 74), using four samples: (A) pool of normal plasma; (B) pool spiked with rivaroxaban (200 ng/mL); (C) rivaroxaban sample subsequently treated with 'DOAC Stop' and; (D) rivaroxaban sample treated with andexanet alfa (200 μg/mL). Testing performed blind to sample type.

Results: All laboratories reported normal FIX and 94% reported normal FVIII in the pool sample. Instead, 55% and 95%, respectively, reported abnormal FIX and FVIII levels for the rivaroxaban sample. DOAC Stop treatment evidenced a correction in most laboratories (100% reported normal FIX and 86% normal FVIII). Andexanet alfa provided intermediate results, with many laboratories still reporting abnormal results (59% for FVIII, 18% for FIX). We also identified reagent-specific issues.

Conclusions: As expected, rivaroxaban caused false low values of FVIII and FIX. This might lead to increased testing to identify the cause of low factor levels and potentially lead to false identification of (mild) haemophilia A or B if unrecognized by clinicians/laboratories. DOAC Stop effectively neutralized the rivaroxaban effect, but andexanet alfa less so, with reagent-related effects evident, and thus, false low values sometimes persisted.
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http://dx.doi.org/10.1111/hae.13930DOI Listing
March 2020

Semi-automated von Willebrand factor multimer assay for von Willebrand disease: Further validation, benefits and limitations.

Int J Lab Hematol 2019 Dec 11;41(6):762-771. Epub 2019 Sep 11.

Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia.

Introduction: Accurate diagnosis of von Willebrand disease (VWD) enables effective patient management. von Willebrand factor (VWF) multimer analysis provides useful information regarding VWF multimer structure, thereby aiding VWD subtyping and management; however, historically technically challenging assays have had limited utility. This study evaluates the Sebia Hydrasys Hydragel-11 semi-automated VWF multimer assay and further validates the Hydragel-5 gel system, as primarily pertaining to VWD diagnostics and monitoring of therapy.

Methods: Provisionally diagnosed (via a reference assay test panel) archived patient samples and prospective test patient samples, including those undergoing desmopressin trial or therapy monitoring, along with commercial and in-house control material and various external quality assessment (EQA) samples, were analysed. VWF multimers were evaluated for presence, loss or partial loss of high molecular weight (HMWM) and intermediate molecular weight (IMWM) multimers by both visual inspection and densitometric scanning, and comparison with reference assay results.

Results: All anticipated multimer patterns were reproduced, with patients generally showing multimer profiles matching expected patterns according to VWD type based on reference test panel 'diagnosis'. Occasional discrepancies were resolved by retesting. The increase in plasma VWF following desmopressin therapy was also clearly demonstrated. Multimer profiles of EQA samples complemented reference test panel results and matched EQA targets. There were some 'technical' limitations noted.

Conclusion: This easy to use, standardised, semi-automated multimer analysis system can demonstrate the multimer profile of VWD patients, thus representing an additional laboratory tool for improved diagnosis, thereby facilitating appropriate patient management.
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http://dx.doi.org/10.1111/ijlh.13107DOI Listing
December 2019

A diagnosis of von Willebrand disease despite normal test results for factor VIII and von Willebrand factor antigen and activity.

Am J Hematol 2019 12 30;94(12):1425-1432. Epub 2019 Aug 30.

Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, New South Wales, Australia.

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http://dx.doi.org/10.1002/ajh.25618DOI Listing
December 2019

Development and implementation of an expert rule set for automated reflex testing and validation of routine coagulation tests in a large pathology network.

Int J Lab Hematol 2019 Oct 4;41(5):642-649. Epub 2019 Jul 4.

Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, New South Wales, Australia.

Introduction: Haemostasis laboratories play a critical role in diagnosis and treatment of individuals with bleeding or thrombotic disorders. Routine coagulation assays such as prothrombin time (PT)/ international normalized ratio (INR), and activated partial thromboplastin time (APTT), are used in monitoring of anticoagulant therapy, as provided for treatment/prevention of thromboembolic disease, and also inform on potential haemostasis dysfunction. Increasing pressure is applied on clinical laboratories to improve response (test turnaround) times, reduce error rates and standardize policies. To this end, we describe our experience with the development and implementation of an automated process for reflex testing and validation of routine coagulation test results in a large pathology network compromising 27 laboratories.

