Initial flow cytometric evaluation of the Clearllab lymphoid screen.

INTRODUCTION: Flow cytometric immunophenotyping (FCI) is an integral part in the diagnosis and classification of hematologic malignancies. FCI results also influence therapeutic decisions and disease prognosis. ClearLLab LS is a 12-antibody 10-color cocktail provided in dry format designed as a screen for patients suspected of having hematolymphoid disease. METHODS: A blinded comparison between ClearLLab LS, (CD8-FITC, Kappa-FITC,CD4-PE, Lambda-PE, CD19-ECD, CD56-PE-Cy5.5, CD10-PE-Cy7, CD34-APC, CD5-APC-A700, CD20-APC-A750, CD3-PB, and CD45-KrO), ClearLLab Reagents (five-color, 17-antibodies) and individual Laboratory Developed Tests (LDTs), was conducted at four laboratories. Evaluation of ClearLLab LS was performed on 210 specimens, compared to the five-color ClearLLab Reagents (IVD and CE-IVD), and a subset (n = 167) to LDTs. RESULTS: ClearLLab LS showed good agreement to ClearLLab Reagents in detecting the absence (104/104) or presence (106/106) of abnormal populations. Of specimens with abnormal populations the ClearLLab LS agreed with the ClearLLab Reagent for neoplasm maturity assessment (70/70 mature and 36/36 immature). Out of 167 specimens with LDTs results, 86 contained abnormal population(s), ClearLLab LS detected 82 (95.3%) of cases. Of the 4 cases not detected by ClearLLab LS, 3 were plasma cell neoplasms and 1 was a mature T cell malignancy. Eighty-one samples with no hematological malignancy as analyzed by LDT were also negative by ClearLLab LS (100% agreement). ClearLLab LS agreed with LDTs assessment of neoplasms' maturity (55/55 mature and 27/27 immature). CONCLUSION: ClearLLab LS screening tube showed excellent agreement between ClearLLab Reagents and with LDT's. The presence of CD34 and CD10 in the tube allowed the detection of blast populations in several acute leukemias and myeloid neoplasms that were tested. © 2017 International Clinical Cytometry Society.

Flow cytometry-Recognizing unusual populations in leukemia and lymphoma diagnosis.

Flow cytometry is an invaluable technology in the examination of blood, bone marrow, tissue and body fluids for the presence or absence of hematological disease. It is used in both diagnostic and follow-up testing, with an increasingly important role in the detection of very small residual disease populations (Minimal Residual Disease, MRD) However, flow cytometry immunophenotyping of leukemia and lymphoma is highly dependent on interpretation of results and with the increased complexity of 8-10 color instruments routinely used in clinical laboratories, knowledge of disease-defining populations is increasingly important as is recognizing normal and reactive patterns. This manuscript presents case studies with flow cytometric patterns encountered in routine screening of samples sent for leukemia and lymphoma immunophenotyping, focusing mainly on B-cell disorders which may be missed or incorrectly interpreted by the laboratory (including a hematopathologist) performing the test. Case studies are used to illustrate our laboratory's standardized approach to the interpretation of flow cytometric data. In addition to a standardized approach, these cases emphasize the importance of interpretative skills of technologist and hematopathologists in recognizing abnormal patterns in detecting hematological malignancies.

Patient preferences for stopping tyrosine kinase inhibitors in chronic myeloid leukemia.

BACKGROUND: We used an interview-assisted survey of patients with chronic myeloid leukemia (cml) at a single tertiary care centre to explore patient reactions to and preferences for, and the risk-acceptability of, stopping tyrosine kinase inhibitor (tki) treatment. METHODS: The study included patients with confirmed cml currently being treated with a tki. The survey was conducted by structured interview using a standard form. Patient preferences were explored in a case-based scenario using 0%-100% visual analog scales and 5-point Likert scales. Data were analyzed using proportions for dichotomous variables and medians and interquartile ranges for continuous variables. RESULTS: Of 63 patients approached, 56 completed the survey. Participant responses suggest that the idea of stopping tki use is appealing to many patients if there is a chance of long-term stable disease and a high probability of response upon restarting a tki. Participants were more likely to stop their tki as the risk of relapse decreased. Participants reported loss of disease control and failure of disease to respond to treatment as important concerns if they chose to stop their tki. CONCLUSIONS: Given the current 60% estimated rate of relapse after discontinuation of tki therapy, most patients with cml chose to continue with tki. However, at the lower relapse rates reported with second-generation tkis, participants were more undecided, demonstrating a basic understanding of risk. Contrary to our hypothesis, neither compliance nor occurrence of side effects significantly affected patient willingness to stop their tki.

Enhanced flagging and improved clinical sensitivity on the new DxH 300(TM) Coulter(®) cellular analysis system.

INTRODUCTION: The Beckman Coulter DxH 300™ is a hematology analyzer that performs a CBC and 3-part WBC differential and incorporates new electronic, algorithm and mechanical design. METHODS: This instrument was compared with the predicate analyzer (Coulter(®) A(c) ·Tdiff2) and the larger format Coulter LH780 analyzer. Of interest were flagging rates, clinical sensitivity, and accuracy of the WBC in the presence of interfering particles. The total sample set (n = 404) consisted of morphologically normal and hematologically abnormal patients. RESULTS: Correlation of the DxH 300 with A(c) ·Tdiff2™ showed good agreement with all directly measured parameters. When compared with the LH780, WBC and platelets (PLT) counts showed good agreement with small biases. Importantly in the low range (PLT <50 × 10(9) /L), there was a small positive bias of only 2 × 10(9) /L PLT. Interfering particles did not affect the DxH 300 WBC count (P > 0.05) with strong correlations to the LH780 (r(2) values >0.95). Importantly, overall and specific flagging rates as well as false-negative and false-positive rates were significantly reduced on the DxH 300 compared with the A(c) ·Tdiff2 (27% and 41% reduction, respectively, P < 0.001). CONCLUSIONS: The DxH 300 offers significant improvement over the predicate Coulter analyzer in flagging rates and improved correlation with larger format analyzers for WBC and PLT counts. Reduced false negatives and false positives significantly improved sensitivity and specificity compared to the predicate analyzer. The 28% improvement in flagging efficiency together with numerous software and data handling enhancements should translate into reduced need to perform follow-up analysis on a significant number of samples.

Initial performance evaluation of the UniCel® DxH 800 Coulter® cellular analysis system.

The Beckman Coulter UniCel® DxH 800 is a hematology analyzer incorporating new electronic and mechanical design with advanced algorithm technology to perform CBC, white blood cell (WBC) differential, nucleated red blood cell (NRBC), and reticulocyte analysis. Evaluation of this instrument was performed in our 800-bed tertiary care hospital and specifically centered upon the correlation of WBC, NRBC, and platelet (PLT) enumeration when compared to a predicate analyzer, the Coulter® LH 780, and flow cytometry (FCM) reference methods. Of particular interest were those samples with morphologically confirmed interference and extreme leukocytosis (evaluated with respect to red blood cell parameter correction). The sample set (n=272) consisted of morphologically normal and hematologically abnormal patients. Correlation of the WBC, PLT, and NRBC showed r(2) values of 0.994, 0.985, and 0.910 for the DxH 800 vs. FCM, respectively. The presence of interfering particles did not affect the accuracy of the DxH 800 with respect to WBC counts. The DxH 800 showed accurate PLT and NRBC counts in the clinically significant low range when compared to FCM. Compared to the LH 780, flagging rates were significantly reduced (NRBC flag), or equivalent (WBC, PLT flag) on the DxH 800. The DxH 800 demonstrated higher sensitivity and specificity for PLTs and NRBCs and achieved a lower NRBC false negative rate compared to the LH 780. The UniCel® DxH 800 represents a significant improvement to previous impedance analyzers in accurately detecting the presence of NRBCs at counts >1/100 WBC. Furthermore, it provides accurate PLT and WBC counts in the presence of interference and improved NRBC flagging efficiency when compared to the LH 780. Correction of red blood cell parameters is appropriate and accurate in cases of extreme leukocytosis.

Access to thalidomide for the treatment of multiple myeloma in Canada: physician behaviours and ethical implications.

BACKGROUND: Multiple myeloma is an incurable malignancy. Since the late 1990s, its management has changed with the introduction of novel agents. Thalidomide, which is often called a "novel" therapy, has significantly prolonged survival in multiple myeloma and is considered worldwide to be part of standard of care in this disease. However, thalidomide is not approved in Canada, leading to problems with drug access for patients. METHODS: Our study surveyed Canadian hematologists on their thalidomide prescribing practices and difficulties with drug access. We address some of the ethical issues facing patients and their doctors who are unable to obtain or afford the drug, and who therefore resort to alternative means such as illegal importation. RESULTS: Of the 411 Canadian hematologists contacted, 122 completed the survey, 97 reported that they did not treat myeloma, and 192 did not respond. Assuming that all non-responders treat myeloma, our estimated overall response rate from physicians who treat this disease was 39%. Survey participants indicated that, in Canada, access to thalidomide is a major issue for physicians and myeloma patients alike, and that 81% of respondents are dissatisfied or very dissatisfied with the drug access process. Many physicians felt that the special access process for thalidomide is unduly onerous, influences treatment decisions, and invades patient privacy. We found that 20% of physicians were unaware of the legal implications of obtaining thalidomide from other countries and that at least 23% overtly or covertly support patients in obtaining the drug from a non-Health-Canada-approved source. CONCLUSIONS: The current lack of access to thalidomide in Canada is a concerning problem for patients and health care providers dealing with myeloma. Regulatory changes at the federal level (Health Canada) need to be re-examined to promptly resolve this issue.

Recombinant activated factor VII in the treatment of non-haemophilia patients: physician under-reporting of thromboembolic adverse events.

The objective of this study was to determine if clinically important thromboembolic adverse events (TAEs) because of recombinant activated factor VII (rFVIIa) administration are being under-reported. rFVIIa is a potent haemostatic agent with a short half-life of 2.6 h that is increasingly used in 'off-label' situations. Retrospective review of 94 patients who received rFVIIa during 1 January 2003 to 30 June 2007 was carried out at a tertiary care centre. Sixty-nine patients, 32 females and 37 males, mean age 55 years (18-84 years), satisfied study criteria of off-label usage. This was a high-risk population with 33 (48%) deaths. A mean dose of 8.2 mg (2.4-19.2 mg) was administered in two average divided doses. Thirty-six potential TAEs were identified in 29 patients, and of these, 12 patients had TAEs deemed to be rFVIIa related and were identified on average 8.8 days after exposure to rFVIIa. Forty-eight (70%) physician questionnaires were completed; however, no TAEs were reported in these questionnaires or on chart review. Potential clinically significant TAEs are being under-reported by treating physicians. Until further evidence, we suggest the urgent need to develop consensus recommendations for utilization and required follow up to monitor the safety of rFVIIa and that at a minimum, all use of rFVIIa should be regulated through a gate-keeping mechanism that ensures adherence to these policies. Furthermore, prospective registries and trials are necessary to evaluate the efficacy and safety of rFVIIa in off-label settings.

Consenting to blood: what do patients remember?

We sought to characterize the consent process for transfusion and determine its impact on patients' knowledge and level of comfort with receiving blood. We identified all adult patients who had received blood transfusion at a tertiary care centre over 3 months. Patients who were discharged each received a survey that assessed their (1) recall of the consent process, (2) recall of information conveyed, (3) assessment of the discussion's understandability and (4) perceived knowledge of as well as comfort level with transfusion as a result of the discussion. Overall, 80% of respondents recalled discussing and signing an informed consent. Information was mostly conveyed by attending physicians (35%) and consent obtained in the patient's hospital room (38%) or the preadmission clinic (19%). Although the majority recalled the consent process, many did not recall the discussion of specific transfusion risks or alternatives to donor blood (88%). Although the majority felt the discussion was at least somewhat understandable (77%), only 35% felt better informed and more comfortable with accepting blood. Despite implementation of written informed consent for transfusion, patients' recollection and understanding of risks and alternatives remain poor. This suggests the need for improving risk communication.

A retrospective study evaluating single-unit red blood cell transfusions in reducing allogeneic blood exposure.

Although guidelines recommend the use of single-unit red blood cell (RBC) transfusions to minimize allogeneic blood exposure, clinical practice remains dominated by two-unit transfusions. This study assesses the potential impact of a single-unit transfusion policy on reducing RBC utilization. We performed a retrospective analysis of adult patients admitted to a tertiary care hospital who received one or two RBC units. In subjects transfused two units, the effect of one unit was estimated by dividing the change in haemoglobin by 2. The proportion of patients reaching a haemoglobin threshold of 70, 75, 80, 85 and 90 g L(-1) with a single RBC unit was estimated. Of 302 included patients, only 65 received a one-unit transfusion. Based on thresholds of > or = 90, > or = 80 and > or = 70 g L(-1), a single-unit transfusion would be sufficient in 42.0% (RRR = 0.54), 79.6% (RRR = 0.23) and 98.0% (RRR = 0.02) of cases, respectively. This corresponds to 0.21, 0.57 and 0.82 mean RBC units saved per patient. In the orthopaedic subpopulation, the mean RBC units saved are 0.53, 0.88 and 1.00 for the same haemoglobin targets. Adopting a policy of transfusing RBC in single-unit aliquots could significantly improve RBC utilization and decrease patient exposure to allogeneic blood.

Effect of haemodilution with diaspirin cross-linked haemoglobin on the oxygen reserve in a rodent model of surgical blood loss.

The efficacy of pre-operative haemodilution is limited by the reduction in haemoglobin concentration. Acellular haemoglobin-based oxygen carriers provide an alternative to colloid as a haemodiluent, potentially extending the safe limits of this procedure. The aim of this investigation was to determine whether haemodilution with a cross-linked haemoglobin solution, diaspirin cross-linked haemoglobin solution (DCLHb), would enhance the oxygen reserve compared to pentastarch. Sprague Dawley rats were placed in a metabolic box to directly measure systemic oxygen consumption (VO2). Rats were randomized to be haemodiluted to a cellular haemoglobin of 80 g L(-1) with either DCLHb or pentastarch. Oxygen reserve was assessed during isovolemic haemorrhage by determining the critical oxygen delivery (DO2crit) and haemoglobin concentration at the point of oxygen supply dependency (OSD). Following haemodilution and for the duration of the experiment, cardiac index (CI) was significantly lower and systemic vascular resistance was significantly higher in the DCLHb than the pentastarch group. The DO2crit (3.2 +/- 0.4 mL minAg(-1) and 3.4 +/- 0.5 mL minAg(-1), DCLHb versus pentastarch) and cellular haemoglobin concentration (51 +/- 9 g L(-1) and 48 +/- 9 g L(-1)), at which rats entered OSD were similar in both groups. Total haemoglobin concentration (cellular and plasma DCLHb) and arterial oxygen content were significantly higher in the DCLHb group (total haemoglobin, 66 +/- 8 g L(-1) and arterial content, 9.2 +/- 1.4 mL dL(-1)) compared to the pentastarch group (total haemoglobin, 48 +/- 9 g L(-1) and arterial content, 7.3 +/- 1.4 mL dL(-1)). Oxygen extraction ratios increased from baseline levels to 0.53 +/- 0.07 and 0.56 +/- 0.1, for the DCLHb and pentastarch groups, respectively, and were not significantly different. The increase in arterial oxygen content from DCLHb in plasma was offset by the decrease in CI observed in this group. Plasma DCLHb did not extend the limits of haemodilution beyond the capacity of the cellular haemoglobin concentration.

Optimising parameters for peripheral blood leukapheresis after r-metHuG-CSF (filgrastim) and r-metHuSCF (ancestim) in patients with multiple myeloma: a temporal analysis of CD34(+) absolute counts and subsets.

Patients (n = 69) with multiple myeloma undergoing peripheral blood stem cell collection (PBSC) were treated with cyclophosphamide and a combination of recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) and recombinant methionyl human stem cell factor (r-metHuSCF, ancestim). The objectives of this study were to determine: (1) The proportion of patients reaching a target yield of >or=5 x 10(6) CD34(+) cells/kg in one or two successive large-volume (20 liter) leukapheresis procedures; (2) the optimal collection time for leukapheresis; (3) mobilization kinetics of CD34(+) subsets in response to G-CSF/SCF. All patients were mobilized with cyclophosphamide (2.5 g/m(2)) on day 0 followed by filgrastim (10 microg/kg ) plus ancestim (20 microg/kg) commencing day 1 and continuing to day 11 or 12. Of the 65 evaluable patients, 57 were considered not heavily pretreated and 96.5% obtained a target of >or=5 x 10(6)/kg in one collection. The median CD34(+) cells/kg was 39.5 x 10(6) (range: 5.2-221.2 x 10(6)). Subset analysis demonstrated the number of CD38(-), CD33(-), and CD133(+) peaked at day 11; and CD34(+), CD90(+) cells peaked at day 10. The optimum day for leukapheresis was determined to be day 11. The median absolute peripheral blood CD34(+) cell numbers on day 11 was 665 x 10(6)/l (range: 76-1481 x 10(6)/l). Eight of the 10 heavily pretreated patients were evaluable: three achieved the target dose in one leukapheresis (37.5%) and three (37.5%) achieved the target dose with two leukaphereses. Use of this mobilization strategy allowed the collection of high numbers of CD34(+) cells and early progenitors and the ability to predictably schedule leukapheresis.

Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation.

Reduced CD34(+) cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34(+) cell numbers at the time of stem cell collection and prior to re-infusion. Viable CD34(+) cells were enumerated using single platform flow cytometry and the molecular exclusion dye 7-amino actinomycin D. The median number of viable CD34(+) cells was 3.6 x 10(6)/kg at the time of harvest and 2.0 x 10(6)/kg after thawing. When viable CD34(+)cells enumerated after thawing were <2.0, 2.0-5.0, or >5.0 x 10(6)/kg, the median time to platelet engraftment was 17, 12 and 10 days, respectively (P < 0.05 for comparison of the group with <2.0 x 10(6)/kg and the other two groups), and the median time to neutrophil engraftment was 13, 14 and 12 days, respectively (P = NS). A minimum of 2.0 x 10(6) CD34(+) cells/kg was harvested in 33 of 36 patients (92%) but only 19 of 36 (52%) patients met this threshold at the time of reinfusion. The reduced numbers of viable CD34(+) cells measured prior to re-infusion is associated with time to platelet engraftment and may be useful in monitoring stem cell loss during processing and identifying patients at risk of graft failure.

Effect of prophylactic transfusion of stored RBCs on oxygen reserve in response to acute isovolemic hemorrhage in a rodent model.

BACKGROUND: The storage of RBCs results in a time-related decline in 2,3 DPG that may reduce the ability to unload oxygen (O(2)) to tissue. The objective of this study was to compare the effect that transfusion of stored 2,3 DPG-depleted rat blood (7 days in CPDA-1) had on the O(2) reserve in conscious rats, with that of the transfusion of fresh blood (<2-hour storage). STUDY DESIGN AND METHODS: Anemic rats (Hb, 80 g/L) received either fresh packed RBCs or stored RBCs to raise Hb levels to 140 g per L. They then underwent isovolemic hemorrhage mimicking surgical blood loss to the point of O(2) supply dependency (OSD). Critical O(2) delivery (DO(2)crit), Hb concentration, and O(2) extraction at OSD were measured in a metabolic chamber. RESULTS: After transfusion, RBC DPG decreased by 50 percent in the stored-blood group, and the p50 value decreased by 5 mmHg (32.1 +/- 2.5 mmHg vs. 37.5 +/- 3.0). DO(2)crit was similar in the two groups (fresh blood: 2.79 +/- 0.44 mL/min x g(-1); stored blood, 2.99 +/- 0.76 mL/min x g(-1)). The critical Hb concentration at DO(2)crit was higher in the stored-blood group (44 +/- 4 g/L) than in the fresh-blood group (38 +/- 5 g/L); the cardiac index and O(2) extraction ratio in the two groups were not different. Under conditions of severe normovolemic anemia in rats, depletion of DPG and a decrease in p50 had only minor effects on the O(2) reserve. At OSD, under these conditions, O(2) consumption is not limited by diffusion. CONCLUSION: The physiologic impact of DPG depletion in transfused stored blood on oxygen availability in normal rats appears to be small and may be clinically inconsequential.

Inactivation of p53 sensitizes U87MG glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea.

We examined the effect of p53 inactivation on the response of U87MG glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These studies were motivated by three observations: (a) some human astrocytomas are sensitive to BCNU and some are resistant; (b) chemosensitive astrocytomas are more likely to be found in young adults whose tumors are more likely to harbor a p53 mutation; and (c) mouse astrocytes lacking the p53 gene are more sensitive to BCNU than wild-type cells. Here, we observed that p53 inactivation by transfection with pCMV-E6 sensitized U87MG cells to BCNU. Compared with control U87MG-neo cells with intact p53 function, the clonogenic survival of U87MG-E6 cells exposed to BCNU was reduced significantly. In U87MG-E6 cells, sensitization to BCNU was associated with failure of p21(WAF1) induction, transient cell cycle arrest in S phase, accumulation of polyploid cells, and significant cell death. In contrast, resistance to BCNU in U87MG-neo cells was associated with up-regulation of p53, prolonged induction of p21(WAF1), sustained cell cycle arrest in S phase, and enhancement of DNA repair. U87MG cells with disrupted p53 function were less able to repair BCNU-induced DNA damage and survive this chemotherapeutic insult. The question arises of whether p53 dysfunction might be a chemosensitizing genetic alteration in human astrocytic gliomas.