International Council for Standardisation in Haematology (ICSH) recommendations for collection of blood samples for coagulation testing

This guidance document has been prepared on behalf of the International Council for Standardisation in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for collection of blood samples for coagulation tests in clinical laboratories throughout the world. The following processes will be covered: ordering tests, sample collection tube and anticoagulant, patient preparation, sample collection device, venous stasis before sample collection, order of draw when different sample types need to be collected, sample labelling, blood-to-anticoagulant ratio (tube filling) and influence of haematocrit. The following areas are excluded from this document, but are included in an associated ICSH document addressing processing of samples for coagulation tests in clinical laboratories: sample transport and primary tube sample stability; centrifugation; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots—transport and storage; and preanalytical variables for platelet function testing. The recommendations are based on published data in peer-reviewed literature and expert opinion.     

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots—transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.     

Quality standards for sample collection in coagulation testing

Preanalytical activities, especially those directly connected with blood sample collection and handling, are the most vulnerable steps throughout the testing process. The receipt of unsuitable samples is commonplace in laboratory practice and represents a serious problem, given the reliability of test results can be adversely compromised following analysis of these specimens. The basic criteria for an appropriate and safe venipuncture are nearly identical to those used for collecting blood for clinical chemistry and immunochemistry testing, and entail proper patient identification, use of the correct technique, as well as appropriate devices and needles. There are, however, some peculiar aspects, which are deemed to be particularly critical when collecting quality specimens for clot-based tests, and these require clearer recognition. These include prevention of prolonged venous stasis, collection of nonhemolyzed specimens, order of draw, and appropriate filling and mixing of the primary collection tubes. All of these important preanalytical issues are discussed in this article, and evidence-based suggestions as well as recommendations on how to obtain a high-quality sample for coagulation testing are also illustrated. We have also performed an investigation aimed to identify variation of test results due to underfilling of primary blood tubes, and have identified a clinically significant bias in test results when tubes are drawn at less than 89% of total fill for activated partial thromboplastin time, less than 78% for fibrinogen, and less than 67% for coagulation factor VIII, whereas prothrombin time and activated protein C resistance remain relatively reliable even in tubes drawn at 67% of the nominal volume.     

Impact of evacuated collection tube fill volume and mixing on routine coagulation testing using 2.5-ml (pediatric) tubes

BACKGROUND: Anecdotal observations by pharmacists monitoring anticoagulated patients indicate that blood samples collected in 2.7-mL (pediatric) evacuated tubes frequently produced falsely elevated international normalized ratio (INR) results. OBJECTIVE: To evaluate the impact of various preanalytical variables (fill volume, sample mixing, and elapsed time between sample collection and mixing) on INR test results using pediatric collection tubes in healthy volunteers and patients receiving warfarin anticoagulation therapy. Fifteen patients receiving warfarin and the 15 healthy volunteers participated in each study arm. METHODS: Multiple blood samples for coagulation testing were obtained from study subjects in full-draw pediatric collection tubes made of siliconized glass. The impact of sample mixing was evaluated by randomly varying the number of times each tube (five tubes total) was inverted following sample collection between one and five. The impact of timely sample mixing was evaluated by randomly varying the elapsed time between sample collection and mixing between 0 min and 4 min in each of five samples. The impact of incomplete collection tube filling was evaluated by randomly varying the volume of six tubes between 50% and 100%. Duplicate coagulation assays were performed on each sample by a centralized hematology laboratory, and the average result was reported. RESULTS: Statistical analysis revealed that neither sample mixing nor the elapsed time between sample collection and mixing had a statistically significant effect on INR test results. For patients receiving warfarin, tube fill volume had a statistically significant effect on the reported INR results (p < 0.001). The mean (+/- SD) INR derived from sample tubes filled 100% was 3.2 +/- 1.2, compared to 9.9 +/- 4.2 for tubes filled only 50% full (p < 0.01). Statistically significant INR elevations became apparent for sample tube fill volumes of < 90%. CONCLUSION: Pediatric blood collection tubes should be filled at least 90% full to ensure accurate INR test results. Anticoagulation therapy providers should routinely inquire about the type of collection tube used (adult vs pediatric) and the adequacy of sample collection volume before deriving therapeutic plans in asymptomatic excessively anticoagulated patients.     

Quality and reliability of routine coagulation testing: can we trust that sample?

