Opportunities and Challenges in Haemostatic Tests

College of PathologistsASM 201927th June 2019Kuching, Sarawak

DR WAN ZAIDAH ABDULLAH

Contents

• Introduction - overview on the practice of haemostatic investigation

• Current haemostasis diagnostic opportunities

• Changing face of hemostasis testing in modern laboratories

• Challenges (from the practical perspective) in hemostatic investigation

Disclaimer: No conflict of interest to disclose

2

• Haemostasis and thrombosis – a subspecialty in Haematology

• Spectrum of disorders from bleeding manifestation to thrombotic event

• Critical laboratory role in the diagnosis of haemostaticdisorder

• Haemostatic investigation requires competencies in many aspects including technical/interpretative skills

3

INTRODUCTION

Haemostatic investigation - Background

• The need for investigation especially specific diagnosis of congenital bleeding disorders ranges from quite uncommon to rare

• Thrombophilia tests are limited to certain patients and in general restricted to unprovoked thrombotic events (guidelines available on indication of the test)

• Most of newer agents do not require drug monitoring

• However the definitive diagnosis of haemostaticconditions is critical for patient management and can be harmful if lab diagnosis is delayed

4

… continue

• Noteworthy, majority of hematologicalemergencies are associated with haemostatic disorders

• Managing lab operations in a single centre pertaining to haemostatic tests eg reagent inventories for specialized investigations and technical skills training are not easy as compared to other haematological tests due to infrequent and unpredictable requests

5

List of Emergency Conditions- in

Hematology

APML

Neutropenic sepsis

Massive blood transfusion

DIC – *bleeding/thrombosis

TTP or HUS

Sickle cell disease (crisis)

HIT

Hyperviscosity syndrome

Tumor lysis syndrome

Hemolytic crisis

Unknown cause of Bleeding (suspected hemostatic problem)

Thrombosis – Congenital - neonates (Homozygous def of PrC/S with purpura fulminans)

Haematological Emergency

• Bleeding (new and known cases & congenital and acquired conditions, included APML – acute promyelocytic leukaemia)

• TTP/HUS

• DIC

• HIT

→Haemostatic Disorders7

Diagnosis of haemostatic disorders

• The diagnostic approach to patients with hemostasis disturbances is multifaceted and involves accurate collection of clinical history and physical examination

• Combined with results of an appropriate number and type of laboratory investigations

• Investigation can be arbitrarily classified as first-line (i.e., screening), second-line (i.e., for the etiological diagnosis) and third-line (i.e., for biochemical or even molecular characterization) analyses.

8

…. Continue…

• The laboratory diagnostics as a whole, and hemostasis testing in particular, is evolving under new paradigms of efficiency to improve the laborious testing

• Instrumentation continues to help reduce labor costs but in hemostasis, it is still "quite” labor intensive, particularly in special coagulation testing

• The paradigms under which these changes are being developed include a variety of needs:

-minimizing pre-analytical issues and laborious method

-improving diagnostic needs by applying the technological advances (in the newer generation analyzers)

- coping with evolution in the investigation trends in demand for newer & reliable tests eg drug monitoring, POCT

• Over the past 20-30 years, remarkable advances have been made in haemostasis globally and hence inviting a lot of opportunities

In various aspects,

- understanding of the mechanisms of blood coagulation and pathophysiology of the related diseases

- introduction of new technologies: from simple laboratory tests to high tech procedures: protein chemistry, DNA …etc

- growing of fundamental researches which have been successfully translated into improved methods for diagnosis

- development of more effective therapy – recombinant clotting factors for replacement therapy and new promising anticoagulants

→ Significant improvement in the patient care

including laboratory services for haemostatic tests

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COAGULATION LABORATORY

• Developments are rapidly improving the patient care and reducing labor-intensive costs for laboratories

• In reality it is undergoing a substantial reorganization, with emergence of new tests under various settings such as- Bedside testing, instrument automation ( eg emergence of new models and softwares) and other on demand investigations

-Thrombin generation assays, TEG and other POCTs are now available as a shift for global hemostasis assessment and therapeutic drug monitoring for efficient patient-care

Summary of Oppotunities in haemostasis investigation

-Diagnostic Efficiency

Recent advances in mainstream hemostasis diagnostics and coagulation testing are changing at light speed– coagulation analyzers and wide range of tests of different principles

- Availability of tests for monitoring drugs/treatment – fromvarious therapeutic choices relevant to hemostatic disorders

-Better understanding on hemostatic system and pathophysiologyof haemostatic related diseases – accurate diagnosis

- Increased in related research activities - Innovations and new discovery have been translated to clinical practice

OPPORTUNITIES

• Subspeciality in Haemostasis – clinical/lab specialist (Sub-sub)

• Growing Emergency specialist and Anaesthetist interest -Communication across specialties have improved patient care and outcomes.

