ALBERT

Description: Introduction: Processing blood samples for full blood count may be delayed for a multitude of reasons. There is little published information on the effect of sample storage on platelet count. This study compares the effect of storing normal samples at room temperature (RT) and at 4°C prior to processing using different analysers with different technologies. Sysmex SF 3000 and XE2100 analysers are capable of providing impedance (IMP) platelet counts and the latter can also perform fluorescence based optical (OPT) platelet counts. The Coulter LH750 and the ABX Pentra DX120 use IMP technology with enhanced data extraction techniques and algorithms to eliminate interference. Method: Intravenous blood samples for routine blood counts were taken into 4ml K2 EDTA Greiner Vacuette containers from 20 out-patients and the platelet counts measured at 0, 6 and 24 hrs on Coulter LH750 (IMP), ABX Pentra DX120 (IMP), Sysmex SF3000 (IMP) and Sysmex XE2100 (both IMP and OPT) blood count analysers. Samples were stored at RT. Samples were also processed on the XE2100 following storage at 4°C. Results: Mean changes in platelet counts (n = 20) expressed as percentages of the original value at 0 hrs and paired t-test values of significance are shown in the Table. The most significant changes occurred with the XE2100 where the IMP value increased by 7% over 24 hrs but the OPT value fell by 5%. No significant changes occurred with the Coulter or the ABX analysers at 0 and 24 hrs or with the SF3000 at 6 hrs. 6 hr v 0 hr 24 hr v 0 hr % change p % change p Coulter RT + 0.19 0.873 −0.12 0.908 ABX Pentra DX120 RT + 0.02 0.985 + 0.57 0.380 SF 3000 RT + 1.25 0.281 + 2.52

Description: Introduction: Elevated fibrinogen levels are well recognized as an independent risk factor for cardiovascular events in adults. Current research highlights the need to understand the mechanisms that influence fibrinogen levels in adolescents in order to elucidate its role in early onset ischaemic heart disease in young people. Depressed levels of fibrinogen are observed in a range of pathological conditions including acquired and congenital hypo- and afibrinogenaemias, consumptive coagulopathies, carcinoma and liver disease. Appropriate determination of the status of fibrinogen levels in patients is vital in identifying fibrinogen as a risk factor for cardiovascular events and in the investigation of coagulopathy. Ascertaining the meaningful status of the fibrinogen level in a patient relies on comparison with a reference range determined by the same methodology using an analogous population from which the patient originates. We present a reference range for Clauss fibrinogen determination in adolescents aged 12–14 years using the Sysmex CA-1500 coagulometer (Sysmex Corp., Kobe; Japan). Methodology: Blood samples for fibrinogen determination were collected from 240 adolescent schoolchildren aged between 12 and 14 years (M=119; F=121). All of the children were healthy with no apparent underlying pathology. Early morning samples were collected into siliconised glass BD Vacutainers containing tri-sodium citrate (Ref: 367691) and analysed within 4 hours of collection. Fibrinogen determination was performed using Dade-Behring thrombin and Owrens Veronal buffer reagents. Calibration of the Clauss fibrinogen assay was performed using NIBSC WHO International reference plasma for human fibrinogen (product number 98/612). Results: Fibrinogen results for males and females were examined for normality using Anderson-Darling and Kolmogorov-Smirnov tests. Results were found to be normally distributed and reference ranges constructed using the arithmetic mean +/− 1.96SDs. Male and female results were examined using the two-sample T- test for gender differences where p

Description: We report the complex coagulation profile in a 74 year old female with progressive CLL and IgM paraproteinaemia and discuss the possible implication of these results in such a clinical setting. A 74 year old female with CLL was investigated for progression to high grade lymphoma and abnormal liver profile. Blood count parameters measured on a Sysmex 2100 were: Hb 10.5 g/dL (12–15.2); WBC 9.5 × 109/L (3.6–9.2) with lymphocytosis; PLT 71 × 109/L (180–380). She had deranged liver function with cholestatic picture thought to be due to NHL. Liver enzymes (U/L) were: Alk Phos 718 (30–250); AST 83 (80.0 MPLU/mL (0–7). IgG and IgA ACAs were normal. She had no bleeding diatheses. Coagulation parameters measured on a Sysmex CA1500 using Dade-Behring reagents and mixing with normal plasma showed markedly prolonged prothrombin time (PT) (Innovin) of 92 sec with no correction with normal plasma. The APTT was also markedly prolonged using (Actin FS) of 76sec and less prolonged at 48sec using lupus anticoagulant sensitive agent (Actin FSL) with no correction on mixing with normal plasma. Fibrinogen (Clauss) and TCT were normal. Thrombotest INR 1.2 (