Poor standardization of preanalytic variables exerts a strong influence on the reliability of coagulation testing, consuming valuable health care resources and compromising patient outcome. Most uncertainties emerge from patient misidentification and the procedures for specimen collection and handling. Location of unsuitable venous access or problematic phlebotomies may produce spurious activation of the hemostatic system and hemolytic specimens. Prolonged venous stasis is associated with hemoconcentration and spurious variations of most coagulation assays. Additional pitfalls can be introduced by inappropriate phlebotomy tools and small-gauge needles. Inappropriate filling and mixing of the tube, unsuitable procedures for centrifugation and storage of the specimens are additional aspects that need accurate standardization. Besides traditional preanalytic variables affecting routine coagulation testing, thrombin-generation assays require specific criteria to be accurately fulfilled. These aspects include the type of specimen (platelet-poor plasma, platelet-rich plasma or whole blood), blood collection tubes, storage conditions and the presence of residual platelets. Compliance with new international quality assessment programs, which will also involve coagulation laboratories, encompasses the adoption of suitable strategies for reducing undue variability throughout the whole testing process. Such strategies would not entail extraordinary costs and are affordable with a structured outlay of existing resources, educational policies and compliance with reliable guidelines.     

Influence of using a roller mixer on rejected samples in coagulation tests

Introduction: The influence of using a roller mixer just after blood collection on the ratio of rejected samples in coagulation tests was investigated retrospectively. Methods: Numbers of blood samples collected in centralized units of outpatient clinics for prothrombin time (PT) were 24 050 in group I and 24 500 in group II (H. Aral, unpublished data). In group I, the tubes were inverted manually. In group II, the tubes were left standing on roller mixer with 30 rpm. The ratios of rejected samples of the two groups were compared statistically with ki‐square analysis. Results: Using a roller mixer, rate of rejection decreased from 0.79% (group I) to 0.20% (group II) (P < 0.001). Conclusion: We suggested using roller mixer to improve the reliability of coagulation testing. Such standardization in preanalytical phase may be helpful in preventing laboratory errors and obtaining correct test results in coagulation tests.     

Effect of thrombin tube on PSA determination, a clue for false negative in screening for prostate cancer

Prostate cancer is an important male carcinoma. Laboratory screening for tumor marker is the routine preventive strategy for control of this cancer. For screening, prostate specific antigen (PSA) has been proposed as a marker in the serum. In this article, the author assesses the effect of new blood collection tube, thrombin tube, on the analysis of the PSA by bioinformatics technique. Using GoFigure server, the molecular functions of thrombin, PSA and thrombin-PSA were predicted. A significant change of molecular function due to the interaction between thrombin and PSA can be found. Increased of coagulation due to the synergistic clot activation can be expected. If this occurs, the false negative can be expected and it can bring the underdiagnosis of early prostate cancer. Therefore, the recommendation for usage of thrombin tube for PSA test should be modified from general usage. Shortening of the period between blood collection and sample analysis than general case must be done.     

Importance of the pre-analytical phase in blood glucose analysis

Blood glucose levels are characterized by a relatively large intra-individual biological variability due to food intake, physical activity and the body's homeostatic response. Careful attention to the pre-analytical phase is essential to ensure accurate glucose measurements. Blood samples should be drawn in the morning after an overnight fast. Proper sample processing after blood collection is crucial. When fast separation of the cells is not possible, blood should be collected into a tube containing a glucose preservative. Glucose concentrations may also differ according to the blood sampling site (venous, arterial or capillary blood). Plasma and whole blood glucose values are not interchangeable. The International Federation of Clinical Chemistry and Laboratory Medicine recommends reporting the glucose concentration in plasma to avoid clinical misinterpretations irrespective of the sample type and method of measurement. Point-of-care testing (POCT) glucose meters are widely used by both health professionals and diabetic patients to monitor blood glucose levels. However, one should take into account that the reliability of POCT glucose measurements depends upon a variety of factors including underlying disease, patient drug regimens and interfering substances as well as instrument analytical performance and user proficiency. It is recommended to perform a laboratory blood glucose analysis if the POCT glucose value is in the critical hypoglycaemic or hyperglycaemic range.     

