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Background Patients' experience of symptoms often goes undetected during consultation in an outpatient clinic, and the use of a patient-reported outcome measure (PRO) in such a setting could be useful to aid treatment decision-making. A new PRO measure, the HM-PRO (Hematological Malignancy Specific Patient-Reported Outcome Measure) has been recently developed to evaluate hematological malignancy (HM) patients' health-related quality of life (HRQoL) and their symptom experience in daily clinical practice as well as in research. The objectives of the study were to assess: the internal consistency of the scores for Part A (impact) and its four domains (physical behavior; social well-being; emotional behavior; and eating and drinking habits) and Part B (signs and symptoms); and the test-retest reliability of the individual items of the newly developed hematological malignancy specific composite measure, the HM-PRO. Methods This was a prospective longitudinal observational study where 150 patients with different HMs and different stage of disease (male n = 98 (65.3%); mean age 64.9 ± 14.4 years, range 17.9-89.2 years; mean time since diagnosis 3.7 ± 4.9 years, range 0.04-25.8 years) completed the HM-PRO at baseline (assessment 1 at t1) and after 7 days (assessment 2 at t2). Data analysis was performed using IBMSPSS 23 statistical software. Results The Cronbach's alpha estimates of the HM-PRO for both assessment points (t1 and t2) were above 0.9 for Part A, and above 0.8 for Part B, showing strong stability of the measurement. The level of agreement for the reproducibility between the two assessments, using intra-class correlation coefficients (ICC), was very strong with Part A: ICC = 0.93 (95% CI = 0.90-0.95), and Part B: ICC = 0.91 (0.88-0.93). The ICC for the four domains of Part A ranged from 0.85-0.91. The ICC was greater than 0.8 for overall score of Part A and Part B for all the 10 diagnoses, confirming strong reliability. Conclusion This study clearly indicates that the HM-PRO possesses strong test-retest reliability for both Part A and Part B. The Cronbach's alpha confirmed acceptable internal consistency. The extensive reliability testing described in this study supports the generic nature of the HM-PRO for use in hematological malignancies in both routine clinical practice, to aid treatment decisions, as well as in research.

Background: Our aim was to identify health-related quality-of-life (HRQoL) issues and symptoms in patients with haematological malignancies (HMs) and develop a conceptual framework to reflect the inter-relation between them. Methods: A total of 129 patients with HMs were interviewed in a UK multicentre qualitative study. All interviews were audio recorded, transcribed and analysed using NVivo-11. Results: Overall, 34 issues were reported by patients and were grouped into two parts: quality of life (QoL) and symptoms. The most prevalent HRQoL issues were: eating and drinking habits; social life; physical activity; sleep; and psychological well-being. Furthermore, most prevalent disease-related symptoms were: tiredness; feeling unwell; breathlessness; lack of energy; and back pain. The most prevalent treatment side effects were: tiredness; feeling sick; disturbance in sense of taste; and breathlessness. Conclusions: Both HMs and their treatments have a significant impact on patients’ HRQoL, in particular on issues such as job-role change, body image and impact on finances.

Background: Validity is the ability of an instrument to measure what it claims to measure. It means the degree to which the empirical evidence supports the trustworthiness of interpretations based on the calculated scores. The hematological malignancy (HM) specific patient reported outcome measure (HM-PRO), is a newly developed instrument for use in daily clinical practice as well as in research. This study, provides the evidence for construct validity of the HM-PRO, specifically focusing on the convergent and divergent validity compared to the other established instruments used in hematology. Methods: This validation study adopted a prospective cross-sectional design where a heterogeneous group of patients diagnosed with different HMs and different disease state were recruited. A total of 905 patients were recruited from seven secondary care hospitals in the UK and online through five patient organizations. Patients were asked to complete the HM-PRO and other cancer specific PRO’s, FACT-G and EORTC QLQ C-30. Data analysis was performed using IBM SPSS 23 statistical software. Results: A total of 486 males (53.7%) and 419 females (46.3%), with a mean age of 64.3 (± 12.4) years and mean time since diagnosis of 4.6 (± 5.2) were recruited. The total score of Part A of the HM-PRO highly correlated with the five functional scales of the EORTC QLQ-C30 (Physical = −0.71, Role = −0.72, Emotional = −0.64, Cognitive = −0.58, Social = −0.74—p < 0.001). With respect to correlation with FACT-G, the total score of Part A of the HM-PRO highly correlated with Physical (−0.74), Emotional (−0.57), Functional (−0.66) domains and overall score of FACT-G (−0.74). Similarly, the total score of Part B of the HM-PRO highly correlated with three symptoms scales of EORTC QLQ-C30 (Fatigue scale = −0.74, Nausea and Vomiting = −0.52, Pain = −0.59—p < 0.001) and individual symptom items (Dyspnea = 0.51, Insomnia= 0.43, Appetite loss = 0.54—p < 0.001). Conclusion: The construct validity evidence presented in this research is a testimony to the HM-PRO’s ability to measure HRQoL issues which it intends to measure. This is of utmost importance when a PRO is used in routine clinical practice so that the interpretation of the scores or response to an individual item is understood by the clinicians/nurses as intended by the patients.