Methods: Custom-built expert rules were created to perform reflex testing and fully automate routine test validation. These rules were developed/implemented over a 15-month period, including 6 months for development/ testing and 9 months for training/implementation of >100 personnel at 27 sites.

Results: These rules have enabled adherence of standardized pre-analytical (sample integrity) checks, automated reflex decisions, automated verification and overall alignment of network practices. In addition, clinically significant results are immediately referred to haematologists. We report an improvement in test turnaround times, also reflecting savings in operator time.

Conclusion: The process was generally well received and generally beneficial to most laboratories in the network.
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http://dx.doi.org/10.1111/ijlh.13078DOI Listing
October 2019

Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa.

Thromb Res 2019 Aug 24;180:10-19. Epub 2019 May 24.

Western Australian Centre for Thrombosis and Haemostasis (WACTH), Murdoch University, Perth, WA, Australia.

Introduction: Lupus anticoagulant (LA) investigation in patients on anticoagulant therapy is problematic. Rivaroxaban in particular causes significant interference, prolonging both LA screening and confirmation tests, and falsely raising LA screen/confirm ratios, leading to potential false identification of LA. The Russell Viper Venom Time (RVVT) assay, key to the investigation of LA, is especially sensitive to rivaroxaban.

Materials And Methods: We assessed cross laboratory (n = 82) testing of four samples to investigate whether rivaroxaban induced interference in LA testing could be neutralised. Testing was performed blind to sample type. The samples comprised: (A) A pool of normal plasma (LA-negative control); (B) sample A spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (LA 'false' positive sample); (C) sample B subsequently treated with a commercial 'DOAC-neutraliser' (DOAC Stop); (D) sample B treated with andexanet alfa (200 μg/mL).

Results: As expected, the rivaroxaban-spiked sample (B) caused prolongation of most LA-tests, and also generated a falsely prolonged RVVT screen/confirm ratio (median 1.37, compared to 0.97 for sample A). The sample (C) treated with DOAC Stop evidenced a correction in LA-test clotting times, as well as neutralising the false positive LA (median RVVT screen/confirm ratio of 0.99). Although the andexanet alfa treated sample (D) also yielded a low median RVVT screen/confirm ratio of 0.88, it did not fully correct LA-test clotting times. Consistent with test findings, all laboratories interpreted samples A and C as being LA-negative. For sample B (rivaroxaban), 45.3% identified this as LA positive, and 38.7% identified LA interference. Most (61.3%) also identified sample D as LA negative, with the remainder (38.7%) identifying LA interference.

Conclusions: DOAC Stop was able to neutralise the false LA activity induced by rivaroxaban, both in terms of clot-times and LA ratios. In contrast, whilst andexanet alfa negated the rivaroxaban-prolonged LA-ratio, it did not fully correct clot-times, leaving some residual LA interference, and requiring additional testing to investigate prolonged clotting times.
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http://dx.doi.org/10.1016/j.thromres.2019.05.013DOI Listing
August 2019

Laboratory testing for lupus anticoagulant (LA) in patients taking direct oral anticoagulants (DOACs): potential for false positives and false negatives.

Pathology 2019 Apr 18;51(3):292-300. Epub 2019 Jan 18.

Department of Laboratory Haematology, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia; Sydney Centres for Thrombosis and Haemostasis, Sydney, NSW, Australia.

