A multicentred study to validate a consensus bleeding assessment tool developed by the biomedical excellence for safer transfusion collaborative for use in patients with haematological malignancy.

BACKGROUND: There continues to be uncertainty about the optimal approach to documenting bleeding data in platelet transfusion trials, with a desire to apply a common assessment tool across all trials. With this in mind, a consensus bleeding assessment tool (BAT) has been developed by the Biomedical Excellence for Safer Transfusion (BEST) collaborative, based on review of data collection forms used in published randomized trials and following content validation with a range of healthcare professionals at seven haematology centres through BEST members. This study aimed to evaluate reliability and reproducibility of the consensus BAT. METHODS: Replicated clinical assessments of bleeding were undertaken by participants with haematological malignancies recruited at four haematology centres in an international, multicentred, observational study. Concordance of repeat assessments was calculated for agreement in site and grade of bleeding observed. RESULTS: Forty patients consented to participate, and 13 trained bleeding assessors collected these data. Bleeding assessments were carried out on 113 separate days. Of all 225 bleeding assessments, 204 were compared for grade concordance, and 160 were compared for site concordance. There was very good grade concordance (83%, 95% confidence interval 74-93%) and good bleeding site concordance (69%, 95% confidence interval 57-79%) in observations of bleeding. Discordance was primarily in relation to assessing skin bleeding. CONCLUSIONS: Alongside a structured training programme, levels of concordance for a consensus BAT were high. Researchers using assessment tools for bleeding need to balance comprehensive data collection against potential loss of accuracy for some types of bleeding, such as skin findings.

Evaluation of clinical coding data to determine causes of critical bleeding in patients receiving massive transfusion: a bi-national, multicentre, cross-sectional study.

OBJECTIVES: To evaluate the use of routinely collected data to determine the cause(s) of critical bleeding in patients who receive massive transfusion (MT). BACKGROUND: Routinely collected data are increasingly being used to describe and evaluate transfusion practice. MATERIALS/METHODS: Chart reviews were undertaken on 10 randomly selected MT patients at 48 hospitals across Australia and New Zealand to determine the cause(s) of critical bleeding. Diagnosis-related group (DRG) and International Classification of Diseases (ICD) codes were extracted separately and used to assign each patient a cause of critical bleeding. These were compared against chart review using percentage agreement and kappa statistics. RESULTS: A total of 427 MT patients were included with complete ICD and DRG data for 427 (100%) and 396 (93%), respectively. Good overall agreement was found between chart review and ICD codes (78·3%; κ = 0·74, 95% CI 0·70-0·79) and only fair overall agreement with DRG (51%; κ = 0·45, 95% CI 0·40-0·50). Both ICD and DRG were sensitive and accurate for classifying obstetric haemorrhage patients (98% sensitivity and κ > 0·94). However, compared with the ICD algorithm, DRGs were less sensitive and accurate in classifying bleeding as a result of gastrointestinal haemorrhage (74% vs 8%; κ = 0·75 vs 0·1), trauma (92% vs 62%; κ = 0·78 vs 0·67), cardiac (80% vs 57%; κ = 0·79 vs 0·60) and vascular surgery (64% vs 56%; κ = 0·69 vs 0·65). CONCLUSION: Algorithms using ICD codes can determine the cause of critical bleeding in patients requiring MT with good to excellent agreement with clinical history. DRG are less suitable to determine critical bleeding causes.

Ibrutinib inhibits collagen-mediated but not ADP-mediated platelet aggregation.

The BTK (Bruton's tyrosine kinase) inhibitor ibrutinib is associated with an increased risk of bleeding. A previous study reported defects in collagen- and adenosine diphosphate (ADP)-dependent platelet responses when ibrutinib was added ex vivo to patient samples. Whereas the collagen defect is expected given the central role of BTK in glycoprotein VI signaling, the ADP defect lacks a mechanistic explanation. In order to determine the real-life consequences of BTK platelet blockade, we performed light transmission aggregometry in 23 patients receiving ibrutinib treatment. All patients had reductions in collagen-mediated platelet aggregation, with a significant association between the degree of inhibition and the occurrence of clinical bleeding or bruising (P=0.044). This collagen defect was reversible on drug cessation. In contrast to the previous ex vivo report, we found no in vivo ADP defects in subjects receiving standard doses of ibrutinib. These results establish platelet light transmission aggregometry as a method for gauging, at least qualitatively, the severity of platelet impairment in patients receiving ibrutinib treatment.

Prevalence and predictors of fatigue in haemo-oncological patients.

BACKGROUND: Fatigue is a common symptom in patients with advanced malignancy, and has been associated with both physiological and psychological factors in patients with solid tumours. AIM: This study sought to explore the predictors of fatigue in a population with haematological malignancy. METHODS: Consecutive outpatients and inpatients attending a haematology centre completed the Memorial Symptom Assessment Scale, and clinical, treatment and demographic information were noted. RESULTS: Of 180 patients, fatigue was present in 69%, and causing considerable distress in 26%. Univariate analysis revealed fatigue was associated with poor performance status, low haemoglobin, feeling sad, worried, irritable and nervous. Multivariate modeling revealed that those factors predictive of fatigue were poor performance status, having active disease, feeling sad and irritable, while haemoglobin level was not predictive of fatigue. CONCLUSIONS: Fatigue is a multidimensional symptom in patients with haematological malignancy whose presence must prompt a holistic assessment of potential contributors that goes beyond correction of haemoglobin levels.

A 'dangerous' group O donor: severe hemolysis in all recipients of organs from a donor with multiple red cell alloantibodies.

Alloimmune hemolysis is a recognized but infrequent complication of solid organ transplantation, particularly where there is incompatibility within the ABO blood group system. We describe severe hemolysis due to passenger lymphocyte syndrome (PLS) in all three recipients of organs from a single donor with multiple red cell (RC) alloantibodies. The first patient, a liver transplant recipient, required augmentation of immunosuppression to treat immune hemolysis due to anti-B, -D, -C and -Cellano (k). This is the first description of PLS caused by alloantibody to the high incidence RC antigen, k. The two single lung transplant recipients developed hemolysis due to anti-D. Both required escalation of immunosuppression and early transfusion support. Three months posttransplant, all three patients have ongoing evidence of compensated hemolysis. This series highlights the potential for severe non-ABO-mediated immune hemolysis following solid organ transplantation. A positive donor RC antibody screen should prompt careful monitoring of organ recipients for hemolysis.

Linezolid-induced dyserythropoiesis: chloramphenicol toxicity revisited.

We report here two cases of dyserythropoietic anaemia associated with long-term linezolid use that share striking similarities to chloramphenicol-associated myelotoxicity.

Myeloma cells can directly contribute to the pool of RANKL in bone bypassing the classic stromal and osteoblast pathway of osteoclast stimulation.

Summary The ratio of osteoprotegerin [OPG, tumour necrosis factor receptor superfamily, member 11b (TNFRSF11B)] to receptor activator of nuclear factor kappaB ligand [RANKL, tumour necrosis factor (ligand) superfamily, member 11 (TNFSF11)] in bone is critical for the regulation of bone remodelling. Myeloma cells can home to bone, triggering increased RANKL and decreased OPG expression by stromal cells, leading to osteolysis. Whether myeloma cells contribute directly to the pool of RANKL or OPG in bone has been contentious. Here we provide evidence of RANKL expression by reverse transcription polymerase chain reaction and in situ hybridization, demonstrating transcripts encoding both the membrane-bound and secreted forms of RANKL in five human multiple myeloma cell lines (LP-1, NCI-H929, OPM-2, RPMI8226, U266) and myeloma cells purified from bone marrow aspirates of myeloma patients. We demonstrated that RANKL encoding mRNAs are translated to protein by antibody detection of RANKL. In vitro assays showed that myeloma cells induced bone marrow derived mononuclear cells to differentiate into adherent tartrate-resistant acid phosphatase positive multinucleated cells, indicative of the formation of functional osteoclasts. This differentiation could also be achieved with passaged myeloma media alone, implicating secreted products. Finally, we provide evidence that the differentiation observed is at least in part the result of myeloma cell expression of RANKL. We therefore conclude that myeloma cells can directly contribute to the pool of RANKL in bone.

MYC amplification in two further cases of acute myeloid leukemia with trisomy 4 and double minute chromosomes.

We report two cases of trisomy 4 with double minute chromosomes (dmin): one in a woman with acute myeloid leukemia (AML), French-American-British subtype M2, the other in a man with chronic myelomonocytic leukemia. In the former case, many cells without trisomy 4 but with dmin were present, a finding not observed in previously reported cases. In both cases, fluorescence in situ hybridization studies demonstrated the double minutes to be MYC amplicons. Ten cases of AML with trisomy 4 and dmin have now been described; in the five cases investigated, the dmin have been shown to be amplified MYC gene sequences.

B-cell chronic lymphocytic leukaemia with CD8 expression: report of 10 cases and immunochemical analysis of the CD8 antigen.

We report 10 cases of B-cell chronic lymphocytic leukaemia (B-CLL) with expression of the T-cell antigen CD8. The majority of patients had typical B-cell CLL with stable and non-progressive stage A(O) disease except for more common expression of lambda light chain and CD25. Two patients had progressive disease and required therapy, one with atypical morphological and phenotypic features. The incidence of CD8 expression was approximately 0.5% of B-CLL patients from our institutions. Immunoprecipitation of the CD8 antigen from four of these B-CLLs showed identity to the CD8 antigen expressed on T cells with precipitation of CD8alpha bands of molecular weight approximately 34 kD. In view of the known intracellular signalling mechanism of CD8 using the tyrosine kinase p56-lck, we studied p56-lck expression by Western blot and found lack of consistent expression of the CD8 surface antigen, with most lacking p56-lck. Our report indicates that CD8 expression in B-CLL is probably underrecognized but is not a marker of disease progression. The CD8 on the B-CLL surface is immunochemically identical to the antigen on T cells, but is not accompanied by its usual signalling mechanism of p56-lck tyrosine kinase and therefore is unlikely to be a functionally active receptor.

The use of IgH fingerprinting and ASO-dependent PCR for the investigation of residual disease (MRD) in ALL.

In acute lymphoblastic leukaemia (ALL), investigation of minimal residual disease by conventional morphology and immunology fails to detect levels of residual disease of < 1 leukaemic in 10-100 normal cells. The use of polymerase chain reaction (PCR) to exploit the diversity of the complementarity determining region (CDR) and immunoglobulin variable heavy chain (VH) family specific usage has greatly improved the sensitivity up to one leukaemic cell in 10(5)-10(6) normal bone marrow cells. Here we report on a prospective study of 14 patients with ALL of B-cell lineage by using a combined PCR approach which estimates levels of disease between 1:10(3) and 1:10(5). The sequential use of allele-specific oligoprimer (ASO) independent tests (using framework 1. FR1 and 3, FR3 primers with a JH consensus primer, sensitivity up to 1:5 x 10(3)) and ASO-dependent PCR (sensitivity up to 1:10(5)) assays were applied to 64 bone marrow (BM) follow-up samples in a sequential array of tests. Results presented in this study indicate high concordance of MRD among different tests for samples with level of residual disease > 1:5 x 10(3). Consequently, samples positive by the FR1 and FR3 fingerprinting tests were confirmed by the more sensitive ASO-dependent tests, as expected. However, the ASO-dependent assays revealed levels of disease undetected by the FR1 and FR3 test. Although a higher level of sensitivity is provided by the ASO-dependent tests, the FR1 and FR3 fingerprinting tests allow MRD investigation in patients with oligoclonal B cell proliferations, CDR3 region of size < 15 bp or with ASO primers unsuitable for PCR investigation on technical grounds (i.e. background signal). If a sequential order of investigation from less (e.g. FR1 and FR3 fingerprinting) to more sensitive tests (ASO-dependent) is applied, an indirect estimate of MRD is obtained for patients with level of disease < 1:10(3).