Background: GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic t...

Background The quality of life of patients at all stages of hematological malignancy is greatly affected by the disease and its treatment. There is a wide range of health-related quality of life (HRQoL) issues important to these patients. Any new instrument developed to measure HRQoL of such patients should be content valid, i.e., the items should be comprehensively relevant to the patients and their health condition. The aim of the present study was to examine content validity of a hematological malignancy specific patient reported outcome measure (HM-PRO) developed for use in routine clinical practice. Methods Following literature review and semi-structured interviews, the generated themes and sub-themes were discussed to develop the prototype version of the HM-PRO. A 4-step approach was used for content validation: initial testing and cognitive interviewing; item rating; content validity panel meeting; final field testing and cognitive interviewing. Additional questions related to patients’ perception of recall period and preferred sentence structure (i.e., question or statement) of the items were also asked during cognitive interviews. Results The content analysis of 129 transcribed semi-structured interviews resulted in the prototype version of the instrument consisting of 58 items grouped into two parts: Part A (impact/HRQoL – 34 items) and Part B (signs and symptoms – 24 items). The initial testing showed intra-class correlation coefficient (ICC) of >0.8 for both Part A and Part B. Item rating for language clarity, completeness, relevance, and response scale by experts and patients showed content validity index for scales average >0.8 for both Part A and Part B, except 0.64 for relevance for Part A by the patient panel. The final testing of the revised version of the instrument showed the Cronbach’s alpha value of 0.91 for Part A and 0.76 for Part B, suggesting high internal consistency, and ICC of 0.91 for Part A and 0.76 for Part B. The recall period of “today” for Part-A and “last 3 days” for Part-B were the patients’ preferred “recall period.” Furthermore, the patients expressed preference to the HM-PRO items as statements. Conclusion The findings of this study confirm that the HM-PRO possesses a strong content validity, includes all the issues important to patients and is easy to read, understand and respond to spontaneously.

BACKGROUND: Initial results from the ongoing GALLIUM trial have shown that patients with follicular lymphoma have a longer progression-free survival after first-line immunochemotherapy with obinutuzumab than with rituximab. The aim of this secondary analysis was to evaluate the prognostic value of PET-CT responses after first-line immunochemotherapy in the GALLIUM study.METHODS: GALLIUM is an open-label, parallel-group randomised, phase 3 trial, which recruited previously untreated patients with CD20-positive follicular lymphoma (grades 1-3a; disease stage III/IV, or stage II with largest tumour diameter ≥7 cm) who were aged 18 years or older and met the criteria for needing treatment. Eligible patients were randomly assigned in a 1:1 ratio to receive intravenous administration of obinutuzumab (1000 mg on days 1, 8, and 15 of cycle 1, then day 1 of subsequent cycles) or rituximab (375 mg/m2 on day 1 of each cycle), in six 21-day cycles with cyclophosphamide, doxorubicin, vincristine, and prednisone (known as CHOP; oral administration) followed by two 21-day cycles of antibody alone, or eight 21-day cycles cyclophosphamide, vincristine, and prednisone (known as CVP; oral administration), or six 28-day cycles with bendamustine, followed by maintenance antibody every 2 months for up to 2 years. The primary endpoint of the trial, investigator-assessed progression-free survival, has been reported previously. This secondary analysis reports PET and CT-based responses at end-of-induction therapy and explains their relation with progression-free and overall survival outcomes in patients with available scans. As per protocol, during the trial, PET scans (mandatory in the first 170 patients enrolled at sites with available PET facilities, and optional thereafter), acquired at baseline and end of induction (PET population), were assessed prospectively by investigators and an independent review committee (IRC) applying International Harmonisation Project (IHP) 2007 response criteria, and retrospectively by the IRC only applying current Lugano 2014 response criteria. IRC members (but not study investigators) were masked to treatment and clinical outcome when assessing response. The landmark analyses excluded patients who died or progressed (contrast enhanced CT-based assessment of progressive disease, or started next anti-lymphoma treatment) before or at end of induction. GALLIUM is registered at ClinicalTrials.gov, number NCT01332968.FINDINGS: 1202 patients were enrolled in GALLIUM between July 6, 2011, and Feb 4, 2014, of whom 595 were included in the PET population; 533 (IHP 2007; prospective analysis), and 508 (Lugano 2014; retrospective analysis) were analysed for progression-free survival (landmark analysis). At end of induction, 390 of 595 patients (65·5% [95% CI 61·6-69·4]) achieved PET complete response according to IHP 2007 criteria, and 450 (75·6% [95% CI 72·0-79·0]) obtained PET complete metabolic response according to Lugano 2014 criteria. With a median of 43·3 months of observation (IQR 36·2-51·8), 2·5-year progression-free survival from end of induction was 87·8% (95% CI 83·9-90·8) in PET complete responders and 72·0% (63·1-79·0) in non-complete responders according to IRC-assessed IHP 2007 criteria (hazard ratio [HR] 0·4, 95% CI 0·3-0·6, pINTERPRETATION: Our results suggest that PET is a better imaging modality than contrast-enhanced CT for response assessment after first-line immunochemotherapy in patients with follicular lymphoma. PET assessment according to Lugano 2014 response criteria provides a platform for investigation of response-adapted therapeutic approaches. Additional supportive data are welcomed.FUNDING: F Hoffmann-La Roche.

