Simplified murine multipotent progenitor isolation scheme: Establishing a consensus approach for multipotent progenitor identification.

The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.

Rapid, sensitive and cost-effective determination of immune checkpoint inhibitor activity using a magnetic bead-based binding assay.

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

Intelligent whole-blood imaging flow cytometry for simple, rapid, and cost-effective drug-susceptibility testing of leukemia.

Drug susceptibility (also called chemosensitivity) is an important criterion for developing a therapeutic strategy for various cancer types such as breast cancer and leukemia. Recently, functional assays such as high-content screening together with genomic analysis have been shown to be effective for predicting drug susceptibility, but their clinical applicability is poor since they are time-consuming (several days long), labor-intensive, and costly. Here we present a highly simple, rapid, and cost-effective liquid biopsy for ex vivo drug-susceptibility testing of leukemia. The method is based on an extreme-throughput (>1 million cells per second), label-free, whole-blood imaging flow cytometer with a deep convolutional autoencoder, enabling image-based identification of the drug susceptibility of every single white blood cell in whole blood within 24 hours by simply flowing a drug-treated whole blood sample as little as 500 µL into the imaging flow cytometer without labeling. Our results show that the method accurately evaluates the drug susceptibility of white blood cells from untreated patients with acute lymphoblastic leukemia. Our method holds promise for affordable precision medicine.

Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Efficiency and Health Economic Evaluations of BD OneFlow™ Flow Cytometry Reagents for Diagnosing Chronic Lymphoid Leukemia.

REASON FOR THE STUDY: To standardize the use of flow cytometry for classifying hematological malignancies and make the results reliable and reproducible across laboratories, the EuroFlow™ Consortium published a comprehensive specification of antibody-fluorochrome conjugates, standard protocols, and algorithms for analysis. The BD OneFlow™ system builds on, and further standardizes, the EuroFlow protocols. We aimed to assess the effects on safety, efficiency, and costs for laboratories of adopting the BD OneFlow reagent tubes (LST and B-CLPD T1) for diagnosing chronic lymphocytic leukemia. METHODS: We compared in-house laboratory processes and results with those using the LST and B-CLPD T1 reagent tubes with, and without, blood film morphology. Outcome measures included concordance in classification results, and efficiency within the laboratory, that is, resource usage, staff time, unwanted events, and cost-consequences. RESULTS: There was 100% concordance between the classifications made with in-house flow cytometry and those with the BD OneFlow reagent tubes. Using BD OneFlow tubes required 13 hours less staff time per month (i.e. for 100 samples) than the in-house process. Sensitivity analyses explored the effects of uncertainties in the price of the BD OneFlow tubes and the prevalence of CLL and identified the thresholds at which laboratories might expect cost-savings from adopting the BD OneFlow system. Laboratory and clinical staff considered the BD OneFlow system to be safe and effective. CONCLUSIONS: Laboratories adopting the BD OneFlow system for classifying patients with suspected CLL can expect safe, efficient processes that can be cost saving if the discount on the list price, and prevalence of CLL (which will both vary between sites and countries), is within the thresholds suggested by the health economics sensitivity analysis. © 2019 International Clinical Cytometry Society.

Managing costs in primary immunodeficiency: minimal immunophenotyping and three national references.

Our aim was to evaluate the cost-effectiveness of a minimal lymphocyte subset quantification (LSQ) by flow cytometry as the first screening in children with clinically suspected primary immunodeficiency (PID). Two hundred sixty-eight Brazilian patients (0-21 years old) were studied. They were divided by clinical and phenotypical features into those fulfilling criteria for PID (PID phenotype) according to the 2017 International Union of Immunological Societies (IUIS) classification and those not fulfilling these criteria (non-PID phenotype). We evaluated how many patients had values below the 10th percentile for five lymphocyte subsets in peripheral blood, (suggestive of PID) according to reference values for Brazil, Italy and USA. Three lymphocyte subsets (T CD3/CD4, B CD19 and NK CD16/CD56) had p-value < 0.05 and Odds Ratio (OR) indicating a risk at least two times higher for the diagnosis of a PID phenotype. The application of Kappa coefficient (k) on Brazilian vs Italian and Brazilian vs US data sets resulted in k compatible with strong or excellent level of agreement between the three classification systems. The authors conclude that a number of CD3+ /CD4+ , CD19+ and CD16+ /CD56+ (NK) cells in peripheral blood <10th percentile represented a significant risk for the diagnosis of PID in this cohort. Natural killer (NK) deficiency is quite rare and has a very specific clinical profile. So, the analysis of these cells could be requested only in some cases, saving even more costs. The minimal immunophenotyping, with quantification of T CD4+ , CD19+ and in some cases CD16+ /CD56+ cells, may be a useful tool for the first screening of PID, saving costs, especially in developing countries.