We and others have previously highlighted the potential problems with testing of lupus anticoagulants (LA) in patients on anticoagulant therapy, including most recently as related to the direct oral anticoagulants (DOACs). Thus, current DOACs in use (e.g., dabigatran, a direct thrombin inhibitor, and apixaban and rivaroxaban, both direct Xa inhibitors), affect a wide variety of coagulation assays, including those used in LA investigation. The Russell viper venom time (RVVT) assay in particular, key to the investigation of LA, is highly sensitive to DOACs. LA is a marker of thrombophilia, and patients who have had a thrombosis may be placed on a DOAC. Thus, there is a high likelihood that LA testing will be requested on patients whilst they are on DOACs. In the current report, we have assessed data from our facility for the past two and a half years for all LA tests performed by RVVT testing, and have evaluated this data with respect to patient anticoagulant status. In total, there were 7170 test requests for RVVT associated testing during the period of data capture. Most LA-RVVT screen results (5008; ∼70%) were within normal limits, thereby excluding LA by RVVT method in most of the patient cohort. All DOACs led to a prolongation in both RVVT screen and confirm assays. However, rivaroxaban affected the screen more than the confirm, leading to higher RVVT ratios, whereas apixaban affected the confirm more than the screen, leading to lower RVVT ratios. LA testing in the presence of DOACs also led to lower intra-patient consistency in LA test results. We conclude that ex-vivo data appears to confirm the potential for false positive (with rivaroxaban) and potential for false negative (with apixaban) identification of LA in patients on DOAC treatment. We also make some recommendations in regards to such testing.
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http://dx.doi.org/10.1016/j.pathol.2018.11.008DOI Listing
April 2019

How to Optimize Activated Partial Thromboplastin Time (APTT) Testing: Solutions to Establishing and Verifying Normal Reference Intervals and Assessing APTT Reagents for Sensitivity to Heparin, Lupus Anticoagulant, and Clotting Factors.

Semin Thromb Hemost 2019 Feb 10;45(1):22-35. Epub 2019 Jan 10.

Department of Haematology, NSW Health Pathology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia.

The activated partial thromboplastin time (APTT) assay is a very common coagulation test, used for several reasons. The test is conventionally used for assessing the contact factor (intrinsic) pathway of blood coagulation, and thus for screening deficiencies in this pathway, most typically factors VIII, IX, and XI. The APTT is also sensitive to contact factor deficiencies, including factor XII, prekallikrein, and high-molecular-weight kininogen. The APTT may also be elevated in a variety of conditions, including liver disease, vitamin K deficiency, and disseminated intravascular coagulation. The APTT can also be used for monitoring unfractionated heparin (UFH) therapy, as well as for screening lupus anticoagulant (LA) or for assessing thrombosis risk. Which of these separate uses is important to a given laboratory or clinician depends on the laboratory and the clinical context. For example, UFH sensitivity is important in hospital-based laboratories, where UFH therapy is used, but not in hospital-based laboratories where low-molecular-weight heparin (LMWH) is largely employed or where UFH may be assessed by anti-factor Xa testing, or in private/community laboratories not associated with a hospital system. High sensitivity to (low levels of) factors VIII, IX, and XI is generally preferred, as their deficiencies are clinically significant. Also preferred, but not usually achieved, is low sensitivity to factor XII and other contact factors, as these deficiencies are usually asymptomatic. Nevertheless, a good knowledge of factor sensitivity is usually needed, if only to help explain the reasons for a prolonged APTT in a given patient, or whether factor testing or other investigation is required. A good working knowledge of reagents sensitivity to LA is also advisable, especially when the reagent is used as part of a LA test panel, or else as a "general-purpose screening reagent." The current report is aimed at providing some guidance around these questions, and is intended as a kind of "how to" guide, that will enable laboratories to assess APTT reagents in regard to their sensitivity to heparin, LA, and clotting factors. The report also provides some advice on generation of normal reference ranges, as well as solutions for troubleshooting prolonged APTTs, when performing factor testing or searching for inhibitors.
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http://dx.doi.org/10.1055/s-0038-1677018DOI Listing
February 2019

Postanalytical considerations that may improve the diagnosis or exclusion of haemophilia and von Willebrand disease.

Haemophilia 2018 Nov 19;24(6):849-861. Epub 2018 Jul 19.