Genetic changes: relevance for diagnosis and detection of minimal residual disease in acute lymphoblastic leukaemia.

Cure can now be achieved in a proportion of patients with ALL. However, relapse and eventual treatment failure occur in many cases receiving identical treatment, presumably as a result of failure to eradicate MRD. While for many years marrow morphology has been the standard by which leukaemic remission has been assessed, more sensitive techniques have been developed for detection of MRD including immunophenotypic analysis, and as discussed in this chapter, methods which detect leukemia-associated clonal genetic changes at the karyotypic and genomic levels. Table 10 lists the applicability and sensitivity of various markers used in MRD analysis in ALL. It is apparent that of the karyotypic and molecular approaches described, only PCR-based strategies for detection of either leukaemia-specific translocations or clonal Ag receptor rearrangements are reliably applicable to a high proportion of both B- and T-ALL at sufficiently high sensitivity. Initial clinical studies of patients undergoing therapy for ALL using a variety of PCR-based methods suggest that in some cases a persistent or increasing level of residual disease may be predictive for clinical relapse, although a number of technical factors and the phenomena of oligo-clonality and clonal evolution may limit the usefulness of this analysis in a few instances. From current available data it appears that in order to define the potential predictive value of PCR detection of MRD a large number of patients will need to be prospectively assessed over several years at multiple time points during and after therapy, preferably using more than one semi-quantitative PCR approach. In addition to reliable prediction of clinical relapse allowing appropriate individual treatment modification, progress in the molecular detection of MRD in ALL is also likely to be of benefit in the assessment of the efficacy of autograft purging and the evaluation of new therapeutic strategies such as the use of biological response modifiers to eliminate a low tumour burden.

Minimal residual disease in acute lymphoblastic leukaemia--PCR analysis of immunoglobulin gene rearrangements.

Rearrangement of the immunoglobulin heavy chain (IgH) gene can be utilized as a marker of clonality in a number of B-lineage lymphoproliferative disorders including acute lymphoblastic leukaemia (ALL). We have used a PCR technique involving a panel of amplimers for the 6 different Variable (VH) region families and for a consensus sequence of the Joining (JH) segment to detect clonal IgH rearrangements in the peripheral blood (PB) and/or bone marrow (BM) of 28 patients (17 children and 11 adults) with B-lineage ALL at presentation (20 patients) or with overt relapse (8 patients). The age range of the patients was 2-65 years (mean 15.7 years). Follow up remission BM samples were analysed in 22 patients during and after therapy (2-7 samples per patient), 1-50 months after presentation or relapse. In 1 relapsed case, previously stored complete remission (CR) samples were analysed retrospectively. Clonal IgH chain rearrangements were detected by PCR in 90% of patients studied initially. The 2 VH region families most commonly used were the large VH3 family (65%) and the smaller more JH-proximal VH4 family (22%). More than one VH clone was detectable in 25% of the cases. A gene "fingerprinting" modification of a previously described method was applied to the detection of minimal residual disease (MRD) in follow up BM samples with a sensitivity of 10(-3) to 10(-4). In 8 of 14 patients remaining in complete remission (CR) during the time of study, all PCR analyses on BM samples in the first 6 months were negative, in some cases as early as 2 weeks post-induction therapy, and a further patient reverted from being PCR positive in the first month after the commencement of therapy to sustained PCR negativity. One adult remains in CR at 50 months after presentation and has been PCR negative at 2 time points after cessation of maintenance therapy (30 and 50 months). Eight patients relapsed in the study period comprising 6 BM and 2 isolated CNS relapses. In 4 cases of BM relapse occurring within 7 months of the start of therapy, all BM remission samples tested in this period were PCR positive. In 2 other patients BM samples tested 7 and 2 months respectively prior to relapse were PCR negative. In the 2 patients with isolated CNS relapse, PCR of BM samples from 2 and 10 months before the relapse were negative.(ABSTRACT TRUNCATED AT 400 WORDS)