Improving outcomes of biochemical testing in the pathology laboratory through improved sample quality

Dimeski, Goce (2011). Improving outcomes of biochemical testing in the pathology laboratory through improved sample quality PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

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Author Dimeski, Goce
Thesis Title Improving outcomes of biochemical testing in the pathology laboratory through improved sample quality
Formatted title
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2011-09
Thesis type PhD Thesis
Supervisor John de Jersey
Paul Masci
Luke Guddat
Total pages 278
Total colour pages 53
Total black and white pages 225
Language eng
Subjects 110106 Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics)
110202 Haematology
100402 Medical Biotechnology Diagnostics (incl. Biosensors)
Formatted abstract
Interferences and subsequent errors in biochemistry results can be generated in any part of the testing cycle and have significant impact on patient care outcomes. Irrespective of how well a laboratory adheres to accreditation/certification protocols and irrespective of whether it uses the latest automation and information systems, a laboratory will not be immune from such errors. Sample quality produced from current blood collection tubes is an area that has not received the intense scrutiny it deserves which will be the scope of this thesis.

The biochemistry laboratory continues to be plagued by variability in analytical sample quality. The distinct differences that exist between sample types (e.g. serum and lithium heparin plasma), the extent to which “true” serum is formed in healthy and anticoagulated patient blood samples, and maintaining the sample integrity remain problematic with current blood collection tubes, even with adherence to recommended collections/processing/storage procedures. The use of lithium heparin plasma as an alternative to serum resolves one major clinical demand, improvement in turn-around time (TAT) in critical hospital situations, but continues to cause problems. It is not cell-free, and thus suffers from poor stability of some analytes on prolonged storage and is unsuitable for some assays. Serum from a completely coagulated blood sample is the preferred specimen type for most biochemical assays as it is theoretically cell-free and coagulation factor free, especially of fibrinogen (plus partly degraded fibrinogen and insoluble fibrin) which is the “fuel” for latent clotting. However, 30 minutes is the recommended clotting time for blood to clot completely, adding to increased TAT if serum is used instead of plasma. Furthermore complete clotting may not be achieved in anticoagulated patients using current commercial tubes. Incomplete clotting can lead to undesirable outcomes; including inaccurate results, analytical system problems and patient outcomes. In turn these impact on costs for service providers and reputation. With medical care decisions being so reliant on pathology data it is important that sample quality be improved.

The thesis research in Chapter 2 has provided some examples of critical pathology errors linked to sample type and quality. The errors are with two of the most critical analytes, troponin and potassium and on the deficiencies of plasma as well as how to determine the most appropriate centrifugation setting to obtain highest quality lithium and citrate plasma samples.

In response, the research has identified and evaluated snake venom prothrombin activators(s) as a clotting tube additive capable of providing highest quality serum sample produced in a very short time, (< 5 minutes) to meet TAT demands of any laboratory while assuring quality in serum from healthy and heavily anticoagulated patients. The incorporation of these materials in collection containers to produce serum for biochemistry and other pathology testing will allow greater confidence of laboratory staff, medical staff, service providers and patients in the results and their accuracy, and in turn improve patient care plus reduce inappropriate use of scare resources.

In Chapters 3 and 4, enzyme activity and clotting studies with pure prothrombin, plasma, and whole blood have provided a detailed overview of the ability of the snake procoagulants to clot blood rapidly even in the presence of different anticoagulants and concentration levels. The kinetic studies using the four groups of snake venom prothrombin activators (PAs) demonstrated that the group C (Pseudonaja textilis prothrombin activator - PtPA and Oxyuranus scutellatus prothrombin activator - OsPA) were very effective in clotting plasma and blood with and without anticoagulants. Clotting was achieved with ~6 nM concentration of PtPA and OsPA in all samples including those on higher heparin doses within 5 minutes. PtPA was the procoagulant most extensively evaluated in this thesis. Use of these procoagulants offers the opportunity to provide a superior serum product in almost all patient samples than is provided by current routinely used commercial tubes including the recently released Becton Dickinson rapid serum tube (BD RST).

In Chapter 5 the BD RST tube which contains added thrombin was evaluated demonstrating it is a step in the right direction towards meeting the TAT demands with the recommended clotting time of 5 minutes. However, it is not able to clot heparinised blood samples, especially those containing > 2 IU/mL heparin, resulting in no clotting at all or latent clotting. To improve its effectiveness in heparinised patients it requires much higher thrombin concentration.

In Chapter 6 the quality of serum produced by using the venom prothrombin activators was evaluated. The experiments demonstrated that high quality serum with significantly reduced cell numbers, less haemolysis, and consistently low residual fibrinogen concentration, < 30 mg/L. Serum with these properties was shown to provide a vastly improved stability in analytes, and is unlikely to result in analytical and patient care errors contributed to cellular activity, or cellular content. The PAs (PtPA, OsPA and ecarin) being proteases have been shown not to affect any of 10 the analytical principles or analytes tested that are proteins or using proteins as testing reagents, and do not alter cellular membranes that may lead to cellular lysis despite the fact that they are proteolytic enzymes. Simply, they offer biochemistry laboratories standardisation on a single specimen type, serum. With such rapid clotting there is an opportunity to perform an extended range of testing from a single tube for both urgent and non-urgent tests. These products not only meet the World Health Organisation definition of serum from the healthy to the most extreme example of anticoagulated patients, but exceed it. Based on the findings here the suggested definition of high quality serum is “serum is the undiluted, extracellular portion of blood after complete coagulation, that is devoid of consumable coagulation factor (fibrinogen < 30 mg/L) and it’s cell numbers and cell content is low such that the lactate dehydrogenase (LD) activity change is < 2% when measured immediately post cell separation (centrifugation) and upon 6-8 hours of storage at room temperature”.

The studies reported in this thesis provide convincing evidence for use of snake venom prothrombin activators to produce high quality serum in blood containers and devices. Producing the highest quality serum sample for biochemical, other pathology and research analyses will lead to in more accurate results and improved patient healthcare outcomes.
Keyword Laboratory errors
Sample quality
Prothrombin Activator

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Created: Wed, 23 May 2012, 11:16:07 EST by Goce Dimeski on behalf of Scholarly Communication and Digitisation Service