Section of Clinical Biochemistry, University of Verona, Verona, Italy.

von Willebrand disease (VWD) and haemophilia represent the most common inherited or acquired bleeding disorders. However, many laboratories and clinicians may be challenged by their accurate diagnosis or exclusion. Difficulties in diagnosis/exclusion may include analytical issues, where assays occasionally generate an incorrect result (ie representing an analytical error) or have limitations in their measurement range of and/or low analytical sensitivity. Also increasingly recognized is the influence of preanalytical issues on the diagnosis of VWD or haemophilia. Unfortunately, postanalytical considerations are often not well considered in the diagnostic process. Therefore, this narrative review aims to provide an overview of some important postanalytical considerations that may help improve the diagnosis of VWD and haemophilia. This review primarily discusses aspects around reporting of test results. However, we also discuss other less well-recognized postanalytical considerations, including the use of assay ratios to help identify differential diagnoses and then guide further investigation.
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http://dx.doi.org/10.1111/hae.13587DOI Listing
November 2018

Mathematical rounding as a post-analytical issue in pathology reporting: generation of bias in INR resulting.

Pathology 2018 Jun 3;50(4):459-461. Epub 2018 May 3.

NSW Health Pathology, Blacktown Hospital, Blacktown, NSW, Australia.

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http://dx.doi.org/10.1016/j.pathol.2017.11.090DOI Listing
June 2018

Differential sensitivity of von Willebrand factor activity assays to reduced VWF molecular weight forms: A large international cross-laboratory study.

Thromb Res 2018 06 19;166:96-105. Epub 2018 Apr 19.

Department of Haematology and Cell Biology, University of the Free State and National Health Laboratory Services, Bloemfontein, South Africa.

Introduction: von Willebrand disease (VWD), the most common inherited bleeding disorder, is due to deficiencies/defects in von Willebrand factor (VWF). Effective diagnosis requires testing for FVIII, VWF antigen and one or more VWF 'activity' assays. Classically, 'activity' is assessed using ristocetin cofactor (VWF:RCo), but collagen binding (VWF:CB) and/or other assays are used by many laboratories. This extensive international cross-laboratory study has specifically evaluated contemporary VWF activity assays for comparative sensitivity to reduction in high molecular weight (HMW) VWF, and their ability to differentiate type 1 vs 2A VWD-like samples.

Materials And Methods: A set of four samples representing step wise reduction in HMW VWF were tested by over 400 laboratories worldwide using various assays. A second set of two samples representing type 1 or type 2A VWD-like plasma was tested by a subset of 251 laboratories.

Results: Combined data identified some differences between VWF activity assays, with sensitivity for reduction of HMW being highest for VWF:CB and VWF:GPIbM, intermediate for VWF:RCo and VWF:GPIbR, and lowest for VWF:Ab. 'Within' method analysis identified the Stago method as the most sensitive VWF:CB assay. A large variation in inter-laboratory CV (e.g., 7-24% for the normal sample) was also demonstrated for various methods. Although performance of various methods differed significantly, most laboratories correctly differentiated between type 1 and 2 samples, irrespective of VWF activity assay employed.

Conclusions: These results hold significant clinical implications for diagnosis and therapy monitoring of VWD, as well as potential future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura (TTP).
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http://dx.doi.org/10.1016/j.thromres.2018.04.015DOI Listing
June 2018

HIT or miss? A comprehensive contemporary investigation of laboratory tests for heparin induced thrombocytopenia.

Pathology 2018 Jun 17;50(4):426-436. Epub 2018 Apr 17.

Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, NSW, Australia; NSW Health Pathology, NSW, Australia; Sydney Centres for Thrombosis and Haemostasis, Westmead, NSW, Australia.

Heparin induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy, which in a proportion of patients causes platelet activation and thrombosis. Initial clinical assessment of the likelihood of HIT is facilitated by laboratory testing to confirm or exclude HIT. This prospective investigation was performed over an 18-month period, and has involved testing of over 300 test samples from over 100 consecutive patients. Clinical assessment by 4T score was supplemented by laboratory tests that comprised both immunological [lateral flow ('STiC'), chemiluminescence (AcuStar; HIT-IgG), ELISA (Asserachrom HPIA IgG)] and functional assays [SRA, platelet aggregation using whole blood ('Multiplate') and platelet rich plasma ('LTA')]. We observed both false positive and false negative test findings with most assays. Overall, the whole blood aggregation method provided a reasonable alternative to SRA for identifying functional HIT. STiC, AcuStar and ELISA procedures were fairly comparable in terms of screening for HIT, although STiC and AcuStar both yielded false negatives, albeit also resulting in fewer false positives than ELISA. The 4T score had less utility in our patient cohort than we were expecting, although there was an association with the likelihood of HIT. Nevertheless, we accept that our observations are based on limited test numbers. In conclusion, no single approach (clinical or laboratory) was associated with optimal sensitivity or specificity of HIT exclusion or identification, and thus, a combination of clinical evaluation and laboratory testing will best ensure the accuracy of diagnosis.
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http://dx.doi.org/10.1016/j.pathol.2017.11.089DOI Listing
June 2018