Purpura fulminans usually consists of large, often symmetrical, spreading ecchymosis, which may later develop into extensive areas of skin necrosis and peripheral gangrene. Postinfectious purpura fulminans associated with an autoantibody directed against protein S has been described. The interaction and the contribution of recently described mutations such as factor V Leiden and prothrombin G20210A to the development and progression of postinfectious purpura fulminans and venous thrombosis is not known. The authors describe a patient heterozygous for prothrombin G20210A who developed purpura fulminans and extensive venous thrombosis secondary to acquired protein S deficiency.

Aims: The impact of haematological malignancies (HM) on patients' health-related quality of life (HRQoL) is still not well understood. The aim of this study was to identify HRQoL issues and symptoms in patients with HM to be included in a new patient-reported outcome measure for use in routine clinical practice. Methods: In a multicentre observational study carried out in the UK, adult patients with various HM, capable of reading English and give written informed consent were recruited from five hospitals in England and Wales. This qualitative study employed semi-structured face-to-face interviews with open-ended questions related to the impact of haematological malignancy and its treatment on HRQoL and symptoms. All the interviews were audio recorded and transcribed verbatim. Content analysis was carried out using the NVivo 11 qualitative analysis software. The themes and the sub-themes generated from the transcribed interviews were discussed during a 2-day "data definition" panel meeting by 2 hematologists, 1 patient research partner, 1 representative of a hematology patient organisation and 3 QoL research experts to select items for inclusion in the prototype instrument. These items will be further re-grouped and refined using cognitive debriefing, content validity and factor analysis. Results: 127 (male=75; mean age = 61.6 years; SD=15.1; median age 65.4 years; and age range =18-88 years) with mean duration of the HM of 3.7 years (SD=4.3; median=2.1 years; and range= 19 days-23 years) were recruited into the study. Diagnoses were: Acute Myeloid Leukaemia (18); Acute Lymphoid Leukaemia (7); Chronic Myeloid Leukaemia (12); Chronic Lymphatic Leukaemia (11); Aggressive Non-Hodgkin Lymphoma (16); Indolent Non-Hodgkin Lymphoma (14); Hodgkin Lymphoma (10); Multiple Myeloma (21); Myeloproliferative Neoplasm (10); and Myelodysplastic Syndrome (8). 383 items were reported by the patients under different themes and subthemes. 117 of these items were reported by more than 5% of the patients. 149 items were selected by the data definition panel to be included in the prototype instrument. The most prevalent QoL issues important to HM patients (Figure 1) were: 'eating and drinking habits (57 patients changed eating and drinking habits; 48 reported loss of appetite; 29 stopped drinking alcohol; and 11 reported increase in appetite); impaired social life and participatory function (86); impaired physical ability or independency (71); disturbed sleep (66); impaired psychological well-being (64); impaired daily activities (61); impaired ability to go on holidays or travelling (60); impaired work life & studies (57 ); impaired sexual life (55); impaired ability to manage finances (34); recreational activities and pastime (32); and relationships (26), from high to low prevalence, respectively. With respect to disease related symptoms, 102 issues were identified, the most prevalent being 'tiredness (65), feeling unwell (28), breathlessness (24), lack of energy (21), back pain (17) and weight loss (17)", from high to low prevalence respectively. Out of 124 treatment related symptoms identified, the most prevalent were: 'tiredness (73); feeling sick (36); lack of energy (20); taste disturbance (20); breathlessness (15); and diarrhoea (15)', from high to low prevalence respectively. Conclusion: The findings of the qualitative and item generation phase clearly indicate that HMs affect patients' QoL significantly. However, in the absence of a validated measure for use in routine clinical practice, this is not captured in a systematic manner. Thus, this highlights the need for the development and validation of a new HM-specific PRO measure for use in such settings. Psychometric testing of the prototype instrument will be carried out to establish the measurement properties of the new HM-specific PRO measure. Figure Figure. Disclosures Salek: EHA: Other: Educational and travel grant. Fielding:Amgen: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Kell:Celgene: Honoraria; Novartis: Honoraria; Sunesis: Equity Ownership. Oliva:Pharma Companies: Honoraria; Italy: Speakers Bureau; Pharma Companies: Consultancy. Ionova:BMS: Research Funding; MSD: Other: lecturer bureau.