• Clinical-Lab communication- electronic lab results and referal procedures - allow rapid diagnosis and treatment

• POC testing at bedside –Screening tests, TEG – beyondthe conventional lab tests

• Simplification of diagnostic algorithm workflow - ?AI

Hemostasis testing is expected to grow at a rate of 3–4% per annum.There are various market dynamics affecting this overall trend:

• Routine testing is slightly decreasing, influencedby cost constraints in healthcare and a move to DOACs.• Specialised testing is increasing as more evidence becomes available to support improved outcomes in haemostatic disorders & new biomarkers• Point of care (POC) is increasing, particularly self-testing and global coagulation testing (e.g., thromboelastography).• New technologies form part of the growth expectations. (For example, recent release of the ST Genesia analyzer which enablethrombin generation to enter routine clinical practice)

TRENDS IN COMING YEARS

Overview of challenges in haemostatic investigation

-The driving forces of haemostasis laboratory for operational

activities are mainly dependent on economic resources. Financial burden on specialized investigation is well recognized in clinical practice.

Cost effectiveness of the service: Uncommon and rare conditions of haemostatic disorders – impact on lab services ie offering small scale tests but costly.

- Preanalytical variables affecting patient results – critical aspect of lab test

- Outsourcing specialized tests and related problems: sample integrity, clinical indications, interpretation correctness (error in reporting), repeat samples, responsibilities and no outsourcing services for certain tests (issue of the distance for referral test)

Challenges in Coagulation Diagnostic Service –Workload (2017)

1616 1637 16981507 1651

1358

1855 17881481

17881622 1720

29902728

2846 2913 28052850

3092 31863658 3442

33973566

0

1000

2000

3000

4000

5000

6000

Jan Feb Mac Apr May Jun Jul Aug Sep Oct Nov Dec

PT & APTT tests workload

PT & APTT (office hour) PT & APTT (oncall)

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Challenges in Coagulation Diagnostic Service – Operational Cost

• Consumable cost about RM 500,000 (2017), includes:• Reagent cost

• Routine test reagents

• Special test reagents

• Internal Quality control cost

• Test tubes cost

• Cuvettes cost (expensive)

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Challenges

• Some of the haemostatic disorders are quite rare but with significant urgency , issues on the availability of tests →may affect diagnosis and treatment –inadequate test panel or no equipment facility is associated with delay in diagnosis

• Require skills and experience – technical aspects and time consuming tests

• No single test/gold standard to diagnose certain diseases- Lupus anticoagulant, plt disorder and von Willebrand disease=Confusion induced by the complexity of diagnostic tool/working algorithm in haemostatic disorders

• Reference range variability (type of methods, age and overlapping findings among healthy individual and patients –implication of diagnosis esp in mild disorder – vWD, thrombophilia study and platelet function test)

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Challenges..Uncommon Conditions but requiring urgency in Lab diagnosis

• Thrombotic Thrombocytopenic Purpura (TTP)/HUS (prevalence – 1: 200,000)

• Heparin induced thrombocytopenia (HIT) -0.76% in patients receiving heparin (UFH)

• Catastrophic antiphoslipid syndrome (1% of APS)• Acquired inhibitor FVIII (Acquired Hemophilia A)- 0.01

%• Congenital bleeding disorders: Factor deficiencies: All

factors except FVIII and FIX including platelet disorders• Severe von Willebrand disease/Acquired vWD• Congenital thrombophilia- Homozygous protein C and

S deficiency20

Challenges continue..

