Disposable gold coated pyramidal SERS sensor on the plastic platform.

In this paper we investigate suitability of arrays of gold coated pyramids for surface-enhanced Raman scattering (SERS) sensing applications. Pyramidarrays composed of 1000 nm pit size with 1250 nm pitch lengthwerereplicated on a plastic substrate by roll-to-roll (R2R) ultraviolet (UV) embossing. The level of SERS enhancement, and qualitative performance provided by the new substrate is investigated by comparing Raman spectrum of benzenethiol (BTh) test molecules to the benchmark Klarite SERS substrate which comprises inverted pyramid arrays(1500 nm pit size with 2000 nm pitch length) fabricated on a silicon substrate. The new substrate is found to provide upto 11 times increase in signal in comparison to the inverted pyramid (IV-pyramid) arrays fabricated on an identical plastic substrate. Numerical simulation and experimental evidence suggest that strongly confined electromagnetic fields close to the base of the pyramids, are mainly responsible for the Raman enhancement factor, instead of the fields localized around the tip. Unusually strong plasmon fields are projected upto 200nm from the sidewalls at the base of the pyramid increasing the cross sectional sensing volume.

Disposable plasmonic plastic SERS sensor.

The 'Klarite™' SERS sensor platform consisting of an array of gold coated inverted square pyramids patterned onto a silicon substrate has become the industry standard over the last decade, providing highly reproducible SERS signals. In this paper, we report successful transfer from silicon to plastic base platform of an optimized SERS substrate design which provides 8 times improvement in sensitivity for a Benzenethiol test molecule compared to standard production Klarite. Transfer is achieved using roll-to-roll and sheet-level nanoimprint fabrication techniques. The new generation plastic SERS sensors provide the added benefit of cheap low cost mass-manufacture, and easy disposal. The plastic replicated SERS sensors are shown to provide ~10(7) enhancement factor with good reproducibility (5%).

Cord blood monocytes, neutrophils and lymphocytes from preterm and full-term neonates show multiple aberrations in signalling profiles measured using phospho-specific whole-blood flow cytometry.

Immaturity of the immune system renders newborns susceptible to infections. We searched for aberrations in leucocyte signalling profiles, using phospho-specific whole-blood flow cytometry, in cord blood of nine preterm (two born before 32nd gestational week) and nine full-term infants, born by caesarean section. Thirteen adults served as reference subjects. Monocyte NF-κB phosphorylation following tumour necrosis factor (TNF) or bacterial stimulation was higher in preterm neonates than in full-term neonates or adults, p38 phosphorylation following bacterial stimulation was higher in both preterm and full-term neonates than in adults, while STAT1 phosphorylation by IFN-γ or IL-6, STAT3 phosphorylation by IL-6 and STAT5 phosphorylation by GM-CSF were lower in both full-term and preterm neonates than in adults. Neutrophil STAT1 and STAT3 phosphorylation following IFN-γ stimulation and STAT5 phosphorylation following GM-CSF stimulation were lower in newborn neonates than in adults. In both CD3(+) CD4(+) and CD3(+) CD8(+) lymphocytes, NF-κB phosphorylation by TNF was higher and STAT5 phosphorylation by IL-2 was lower in preterm and full-term newborns than in adults. STAT6 phosphorylation by IL-4 was comparable in monocytes and lymphocytes of newborns and adults. The results suggest that innate immune signalling pathways responding to inflammatory stimuli are strongly functional in leucocytes of preterm neonates, which may render these neonates susceptible to inappropriate tissue injury. In leucocytes of both preterm and full-term newborns, responses needed against intracellular pathogens, and regulatory functions show immaturities, possibly contributing to worse control of infections.

Signalling profiles of circulating leucocytes in patients recovered from reactive arthritis.