Accurate, reliable laboratory reference ranges are essential for effective clinical evaluation and monitoring. We present robust reference ranges established for haematology, coagulation and haematinic parameters using the Sysmex XE 2100, CA 1500 and Beckman-Coulter Access analysers. Blood samples were taken from 250 healthy laboratory personnel and routine haematology, coagulation and haematinic parameter analysis performed. Our data represent findings from an extensive study to establish reference ranges in healthy adults.

New-onset pancytopenia in pregnancy is challenging in the clinical setting particularly as the management and outcome of pregnancy are entirely dependent on the underlying aetiology. In the absence of increased peripheral destruction, for example, hypersplenism, bone marrow (BM) failure should be considered as the cause of pancytopenia. Profound folate or B12 deficiency may result in BM failure and are relatively easy to diagnose and manage. Other causes include aplastic anaemia (AA), infiltration by a haematological malignancy and other non-haematological disorders. We report a 26-year-old woman presenting with severe pancytopenia due to folate deficiency with complete recovery observed after folic acid replacement. This case highlights the importance of recognising folate deficiency as a reversible cause of pancytopenia, since prompt replacement can lead to rapid normalisation of counts with no subsequent clinical sequelae. We also consider the indications for measuring serum folate in pregnancy.

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 N Mean (g/l) SD Ref Range (g/l) Two sample test for gender difference (p) Adolescents Male 119 2.66 0.464 1.76 – 3.58 0.76 Female 121 2.67 0.467 1.76 – 3.59 Total sample 240 2.67 0.465 1.76 – 3.58 Adults Male 58 2.71 0.672 1.37 – 4.05 Conclusion: Reference ranges for Clauss fibrinogen for school children aged 12–14 years (1.8–3.6g/l) determined using the Sysmex CA-1500 coagulometer differ from the reference range for the healthy adult population (1.6–4.3g/l) determined using the same analyzer and methodology. No statistically significant difference was observed between adolescent male and female cohorts in contrast to significant gender differences observed within the healthy adult population. Reference ranges described in our study show the upper limit in adolescents to be lower than in the healthy adult population. The meaningful assessment of fibrinogen as a risk factor for cardiovascular events in young people should take cognizance of the age specific reference interval. The use of an appropriate, robust reference range for young people is essential in evaluating the influence of contributory risk factors for cardiovascular disease such as diet, body mass and exercise. Assessment of haemostatic function and investigations of coagulopathies in adolescents should be undertaken with reference to age specific ranges where available. This study was approved by the LREC and informed consent was obtained from parents/guardians.