Approaches to investigating common bleeding disorders: an evaluation of North American coagulation laboratory practices

Bleeding disorders commonly result from deficiencies or defects in von Willebrand factor (VWF), platelets, coagulation factors, or fibrinolytic proteins. The primary goal of our study was to assess current North American coagulation laboratory practices for diagnosing bleeding disorders, using an on-line patterns-of-practice survey of diagnostic laboratory members of the North American Specialized Coagulation Laboratory Association. The survey examined laboratory approaches to evaluating bleeding disorders, with specific questions about the tests and test panels offered and compliance to recent guideline recommendations on diagnosing von Willebrand disease (VWD) and platelet function disorders. All laboratories responding to the survey performed a prothrombin time/international normalized ratio, an activated partial thromboplastin time, and coagulation factor assays, and many tested for VWD and platelet disorders. However, few laboratories had test panels that evaluated the more common bleeding disorders and few performed some assays, including VWF multimer assessments and assays for fibrinolytic disorders. Additionally, the cutoffs used by laboratories to diagnose type 1 VWD varied considerably, with only a minority following the National Heart Lung Blood Institute recommendations. In contrast, laboratories that tested for platelet function disorders mostly complied with aggregation testing recommendations, as published in the recent North American guidelines. Our results indicate that there are some gaps in the strategies used by laboratories to diagnose bleeding disorders that might be addressed by development of further guidelines and test algorithms that emphasize evaluations for common bleeding disorders. Laboratories may also benefit from guidelines on test interpretation, and external evaluation of their bleeding disorder testing strategies.     

Development of the Global Measles Laboratory Network

The routine reporting of suspected measles cases and laboratory testing of samples from these cases is the backbone of measles surveillance. The Global Measles Laboratory Network (GMLN) has developed standards for laboratory confirmation of measles and provides training resources for staff of network laboratories, reference materials and expertise for the development and quality control of testing procedures, and accurate information for the Measles Mortality Reduction and Regional Elimination Initiative. The GMLN was developed along the lines of the successful Global Polio Laboratory Network, and much of the polio laboratory infrastructure was utilized for measles. The GMLN has developed as countries focus on measles control activities following successful eradication of polio. Currently more than 100 laboratories are part of the global network and follow standardized testing and reporting procedures. A comprehensive laboratory accreditation process will be introduced in 2002 with six quality assurance and performance indicators.     

The sensitivity and specificity of a red blood cell agglutination D-dimer assay for venous thromboembolism when performed on venous blood.

BACKGROUND: Studies evaluating the accuracy of the SimpliRED D-dimer assay for venous thromboembolism (VTE) have used a capillary fingerstick blood sample, which requires the test to be performed immediately at the bedside. Initial studies showed a sensitivity for VTE of 90% to 95% when the assay was performed by a finite number of experienced health care workers. However, because of the test's subjectivity, misinterpretation of the result is possible when performed by inexperienced staff. Recent reports by other investigators indicated a low sensitivity of this assay for VTE and noted a reduction in sensitivity (84%) for pulmonary embolism. OBJECTIVE: To determine the sensitivity and specificity of the D-dimer test performed in the laboratory by experienced technologists on venous whole-blood samples in routine collection tubes. If D-dimer testing results accurately detect VTE when performed in this manner, concerns about the sensitivity of this assay would be solved. METHODS: One hundred forty-eight consecutive patients with suspected VTE underwent D-dimer testing at the bedside using a fingerstick sample and venous blood collected into a plain tube. Venous blood was also collected into tubes containing tri-potassium EDTA, sodium citrate, or a combination of lithium and heparin for D-dimer testing in the laboratory. In addition, the EDTA tube was refrigerated overnight at 4 degrees C for retesting at approximately 24 hours. The presence or absence of VTE was determined by means of objective results of testing and a 3-month follow-up. RESULTS: Thirty-four subjects (23%) had confirmed VTE (25 with deep vein thrombosis; 9 with pulmonary embolism). All laboratory venous blood D-dimer results showed sensitivities of 97%, specificities of 61% to 64%, and negative predictive values of 99%, compared with 88%, 71%, and 95%, respectively, when the results were obtained by means of fingerstick at the bedside. CONCLUSIONS: The SimpliRED D-dimer assay performed in the laboratory on venous blood, collected into any of 3 routine laboratory tubes, is sensitive and moderately specific for VTE. Based on this study, immediate bedside testing (particularly by inexperienced personnel) under suboptimal conditions is unnecessary. Furthermore, the high sensitivity of refrigerated EDTA samples allows specimens to be stored or transported (on ice at 4 degrees C) for testing for 24 hours after collection.     