Description: Introduction: Paraproteins cause interference in many assays systems due to increased viscosity and/or by non-specific binding to either analytes or reagents which may variably affect the results. There is abundant evidence to indicate that performance characteristics of automated, competitive protein-binding assays for folate assays are normally sound, but there is a paucity of data comparing folate assays in patients with paraproteinaemia compared to normals. Method: Serum samples were prepared from venous blood taken into Greiner Vacuette containers (code: 456018) in fifty patients with paraproteinaemia (IgG Kappa, n = 20: IgG Lambda, n = 9: IgM Kappa, n = 6: IgM Lambda, n = 4: IgA Kappa, n = 5: IgA Lambda, n = 3: multiclonal, n = 3). Serum folate assays were measured according to manufacturer’s instructions on Beckman-Coulter Access and Abbott Architect haematinics analysers on the day of sample collection. Results: The ratio of means of serum protein concentrations in the paraprotein sub-groups compared to the mean values of age/gender related reference ranges were: IgG = 1.57: IgM = 7.8: IgA = 6.3. Serum albumin concentration was normal in all patients. Mean serum folate results (±1SD) for paraprotein patients and normals controls (laboratory personnel) are shown in the table below: Serum Folate (μg/L) Access Architect paired t-test Normal controls (n = 50) 5.98 (±3.57) 5.01 (±3.14) p0.05 in each case). Conclusions: The complex and variable mixture of proteins present in paraproteinaemias pose potential erroneous measurement in immunoassay systems. This is because they may interfere with the associated antigen-antibody reactions which are dependant on complimentary matching shapes being assumed by the antibody variable regions of the immunoglobulin. Despite this, our data using two distinct chemiluminescence analysers for folate measurement indicate no statistically significant difference for this parameter between patients with paraproteinaemia compared to normal controls. However, it has yet to be determined whether differing results between these two groups are generated using different analytical techniques.

Description: A 58 year old lady with Metabolic Syndrome of 10 years duration presented with post-menopausal PV bleeding, haematuria, occasional epistaxis and ecchymoses. Prescribed medication which had remained unchanged for the preceding two years included daily doses (mgms) of Aspirin (150), Atenelol (50), Metformin (500 × 3), Bendroflumethiazide (2.5), Losartan (50) and Simvastatin (20). Intravenous urogram, cystoscopy, cytological examination of urine sediment, hysteroscopy, a cervical scan and endometrial biopsies showed neither evidence of overt pathology nor any physiological indication for the cause of haematuria or PV bleeding. Tests for urinary infection were negative. Apart from the raised blood glucose (9.1: NR 3.3 – 6.0 mmol/L), the biochemistry profile including liver enzymes, coagulation profile and blood count were normal (Platelets = 265 × 109/L). Bleeding episodes were observed after commencement of a daily intake of 7–8 cups of green tea for a period of six months. Green tea intake was self-instigated in response to reported amelioration of risk factors associated with Metabolic Syndrome (reduction in LDL cholesterol and serum triglyceride levels; elevation of protective HDL; potent antioxidant activity; ACE inhibition and promotion of glucose metabolism). Hot water extract of green tea specifically inhibits platelet adhesion and lowers sub-maximal platelet aggregation and prolongs the lag time in a dose-dependent manner. Previous fractionation studies of these hot water extracts, has revealed that the tea catechins (tannins) actively inhibit thromboxane A2 production and that ester-type catechins are more effective than free-type catechins. One of the ester-type catechins, epigallocatechin gallate (EGCG), suppresses thrombin and collagen-induced platelet aggregation completely at a concentration of 0.2 mg/ml. EGCG also inhibits aggregation by a mechanism which differs from that of aspirin by inhibiting platelet activating factor (PAF). The IC50 values of EGCG and aspirin indicate that their potencies are comparable. Bleeding symptoms ceased two weeks after the patient stopped drinking green tea. Our assumption of the causal effect of green tea on anomalous bleeding in this patient needs to be confirmed by structured platelet function tests in both aspirinised and non-aspirinised patients. Since inhibition of cyclo-oxygenase is an additional anti-thrombotic property of aspirin which differs from that of green tea, diabetic patients taking prophylactic low-dose aspirin should continue to do so and potentially beneficial ingestion of green tea should not be considered without consultation with an appropriate health professional in view of its synergistic potential on the effect of aspirin and the associated haemorrhagic risks.