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 EDTA exposure and the formation of membrane lesions during sample storage leads to changes in platelet structure, volume, function and shape due to decreased optical density and the beginning of degranulation. All these have a potential impact on the accurate assessment of platelet numbers. If these numbers are to be used accurately as thresholds for platelet transfusion or for monitoring progressive loss or increases of platelets associated with underlying pathology, it is vital that the pre-analytical limitations of current analysers are fully understood. The way in which discriminators are used on various analysers to make distinction between the characteristics of platelets and other blood cells in individual, stored samples will vary in their effectiveness. Conclusions: Our data indicate that various combinations of pre-analytical sample storage and analyser technology should be considered as potential influences on the outcome of platelet counting in the clinical laboratory.

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) p When analysed using the paired-t-test serum folate was significantly different in normal subjects using the Access compared to the Architect. However, no such significant difference between the two systems was observed in the paraproteinaemia group. Although the mean serum folate in normals was higher compared to the paraprotein group as analysed by the Access, this difference was not significant when analysed using the two-sample t-test. Interestingly the mean serum folate when analysed in the two groups using the Architect gave identical mean values. There were no statistically significant differences between the two methods in any of the paraprotein sub-types (p>0.05 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.

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 ( The inhibitor in our patient did not satisfy the criteria for diagnosis of a lupus-type anticoagulant (LTA). Furthermore, the more prolonged APTT result with Actin FS rather than with the more LTA-sensitive reagent Actin FSL mitigates the presence of LTA. The findings of a normal Thrombotest result and normal Thromborel-S derived coagulation factor assays suggest that the prolongation of the rTP-derived PT and the prolonged APTTs were not due to deficiencies of vitamin K dependent coagulation factors. We have previously described the phenomenon of non-correctable inhibition of rTP-derived PTs associated with anti-cardiolipin antibodies (ACA) in patients with lymphoproliferative disease and also in one patient with IgM lambda paraproteinaemia. This case showed more complex coagulation results. It is possible that the combined presence of IgM ACA and IgM Lambda paraproteins in this patient may have resulted unusually aggressive activity directed against the limited number of phospholipids used for relipidation of rTPs. We believe that the finding of prolonged rTP-derived PT in the presence of a normal conventional thromboplastin-derived PT should be followed up by routine screening for the presence of unsuspected ACA or paraproteinaemia in patients with lymphoproliferative disease. Further investigations are required to elucidate the nature of the antibodies produced in these patients and to determine the extent to which their inhibitory effects may be the consequence of a laboratory epiphenomenon.

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: Results: The T-test for independent samples using the separate variance estimate showed that there was a statistically significant difference in the mean fibrinogen between patients on warfarin and those not on warfarin (p Conclusion: Differences or correlations detected in this analysis are of statistical significance but not necessarily clinically significant. Placing age and warfarin treatment in the same model shows that variations in fibrinogen have to be explained by other factors (e.g. technical) not included in the study as only 12% of the error in predicting fibrinogen levels can be reduced by knowing both the age and status of warfarin treatment in individual patients.

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 = Conclusion Our data suggest that DF/CF ratios cannot be used as an adjunctive marker of DVT when used in combination with D-d values. DF/CF ratios are significantly higher in patients attending for clinical assessment irrespective of whether they are DVT positive or DVT negative. This may be because fibrinogen is an acute phase protein which increases with various pathological states and stressful events such as the experience of attending hospital for investigation and assessment. Further work is required to determine whether DF/CF ratios can be used adjunctively when the D-d cut off value is significantly higher than that used conventionally.

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). | 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) | Coagulation Parameter Results and Statistical Analysis 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.