Not as sweet as honey: A rare case of an apparent factor V "inhibitor" in association with bee sting anaphylaxis.

Am J Hematol 2018 07 11;93(7):965-970. Epub 2018 May 11.

Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia.

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http://dx.doi.org/10.1002/ajh.25121DOI Listing
July 2018

Laboratory Testing for von Willebrand Factor: Factor VIII Binding (for 2N VWD).

Methods Mol Biol 2017 ;1646:461-472

Haematology Department, Sydney Centres for Thrombosis and Haemostasis, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW Health Pathology, Westmead, NSW, 2145, Australia.

Von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders develop due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for the VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. The current paper describes a protocol for assessment of VWF activity by means of VWF: factor VIII binding (VWF:FVIIIB). Such assays identify VWF activity by quantitative assessment of VWF protein adhesion to FVIII, which is the activity lost in type 2N VWD. This assay is therefore a critical assay for identification or exclusion of 2N VWD. The most commonly performed assays for VWF:FVIIIB comprise enzyme-linked immunosorbent assays (ELISA), and such an assay is described in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-7196-1_34DOI Listing
May 2018

Laboratory Testing for von Willebrand Factor Ristocetin Cofactor (VWF:RCo).

Methods Mol Biol 2017 ;1646:435-451

Haematology Department, Sydney Centres for Thrombosis and Haemostasis, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW Health Pathology, Westmead, NSW 2145, Australia.

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders develop due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for these VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. The current paper describes several protocols for assessment of VWF activity by means of VWF ristocetin cofactor (VWF:RCo). These assays identify VWF activity by quantitative assessment of VWF protein adhesion to platelets or other particles and subsequent detection of the adhered VWF as facilitated by inclusion of ristocetin. The most commonly performed assays for VWF:RCo comprise platelet agglutination assays, latex agglutination assays, and chemiluminescent assay (CLIA), with three of these described in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-7196-1_32DOI Listing
May 2018

Laboratory Testing for von Willebrand Factor Collagen Binding (VWF:CB).

Methods Mol Biol 2017 ;1646:417-433

Haematology Department, Sydney Centres for Thrombosis and Haemostasis, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW Health Pathology, Westmead, NSW, 2145, Australia.

Von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders develop due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for the VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. This chapter describes several protocols for assessment of VWF activity by means of VWF collagen binding (VWF:CB). These assays identify VWF activity by quantitative assessment of VWF protein adhesion to collagen or collagen peptides and subsequent immunological detection of the adhered VWF. The most commonly performed assays for VWF:CB comprise enzyme-linked immunosorbent assays (ELISA) and chemiluminescent assay (CLIA), as described in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-7196-1_31DOI Listing
May 2018

Laboratory Testing for von Willebrand Factor Antigen (VWF:Ag).

Methods Mol Biol 2017 ;1646:403-416

Siemens Healthcare Diagnostics Products GmbH, Assay Development, Marburg, Germany.

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders arise due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for the VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. The current paper describes protocols for assessment of VWF level by means of VWF antigen (VWF:Ag). These assays identify VWF levels by quantitative assessment of VWF protein by means of immunological assays. The most commonly performed assays for VWF:Ag comprise enzyme-linked immunosorbent assays (ELISA) and latex-enhanced immunoassays (LIA), as described in this chapter.
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http://dx.doi.org/10.1007/978-1-4939-7196-1_30DOI Listing
May 2018

Laboratory Testing Protocols for Heparin-Induced Thrombocytopenia (HIT) Testing.

Methods Mol Biol 2017 ;1646:227-243

Haematology Department, Sydney Centres for Thrombosis and Haemostasis, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW Health Pathology, Westmead, 2145 NSW, Australia.

Heparin-induced thrombocytopenia (HIT) represents a significant high morbidity complication of heparin therapy. The clinicopathological diagnosis of HIT remains challenging for many reasons; thus, laboratory testing represents an important component of an accurate diagnosis. Although there are many assays available to assess HIT, these essentially fall into two categories-(a) immunological assays, and (b) functional assays. The current chapter presents protocols for several HIT assays, being those that are most commonly performed in laboratory practice and have the widest geographic distribution. These comprise a manual lateral flow-based system (STiC), a fully automated latex immunoturbidimetric assay, a fully automated chemiluminescent assay (CLIA), light transmission aggregation (LTA), and whole blood aggregation (Multiplate).
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http://dx.doi.org/10.1007/978-1-4939-7196-1_19DOI Listing
May 2018

Laboratory Testing for Activated Protein C Resistance (APCR).

Methods Mol Biol 2017 ;1646:137-143

Haematology Department, Sydney Centres for Thrombosis and Haemostasis, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW Health Pathology, Westmead, NSW, 2145, Australia.

Activated protein C resistance (APCR) describes a hemostatic disorder characterized by a poor anticoagulant response to activated protein C (APC). This results in an increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. Protein C is a natural anticoagulant that is synthesized in the liver and is activated to APC via proteolysis. APC then degrades Factors Va and VIIIa. APCR describes the reduced inability of APC to cleave Factors Va and VIIIa, which therefore promotes increased thrombin generation and potentially leads to a prothrombotic state. APCR may be hereditary or acquired. The most common hereditary defect is due to mutations in Factor V, predominantly the Factor V Leiden [FVL] mutation-a G1691A missense mutation at Arginine 506 that results in its replacement by a glutamine [R506Q] and the abolition of an APC inactivation cleavage site in Factor Va. Laboratory testing for APCR may be undertaken by a variety of methods, but this chapter describes an automated procedure using a commercial Russell Viper Venom-based clotting assay, and using CS-5100 and STA-R analyzers.
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http://dx.doi.org/10.1007/978-1-4939-7196-1_10DOI Listing
May 2018

The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples.

Pathology 2016 Jan 17;48(1):60-71. Epub 2015 Dec 17.

Pathology Services, Royal Hobart Hospital, Hobart, Tas, Australia.

The direct oral anticoagulants (DOACs), now including dabigatran, apixaban and rivaroxaban, have given clinicians alternative options to low molecular weight heparins (LMWHs) and vitamin K antagonist therapy, including warfarin, for the treatment of atrial fibrillation and treatment and prevention of venous thromboembolic (VTE) disease. DOACs have been successfully marketed as not requiring monitoring; however, there will be situations where clinicians will request laboratory testing, including emergency department admissions for haemorrhage or thrombosis, or emergency surgical interventions. We report the results of several Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) surveys using apixaban and rivaroxaban spiked samples to either assess the suitability of certain potential screening or drug-quantifying assays, for assessment of drug presence or absence or measurement of levels, as well as assessing potential interference in a wide variety of haemostasis assays. We also include additional evaluations using ex vivo samples from patients given apixaban and rivaroxaban for various therapeutic reasons. The prothrombin time (PT) and activated partial thromboplastin time (APTT) show better sensitivity with rivaroxaban than apixaban. Anti-Xa assays show good concordance and reproducibility with expected drug levels; however, availability of these assays may be limited to larger institutions. Interference of apixaban and rivaroxaban on haemostasis testing extends beyond routine coagulation assays to encompass a plethora of specialised assays, including factor assays, lupus inhibitor, and FVIII inhibitor estimation. In conclusion, this report highlights the influence of these drugs on most tests performed in haemostasis laboratories, and the potential for some tests to predict the presence, absence or level of these drugs in plasma.
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http://dx.doi.org/10.1016/j.pathol.2015.11.025DOI Listing
January 2016