Description: Introduction Haematophages, animals evolved to a blood sucking lifestyle as their exclusive mode of feeding secrete compounds capable of arresting haemostasis in the host. Since the discovery and subsequent characterisation and engineering of hirudin, attention is being focused on the potential anticoagulant and platelet aggregation inhibitors from an array of different species of leech from both the Rhynchobdellid and Arhynchobdellid orders. The purpose of this study was to survey the presence of inhibitors of platelet aggregation in cephalic extracts prepared from phylogenetically diverse proboscis-bearing and jawed blood-sucking leeches compared to those which are predaceous in nature. Methods Cephalic extracts from specimens of each species were prepared by homogenising in tris buffer the anterior one-third region of the bodies containing the salivary glands. In the case of the giant leech, Haementeria ghilianii, paired anterior salivary glands were excised through the oesophagus. The posterior musculature served as control material. Homogenates were double centrifuged and supernatants micro-filtered. Individual extracts or imidazole buffer as control were pre-incubated 1:4 with platelet rich plasma for 5 minutes prior to testing for inhibition of platelet aggregation. Inducers of aggregation were: ADP (5 and 10 μM/ml), Collagen (COL) (4μg/ml), Thrombin (THR) (0.2 U/ml), Ristocetin (RIS) (1.5 mg/ml) and Adrenaline (ADR) (1 μM). Maximal aggregation responses were measured over 10 minutes. All extracts were kept on ice and tested within 1 hour of preparation. Results The species of leeches examined and maximal aggregation responses (%) after 10 minutes are shown in table 1. Maximal Responses (%) to Aggregation Inducers in Haematophagous and Predaceous Leeches Species Source ADP COL THR RIS ADR Haematophagus Haementeria ghilianii Brazil

Description: Introduction: There is published evidence which indicates that advancing age may be associated with higher plasma concentrations of fibrinogen. There is also evidence that derived fibrinogen values are significantly higher than Clauss measurements and that this discrepancy is greater in patients receiving warfarin. The purpose of this study was to determine whether age related derived fibrinogen levels are similar in both warfarin and non-warfarin groups. Methods: Venous samples were collected into siliconised glass B-D Vacutainers containing tri-sodium citrate (Ref: 367691) from 1000 patients receiving long term warfarin treatment and an equal number of age-matched patients not receiving warfarin. Genders were equally represented in both groups. Patients in both groups were categorized into 5 years age bands as follows:

Description: Introduction Along with clinical assessment, D-dimer (D-d) assays are routinely used to exclude DVT. It has been suggested that measurement of derived fibrinogen (DF) may be an effective reflection of endogenously-derived thrombin generation and that ratios between this and Clauss-derived fibrinogen (CF) may be useful in determining whether patients have experienced, or are vulnerable to thromboembolic events. Some studies indicate that a D-d/fibrinogen ratio is significantly higher among patients with confirmed DVT. The purpose of this study was to determine whether DF/CF ratios in individuals are a useful adjunct to D-d assays in the detection of DVT compared to D-d assay in isolation. Methods Venous samples were collected into glass B-D Vacutainers containing tri-sodium citrate (Becton-Dickinson, Plymouth, UKRef: 367691) from 162 out-patients presenting to the medical admissions unit with suspected DVT. Laboratory staff (N=100) served as the normal control group. D-dimer (MiniVidas, BioMerieux), DF and CF (Dade-Behring reagents in combination with a CA1500 coagulometer) were measured on all patients and normal control samples within two hours of collection. Results Following clinical assessment (Wells scoring), 85 patients were considered not to have had a DVT. Doppler scanning confirmed DVT in 38 of the remaining patients and 39 were shown to be negative. Two sample t-test analysis of the data showed significant differences between DF and CF levels in the normal group (n=100), patients who did not have a DVT (n=124) and those who did have a DVT (n=38), (p =