• Maintenance of continued quality activities in the lab service: Problems in the chain of test request process ( indication, timing of sample, types of samples, analytical performance, other quality management requirements and etc)

• Delayed lab results (unmet expectation) – batches analysis

• Post analytical issues- interpretation accuracy ( patient factor)

• International standardization of reagent materials working parties by recognized bodies

Summary of Challenges - Practical Perspective

• Relatively modest knowledge that many laboratory professionals have for hemostasis in health and disease

• The unacceptable heterogeneity of available diagnostic algorithms for both diagnosis and therapeutic management of hemostatic diseases

• The grey zone of reference ranges

• The identification and communication of critical results, (post examination : Report and release of result )

• Unsatisfactory harmonization of many preanalytical and analytical tests and procedures

Advances and Opportunities in Haemostasis

Examples

Understanding of the mechanisms of blood

coagulation and pathophysiology of the related

diseases

Expansion of sub specialty training and improvement in patient care

Inflammation

Venous Thrombosis & Atherosclerosis Share Common Risk Factor

Shared Risk Factors:

1. Obesity2. Hypertension3. Tobacco use4. Dyslipidemia5. Diabetes6. Diet7. Stress8. Hormone replacement/contraceptive therapy

Hypercoagulability

Endothelial Injury

Piazza G, et al. Circulation. 2010;121:2146-2150

VENOUS THROMBOSIS ATHEROTHROMBOSIS

Hypercoagulability

The Link Between CVD Risk Factors & Outcomes: Hypercoagulability & Inflammation

Obesity

Smoking

HTN

Diabetes

Dyslipidemia

Inflammation

Risk Factors:

Outcomes:

Venous Thrombosis and Atherosclerosis

Abandon “Silo Thinking”

VTE

Stroke + PADMI

Silo

Silo

Silo

Knowledge on coagulation system• The cascade model of coagulation divided coagulation into extrinsic

and intrinsic pathways. Factor VIIa/tissue factor complex of the extrinsic system is the major initiating event of hemostasis in vivo.

• The intrinsic pathway (the contact system) does not play a major physiologic role in hemostasis.

• The cascade model has been replaced with a cell-based model with three overlapping phases: initiation, amplification, propagation, stabilization and inhibition.

• Although Factors XI and XII of the intrinsic system are not involved as major role in hemostasis, they may have a key role in abnormal hemostasis or thrombosis.

• Linked to cardiovascular diseases

Traditional Water fall cascade

Coagulation cascade

Coagulation system…continue

• The pathways of coagulation and inflammation are intertwined at numerous points.

• The procoagulant factors, VIIa, Xa, and thrombin can activate members of the protease-activated receptor family, which play an important role in coagulation, inflammation, and vascular hemostasis.

• Factor V plays a crucial role in both the procoagulant and anticoagulant systems.

• Bringing together experts in biology, patho-biology of thrombosis/hemostasis, and vascular biology

Microparticles – new biomarkers

• MPs have been proposed to play roles in thrombosis, inflammation, and angiogenesis.

• The elevated levels of monocyte-derived TF+

MPs observed in hyperlipidemia patients may also contribute to arterial thrombosis after rupture of atherosclerotic plaques.

• New insights into hemostasis provide greater understanding of the causes of both venous and arterial thrombosis.

Cancer-associated venous thrombosis. Tumors release TF+ MPs into the

circulation, where they are proposed to bind to activated endothelium and trigger

venous thrombosis.

Adapted from: Microparticles in Hemostasis and Thrombosis

A. Phillip Owens III,

Nigel Mackman

Trauma/Battle field- platinum 5 minutes

• - Intracavitary hemostatic agent

• - High Intensity Focused Ultrasound (HIFU)

• - Drugs to enhance hemostatic function

-rFVIIa

-Factor Xa-PCPS (phosphatidyl choline phosphatidyl serine vesicles)

• - Treat early trauma coagulopathies

Hypothermia, hemodilution and acidosis

Technology and researches which have been successfully translated into

improved methods and diagnosis for Haemostatic conditions

opportunities

The generation of thrombin is a fundamental part of the clotting cascade and as such an

estimation of an how well a particular individual can generate thrombin may correlate with either

a risk of bleeding or thrombosis.

Historically coagulation tests such as the PT and APTT, have a number of intrinsic problems:

- they look at the clotting cascade in isolation

- they are non-physiological

- they show a poor correlation with the clinical phenotype i.e. the PT or APTT may be

prolonged but this does not necessarily predict the bleeding phenotype

- they are, in general, insensitive to prothrombotic states

Furthermore the PT and APTT use the formation of a fibrin clot as the endpoint of the test and yet

this occurs when only ~5% of the total amount of thrombin that is generated.

