Giovanni Poli

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.

Prevalence and type of pre-analytical problems for inpatients samples in coagulation laboratory

RATIONALE, AIMS AND OBJECTIVES Total quality in coagulation testing is a necessary requisite to achieve clinically reliable results. Evidence was provided that poor standardization in the extra-analytical phases of the testing process has the greatest influence on test results, though little information is available so far on prevalence and type of pre-analytical variability in coagulation testing. METHODS The present study was designed to describe all pre-analytical problems on inpatients routine and stat samples recorded in our coagulation laboratory over a 2-year period and clustered according to their source (hospital departments). RESULTS Overall, pre-analytic problems were identified in 5.5% of the specimens. Although the highest frequency was observed for paediatric departments, in no case was the comparison of the prevalence among the different hospital departments statistically significant. The more frequent problems could be referred to samples not received in the laboratory following a doctors order (49.3%), haemolysis (19.5%), clotting (14.2%) and inappropriate volume (13.7%). Specimens not received prevailed in the intensive care unit, surgical and clinical departments, whereas clotted and haemolysed specimens were those most frequently recorded from paediatric and emergency departments, respectively. The present investigation demonstrates a high prevalence of pre-analytical problems affecting samples for coagulation testing. CONCLUSIONS Full implementation of a total quality system, encompassing a systematic error tracking system, is a valuable tool to achieve meaningful information on the local pre-analytic processes most susceptible to errors, enabling considerations on specific responsibilities and providing the ideal basis for an efficient feedback within the hospital departments.

Influence of the needle bore size on platelet count and routine coagulation testing.

The phlebotomy technique, particularly the use of small-bore needles, may influence the reliability of coagulation testing and platelet count. Routine coagulation tests were assayed in blood specimens collected from 22 consecutive patients in three separate, sequential phlebotomies, using butterfly devices with different needle sizes. Test results of samples collected with 23 and 25 G needles were compared with those obtained with the currently recommended 21 G needle. Although both the prothrombin time and activated partial thromboplastin time displayed a trend towards lower values employing the smaller 23 and 25 G needles, results did not differ significantly from the reference 21 G needle specimen, with the exceptions of D-dimer (25 G versus 21 G needle, 186 ± 70 versus 178 ± 66/ml, P < 0.01) and platelet count (23 G versus 21 G needle, 246 ± 55 versus 254 ± 56 × 10−3/l, P < 0.01; 25 G versus 21 G needle, 240 ± 55 versus 254 ± 56 × 10−3/l, P < 0.01). None of the mean biases recorded for the parameters was clinically meaningful, nor did they exceed the current desirable analytical quality specifications for desirable bias. Results of the present investigation suggest that, when a proper technique is used and within certain limitations, butterfly devices with small-bore needles may be a reliable alternative to draw venous blood for platelet count and coagulation testing.

Influence of temperature and time before centrifugation of specimens for routine coagulation testing

The accurate standardization of the preanalytical phase is of pivotal importance for achieving reliable results of coagulation tests. Because information on the suitable storage conditions for coagulation testing is controversial, we aimed at investigating the sample stability with regard to the temperature and time before centrifugation. The activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and D‐dimer were assayed in specimens collected from 26 consecutive patients on antivitamin K therapy on the ACL TOP analyzer. Three primary 3.6‐ml siliconized evacuated tubes containing 0.109 mol/l buffered trisodium citrate were sequentially collected from each patient. These three tubes were mixed, pooled and divided into seven identical aliquots. The first aliquot was immediately centrifuged according to the standard protocol [1500 g for 15 min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24 h, respectively, at RT or 4 °C, and then centrifuged and analyzed. Test results were compared with those obtained on the reference specimen. Statistically significant prolongations were observed for aPTT in all the samples. Such differences exceeded the analytical quality specifications for desirable bias in the samples stored for 24 h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6 h. No significant biases could be recorded in D‐dimer. In conclusion, a 6‐h storage of uncentrifuged specimens at either RT or 4 °C may still be suitable to achieve results of routine coagulation testing comprised within the analytical quality specifications for desirable bias.

Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation

Summary.  In order to describe the haemostatic role of a variation in inhibitor reactivity with different factor VIII (FVIII) concentrates, we have compared inhibitor titres against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma‐derived concentrates were tested in vitro in mixing experiments with inhibitor plasmas from 11 patients with severe haemophilia A: Fanhdi, which contains von Willebrand factor (VWF) with a final ratio of approximately 1:1 (VWF IU per IU FVIII:C); Haemate‐P with a ratio of 2.5:1 and Hemofil‐M containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer containing no VWF was included. Inhibitor titres and the capacity to generate thrombin were measured. A statistically significant difference in measured titres was found with the highest titres recorded against Hemofil‐M. The inhibitor titres needed to inhibit 50% maximum thrombin generation were the lowest for Kogenate Bayer and the highest and similar for Fanhdi and Haemate‐P with intermediate titres needed for inhibition of Hemofil‐M. In this study, the thrombin generation assay provides additional indications for the role of VWF in the treatment of patients with inhibitors. The VWF‐containing concentrates Fanhdi and Haemate‐P, added to FVIII‐deficient plasma with the presence of inhibitor, generate more thrombin than do the purified concentrates Hemofil‐M and Kogenate Bayer.

Selenium and glutathione peroxidase variations induced by polyunsaturated fatty acids oral supplementation in humans.

Serum and erythrocyte selenium, erythrocyte and platelet glutathione-peroxidase (GSH-Px) activities, and erythrocyte reduced glutathione (GSH) content were measured in 25 healthy adult individuals before and after daily supplementation with 20 ml of fish oil for 10 weeks. Serum-Se decreased from 0.83 +/- 0.01 mumol/l to 0.75 +/- 0.02 mumol/l (mean +/- S.E.M.) (P less than 0.01); erythrocyte-Se decreased from 4.39 +/- 0.17 nmol/g hemoglobin (Hb) to 2.83 +/- 0.15 nmol/g (P less than 0.001). GSH-Px activities increased both in erythrocytes (6.93 +/- 0.24 iu/g vs 8.18 +/- 0.27 iu/g Hb, P less than 0.01) and in platelets (69.2 +/- 2.8 iu/g vs 90.9 +/- 3.6 iu/g protein, P less than 0.001). The concentration of GSH in erythrocytes fell from 9.56 +/- 0.29 mumol/g Hb to 5.90 +/- 0.30 mumol/g Hb (P less than 0.001). The effects on plasma lipids were evident only for triglycerides (before 1.96 +/- 0.16 mmol/l, after 1.75 +/- 0.14 mmol/l, P less than 0.001). We hypothesise the enrichment of erythrocyte and platelet membranes with polyunsaturated fatty acids (PUFAs), following fish oil intake, can generate increased amounts of lipid peroxides and thus allosterically activate GSH-Px: with time this is harmful for the integrity of the enzyme molecule and Se release may result. We suggest that the Se status of individuals given PUFAs is assessed before and during intake; Se supplements should only be given when serum and/or erythrocyte Se are reduced.

Evaluation of platelet turnover by flow cytometry

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K2EDTA, was incubated with 0.6 µg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 ± 3.09%. RPs were 10.41 ± 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 ± 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 ± 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 ± 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 ± 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

A study on thrombophilic factors in Italian Behcet's patients

BACKGROUND Behcets disease (BD) may complicate with arterial and venous thrombosis. The purpose of this work is to evaluate in an Italian group of BD patients with thrombotic events a large panel of inherited and acquired thrombophilic factors. METHODS Thirty BD patients, of which nine with previously arterial or venous thrombosis and 21 without, underwent the following investigations: plasma antithrombin activity, protein C activity, free protein S level, sensitivity to APC, total plasma homocysteine concentration, serum folate level, determination of anti-phospholipid antibodies, serum Lp(a) levels, tests for gene polymorphisms of factor V Leiden, prothrombin and methylenetetrahydrofolate reductase genes. Tests for the gene polymorphisms were also performed in a group of healthy control subjects. RESULTS All the six patients with arterial or deep venous thrombosis showed thrombophilic conditions such as protein C or protein S deficiency (one case each), hyperhomocysteinemia (two cases), positivity of anti-phospholipid antibodies associated with APC resistance or hyperhomocysteinemia (one case each). Among three subjects with superficial thrombophlebitis only one showed a mild hyperhomocysteinemia. No differences were found between BD patients and control subjects concerning polymorphisms of the genes considered. Among BD patients the Factor V H1299R mutation showed a weak association with venous thrombosis (P=0.048). CONCLUSION In BD patients different concomitant significant thrombophilic risk factors may contribute to the development of thrombotic events. Patients affected by vasculo-Behcet should be evaluated for the presence of coexisting major thrombophilic conditions.

Development and implementation of an automatic system for verification, validation and delivery of laboratory test results

Abstract Background: The verification/validation of laboratory test results is one of the most critical aspects of the total testing process, which may produce conflicts between competencies and duties at the point of professional crossroads. This process has centered for decades on the human component, with positive effects as well as potential adverse consequences (postanalytical errors). Manual validation of data is a time-consuming activity, is inherently subjective and arbitrary, and requires the constant presence of postgraduate physicians or biologists within the laboratory with adverse economical and organizational impacts. To overcome these inherent limitations, we have developed and implemented in our stat department an automatic system for verification, validation and delivery of laboratory results. Methods: The procedure is based on automatic validation of test results by an expert system, coupled with remote wireless connection, which allows the laboratory professional “on call” to access, visualize, analyze, validate and deliver alert values (suspect, erroneous or critical) using a small laptop. This system also provides five phases where preanalytical and analytical errors can be identified and handled. Results and conclusions: Six months following implementation of this innovative system, which can be customized to facilitate a wide variety of laboratory workflow models, the reporting efficiency of our stat laboratory has greatly improved, reducing manual data entry, and increasing the timeliness and utility of test results. Clin Chem Lab Med 2009;47:1355–60.

K(3)EDTA Vacuum Tubes Validation for Routine Hematological Testing.

Background and Objective. Some in vitro diagnostic devices (e.g, blood collection vacuum tubes and syringes for blood analyses) are not validated before the quality laboratory managers decide to start using or to change the brand. Frequently, the laboratory or hospital managers select the vacuum tubes for blood collection based on cost considerations or on relevance of a brand. The aim of this study was to validate two dry K3EDTA vacuum tubes of different brands for routine hematological testing. Methods. Blood specimens from 100 volunteers in two different K3EDTA vacuum tubes were collected by a single, expert phlebotomist. The routine hematological testing was done on Advia 2120i hematology system. The significance of the differences between samples was assessed by paired Students t-test after checking for normality. The level of statistical significance was set at P < 0.05. Results and Conclusions. Different brands tubes evaluated can represent a clinically relevant source of variations only on mean platelet volume (MPV) and platelet distribution width (PDW). Basically, our validation will permit the laboratory or hospital managers to select the brands vacuum tubes validated according to him/her technical or economical reasons for routine hematological tests.