Description: Introduction: The recommended order of draw for multiple tube collections (NCCLS [CLSI] H3-A5) clearly indicate that citrate tubes for coagulation tests should be taken before any other tubes (except blood cultures) and that a discard tube should be used if specialised coagulation tests are to be performed. This is to avoid the possibility of tissue activation and the theoretical risk of additive carryover on the parameters being measured. Because of the paucity of published evidence, this study was performed to determine the effect of the order of draw and whether the use of a discard tube is really necessary. Methods: Three consecutive early morning venous samples were collected into siliconised glass B–D Vacutainers containing tri-sodium citrate (Ref: 367691) from 116 healthy laboratory personnel (F= 74; M = 42) aged 20–63 yrs. Age groups were equally represented. Samples were processed on a Sysmex CA1500 analyser within 1 hour of collection. Appropriate CLSI guidelines were followed throughout. All parameters were measured using Dade-Behring reagents: Activated partial thromboplastin time (APTT) (Actin FSL), prothrombin time (PT) and derived fibrinogen (DF) (Innovin), thrombin clotting time (TCT) (Thromboclotin) and Clauss fibrinogen (CF) (Bovine thrombin and Owren’s veronal buffer). For each parameter, the data from each of the three samples were analysed for significant differences by one way analysis of variance (ANOVA). Results: Data obtained on measurements of basic coagulation parameters are shown in the table below. SDs are shown in parenthesis. (ns = not significant). Coagulation Parameter Results and Statistical Analysis Parameter First Sample Second Sample Third Sample ANOVA (p) ns=not significant APTT (secs) 28.3 (1.73) 28.3 (1.73) 27.9 (1.64) 0.230 (ns) PT (secs) 10.9 (0.47) 10.9 (0.47) 10.8 (0.45) 0.368 (ns) TCT (secs) 15.8 (1.03) 15.8 (1.02) 15.7 (1.02) 0.740 (ns) DF (g/L −1) 2.44 (0.54) 2.47 (0.55) 2.48 (0.55) 0.866 (ns) CF (g/L −1) 3.03(0.67) 3.04 (0.67) 3.10 (0.67) 0.825 (ns) No statistically significant differences were found between the first, second or third samples for any of the measured parameters. Conclusions: The CLSI recommends an order of draw for evacuated blood collection tubes in order to reduce the possibility of tissue activation in coagulation samples and the theoretical risk of additive carryover on the parameters being measured. Until now, this was based largely on theoretical probability. This comprehensive study demonstrates that the use of a discard tube is probably unnecessary since there is no statistical difference in any of the parameters measured between the first, second or third samples. Although this potentially obviates the expensive use of a discard tube in normal subjects, further work is required to determine whether it is necessary when measuring abnormally prolonged parameters in various pathological states.

Description: Introduction Haematophages, animals evolved to a bloodsucking lifestyle as their exclusive mode of feeding secrete compounds capable of arresting haemostasis in the host. It is clear that exploitation of host haemostasis is an absolute requirement for the survival of these species. Since the discovery and with the subsequent characterisation and engineering of Hirudin a potent thrombin inhibitor from the European medicinal leech Hirudo medicinalis, attention has been focused on the potential anticoagulant and platelet aggregation inhibitors derived from an array of different species of leech from both the Rhynchobdellid and Arhynchobdellid orders. Haematophagous leeches of the genus Theromyzon sp. of the Rhynchobdellid order, also termed duck leeches, feed directly on the nasal passages, trachea and nictating membranes of migratory birds. We present the novel observation of inhibition of aggregation of human platelets by the salivary secretion extracts of the avian leech Theromyzon tessulatum. Methods Twelve adult leeches of the species T. tessulatum (total weight 2.828g) were anaesthetised with ethanol vapour. The leeches were severed at the anterior end and a homogenate produced containing salivary gland secretions. The posterior two thirds of the leeches were treated identically to serve as control material. Platelet rich plasma (PRP) was prepared from blood from a normal individual (free from known platelet modifying medicines) mixed 9:1 (v:v) with 0.105 M trisodium citrate in siliconised glass vacutainers. Platelet numbers were adjusted with autologous platelet poor plasma (PPP) to obtain concentrations of approximately 300 × 109/L. Leech extracts (anterior or posterior control) were added to PRP at a ratio of 1:4. Aggregation studies were performed using thrombin (10units/ml), collagen 10μg/ml, Ristocetin (1.5mg/ml) and ADP (5μm/ml). Results Data from this study shows that platelet aggregation was completely inhibited when stimulated by thrombin, collagen and ADP and partially inhibited (40%) on the addition of ristocetin. Conclusion Our observations contradict the belief that the anti-thrombocyte properties of this species of haematophagous leech are restricted to duck thrombocytes. We suggest the presence of one or more inhibitory molecules acting by various mechanisms including inhibition of vWF and platelet integrin mediated collagen interactions, inhibition of ristocetin mediated vWF and platelet GPIb receptor binding and salivary secretion derived apyrase inhibition of arachidonic acid mediated platelet aggregation. These findings provide conclusive evidence that this blood sucking bird leech has the ability to overcome thrombocyte function in higher vertebrates.