100 Years of Milestone Developments in Hemostasis and Thrombosis

By Geoffrey Wool - March 08, 2022

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Editor's note: This essay is part of an ongoing series about the evolution of the laboratory over the past century, and part of ASCP's 100th Anniversary celebration.


The 100 years between 1922 to 2022 have witnessed revolutionary improvement in both our scientific understanding of the physiology of hemostasis and pathophysiology of thrombosis as well as the routine availability of high-quality diagnostic and monitoring testing for bleeding and clotting disorders. 

Expanding scientific understanding of hemostasis 
Fibrinogen, thrombin, and calcium were known to play a role in coagulation as early as 1905, as described by Morawitz.1,2  In 1926, Dr. von Willebrand described patients with an autosomal mucosal bleeding disorder, but his criteria identified both patients with what we would now call von Willebrand disease as well as platelet dysfunction.3  Roman numerology for coagulation factors began in 1943 when Paul Owren described factor V.1,4  Additional clotting factors were identified in the 1940s and 50s and coagulation factor nomenclature was rationalized and standardized in 1959 by consensus agreement.5  The familiar cascade/waterfall theories of coagulation were promulgated in the 1960s.6,7 

Since these foundational discoveries, the hemostasis field has seen the benefits of factor concentrates for patient care, the devastation of HIV and hepatitis in the hemophilia patient community, the cloning of coagulation factors, and the true revolution of current hemophilia care advancements.8-10  Additionally, the cell-based model of coagulation has been accepted to capture the accurate in vivo interplay of plasmatic coagulation factors as well as platelets and tissue factor (TF)-bearing cells.11 

Advances in diagnostic testing for bleeding and clotting disorders 
Primary hemostasis 
The quintessential point-of-care and whole blood clotting test, bleeding time was first described in 1901 as a screen for platelet dysfunction or coagulation factor deficiency. In 1982, approximately 1 million bleeding time determinations were performed annually in the United States.12  Rodgers and Levin published a landmark meta-analysis in 1990 questioning the use of the bleeding time as an accurate screen for bleeding disorders; particularly in a low-risk group such as pre-operative patients with no documented abnormal bleeding events, the bleeding time was no better than a coin flip at predicting bleeding events.12 Since that time, bleeding times have become nearly unheard of in the United States as a routine clinical test.13-15 Correspondingly, whole blood laboratory assays such as the PFA-100 have increased in utilization as a screen for disorders of primary hemostasis.16 

In 1962, Born and O’Brien developed platelet light-transmission aggregometry (LTA), the gold standard platelet function assay.17  Automated instruments for combined detection of platelet aggregation and dense granule secretion were developed in the 1970s18 and computer-assisted aggregation/secretion trace analysis was described in 1984.19  Whole blood impendence aggregometry was shown to be an accurate method that could reduce the preanalytical work and volume of blood required, compared to LTA.20,21 

Secondary hemostasis 
Citrated plasma-based testing has been the backbone of routine laboratory hemostasis testing. Seminal achievements in laboratory hemostasis testing include Quick’s description of the prothrombin time (PT) in 1935 (using rabbit brain thromboplastin and oxalate plasma) and the development of the activated partial thromboplastin time (aPTT) by Langdell, Wagner, and Brinkhous in 1953.2,22,23 These assays remain the foundation of routine coagulation testing to this day. The growth of the utilization of the PT and aPTT mirrored somewhat the development of anticoagulant medications which could be monitored by these assays: the discoveries of heparin by McLean and Howell in 1916 and dicoumarol by Campbell and Link in 1941.24  