Reference ranges (RRs) for haematinics need to be independently verified by individual laboratories since it has been shown that those quoted by some manufacturers may be inappropriate. The measurement of serum folate, ferritin and B12 remains a first-line investigation in the assessment of several pathologies. Establishing clearly defined, accurate reference ranges facilitates good interpretation and effective discrimination between health and disease thereby avoiding expensive and needless follow-up. Early morning venous samples were collected into Greiner Vacuette serum tubes (Ref: 456018) from 221 healthy laboratory personnel (F= 159;M = 62) aged 20–63 years for both gender. Age groups were equally represented. Serum vitamin B12, folate and ferritin were measured on all samples on a Beckman-Coulter Access analyser on the same day of collection. NCCLS guidelines (C28-A and H3-A4) were followed throughout. Outliers were excluded and data examined for normal distribution. The following normality checks were applied - Kurtosis, Kolmogorov-Smirnov and Shapiro-Wilk tests of normality, T-values (Skewness/SE Skewness), %diff from mean-median, histogram and normal curve, normal Q-Q probability plot and Box plot. Because all three parameters showed non-normal distribution, RRs were calculated using (1) the 2.5-and 97.5- percentiles, (2) the 2.5- and 97.5- percentiles on the transformed scale. The transformations, log natural, log10 and square root were applied to the variables and tested for normality. The transformation giving the best normal distribution was then selected. The non-parametric Mann-Whitney test was used to examine significant differences between males and females. Significant differences (p values shown below) are indicated by an asterisk. New Limits Historical Limits Manufacturers quoted RR Mann-Whitney U test (p value) (a) = Log natural; (b) = Log 10; (c) = square root RR (1) RR (2) Folate ng/mL M 2.1 – 14.7 2.1 (a) – 14.6 2.7–14.0 >3.0 0.016* F 2.7 – 18.1 2.7 (b) – 18.1 2.7–14.0 >3.0 Ferritin ng/mL M 11 – 215 11 (c) – 215 20.0–350 24–336 It is vital that investigators use method-specific RRs in their own laboratories since those quoted by some manufacturers are inappropriate. Although our lower limits for serum ferritin appear to be low, they are in keeping with previously published data. A proportion of the normal population have low serum ferritin but are not anaemic nor symptomatic. This confusion between normality and iron deficiency continues to cause difficulties in interpretation. The range of vitamin B12 and folate concentrations in some healthy individuals overlap with those in symptomatic patients. It is useful therefore to quote “indeterminate” ranges. Our data indicates that such a range for vitamin B12 should be 130 – 160 since our distribution histogram (not shown) shows a clearly defined “flattened shoulder” between these values.

Most current commercial assays for serum folate use folate-binding protein with chemiluminescence detection. Historically serum folate methods have been calibrated using pteroylglutamic acid (PGA) as this is more stable than 5Methyltetrahydrofolate (5MTHF) which is the form present in patients not receiving supplementation. Poor specificity of these binding assays together with different detection technologies and different method designs have highlighted the need for greater standardisation to calibration regimes so that individual folate species can be better identified. We describe here discrepant serum folate results in a patient which appear to be unrelated to the issues described above. Consequent to a procurement evaluation of multiple analytical systems, serum ferritin, vitamin B12 and folate assays were performed on the Beckman Access, Abbott Architect 12000SR, Bayer Centaur and Roche E170 analysers according to manufacturer’s instructions. Method comparison studies using serum samples from 500 individuals showed good agreement between all analytical systems for all three parameters. However, one serum sample from a 44 year old Caucasian male who had presented with weight loss, diarrhoea and a microcytic, hypochromic anaemia (Hb 10.7 g/L) gave discrepant serum folate results and was further investigated. He had no evidence of renal impairment. His only medication was a multivitamin tablet supplement which he had been taking for several weeks. IgA anti-TTG antibodies were 33 U/mL (NR 0 – 10) and IgA endomysial antibodies were weakly positive. There was a polyclonal increase in serum IgA = 4.62 g/L (NR 0.8 – 2.8). Otherwise routine chemistry, thyroid profile and a paraprotein screen were unremarkable. Parietal cell and intrinsic factor antibodies were negative. Serum ferritin = 16 ng/mL (NR 20 – 215); Vitamin B12 = 164 pg/mL (NR 150–600). The patient’s sample showed no discrepant inter-system results for ferritin or Vitamin B12. Serum folate assays (NR 3.0 – 15.0 ng/mL) were as follows: | Technique | Serum Folate | Calibration method | |:---------------- | ------------ | ------------------ | | Beckman Access | >20.0 | PGA | | Roche E170 | 2.2 | Quantaphase II | | Abbott Architect | 1.9 | Quantaphase II | | Bayer Centaur | 1.5 | 5MTHF | This pattern of results was confirmed on a second sample a month later. No discrepancies were found in twenty other patients with positive TTG and endomysial antibodies. Current methods for folate assays give different results and cannot measure the various forms of folate due to lack of calibrant standardisation with different types of automated analysers. In this patient, it may be that the folate species in the Vitamin supplement are measurable by the Beckman Access but are undermeasured with the other assay techniques. This data suggests the need for a reference method capable of identifying individual folate species in all patients. | Technique | Serum Folate | Calibration method | |:---------------- | ------------ | ------------------ | | Beckman Access | >20.0 | PGA | | Roche E170 | 2.2 | Quantaphase II | | Abbott Architect | 1.9 | Quantaphase II | | Bayer Centaur | 1.5 | 5MTHF