THROMBIN GENERATION

POCT: TEG

TEG- Blood management

Development of more effective therapy and test methods – Eg recombinant clotting factors and new promising

anticoagulants

opportunities

Dabigatran

etexilate Rivaroxaban

Thrombin (FIIa) Factor Xa

Yes No

6.50% 80%

1-2 h 2.5-4 h

9-13 h 7-11 h

No No

Fixed, once or twice

daily

Fixed, once or twice

daily

80%

67% renal, 33%

fecal

DIRECT ANTICOAGULANT DRUGS

Drug

Target

Prodrug

Bioavailability

T (max)

Half-Life

Routine

coagulation

monitoring

Dosing

Elimination

RIVAROXABAN Calibration Curve – anti-Xa assay

DABIGATRAN Calibration Curve-TT

POC: Example: INR test

The results correlate well with laboratory values for

both routine samples and patients on oral

anticoagulants. It is approved by the Medical

Devices Agency/FDA

Its use is well suited to patients established on

warfarin for 6 months or more for general practice or

home-based monitoring of warfarin (PST).

Platelet resistant state- Plavix and aspirin.

• Whole Blood Platelet Aggregation by Multiplate® analyzer

Atypical light transmittance profile on the aPTT

• Has been reported to be associated with DIC and etc

• Allow early diagnosis of DIC and intervention. Provides a practical tool in assigning risk in critical care patients

• The BPW showed a moderate sensitivity but a high specificity for DIC. The association of the BPW with DIC was highly significant, with odds ratios of 29.9 and 19.0 for the ISTH and Japanese DIC criteria, respectively. Limited to specific photo-optical analysers.

Genotypes and drugs- pharmacogenetics

• Example: Variations in warfarin response

• Predict phenotypes of patients receiving therapy- algorithm

• Warfarin sensitivity eg CYP2C9 genotypes and VKORC1 genes

• Managing anticoagulant therapy effectively and responsiveness to the treatment

• Genetic study from simple PCR to microarray etc

Changing face of hemostasis testing in modern laboratories

Recent advances in mainstream haemostasis diagnostics

New Generation Coagulation Analyzer

• Provide higher throughput, more efficient systems with the ability to deliver cost savings, due to healthcare cost constraints.

• Most recent developments have included the launch of the STA R Max 2 (2017) reducing maintenance by up to 77% vs. previous generations

• Including a check for detection of preanalytical interferences and sample volume. Eg Compact Max 3 with HIL and sample volume check and STA R Max 3

… continue

• Advanced cybersecurity features

• A new solution for the low volume automated segment.

Coagulation Expert which assists the laboratory with

• (1) advanced QC management,

• (2) automation of accreditation requirements,

• (3) expert rules to define clinical pathways for routine and specialised investigations,

• (4) management tools for cost and turnaround time monitoring,

• (5) remote monitoring for service applications, remote validation, and cross site visibility

Preanalytical check: HIL detection technology

• HIL - Each of these have the potential to compromise sample and test quality, either because they interfere with optical (“spectral”) detection, or because of resulting biological or biochemical changes to the sample.

• HIL tends to affect optical clot detection test systems, with mechanical test systems being fairly insensitive to HIL spectral events.

• Some optical test systems can identify HIL events, and then “overcome” them by switching the wavelength of optical detection, to that of a wavelength that is relatively insensitive to HIL

5/7/2019Hemostasis Course: Sample Handling in Hematology

HIL Technology – Siemens/Sysmex/IL

Interference: preanalytical error

Mechanical (VBDS) vs Optical

• Both methodologies do have their advantages and disadvantages—however, benefits for the optical detection method provides more insight into the reaction and more information is available and enables different interpretation (e.g., derived fibrinogen, Clot Wave Form Analysis),

• The argument that optical methods are more sensitive to optical interferences is recognized but latest studies have shown that the result can be impacted as well by using mechanical detection, particularly for hemolytic samples.

… continue

• Especially for these cases, an automated check for interfering substances is beneficial for both mechanical and optical detection

• Looking at low fibrinogen samples (weak clots), the optical method shows advantages when measuring at 405 nm

• Therefore, the option of an automated wavelength switch is quite valuable in order to avoid sample re-runs.

• Nevertheless, mechanical detection can be helpful example in cases of highly lipemic samples—therefore, an easy alternative method, ideally on the same system with automated reflex options, would be a good solution.