OBJECTIVES: Reactive arthritis (ReA) is a sterile joint inflammation triggered by a remote infection and associated with human leucocyte antigen (HLA)-B27. Its pathogenesis is unknown, but abnormal response to microbial structures or endogenous inflammatory mediators may be involved. We studied responses in leucocyte signalling profiles in patients with previous ReA after a full recovery. METHOD: The study comprised 10 HLA-B27-positive healthy subjects with a history of Yersinia enterocolitica-triggered ReA (B27+ReA+) and 20 healthy reference subjects, of whom 10 carried HLA-B27 (B27+ReA-) and 10 did not (B27-ReA-). Phosphospecific fluorescent monoclonal antibodies and flow cytometry were used to determine activation of nuclear factor kappa B (NF-κB), signal transducers and activators of transcription (STATs) 1, 3, 5, and 6, and two mitogen-activated protein (MAP) kinases, p38 and extracellular signal-regulated kinase (ERK)1/2, in monocytes, lymphocytes, lymphocyte subsets, and neutrophils. B27+ReA+ and B27-ReA- whole-blood samples were incubated with Yersinia with or without infliximab to study the role of tumour necrosis factor (TNF) in lymphocyte subset activation. Samples of the three subject groups were studied using soluble bacterial or endogenous stimuli. Fluorescence levels were determined as relative fluorescence units (RFU) and the proportion of positively fluorescing cells. RESULTS: The intracellular activation of circulating leucocytes in response to soluble stimuli was consistently comparable in B27+ReA+, B27+ReA-, and B27-ReA- subjects. Infliximab inhibited Yersinia-induced lymphocyte NF-κB phosphorylation similarly in B27+ReA+ and B27-ReA- groups. CONCLUSIONS: ReA susceptibility is not reflected in leucocyte signalling profiles elicited by phlogistic stimuli. However, the possibility remains that aberrations occur in response to combinations of stimuli, such as those associated with leucocyte adhesion.

The impact of early viral infections and graft-versus-host disease on immune reconstitution following paediatric stem cell transplantation.

Viral infections and graft-versus-host disease (GVHD) render an impact on both the clinical and immunological recovery following allogeneic hematopoietic stem cell transplantation (HSCT). We studied the recuperation of the immune defence after transplant in the paediatric setting and assessed the impact of early (<100 days post-HSCT) viral [cytomegalovirus (CMV), Ebstein-Barr virus (EBV) and adenovirus] reactivations/infections and GVHD. Fifty-one paediatric recipients of HSCT were enrolled. T cell recovery was evaluated on lymphocyte subpopulations using flow cytometry and functionally by measuring T cell excision circles (TRECs) and through the analysis of T lymphocyte responses to mitogens. B cell recovery was studied by flow cytometry and functionally by ELISPOT. Acute and mild chronic GVHD allowed for a brisk recovery of both cellular and humoral immunity while moderate to severe chronic graft-versus-host disease (cGVHD) associated with a significant, tampering effect on the immunological recovery after transplant. In the former group, the early viral reactivations/infections seemingly linked with a delayed recovery of T lymphocytes and low TRECs values. Moderate to severe cGVHD appears to associate with an impaired immunological recovery after HSCT. Early viral infections linked with prolonged T cell immunodeficiency and thymic dysfunction may be indicative of the presence of subclinical GVHD.

Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

T-cell acute lymphoblastic leukemia in patients 1-45 years treated with the pediatric NOPHO ALL2008 protocol.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1-45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69-0.81), being 0.82 (0.74-0.88) for patients 1.0-9.9 years, 0.76 (0.66-0.86) for those 10.0-17.9 years, and 0.65 (0.55-0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1-9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1-45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

Correction: Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hepatic neutrophil activation during reperfusion may not contribute to initial graft function after short cold ischemia in human liver transplantation.

BACKGROUND: Experimental models of hepatic ischemia/reperfusion injury have implicated a pathophysiologic role for neutrophils in subsequent hepatocellular damage. In human liver transplantation, however, the effect of reperfusion-induced neutrophil activation on initial graft function is not clear. METHODS: In 38 patients undergoing liver transplantation, neutrophil CD11b and L-selectin expression, neutrophil count, and plasma lactoferrin levels were measured. To assess changes within the graft during initial reperfusion, samples of blood entering and leaving the graft were obtained simultaneously, and transhepatic ratio calculated (hepatic vein/portal vein; 1 denotes no change, <1 a decrease, and >1 an increase across the liver). Graft steatosis, postoperative liver function, and outcome were recorded. Associations between neutrophil activation markers and outcome measures were evaluated. RESULTS: Substantial hepatic neutrophil activation occurred during initial reperfusion, demonstrated by concomitant L-selectin shedding and CD11b upregulation (transhepatic ratios 0.9 [0.7-1.0]; 1.4 [0.9-1.9]; both P < .001; portal vs hepatic vein]. Simultaneously, hepatic neutrophil sequestration and lactoferrin release occurred (0.3 [0.2-0.5]; 1.7 [1.3-3.4]; both P < .001). Neither cold ischemic time (CIT; median 5 hours 36 minutes) nor hepatic neutrophil activation during reperfusion predicted early graft function, nor was there any association between CIT and neutrophil activation. CONCLUSIONS: Despite short CIT, extensive graft neutrophil activation and sequestration occurred. This, however, was not associated with impaired early graft function, suggesting short CIT may protect against severe neutrophil-mediated injury.

Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD < 10-3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10-4-<10-3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10-4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10-4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.

A novel modification of a flow cytometric assay of phosphorylated STAT1 in whole blood monocytes for immunomonitoring of patients on IFN alpha regimen.

We explored whether episodes stimulating leucocytes in vivo could be tracked from whole blood samples by monitoring activation of STAT1 by flow cytometry. The method was tested in hepatitis C patients (n = 9) that were on interferon (IFN)alpha regimen. CD14+ monocytes responded strongly to IFNalpha/gamma being sensitive indicators for recent immune activation. At 45 min after s.c. IFNalpha 91% of monocytes were phosphorylated STAT1+. The frequency of responding cells decreased to a base level within 6 h. Monocytes, however, had a long-term deficient phosphorylated STAT1 response to IFNalphain vitro that in patients on standard IFNalpha regimen lasted for 48 h. In patients on pegylated IFNalpha the phosphorylated STAT1 response was completely absent. We conclude that whole blood analysis of STAT1 activation by flow cytometry is applicable to monitor immune cells in patient material.

T cell regeneration in pediatric allogeneic stem cell transplantation.

Delayed and/or insufficient T cell recovery post hematopoietic stem cell transplantation (HSCT) leads to an increased risk of morbidity and mortality. We evaluated thymic function and its association with T cell regeneration post HSCT and identified factors involved in the process among pediatric stem cell transplant recipients. T cell regeneration in 66 pediatric patients was prospectively followed by naive T cell phenotyping, measuring of T cell receptor excision circles (TRECs) and expression of Foxp3 by regulatory T cells for the first 18 months post HSCT. TRECs were lower pre-HSCT in children with a malignant than non-malignant primary disease or immunosuppressed controls (P=0.001). Naive T lymphocyte reconstitution and thymic recovery were slow in the recipients of allogeneic stem cell grafts post HSCT. Infections caused by herpesviruses had a prognostic impact on mortality. Children with low TRECs had a high mortality (P=0.05) and low TRECs were also associated with extensive chronic graft-versus-host disease from 6 months onwards. Low amount of Foxp3 pre-HSCT was associated with an increased mortality post HSCT (P=0.03). Our study indicates an association between impaired T cell regeneration and thymic dysfunction and the clinical post transplant complications in pediatric allogeneic stem cell transplantation.

Probiotics and respiratory and gastrointestinal tract infections in Finnish military conscripts - a randomised placebo-controlled double-blinded study.

Military conscripts are susceptible to respiratory and gastrointestinal tract infections. In previous studies probiotics have shown potency to reduce upper respiratory and gastrointestinal infections. The aim was to study whether probiotic intervention has an impact on seasonal occurrence of upper respiratory and gastrointestinal infections in two different conscript groups. In a randomised, double-blinded, placebo controlled study (https://clinicaltrials.gov NCT01651195), a total of 983 healthy adults were enrolled from two intakes of conscripts. Conscripts were randomised to receive either a probiotic combination of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis ssp. lactis BB12 (BB12) or a control chewing tablet twice daily for 150 days (recruits) or for 90 days (reserve officer candidates). Clinical examinations were carried out and daily symptom diaries were collected. Outcome measures were the number of days with respiratory and gastrointestinal symptoms and symptom incidence, number and duration of infection episodes, number of antibiotic treatments received and number of days out of service because of the infection. Statistically no significant differences were found between the intervention groups either in the risk of symptom incidence or duration. However, probiotic intervention was associated with reduction of specific respiratory infection symptoms in military recruits, but not in reserve officer candidates. Probiotics did not significantly reduce overall respiratory and gastrointestinal infection morbidity.

Discontinuous deletions at 11q23 in B cell chronic lymphocytic leukemia.

Loss of genomic material in 11q is one of the most common structural chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL). In order to characterize the deletions of 11q23 in B-CLL, we performed fluorescence in situ hybridization (FISH) with eight YAC (yeast artificial chromosome) probes on peripheral leukocytes of 30 patients. These YACs form a contig spanning 7.8 Mb at 11q23.1-q23.3. We found deletions in nine out of 30 cases (30%) and five of them had discontinuous deletions in this region. The region represented by YAC 755b11 (1.6 Mb in size) was involved in all cases with deletions, supporting the hypothesis that this region might contain a novel gene of pathogenic importance to B-CLL. A more distal region represented by YAC 785e12 (760 kb in size) was deleted frequently and specifically. Whether there is another novel gene of pathogenic importance to B-CLL and what is its potential relationship to the deletions in the region represented by YAC 755b11, are issues that require further studies.

Prognostic value of cells with more than 5c DNA content in node-negative breast cancer as determined by image cytometry from tissue sections.

The aim of this investigation was to study the prognostic significance of 5c cells (presence of cancer cells with > 5c DNA content; ie, over 18 pg of DNA per nucleus) in axillary node-negative breast cancer. Tissue sections (3 microns) from 134 tumors were stained for DNA using the Feulgen method and screened for the percentage of 5c cells with the CAS 200 image analysis system (Cell Analysis System, Inc, Lombard, IL). Cancer cells with a DNA content exceeding the 5c level were found in 45% (60 of 134) of the cases, accounting for a median of 0.2% (range, 0.05% to 1.05%) of all cells. The presence of 5c cells was associated with a high histologic grade of the tumor (P = .0001), a large number of mitoses (P < .0001), flow cytometric DNA aneuploidy and high S-phase fraction (P = .0002 and P < .0001, respectively), and c-erbB-2 oncoprotein and p53 tumor suppressor gene product overexpression (P = .0002 and P = .0006, respectively). Patients with 5c cell-positive tumors had a significantly worse 8-year survival rate (P = .003) than those with 5c cell-negative tumors. Subgroup analysis showed that the presence of 5c cells had a prognostic impact in low malignancy tumors, ie, in well-differentiated (grade I or II) and slowly proliferating tumors. Our findings suggest that determination of 5c cells may be a useful additional prognostic factor in axillary node-negative breast cancer. It adds prognostic information, especially in cases that are otherwise thought to have a favorable course.

Intratumor variation in cell proliferation in breast carcinoma as determined by antiproliferating cell nuclear antigen monoclonal antibody and automated image analysis.

Immunohistochemical detection of the proliferating cell nuclear antigen (PCNA) represents a potentially useful tool for the study of tumor proliferative activity. To study the intratumor heterogeneity of tumor growth, 88 breast carcinomas were immunostained with the anti-PCNA antibody 19F4 and analyzed with the CAS 200 image analysis system (Cell Analysis System, Inc., Lombard, IL). For each sample, 12 fields from both the central and the peripheral areas of the tumor were measured. The proportion of PCNA-positive nuclear area in the whole tumor (PCNAt score) varied from 0.7% to 45.2% (median, 14.4%). There was considerable intratumor heterogeneity in the staining for PCNA. In 79% of the specimens, the PCNA score was higher in peripheral areas than in the center of the tumor, the average difference being +3.4% (range, -9.2- +15.1%; P < 0.0001, Student's t-test). The S-phase fraction, determined by DNA flow cytometry of the same tumors, varied from 2.0% to 32.6% (median, 10.0%). The PCNA score showed a significant correlation with the S-phase fraction (r = 0.469, P < 0.001). Most divergent results were those with high PCNA scores and low S-phase fraction; possible explanations for this are discussed. The PCNA score also was related to the histologic grade of the tumors (P = 0.03, analysis of variance). In conclusion, proliferation indices obtained from different areas of a tumor can differ significantly because of intratumor heterogeneity in growth fractions. The PCNA immunostaining correlates with well-known prognostic factors (S-phase fraction and histologic tumor grade) in breast carcinoma.

Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer.