Rabbit brain-derived thromboplastin was the basis of the PT for many years.  The International Normalized Ratio (INR) method of reporting prothrombin time values was introduced in 1983 by the Expert Committee on Biological Standardization of the World Health Organization to standardize laboratory monitoring of oral anticoagulant therapy. Standardization was needed due to the significant variation in PT results derived from different commercial thromboplastin reagents, corresponding to their differential response to deficiency of vitamin K-dependent clotting factors.25,26 With the cloning of the tissue factor (TF) gene in 1987,27 recombinant human TF-based thromboplastins were developed (thromboplastins containing human TF were previously available from processed placentas).28 The availability of recombinant human tissue factor made it easier to produce sensitive thromboplastin reagents in commercial quantities,29 with the additional benefit that recombinant thromboplastins are a highly defined reagent allowing further standardization of PT testing.30 The 1998 College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy published a recommendation that laboratories use thromboplastins with an International Sensitivity Index (ISI) between 0.9 and 1.7, with a preference for reagents with an ISI toward the lower end of the scale.31 Although the effect of using sensitive thromboplastins on precision is modest, data from interlaboratory proficiency testing programs generally support the recommendation for an ISI close to 1.0.26 

The introduction of evacuated tubes in the late 1940s enhanced the precision and accuracy of coagulation test results by easing collection (eliminating the need for syringes) and standardizing blood-to-additive ratios.32 We have further progressed from glass evacuated citrate tubes to plastic evacuated tubes containing liquid citrate with a polypropylene insert tube within the polyethylene outer tube. In such configurations, the inner polypropylene tube is a non-activating surface and prevents the loss of water vapor, while the outer tube preserves the vacuum.32 Moving from glass to plastic blood tubes was a significant safety improvement for phlebotomists, nurses, and medical technologists, and technicians.33 

There has been a continuing increase in the use of semi- and fully automated devices for coagulation testing in the past 60 years. While the methodology of detecting changes in light passage or in the movement of a metal ball in a clotting plasma sample remain similar, automation moves the end-point detection from the human eye to a precise optical detection or electromagnetic circuit.  In 1962 essentially all coagulation tests were done using manual techniques. By 1969, about 40 percent of prothrombin time testing was being done using a semi-automated device, most often the Fibrometer.  Such a device automated endpoint detection while requiring the operator to be continually present for sample application.34 By 1977 more than 80 percent of American hospital laboratories in a CAP survey performed PT and aPTT using some degree of automation.35 We have now progressed to nearly all routine coagulation assays in the developed world being performed by automated techniques; fully automated tests show increased precision and therefore increase the reliability of patient testing results.35 Large laboratories increasingly make use of total laboratory automation with interfacing of hemostasis measurement devices onto automated laboratory delivery lines.36 We continue to attempt to harmonize standard coagulation testing, to further improve laboratory monitoring and patient care.37   

Whole blood testing has received new attention in the past three to four decades with the use of viscoelastic testing (VET).38,39 VET uses a physiologic matrix and captures the interplay of platelets, coagulation factors, tissue factor-bearing cells such as monocytes, and red blood cells.11 For operative or massively bleeding patients, VET-based transfusion algorithms offer benefits over conventional coagulation assays with reductions in blood product utilization, bleeding, costs, and lengths of stay.40  

Finally, thrombin generation assays (TGA) offer the ability to specifically measure the generation of thrombin in platelet-poor plasma, platelet-rich plasma, or whole blood.41,42 The generation of thrombin correlates well with bleeding events and offers the potential to more objectively measure the hemophilic phenotype as well as the response to treatment.43 Additionally, TGA may have a role in thrombosis prediction.44,45 TGA is not currently FDA approved for clinical use in the United States, however. 

Conclusion 
As with most other areas of biology and medicine, the past 100 years have seen truly remarkable advancements in hemostasis science and coagulation laboratory testing. However, as is generally true in medicine, disease will continue to challenge us and show the limits of our knowledge and capabilities. The COVID-19 pandemic has brought these issues to the fore, from the limitations of standard coagulation assays for predicting thrombosis in COVID-19 patients, to the newly described vaccine-induced immune thrombotic thrombocytopenia (VITT).46    

Further progress in scientific knowledge as well as standardization, automation, and new assay development will continue to advance the accuracy and utility of the coagulation laboratory. As we look forward to another successful 100 years in the hemostasis field, we must remember that dedicated, knowledgeable, and skilled laboratory professionals are the foundation of our work.    

 

References  

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Geoffrey Wool

Associate Professor

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