Utility of Flow Cytometry in Diagnosing Hematologic Malignancy in Tonsillar Tissue.

OBJECTIVE: Tonsil surgical biopsy or excision is a very common procedure. However, there exist no consensus guidelines for the pathologic handling of tonsil specimens; gross and/or microscopic evaluation may be used. Diagnosis of tonsillar hematologic malignancy requires histology, immunohistochemistry and/or flow cytometry. Data regarding the utility of flow cytometry in tonsillar tissues are limited. We assessed our experience with flow cytometry for tonsil diagnosis with regard to accuracy and use patterns at a tertiary academic medical center. METHODS: We retrospectively analyzed all surgically biopsied or excised tonsil specimens that underwent flow cytometry evaluation from August 2011 to March 2014. Patient clinical information, intraoperative frozen section, histology, immunohistochemistry, and flow cytometry diagnoses were recorded. RESULTS: The study included 154 tonsil specimens from 89 females and 65 males. Patients averaged 27.4 years old (range 2-87 years); 73 were pediatric. Both histology and flow cytometry were benign for 148 patients (96.1%). Hematolymphoid malignancy was diagnosed in 6 adults by histology/immunohistochemistry: diffuse large B-cell lymphoma (2), small B-cell lymphoma (2), concomitant follicular lymphoma and histiocytic sarcoma (1), and extraosseous plasmacytoma (1). Flow cytometry identified abnormal populations in 5 of 6 cases, and detected clonal populations in 2 reactive follicular hyperplasia cases. CONCLUSION: Tonsillar hematolymphoid malignancy is uncommon, and flow cytometry was less accurate than histology/immunohistochemistry for its diagnosis. Despite the rarity of tonsillar lymphoma in children, nearly half of study patients were pediatric. Intraoperative frozen section diagnosis showed excellent sensitivity for malignancy, and could be used to effectively triage cases for flow cytometry evaluation.

Multicenter validation of a simplified method for paroxysmal nocturnal hemoglobinuria screening.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) diagnostic guidelines recommend single-tube five- to six-color or two-tube four-color assays. PNH clones are detectable in only a fraction of patients at risk, and screening for new PNH cases can be complex and expensive. In this multicenter study, we have validated a simplified, one-tube two-color FLAER-based assay suitable for PNH screening. METHODS: Six laboratories received samples containing spiked PNH leukocyte clones to be analyzed in parallel with a common six-color cocktail (FLAER/CD24/CD45/CD64/CD15/CD14) and a simplified two-color mixture (FLAER/CD15), a shared calibration procedure, and a common analysis protocol. Replicate precision and sensitivity tests were performed on PNH patients, from undiluted to 1:10 000. Specificity tests were performed on normal donors to identify the possible sources of artifacts. RESULTS: The performance comparison between six-color and two-color assays showed an excellent agreement for granulocyte PNH clones. Dilution experiments showed an accurate detectability down to 0.01% sensitivity level for granulocyte PNH clones and to 1% for monocytes. Specificity experiments disclosed that basophils and platelets can contaminate the monocyte gate and generate false PNH events. CONCLUSIONS: A simplified two-color (FLAER/CD15) PNH screening test has been validated in a highly standardized multicenter study and proved feasible and effective in ongoing regional programs. Precision, sensitivity, and specificity of the simplified test for granulocytes were comparable to the more complex and expensive six-color assay and applicable for screening also in peripheral laboratories. The diagnostic confirmation of PNH should be always performed by a reference center using the established technique on all cell lineages.

Aligning the flow cytometric evaluation with the diagnostic need: an evidence-based approach.