E-cadherin (E-cad) is a calcium-dependent, epithelial cell adhesion molecule whose reduced or lost expression has been associated with tumor dedifferentiation and increased metastatic potential in human carcinomas. The authors studied immunohistochemically E-cad expression in frozen sections of 362 breast carcinomas using a monoclonal antibody (HECD-1). The immunohistochemical detection of reduced E-cad expression was confirmed by mRNA in situ hybridization with two different oligonucleotide probes. THe proportion of tumors with reduced or lost E-cad expression increased significantly from pure intraductal carcinomas (20%, 4 of 20) through invasive ductal (IDCs; 52%, 124 of 239) to recurrent carcinomas (64%, 18 of 28; chi square test for trend, P = .004). Invasive lobular carcinomas (ILCs) and IDCs differed from each other in their E-cad expression. None of the ILCs (n=55) retained normal E-cad expression in contrast to 48% (115 of 239) of the IDCs. In 259 primary IDCs, reduced E-cad expression was associated with high histologic grade (chi square test for trend, P < .001), negative estrogen receptor status (ER; Fisher's exact test; P = .042), and marginally with axillary node involvement (Fisher's exact test, P = .063). In a subset of 109 primary IDC patients whose clinical follow-up was available (median follow-up 51 months), reduced E-cad expression was associated with shortened disease-free survival (DFS; Mantel-Cox test, P = .027). In Cox's multivariate regression analysis, progesterone receptor status (P = .018) and E-cad expression (P = .072) were selected as independent predictors of DFS. Our findings provide clinical evidence that loss of normal E-cad expression is an indicator of increased invasiveness and dedifferentiation in breast carcinoma. E-cad is a potentially important prognostic factor in primary IDCs.

Genetic aberrations in pediatric acute lymphoblastic leukemia by comparative genomic hybridization.

Classical cytogenetic analysis plays an important role in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). However, poor in vitro growth of the malignant cells and suboptimal quality of metaphase spreads may sometimes cause false-negative findings (normal karyotype). We used comparative genomic hybridization (CGH) to study whether this new method is able to detect and characterize genetic aberrations not detected by karyotyping. CGH showed clonal genetic aberrations in 8 of 13 cases, most of which showed gains of several chromosomes, indicating hyperdiploidy. The sensitivity of CGH was sufficient to detect a small interstitial deletion of 6q. One karyotypically complex case was resolved by CGH showing a high-level amplification of DNA sequences originating from the 12p12-13. Interphase fluorescence in situ hybridization (FISH) analyses confirmed the CGH findings in 2 cases, validating the accuracy of CGH. In conclusion, CGH experiments established the known fact that hyperdiploidy is the most common finding in pediatric ALLs and that CGH may detect aberrations that are not seen in the G-banded karyotype. CGH was also able to further characterize genetic aberrations such as gene amplification, which is occasionally involved in pediatric ALL as well as in other leukemias.

Predictive value of interleukins 6, 8 and 10, and low HLA-DR expression in acute renal failure.

AIMS: HLA-DR expression and plasma levels of pro- and anti-inflammatory cytokines (IL-6, IL-8 and IL-10) and their predictive value concerning survival of critically ill systemic inflammatory response syndrome (SIRS) patients with and without acute renal failure (ARF) were evaluated. MATERIAL: A total of 103 consecutive adult patients with SIRS from 2 university hospital intensive care units participated in the study. METHOD: Laboratory data for all patients were prospectively collected on the day of admission and 2 days thereafter. Patients with acute renal failure (ARF) and non-ARF patients were compared by Mann-Whitney U-test. Independent predictors of mortality were tested using forward stepwise logistic multiple regression analysis. The discriminative power of different variables was tested using receiver operating characteristic (ROC) curve analysis. RESULTS: ARF developed in 36 patients (35%). ARF patients showed significantly lower HLA-DR expression and higher plasma levels of IL-6, IL-8 and IL-10 than non-ARF patients. In ARF, moderate discriminative power in predicting survival was observed for day 2 IL-6 and IL-10 plasma levels (AUCs 0.703 and 0.749, respectively). CONCLUSIONS: We found no clinically significant discriminative power in predicting survival of ARF patients for monocyte HLA-DR expression or cytokine plasma levels. Therefore, our results do not support the use of HLA-DR expression or cytokine plasma levels for that purpose.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.