Technology advances and reduction of errors

• Instrument: Sample preparation will become a valuable addition through automation

(1) Increase number of detection systems in a single platform(2) Fewer and less skilled staff can operate(3) TLA systems with walk-away capabilities by automation • one single point of entry and continuous loading of samples; • automatic centrifugation;• automatic routing of samples to instruments; • high throughput with centrifugation;• storage

• Liquid ready reagents – reduce operator error from incorrectreconstitution/manual preparation and time dedicated to preparation

Enhancement of Diagnosis

• 1. vWD

• 2. Plt function testing

• 3. Assessing factor levels in new era of extended life factor replacement

• 4. Direct oral anticoagulant agents

• 5. Lupus anticoagulant

Coagulation Diagnostic Service Section Coagulation Analyzers

Stago STA-R Max Coagulation Analyser- Routine tests: PT, APTT, Fibrinogen level, Fibrinogen monomer, DIC screening, D-dimer

Stago STA-R EVO Coagulation Analysers- Routine tests

Stago STA CompactCoagulation Analysers

LA, FVIII, FIX assays, inhibitor assay

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Coagulation Diagnostic Service Section Profile -Analyzers

ACL-Top seriesThrombophilia, vWD, Other Factor assays:

II,V,X,XI XII,VII

Sysmex CS-2100iRiCOF (vWD), Platelet

Aggregation

Chronolog machinePlatelet Aggregation study

59

Opportunities: Haemostasis investigation & Research Project

• Continuous staff development on advanced diagnostic technologies:• Stago STA-R Max analyzer

• Sysmex CS analyzer – optical clot detection and automated platelet aggregation testing capabilities

60

Coagulation Diagnostic Services –Ensuring test quality – opportunities to improve services

• Quality Assurance activities

61

HUSM/HEMA-UPT/QP-05

PROCESS OF HANDLING INTERLABORATORY COMPARISON SAMPLES

HAEMATOLOGY AND TRANSFUSION MEDICINE LABORATORY HOSPITAL UNIVERSITI SAINS MALAYSIA

Prepared by: Ahmad Zakwan Mustafa Mohd Amirudin Sidik

01.08.2016 Checked by: AP Dr. Rapiaah Mustaffa

10.08.2016 Approved by: Prof. Dr. Wan Zaidah Abdullah

15.08.2016 Effective date:

15.08.2016

Hakcipta © Makmal Hematologi & Unit Perubatan Transfusi 2016

Sebarang cetakan dan penggunaan semula mana-mana bahagian dalam dokumen ini tidak dibenarkan

HUSM/HEMA-UPT/QP-04

ASSURING QUALITY OF TEST RESULTS

HAEMATOLOGY AND

TRANSFUSION MEDICINE LABORATORY

HOSPITAL UNIVERSITI SAINS MALAYSIA

Prepared by: Ahmad Zakwan Mustafa

Mohd Amirudin Sidik

01.08.2016

Checked by: AP Dr. Rapiaah Mustaffa

10.08.2016

Approved by: Prof. Dr. Wan Zaidah Abdullah

15.08.2016

Effective date:

15.08.2016

Coagulation Diagnostic Service – MS ISO 15189

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• MS ISO 15189 accreditation

• Accreditation Quality & Competence

ConclusionCurrent Changes in Haemostasis

Open more opportunities and moving towards minimizing challenges

Up-dated mechanics, molecular biology and genetics affect the

clinical implications of haemostatic and thrombotic disorders as

seen in current lab services and treatment strategies.

Significant achievements:

-pathophysiology and recognition of disease in clinical practice

-novel exploitation of diagnostic methodologies and therapeutic

applications

-prevention of thrombosis-hemostasis morbidities & mortalities

- wide application in teaching, learning and research activities

(at national level and world wide)

References

• Historical perspective and future direction of coagulation research H. SAITO, T. MATSUSHITA and T. KOJ IMA . Journal of Thrombosis and Haemostasis, 9 (Suppl. 1): 352–363, 2012

• Recent Advances in Mainstream Hemostasis Diagnostics and Coagulation Testing Emmanuel J. Favaloro and Giuseppe Lippi. Semin Thromb Hemost 2019;45:228–246.

• Hemostasis practice: state-of-the-art Giuseppe Lippi and Emmanuel J. Favaloro. Journal of Laboratory and Precision Medicine. J Lab Precis Med 2018;3:67

THANK YOU

Acknowledgement:

• Hospital Director and Dean School of Medical Sciences. USM

• All staff Hematology Lab and Coagulation Section

• Lecturers and students in Transfusion Medicine Unit and Hematology Department65