AIMS: Elimination of non-value added testing without compromising high-quality clinical care is an important mandate for laboratories in a value-based reimbursement system. The goal of this study was to determine the optimal combination of flow cytometric markers for a screening approach that balances efficiency and accuracy. METHODS: An audit over 9 months of flow cytometric testing was performed, including rereview of all dot plots from positive cases. RESULTS: Of the 807 cases in which leukaemia/lymphoma testing was performed, 23 were non-diagnostic and 189 represented bronchoalveolar lavage specimens. Of the remaining 595 cases, 137 (23%) were positive for an abnormal haematolymphoid population. Review of the positive cases identified minimum requirements for a screening tube as well as analysis strategies to overcome the diagnostic pitfalls noted. It is estimated that 38% fewer antibodies would be used in a screening approach, representing an opportunity for significant cost savings. CONCLUSIONS: We provide a framework for developing an evidence-based screening combination for cost-effective characterisation of haematolymphoid malignancies, promoting adoption of 'just-in-time' testing systems that tailor the evaluation to the diagnostic need.

Cost-effectiveness of a new system in ruling out negative urine cultures on the day of administration.

Urine samples account for a significant part of the workload in clinical microbiology laboratories. However, the culture process is time-consuming and a large proportion is reported as negative. To reduce unnecessary culture procedures and speed up the reporting of negative results, a reliable screening method is needed. For this purpose, urine samples submitted to our clinical microbiology laboratory were simultaneously screened by a flow cytometry method (Sysmex UF-1000i, Japan). During screening, the evaluation of various combinations of leucocytes and bacteria cut-offs demonstrated that cut-offs of 30 and 50/µL, respectively, were the best threshold values to reach a 100% negative predictive value (NPV) with a culture reduction rate of 44.8% in adults and 61.9% in children between the ages of 6 and 17 years. With the culture reduction rates mentioned above, the screening method has provided at least 24% savings in expenditures of the routine clinical microbiology laboratory. Since we did not reach such an NPV with any combinations of screening parameters in children younger than 5 years of age, we recommend cultivation of all urine samples in those patients without a screening step. In conclusion, Sysmex UF-1000i as a screening method was capable of improving the efficiency of the routine microbiology laboratory by providing negative results in a few minutes in children greater than 6 years of age and in adults.

Thirty-five years of CD4 T-cell counting in HIV infection: From flow cytometry in the lab to point-of-care testing in the field.

CD4 T-cell counting was introduced in clinical laboratories shortly after the discovery of the human immune deficiency virus (HIV) in the early eighties. In western clinical laboratories, improvements in the CD4 T-cell counting methods were mainly driven by progress in the field of flow cytometry and immunology. In contrast, the development of dedicated CD4 T-cell counting technologies were needs driven. When antiretroviral treatment (ART) was made available on a large scale by international Acquired Immune Deficiency Syndrome (AIDS) relief programs to HIV+ patients living in low income countries in 2003, there was a distinct need for simplified and affordable CD4 T-cell counting technologies. The first decade of 2000, several compact flow cytometers appeared on the market, mainly to the benefit of low income countries with limited resources. More recently, however, portable point-of-care (POC) CD4 T-cell counting devices have been developed especially to improve access to affordable monitoring of HIV+ patients in low income countries. The accuracy of these POC instruments is not yet very well documented as many are still under development and clinical validation but preliminary evidence is encouraging. The new HIV treatment guidelines released by the World Health Organization in 2016 give CD4 T-cell counting a less central role in the management of HIV infection. It is, therefore, to be expected that CD4 T-cell counting will be phased out as a tool to assess eligibility of HIV+ patients for ART in the future. However, CD4 T-cell counting will remain a valuable tool for directing treatment against opportunistic infections. © 2016 International Clinical Cytometry Society.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

OBJECTIVE: Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method. MATERIALS AND METHODS: In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units. RESULTS: Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant. CONCLUSION: In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

Analysis of the costs for the laboratory of flow cytometry screening of urine samples before culture.