Application of Robotics in Blood Banking1,2

Abstract. To demonstrate the feasibility of using robots in blood banking applications several prototype systems were developed. Activities associated with sample testing and component preparation were examined. In one project, a general-purpose laboratory robot (Zymate Laboratory Automation System, Zymark, Inc., Hopkinton, Mass.) was configured to prepare samples for microplate-based ABO/Rh testing. In a second project, this same robot was configured to carry out specific steps in evaluating bar-coded labels, as part of a quality control procedure. A fluid-handling robot (Sampler 505, Tecan AG, Hombrechtikon, Switzerland) was used to prepare the dilution of serum samples for the evaluation of an anti-HTLV-III test kit. It was then configured to aspirate, dilute and dispense samples for anti-HTLV-III and HBsAg testing. This robotic system is now in field trial. The use of large industrial robots for automating component production was also considered. The key element in this design was the development of a fixture that would hold the blood bag set during the balancing, centrifugation and expressing steps. However, a fixture which was capable of performing these operations and that was equivalent in size and adaptable to a standard centrifuge bucket could not be fabricated.     

Hämostaseologische Diagnostik und Therapie in der Intensivmedizin

Disorders in hemostasis, leading to hemorrhage or thrombembolic diseases, are of significant importance for intensive care unit (ICU) patients. Possible causes for hemostatic disorders are changes in thrombocyte count or function, changes in plasmatic coagulation or changes in fibrinolysis. Laboratory studies of hemostasis in ICU patients include platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT) as well as measurement of fibrinogen, D-dimers and antithrombin. Except for platelet function disorders, for which no suitable screening test is currently available, the vast majority of clinically relevant disorders of hemostasis can be detected by the existing tests. Physicians in ICUs should have knowledge about possible conditions for pathologic test results, as wells as about conditions that interfere with the laboratory studies. Disorders in hemostasis in ICUs are mostly of complex and acquired origin. The differential diagnosis does not only have to consider the pathologic test result but also the underlying disease as well as the trend of the laboratory tests. In recent years, ?point-of-care testing“ has become more and more important in intensive care medicine. This method allows simple and rapid bedside hemostasis analysis. Of clinical importance in intensive care medicine are the following: activated clotting time (ACT) to monitor heparin administration and the so called rotatiol thrombelastography (ROTEG) to analyze clotting formation and stabilization. Platelet concentrates, fresh frozen plasma, blood coagulation factors as well as special pharmacological options are possible tools in the therapy of hemostasic disorders. Therapeutic decisions need to consider clinical aspects as well as the underlying hemostatic disorder and, last but not least, the availability of blood products, factor concentrates and advanced medical treatment.     

Laboratory methods for determining pneumonia etiology in children

Laboratory diagnostics are a core component of any pneumonia etiology study. Recent advances in diagnostic technology have introduced newer methods that have greatly improved the ability to identify respiratory pathogens. However, determining the microbial etiology of pneumonia remains a challenge, especially in children. This is largely because of the inconsistent use of assays between studies, difficulties in specimen collection, and problems in interpreting the presence of pathogens as being causally related to the pneumonia event. The laboratory testing strategy for the Pneumonia Etiology Research for Child Health (PERCH) study aims to incorporate a broad range of diagnostic testing that will be standardized across the 7 participating sites. We describe the current status of laboratory diagnostics for pneumonia and the PERCH approach for specimen testing. Pneumonia diagnostics are evolving, and it is also a priority of PERCH to collect and archive specimens for future testing by promising diagnostic methods that are currently under development.     

Restructuring of international council for standardization in haematology (ICSH) in Asia

Standardization and harmonization in Laboratory testing are a key issue in the midst of globalization era, because most of laboratory testing has been currently achieved with various kinds of automated systems. In the developed countries, automated systems with highly-regulated principles are commonly used in the routine laboratory. However, there are so many undeveloped and developing countries in Asia that diversity of testing levels can be observed in the area. Some laboratories use glass chamber method for blood cell counting, while other laboratory use semi-automated or fully automated analyzers for complete blood count. International standardization on Hematology is focused on the developed system but not for the developing system. Established standardized documents therefore whould not be unsuitable for Asian societies. In the context, International Council for Standardization in Hematology (ICSH) changed its rules to adjust our Asian Societies and ICSH started to restructure the body. International ICSH society is divided into 5 region sub-groups. Asian area is able to possess one new sub-society, ICSH-Asia. Its reconstruction work has been just started with Asain colleagues, and we are now extending the new societies to discuss Asian problems on the quality of hematology testing.     

Transfusion therapy in emergency medicine

Volume replacement is critical to the resuscitation of the hemorrhaging patient, but this usually can be accomplished quickly and safely with crystalloid and/or colloid solutions. Red cells should be used in addition to asanguinous fluids in the treatment of tissue hypoxia due to anemia. The need for whole blood as opposed to packed red blood cells is controversial. However, plasma should not be used as a volume expander, and its use to supplement coagulation factors during the massive transfusion of red cells should be guided by laboratory tests that document a coagulopathy. Similarly, platelet transfusions are indicated to correct documented thrombocytopenia or platelet dysfunction, and routine prophylaxis after fixed volumes of red cells results is unwarranted. Many anticipated complications of massive transfusions, including hemostatic abnormalities, acid-base imbalances, hyperkalemia, and hypocalcemia, are uncommon or of limited clinical significance. The risks of immune hemolysis and transfusion-transmitted diseases, on the other hand, are significant, and argue for judicious use of blood components. In emergencies in which blood is required immediately before compatibility testing can be completed, O-negative uncrossmatched blood can be requested. Careful blood specimen collection and patient identification prior to transfusion are critical. Practices that emphasize blood conservation, including the use of autologous salvaged blood, are always to the patient's advantage.     

老年重型胰腺炎患者机体凝血和抗凝系统的变化情况

目的探讨老年重型胰腺炎患者机体内凝血和抗凝系统的变化情况,分析凝血、抗凝指标对疾病发展程度的意义。方法收集2009年10月-2013年10月湖北医药学院附属太和医院收治的重型胰腺炎老年患者44例作为观察组,同期选择健康人群24例作为对照A组,同期选择老年轻型胰腺炎患者24例作为对照B组,对三组入选受试者进行血液样本采集,检测和记录标本凝血酶原时间(PT)、部分活化凝血酶原时间(APTT)、纤维蛋白原(FIB)D-二聚体。结果观察组PT、APTT、FIB和D-二聚体均明显高于对照A组和对照B组(P0.05)。结论老年重型胰腺炎患者机体内的凝血和抗凝系统明显异于常态时,可以直接影响到正常的微循环,同时凝血和抗凝系统指标患者病情发展的程度,有助于评估。     

The laboratory diagnosis of HIV infections.

HIV diagnostic testing has come a long way since its inception in the early 1980s. Current enzyme immunoassays are sensitive enough to detect antibody as early as one to two weeks after infection. A variety of other assays are essential to confirm positive antibody screens (Western blot, polymerase chain reaction [PCR]), provide an adjunct to antibody testing (p24 antigen, PCR), or provide additional information for the clinician treating HIV-positive patients (qualitative and quantitative PCR, and genotyping). Most diagnostic laboratories have complex testing algorithms to ensure accuracy of results and optimal use of laboratory resources. The choice of assays is guided by the initial screening results and the clinical information provided by the physician; both are integral to the laboratory's ability to provide an accurate laboratory diagnosis. Laboratories should also provide specific information on specimen collection, storage and transport so that specimen integrity is not compromised, thereby preserving the accuracy of laboratory results. Point of Care tests have become increasingly popular in the United States and some places in Canada over the past several years. These tests provide rapid, on-site HIV results in a format that is relatively easy for clinic staff to perform. However, the performance of these tests requires adherence to good laboratory quality control practices, as well as the backup of a licensed diagnostic laboratory to provide confirmation and resolution of positive or indeterminate results. Laboratory quality assurance programs and the participation in HIV proficiency testing programs are essential to ensure that diagnostic laboratories provide accurate, timely and clinically relevant laboratory results.     

The incorrect laboratory result. Part 1: Pre- and postanalytical phase

Laboratory results play a key role in the diagnostic procedure, the decision of treatment and the follow up of diseases. A high validity of the laboratory result is an important precondition for the efficacy in clinical medicine. Analytical standards have been developed under strong quality control criteria, however, there are no sufficiently defined standards for the pre- and postanalytical phase in laboratory diagnostics. Thus, most of laboratory errors are caused by pre- and postanalytical mistakes. The competent knowledge of possible sources for laboratory errors is a critical precondition for their avoidance. Diagnostic sensitivity and specificity play an important role for the choice of a laboratory test, whereas the predictive value should be considered for the medical relevance of the test result. In addition, many interference factors, which may influence the results of the laboratory tests have to be considered as age, sex, race, lifestyle, drugs, pregnancy, specimen collection, quality and handling as well as special factors, which may influence the complex of immunological and molecular diagnostics.