Urine culture samples comprise a large proportion of the workload in clinical microbiology laboratories, and most of the urine samples show no growth or insignificant growth. A flow cytometry-based analyzer (Sysmex Corporation, Japan) has been used to screen out negative urine samples prior to culture in the Päijät-Häme district. We applied decision analytic modelling to analyze, from a laboratory perspective, the economic feasibility of the screening method as compared to culture only (conventional method) for diagnosis of urinary tract infection. Our model suggests that the least costly analytical strategy is the conventional method. The incremental cost of screening is €0.29/sample. Although laboratory costs are higher, considerable savings on workload can be achieved. Furthermore, screening has numerous benefits on the treatment process of a patient that well warrant the use of the screening method. We conclude that the incremental cost of screening the samples is worth the expense.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria / Avanços técnicos no diagnóstico e no monitoramento de hemoglobinúria paroxística noturna por citometria de fluxo

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

RESUMO Objetivo: Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. Métodos: Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. Resultados: Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. Conclusão: A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

BACKGROUND: Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. METHODS: A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. RESULTS: In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. CONCLUSIONS: Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.

Mobile flow cytometer for mHealth.

Flow cytometry is used for cell counting and analysis in numerous clinical and environmental applications. However flow cytometry is not used in mHealth mainly because current flow cytometers are large, expensive, power-intensive devices designed to operate in a laboratory. Their design results in a lack of portability and makes them unsuitable for mHealth applications. Another limitation of current technology is the low volumetric throughput rates that are not suitable for rapid detection of rare cells.To address these limitations, we describe here a novel, low-cost, mobile flow cytometer based on wide-field imaging with a webcam for large volume and high throughput fluorescence detection of rare cells as a simulation for circulating tumor cells (CTCs) detection. The mobile flow cytometer uses a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. For fluorescence detection, a 1 W 450 nm blue laser is used for excitation of Syto-9 fluorescently stained cells detected at 535 nm. A wide-field flow cell was developed for large volume analysis that allows for the linear velocity of target cells to be lower than in conventional hydrodynamic focusing flow cells typically used in cytometry. The mobile flow cytometer was found to be capable of detecting low concentrations at flow rates of 500 µL/min, suitable for rare cell detection in large volumes. The simplicity and low cost of this device suggests that it may have a potential clinical use for mHealth flow cytometry for resource-poor settings associated with global health.

Value-based flow testing of chronic lymphoproliferative disorders: a quality improvement project to develop an algorithm to streamline testing and reduce costs.

OBJECTIVES: Flow cytometry is essential for the evaluation of lymphoproliferative disorders (LPDs) and their classification. Flow panels routinely incorporate a large array of antibodies, making testing complex and expensive; such panels are likely unnecessary in benign cases or those with straightforward diagnoses. Our aim was to develop a more cost-effective testing strategy based on a retrospective analysis of flow studies for possible LPDs in blood. METHODS: We identified LPD frequencies and types, as well as associated results with patient age and absolute lymphocyte count. RESULTS: We found that the likelihood of LPDs increased with patient age and absolute lymphocyte count and that CD5-positive LPD was the most common LPD diagnosed in our institution (71% of LPDs). Using these data, we devised flow-testing algorithms with a screening test for patients at low risk of disease and a focus on CD5-positive LPD detection, with reflexing as needed. CONCLUSIONS: We project this approach will result in a 40% decrease in antibody utilization.

Limited flow cytometry panels on bone marrow specimens reduce costs and predict negative cytogenetics.

OBJECTIVES: To determine the clinical and financial impact and predictive value of a limited flow cytometry strategy in the evaluation of bone marrow specimens. METHODS: Consecutive bone marrow cases (n = 1,242) were reviewed following the independent, prospective application of two flow cytometry protocols: a limited marker strategy and a multimarker strategy. Combined morphologic and flow cytometry findings were also compared with cytogenetic results. RESULTS: A limited flow cytometry strategy did not have a negative impact on disease detection and resulted in reduced utilization and cost. In addition, negative combined morphology and flow cytometry had a 98.4% predictive value for negative cytogenetics (P < .001). CONCLUSIONS: Careful initial evaluation of bone marrow specimens can markedly reduce the costs of bone marrow examination and significantly reduce the need for flow cytometric and cytogenetic studies on these samples.

Clinical applicability and prognostic significance of molecular response assessed by fluorescent-PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study.

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent-PCR (F-PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F-PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D-J, IGK V-J and KDE rearrangements. The applicability of F-PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non-molecular response; median progression-free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5-year survival: 75%) versus 66 months in the non-molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non-immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5-year survival: 95%) versus 69 months (P = 0·004), respectively. F-PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM.