Mean Fluorescence Intensity Ratio Research Articles

Ocular surface assessment in soft contact lens wearers; the contribution of tear osmolarity among other tests

To determine whether tear osmolarity contributes to the assessment of the ocular surface in soft contact lens (CL) wearers. Prospective, case-control series in 44 CL wearers (28 tolerant and 16 intolerant) and 34 healthy subjects. Every patient underwent a thorough ophthalmic examination with a tear osmolarity test (TearLab System), conjunctival impression cytology and meibomian lipid sampling. Symptoms, break-up time (BUT), tear osmolarity, conjunctival expression of HLA-DR and meibomian fatty acid composition were evaluated. Tear osmolarity did not differ between controls and CL wearers (p= 0.23). Flow cytometry results expressed in antibody-binding capacity (ABC) units and percentage of positive cells revealed a significant difference between the intolerant CL wearer group and the control group (p< 0.0001). Comparisons between tolerant and intolerant CL wearers showed only a significant difference for mean fluorescence levels expressed in ABC units (p< 0.0001). The BUT was significantly shorter in intolerant and tolerant CL wearers subjects than in healthy subjects (p< 0.0001), whereas there was no significant difference in meibomian fatty acid composition (p= 0.99) between the two groups. Contact lens wear is responsible for ocular surface alterations whose patterns are very similar to those reported in early dry-eye syndrome. However, tear osmolarity was not modified in these selected CL wearers. The yield of tear osmolarity with TearLab™ in assessing ocular surface disorders in CL wearers deserves further investigation.

Surface markers and gene expression to characterize the differentiation of monolayer expanded human articular chondrocytes.

Autologous chondrocyte implantation (ACI) is a method of cartilage repair. To improve the quality of regenerated tissue by ACI, it is essential to identify surface marker expression correlated with the differentiation status of monolayer expanded human articular chondrocytes and to define the index for discriminating dedifferentiated cells from monolayer expanded human articular chondrocytes. Normal human articular chondrocytes were cultured in monolayer until passage 4. At each passage, mRNA expression of collagen type I, II, and X and aggrecan was analyzed by real-time quantitative PCR, and the surface marker expression of CD14, CD26, CD44, CD49a, CD49c, CD54, and CD151 was analyzed by fluorescence-activated cell sorting (FACS). The ratios of mRNA levels of collagen type II to I (Col II/Col I) represented the differentiation status of chondrocytes more appropriately during monolayer culture. The surface marker expression of CD44, CD49c, and CD151 was upregulated according to the dedifferentiation status, whereas that of CD14, CD49a, and CD54 was downregulated. The most appropriate combination of the ratio of Col II/Col I was CD54 and CD44. Cell sorting was performed using a magnetic cell sorting system (MACS) according to CD54 and CD44, and real-time quantitative PCR was performed for the cell subpopulations before and after cell sorting. The expression of collagen type II and aggrecan of the chondrocytes after MACS was higher than that before sorting, but not significantly. The mean fluorescence intensity (MFI) ratio of CD54 to CD44 could be an adequate candidate as the index of the differentiation status.

Novel Negative Selection Marker CD54 Enhances Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

Due to their multi-differentiation potential and immunosuppressive function, mesenchymal stem cells (MSCs) hold huge promise in regenerative medicine. Lack of specific selection markers to isolate MSCs renders their use at risk of fibroblast contamination. The aim of the study was to identify new surface protein markers that can be used for MSC purification during in vitro expansion. With real-time RT-PCR, we demonstrated that primary human dermal fibroblasts expressed CD54 mRNA 10-fold more than early passage human adipose-derived MSCs (AMSCs). Flow cytometry illustrated 88.0% ± 4.1% of dermal fibroblasts strongly expressed CD54 on their surface with a mean fluorescence intensity ratio of 24.0 ± 0.0 compared to 11.0% ± 0.7% and minimal intensity for AMSCs. Evaluation of CD54 sorted AMSCs revealed CD73 expression was 2.2-fold higher in the CD54- versus CD54+ fraction. CD54- AMSCs demonstrated increased adipogenic and osteogenic differentiation potential relative to CD54+ AMSCs. In conclusion, we identified CD54 as a novel selection marker capable of distinguishing MSCs from fibroblasts and thus enhancing MSC osteogenic and adipogenic differentiation potential.

Identification of CRLF2 Genomic Lesions in Patients with Pediatric B-Precursor Acute Lymphoblastic Leukemia (BCP ALL) by Flow Cytometry or Quantitative RT-PCR: A Children's Oncology Group (COG) Stud.

AbstractAbstract 2529▪▪This icon denotes a clinically relevant abstractGenomic alterations involving the CRLF2 gene lead to over-expression of intact CRLF2 and have significant prognostic value in pediatric BCP-ALL. Not only do patients with these lesions have inferior outcomes, they also have a very high frequency of JAK1 and JAK2 mutations and may be candidates for targeted therapies. The two major CRLF2 lesions include cryptic translocations that produce IgH@-CRLF2 and interstitial deletions of the pseudoautosomal region of X/Y causing P2RY8-CRLF2 fusion. Both lesions can be detected by fluorescence in situ hybridization (FISH), and genomic PCR or RT-PCR can identify P2RY8-CRLF2. To develop rapid and inexpensive assays for detection/screening of these events, we developed a flow cytometry based method to measure CRLF2 expression and compared this assay to quantitative RT-PCR (qPCR) measurement of CRLF2 expression by evaluating their performance in an unselected cohort of 279 newly diagnosed pediatric BCP-ALL patients consecutively enrolled on the COG AALL03B1 biology/classification study between 10/30/09-5/1/10. Flow cytometry was performed first in real time on diagnostic specimens shipped to a central COG reference laboratory and then residual diagnostic material was shipped to a separate laboratory for RNA isolation and qPCR analysis. Of the 279 cases analyzed by flow, 257 (92%) yielded sufficient RNA quality and quantity for qPCR analysis. In our previous studies with qPCR and CRLF2 it was shown that CRLF2 lesions occurred only among those cases with the highest expression (ΔCt < 8). In order to assure that we identified all cases with CRLF2 lesions, we performed FISH and P2RY8-CRLF2 PCR on all cases with qPCR expression ΔCt < 10 (n = 109) and an additional 14 cases with a flow blast/lymph CRLF2 mean fluorescence intensity (MFI) ratio >1.15. Of these 123 cases, 11 were determined by FISH to have the IGH@-CRLF2 translocation and 15 were shown to have P2RY8-CRLF2 fusions by PCR. Figure 1 shows the locations of these genomic lesion-positive cases among the qPCR (panel A) and flow cytometry (panel B) CRLF2 expression data. The overall frequency of CRLF2 lesions among these patients is 10.1% (assuming all lesions were identified among the highest expressing cases) and, surprisingly, the frequencies of IgH@-CRLF2 and P2RY8-CRLF2 were very similar (4.3% and 5.8%, respectively). With both methods, the 11 IgH@-CRLF2 cases were found to be the highest expressing (among the top 12 cases by qPCR and 16 cases by flow). Receiver operating curve analysis of each method identified cutoffs with excellent performance: qPCR cutoff CRLF2 ΔCt = 5.47 with 96.9% specificity and 88.5% sensitivity; flow cutoff MFI CRLF2 ratio of 2.04 with 95.9% specificity and 92.3% sensitivity. The broader dynamic range of the qPCR assay may be necessary for the identification of poor risk cases with high CRLF2 expression that lack genomic lesions, however both methods are rapid, highly effective and very comparable for finding ALL cases that harbor CRLF2 genomic lesions, and suitable for incorporation in large scale clinical trials. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Optimized Flow Cytometry to Measure Anti-ABO Immunoglobulin G

Background: An accurate measurement of ABO antibody levels before and after ABO-incompatible organ transplantations is critical. We verified several assay steps of a previously reported protocol using flow cytometry, improved its shortcomings, and developed a more optimized protocol, which we named Flow ABO Ab, for the anti-ABO immunoglobulin G (IgG). Methods: Fixed red blood cells (RBCs) were sensitized with dithiothreitol-treated sera at 4°C. Residual hemagglutination and hemolysis were monitored on a time-dependent plot. Only singlet RBCs were analyzed, excluding RBC agglutinates, to obtain mean fluorescence intensity (MFI) ratios. Results: The sensitivity for anti-ABO IgG was compared between Flow ABO Ab and the column agglutination technique (CAT) in 34 sera diluted to have borderline reactivity of anti-ABO IgG. Flow ABO Ab yielded a positivity rate of 68% (23/34), whereas CAT yielded 50% (17/34) (P = .18). The CAT titer was highly correlated with the mean fluorescence intensity (MFI) ratio of Flow ABO Ab (r = 0.843, P<.001) in 17 undiluted sera. Using Flow ABO Ab, all group A (30 [100%]) and group B (30 [100%]) healthy individuals tested positive for anti-B and anti-A, respectively. Conclusions: Our protocol markedly improved on the previously reported protocol and provided its analytical performance to be comparable to that of CAT, suggesting its potential as an additional effective tool for the measurement of anti-ABO IgG. Further studies are needed to clarify the target MFI ratio for transplantation and whether Flow ABO Ab is preferable to CAT for a correlation with a clinical outcome.

Abstract 200: Time Course of Macrophages Infiltration and Apha 2 Adrenergic Autoreceptor Impairment in DOCA-salt Hypertensive Rats

The development of DOCA-salt hypertension displayed a phasic phenomenon: pre-hypertension, phase 1, phase 2 and established hypertension. We have reported that in established DOCA-salt hypertension rats there were in increased number of pro-inflammatory macrophages (Mϕ) in the mesenteric arteries (MA) adventitia, and the α2-adrenergic autoreceptor (α2AR) regulating norepinephrine (NE) release from sympathetic nerves supplying arteries is impaired. DOCA-salt rats treated with apocynin, an NADPH-oxidase inhibitor, exhibited reduced inflammation and are protected against established hypertension. The proposed studies tested the hypothesis that as blood pressure increased in DOCA-salt rats, Mϕ infiltrated into the adventitia of MA. Mϕ released O2- that disrupted α2AR function causing increased NE release and further increased in blood pressure. Using immunohistochemistry, we detected the number of Mϕ in MA adventitia of phase 1, 2 and established hypertension was 4-5 higher than control (p&lt;0.05). Similarly, the level of O2-, detected with relative ratio of DOCA-Sham mean fluorescence intensity of dihydroethidium, in DOCA-salt (2-2.5) peaked higher than control (1.0) during the same time period (p&lt;0.05). However, using amperometry we found that the normalized NE current in the presence of Idazoxan, an α2AR antagonist, was significantly difference between DOCA and SHAM MA only during Phase 2 and established hypertension (p&lt;0.05). The data suggests in DOCA-salt hypertension development, as the blood pressure increases to Phase 1 hypertension Mϕ is initially recruited to the MA adventitia where they release O2-. Consequently, over a few days period the O2- disrupts the α2AR function causing increased NE release and further increased in blood pressure.

The effects of Castanospermine, an oligosaccharide processing inhibitor, on mononuclear/endothelial cell binding and the expression of cell adhesion molecules

IntroductionIn this study we aimed to determine whether Castanospermine, a transplant immunosuppressive agent, impaired mononuclear/endothelial cell binding and expression of their cell adhesion molecules. MethodsThe binding of human umbilical vein endothelial cells with peripheral blood mononuclear cells was measured by a binding assay using Chromium 51 label; the membrane expression of cell adhesion molecules was measured by flow cytometry expressed as mean fluorescence intensity ratios. ResultsCastanospermine decreased mononuclear/endothelial cell binding if and only if both cell types were treated with Castanospermine: this impairment occurred if endothelial cells were treated with a range of doses of Castanospermine and mononuclear cells were treated with a constant dose of Castanospermine (p<0.001 versus untreated p=0.978) or vice versa (p=0.004 versus untreated p=0.582). Upon human umbilical vein endothelial cells Castanospermine reduced the mean fluorescence intensity ratios of E-selectin (p=0.003), ICAM-1 (p<0.001), ICAM-2 (p=0.004) and PECAM-1 (p<0.001) but increased it for P-selectin (p<0.001). Upon peripheral blood mononuclear cells Castanospermine reduced the mean fluorescence intensity ratios of L-selectin (P<0.001), LFA-1α (p<0.001), VLA-4 (p<0.001), Mac-1 (P<0.001) and CR4 (p<0.001) but increased the mean fluorescence intensity ratios of PSGL-1 (p<0.001) and PECAM-1 (p=0.001). Similar changes in mean fluorescence intensity ratios were found in the subset of lymphocytes and monocytes but the reductions in LFA-1α and VLA-4 on lymphocytes and Mac-1 and CR4 on monocytes were greater. ConclusionsThe reduction in mononuclear/endothelial cell binding mediated by CAST and the reduction in expression of multiple cell adhesion molecules on these cell types help to explain the mechanism of its established immunosuppressive effect.

Cutoff values and data handling for solid-phase testing for antibodies to HLA: Effects on listing unacceptable antigens for thoracic organ transplantation

Application of single-antigen solid-phase immunoassay (SPI) in virtual crossmatch-based organ allocation has been hindered by continued debate over the biologic relevance of detected antibodies and the relationship between cutoff mean fluorescence intensity (MFI) values with crossmatch testing results. To define SPI parameters accurately predicting crossmatch testing, we analyzed a series of anti-HLA antibodies from highly-sensitized patients awaiting lung or heart transplantation. Serial dilution of serum for SPI and cytotoxic crossmatch (CXM) enabled comparison over a wide spectrum of antibody "strengths". Receiver operating characteristic (ROC) analysis identified predictive cutoff values for HLA Class I and DR-specific antibodies. However, antibodies to HLA-DQ antigens demonstrated a significantly different characteristic, highlighting difficulties in interpretation of clinical significance. We also quantitatively characterized two data handling methods, MFI ratio (MR) and relative ratio (RR), to examine their potential impact on identifying unacceptable antigens. In combination with user defined cutoff values, MFI, MR and RR lead to discordant identification of antibodies. Establishment of cutoff values for MR and RR that are comparable to MFI demonstrated increased consistency in antibody identification. This single laboratory experience is an example of establishing statistically robust cutoff values and validation across different data handling methods for use of SPI in virtual crossmatch.

A Resource for the Conditional Ablation of microRNAs in the Mouse

The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here, we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.

ABCB1 genetic variation and P-glycoprotein expression/activity in a cohort of Brazilian acute myeloid leukemia patients

Polymorphisms in the ABCB1 gene may influence P-glycoprotein (Pgp) expression and/or activity. Because the population in Brazil is markedly heterogeneous, we analyzed the relationship between ABCB1 polymorphisms and Pgp expression/activity in Brazilian acute myeloid leukemia (AML) patients. Acute myeloid leukemia samples from 109 patients were studied. ABCB1 gene variants rs1128503 (C1236T) and rs1045643 (C3435T) were analyzed by PCR-RFLP assay. Pgp expression and Pgp activity were analyzed by flow cytometry. There was a similar distribution of Pgp expression and activity on polymorphisms C1236T, C1236C, and T1236T for exon 12, and C3435T, C3435C, and T3435T for exon 26. An exception was observed in the lowest ratio of mean fluorescence intensity (MFI) median for Pgp expression in the TT genotype for both studied exons, and its correspondence to a low MFI median for Pgp activity. Pgp expression did not show impact on the response to remission induction therapy, but the MFI median of Pgp expression in the remission failure group was higher than that of the complete remission (CR) group of patients (p = 0.04). Overall survival (OS) was significantly influenced by CR (p = 0.0001). Better 5-year OS and 5-year event-free survival rates (p = 0.04 and p = 0.007, respectively) were achieved in patients presenting the genetic variant CC in exon 12 followed by those presenting the variant CT in exon 26 (p = 0.001). Polymorphisms in the ABCB1 gene and the levels of Pgp expression could be useful to identify prognostic in AML patients.

Automated Analysis of Multidimensional Flow Cytometry Data Improves Diagnostic Accuracy Between Mantle Cell Lymphoma and Small Lymphocytic Lymphoma

Mantle cell lymphoma (MCL) and small lymphocytic lymphoma (SLL) exhibit similar but distinct immunophenotypic profiles. Many cases can be diagnosed readily by flow cytometry (FCM) alone; however, ambiguous cases are frequently encountered and necessitate additional studies, including immunohistochemical staining for cyclin D1 and fluorescence in situ hybridization for IgH-CCND1 rearrangement. To determine if greater diagnostic accuracy could be achieved from FCM data alone, we developed an unbiased, machine-based algorithm to identify features that best distinguish between the 2 diseases. By applying conventional diagnostic criteria to the flow cytometry data, we were able to assign 28 of 44 (64%) MCL and 48 of 70 (69%) SLL cases correctly. In contrast, we were able to assign all 44 (100%) MCL and 68 of 70 (97%) SLL cases correctly using a novel set of criteria, as identified by our automated approach. The most discriminating feature was the CD20/CD23 mean fluorescence intensity ratio, and we found unexpectedly that inclusion of FMC7 expression in the diagnostic algorithm actually reduced its accuracy. This study demonstrates that computational methods can be used on existing clinical FCM data to improve diagnostic accuracy and suggests similar computational approaches could be used to identify novel prognostic markers and perhaps subdivide existing or define new diagnostic entities.

Trastuzumab (Herceptin®) for Treatment of Refractory/Relapsed HER2 Positive Adult B-ALL: Results of a Phase II GRAALL Study

Abstract 1525Trastuzumab (rhu-mAb-HER2, Herceptin®, F. Hoffmann-La Roche, Basel, Switzerland) is the humanized equivalent of the murine 4D5 monoclonal antibody targeted against the HER2 cell-surface receptor. In combination with chemotherapy, trastuzumab has significantly improved the outcome of women with HER2+ breast cancer. We have previously shown that HER2 surface antigen is up-regulated in around one third of adult B-ALL and is associated with chemoresistance in these patients (Chevallier et al, Haematologica, 2004). We report here for the first time the results of a Phase II study evaluating the safety and efficacy of trastuzumab in refractory/relapsed HER2 + adult B-ALL patients.Prior to patient inclusion, HER2 positivity was assessed using multicolor flow cytometry with the phycoerythrin-conjugated HER2 Neu 24.7 antibody (BD) and a CD19+ CD45+low blast cell gating strategy. The mean fluorescence intensity (MFI) ratio was obtained by dividing the MFI of HER2 with that of its isotypic control. HER2 positivity threshold was defined by a ratio intensity (RI) >= 2. Also, HER2 oncogene amplification was assessed by FISH analysis using the HER2 DNA probe kit (Vysis, Downers Grove, IL, USA). Relapsed/refractory B-ALL patients aged >=18 years and with a HER2+ expression for at least 30% of the leukemic blast population in peripheral blood (PB) and/or bone marrow (BM) were included. Left ventricular ejection fraction has to be > 50%. All patients gave informed consent and the protocol was approved by the required regulatory authorities. The trial was registered at http://clinicaltrials.gov/ct no.NCT00724360. Trastuzumab was administered according to the approved schedule in breast cancer patients at 4 mg/kg intravenous loading dose followed by 2 mg/kg weekly. There was no corticosteroid premedication. Trastuzumab was provided by Roche.Out of 50 patients screened for the HER2 expression, 15 patients (30%) (male n=8; female n=7), with a median age of 62 years (range: 24–80), have been included in the study between November 2006 and July 2011. This was considered as a very high-risk population: 2 patients had refractory disease after 2 induction courses, 2 patients were in first untreated relapse while 11 patients had a refractory first relapse or were beyond first relapse. Median percentage of HER2+ leukemic blast population was 94% (range: 0–100) in PB and 100% (range: 31–100) in BM. Surprisingly, no HER2 gene amplification was detected in samples assayed by FISH. Normal and complex karyotype were detected in 7 and 3 patients respectively. Three patients had a Phi+ B-ALL, and 1 patient a monosomy 7 (unknown karyotype n=1).Currently, 3 patients are still receiving therapy. Considering the 12 other patients, the median number of trastuzumab infusions was 4 (1 month of treatment) (range: 2–20). No grade 3–4 toxicities were observed, with no cardiotoxic events. The overall response rate (CR or PR (decrease >=50% of blast population) or blast clearance in BM or PB) was 33%. No CR was observed. Two patients achieved partial response in BM (92% vs 12% after 9 infusions with loss of HER2 expression on blast population, total number of infusions (TNI) n=18); 25% vs 11% after 4 injections, TNI n=13) while blast clearance was observed in 2 other patients (96% vs 57% of blasts in BM after 8 infusions with loss of HER2 blast expression, TNI n=20; 20.5% vs 2% of blasts in PB after 3 infusions, TNI n=3).In all, we conclude that trastuzumab in monotherapy can allow for some responses in this very high-risk refractory/relapsed HER2+ adult B-ALL population. Combination of trastuzumab with chemotherapy or other therapeutic monoclonal antibodies should be tested in the future. An updated follow-up of the study will be provided during the meeting. Disclosures:No relevant conflicts of interest to declare.

2.51 CD38, CD44 and β1 Integrins - CD49d, CD49c and CD29 in Different Microenvironment Have Different Pattern of Expression and Relationship to Survival

Background: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by variable clinical presentations with different involvement of various lymphoid compartments, including peripheral blood, bone marrow and lymphoid organs such as lymph nodes and spleen. This variable distribution of tumor mass has strong association with prognosis. There is also well documented intraclonal and interclonal variability in B-CLL cells within different microenvironments regarding a number of surface and intracellular molecules (including molecules with adhesion properties, for example CD38). Aims: 1) to evaluate in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) the pattern of expression of receptors with known adhesion properties and their influence on survival (notably CD38 and CD49d), and to identify adhesion molecules that reportedly co-localize with them forming macromolecular complexes (CD38, CD49d, CD44 and MMP9); 2) to evaluate the mutual relationship between these patterns and 3) to assess their relation to other clinical parameters, including survival. Methods: peripheral blood, bone marrow and lymph node samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression levels of CD38, CD49d, CD49c, CD29 and CD44 on B-CLL cells were analyzed by flow cytometry. Results were expressed as ratio of mean fluorescence intensity (MFI) of specific antibody and negative control (MFI-R) and percentage of positive cells and analyzed by paired tests. Differences between compartments were also quantified by logarithmic ratios of MFI. Results: We analyzed samples taken from 33 typical B-CLL patients with median age of 65 years. There were 18 males and 15 females. Mean TTM size was 10.4 and mean TD was 0.62. There were 11, 14 and 8 patients in Binet stages A, B and C, respectively. Median follow-up of patients was 48 months and maximum 91 months. There was a significantly higher expression of CD38 in LN compared to BM and PB (p 0.05), CD44 expression was lower in LN compared to PB (p 0.05) and CD44v4 higher in LN than in PB and BM (p 0.05). For CD29, CD49d and CD49c there was no significant differences between compartments. We then analyzed statistically significant correlations between these receptors within each lymphoid compartment. In PB correlation was very high between CD29 and CD49d, high between CD38 and CD49d, CD38 and CD29, and CD49d and CD49c, and moderate between CD49c and CD29, CD49c and CD38 (p 0.05). There was a moderate correlation between CD44 and CD29, and low correlation between CD44 and CD49d, and CD44v4 and CD49c (p 0.05). In BM we observed the same pattern of correlations, although to a lesser degree, between CD29, CD49c, CD49d and CD38, and CD44 and CD29 (p 0.05). However, CD44v4 showed moderate correlation with CD44, and low correlation with CD49c, CD49d and CD29 (p 0.05). In LN as in BM there was the same pattern of correlations between CD29, CD49c, CD49d and CD38, and CD44 and CD29 (p 0.05). Higher CD38 expression in all lymphoid compartments, even in this small group of patients, showed a relationship with shorter survival (p 0.05). However, patients with higher expression of CD49d in BM and LN than in PB showed superior survival compared to patients with the opposite pattern of expression (p 0.05). Conclusions: There is a correlation of expression levels between analyzed molecules, although lower than between CD29 and CD49d, which form an obligate heterodimer. Also, these molecules show different level and pattern of expression between lymphoid compartments and show different relationships with survival. These results call into question the extent to which these molecules form macromolecular complex as opposed to standalone molecules, and determining the biological relevance of this putative macromolecular complex warrants further studies.

Protective effects of tert-butylhydroquinone on sodium arsenite-induced cytotoxicity and oxidative injuries

Objective To study the protective effects of tert-butylhydroquinone(tBHQ) on sodium arsenite (NaAsO2)-induced cytotoxicity and oxidative injuries. Methods Chang liver cells were pretreated with tBHQ[0(control), 5, 25 μmol/L]for 24 h, and then co-treated with tBHQ(5 μmol/L) together with NaAsO2[0(control),30, 40, 50, 60 μmol/L] for another 24 h, and Alamar blue reduction rates were used to evaluate cell viability,the results were expressed as the relative ratio of Alamar blue reduction rates between the experimental group and the control group. On the other hand, Chang liver cells were pretreated with tBHQ[0(control), 5, 25 μmol/L] for24 h,and then co-treated with tBHQ(5 μmol/L) together with NaAsO2[0(control), 40, 50 μmol/L] for another 24 h,and the levels of cellular reactive oxygen species(ROS) were detected by staining cells with 2',7'-dichlorofluorescin diacetate(DCFH-DA), the results were expressed as the relative ratio of mean fluorescence intensity between the experimental group and the control group. Results Cell viability decreased dramatically by treatment with NaAsO2(30, 40, 50, 60 μmol/L), while relieved to some extent by pretreatment with 5, 25 μmol/L tBHQ, the main effects of NaAsO2 and tBHQ, as well as their interaction were all statistically significant(F =566.57, 55.09, 14.50,all P ＜ 0.05) ; the cell viability of NaAsO2(30, 40, 50, 60 μmol/L) pretreated with tBHQ(5, 25 mol/L) were 0.75 ±0.02, 0.70 ± 0.04, 0.59 ± 0.03, 0.43 ± 0.03 and 0.75 ± 0.02, 0.73 ± 0.03, 0.65 ± 0.02, 0.50 ± 0.02, respectively,all significantly higher than corresponding NaAsO2 alone groups(0.70 ± 0.03, 0.64 ± 0.03, 0.43 ± 0.03, 0.33 ±0.01, all P ＜ 0.05), the cell viability of NaAsO2(50, 60 μmol/L) pretreated with 25 μmol/L tBHQ was higher than corresponding 5 μmol/L tBHQ pretreatment groups(all P ＜ 0.05). On the other hand, 40, 50 μmol/L of NaAsO2 significantly induced hepatocellular ROS generation, while tBHQ(5, 25 μ mol/L) pretreatment significantly decreased NaAsO2-induced intracellular ROS levels, the main effects of NaAsO2 and tBHQ, as well as their interaction were all statistically significant (F =181.78, 60.55, 4.93, all P ＜ 0.05) ; the ROS levels of NaAsO2(40, 50 μ mol/L) pretreated with tBHQ(5, 25 μmol/L) were 1.87 ± 0.09, 1.80 ± 0.07 and 1.36 ± 0.11, 1.44 ± 0.12,all significantly decreased than corresponding NaAsO2 alone groups(2.30 ± 0.18, 2.18 ± 0.17, all P ＜ 0.05),the ROS levels of NaAsO2(40, 50 μmol/L) pretreated with 25 μmol/L tBHQ decreased than corresponding 5 μmol/L tBHQ pretreatment groups (all P ＜ 0.05). Conclusion tBHQ has a certain antagonism on arsenic induced cytotoxicity and oxidative injuries. Key words: Arsenites; Tert-butylhydroquinone; Reactive oxygen species

Analysis of CD177 neutrophil expression in β-thalassemia patients

Ineffective erythropoiesis plays a well established role in the pathophysiology of disease expression in β-thalassemia major and intermedia. CD177 expression was investigated in different clinical conditions. The study aimed to analyze neutrophil expression of CD177 in β-thalassemia patients and its correlation with serum soluble transferrin receptor (s-TfR) concentration as a marker for the extent of erythropoiesis and hence disease severity in these patients. Flow cytometric analysis of neutrophil CD177 expression and enzyme immunoassay measurement of serum s-TfR concentration were assessed in 45 β-thalassemia patients of whom 36 had β-thalassemia major and nine had β-thalassemia intermedia. They were also assessed in 21 age- and gender-matched control children. Neutrophil mean fluorescence intensity ratio (MFIR) of CD177 expression was significantly higher in patients than in controls (p < 0.001). There was highly significant increase in serum s-TfR concentration in β-thalassemia patients than in controls (p < 0.001). There was a highly significant positive correlation between MFIR of CD177 expression and serum s-TfR concentration in β-thalassemia patients (r = 0.59, p < 0.001). Elevated CD177 expression is not only a specific feature of polycythemia rubra vera (PV) but may be also an indicator of increased erythropoietic activity in thalassemia syndromes.

Detection methods of ZAP-70 in chronic lymphocytic leukemia

The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous, with some patients experiencing rapid disease progression and others living for decades without requiring treatment. Immunoglobulin heavy chain variable region (IGHV) mutation status is a powerful prognostic factor in patients with CLL. The presence or absence of IGHV mutation status is currently the gold-standard prognostic factor, but this technique is labor-intensive and costly. The expression of ζ-chain-associated protein kinase 70kDa (ZAP-70), detected by flow cytometry, has been served as a potential surrogate for the absence of IGHV mutation status since 2003. Given the current problems with the standardization of ZAP-70 assessment by flow cytometry, this chapter reviews the analysis of ZAP-70 expression by flow cytometry and the relative experimental conditions using the associated publications for "ZAP-70" and "detection method" on Medline since 2003. Thus, mean fluorescence intensity (MFI) ratio method yielded a more reproducible and easily adaptable method to routine use in the clinical diagnostic laboratory. In conclusion, though ZAP-70 is a valuable prognostic factor in CLL, detection methods of ZAP-70 remain to be fully standardized.

Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL). However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method. The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods. Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%). Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2. Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of T-cells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5). MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074). The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods. Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012; ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019). In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course. Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient´s prognosis.

B Cell Stimulation through BCR and CD40 Modulates the Response of Chronic Lymphocytic Leukemia Cells to BAFF and APRIL.

AbstractAbstract 1361The two TNF family proteins (B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL]) and their three receptors (transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R]) play a critical role in the process of differentiation, maturation and survival of normal B cells. Additionally, recent studies indicate that activation or inhibitory signals can modulate the sensitivity of normal B cells to BAFF and APRIL through the regulation of their receptors. In chronic lymphocytic leukemia (CLL), BAFF and APRIL have been shown to increase survival of neoplastic B cells in vitro. We investigated whether stimulation of CLL cells through the B cell receptor (BCR) or CD40 ligation could regulate the expression of BAFF-R, TACI and BCMA and enhance BAFF and APRIL sensitivity. Purified B cells were obtained from 23 CLL patients and nine healthy controls. Receptor expression was measured by flow cytometry at baseline and at 48 hours after stimulation with F(ab')2 antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling CD69 and Annexin V/TO-PRO-3, were evaluated at 48, and at 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). Baseline analyses showed that BAFF-R was the most highly expressed receptor in CLL cells and normal B cells (Mean fluorescence intensity (MFI) ratios, 213.5 and 185.8, respectively). TACI and BCMA were also expressed in all CLL cells and normal B cells (MFI ratios TACI: 2.5 and 1.9; BCMA: 14.8 and 6.6, respectively), but at a significantly lower level than BAFF-R (p<0.001). Furthermore, BCMA MFI ratio was significantly higher in CLL than in normal B cells (p=0.015). After 48h of culture, an increase of all three receptors was observed in normal B cells in response to either BCR stimulation or CD40 ligation. In contrast, in CLL cells BCR stimulation induced almost no variation in the receptors expression in all cases. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). By contrast, CD40 ligation in CLL cells induced a significant upregulation of TACI expression (p=0.007) and a significant reduction of BCMA (p=0.007), which correlated with an increase of CLL cell activation and viability (p<0.001). BAFF-R levels did not change. The addition of exogenous soluble BAFF or APRIL showed increase in the viability of normal B cells at 72 hours independently of whether cells were unstimulated or stimulated through the BCR or by CD40 ligation. In CLL cells, however, the viability was significantly increased in CD40-stimulated cells whereas in either unstimulated or BCR-stimulated CLL cells, the addition of BAFF and APRIL had a modest effect on viability (Table).These findings indicate that stimulation of CLL cells through the BCR and CD40 modifies the sensitivity of CLL cells to respond to BAFF and APRIL which reflects the regulation of BCMA, TACI and BAFF-R. In contrast to normal B cells, CD40-ligation in CLL cells upregulated only TACI expression. The fact that the addition of CD40L plus IL-4 and BAFF increased viability in CLL cells while BAFF alone had almost no effect may be related to the ability of CD40 ligation to increase TACI expression. Although BCR stimulation failed to increase the expression of the receptors, co-stimulation by BAFF plus BCR increased viability in CLL cells. Disclosures:No relevant conflicts of interest to declare.

CD133+ Acute Myeloid Leukemia (AML) Cells Exhibit Higher Clonogenic Capacity and Higher Levels of pAKT and Bcl-2 Proteins Than Their Negative Counterpart

AbstractAbstract 1721 Introduction:CD133 is a transmembrane protein expressed by hematopoietic stem cells and other cell types, whose physiological functions remain unknown. To determine its potential prognostic value in AML, we analyzed its expression on blasts from 76 patients at diagnosis. Moreover, since leukemic stem cells play a critical role in the resistance to chemotherapeutic drugs and may be responsible for relapse, we compared the growth potential and activated cellular pathways of CD133+ and CD133- cells from 20 AML patients. Methods:First, bone marrow samples were collected at diagnosis from 76 AML patients (median age: 65, range: 1–84). CD133 expression was evaluated on blasts cells and CD34+ cells using 6-color flow cytometry (FC) and the monoclonal antibody AC133. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the MFI of CD133 by the MFI of its isotypic control. Second, purified CD34+, CD34+CD133+ and CD34+CD133- cells were isolated from 20 AML bone marrows using the magnetic separation system Minimacs (Miltenyi Biotec). These fractions were submitted to an in vitro colony forming assay during 14 days. Finally, the level of expression of survival proteins involved in the PI3K/AKT, Mitogen-Activated Protein Kinase (MAPK) and Bcl-2 pathways, as well as CXCR4 and Focal Adhesion Kinase (FAK) migration proteins, was analyzed by FC in these fractions. Results:The level of CD133 expression on blasts cells was significantly different between AML patients with favorable (n=12), intermediate (n=38) and unfavorable karyotypes (n=23) (p=0.007). In particular, it was significantly higher in patients with unfavorable karyotype than in patients with intermediate karyotype (p=0.032) or favorable karyotype (p=0.005). Moreover, when considering NPM1 and FLT3 mutation status in patients with intermediate karyotype, the level of CD133 expression was significantly higher in patients with an intermediate molecular profile than in patients with a favorable molecular profile (p=0.004). Most interestingly, we showed that the level of CD133 expression has an impact on overall survival in multivariate analysis, independently of leukocytosis, karyotype and achievement of complete remission after induction therapy (p=0.04). Second, in 20 patients, we found that AML progenitor cells (AML CFU) were present in all sorted fractions, but the CD34+CD133+ fraction gave rise to larger and more numerous colonies (74.9±39.6) than the CD34+CD133- fraction (11,25±6). Moreover, the CD34+CD133+ fraction was the only one able to form secondary colonies (52.1±31.8). Finally, we found that the expression of PI3K (mean percentage of positive cells: 78±20.1), pAKT (80±19.8), pERK (80.1±19), CXCR4 (71.6±22.7), pFAK (81.2±19.8), Bcl-2 (72.9±26.1), Bcl-xL (45.7±24.8), and pBad (63.9±36.1) was significantly upregulated in CD34+CD133+ cells compared to CD34+CD133- cells, whereas Bax was downregulated. Discussion:CD133 is known to be expressed in hematopoietic progenitors, but its role in the resistance to chemotherapy is not well studied. In our work, the level of expression of CD133 on AML blasts appeared as an independent immunophenotypic prognostic factor, encouraging its further evaluation in a larger number of cases. We also showed that CD133+ cells have greater replicative properties than CD133- cells, and exhibit activation of various signaling pathways. Since CD133+ cells might escape from apoptosis through activation of PI3K/AKT and ERK, these proteins could represent potential therapeutic targets. In conclusion, our results revealed that CD133+ cells exhibit more aggressive behavior than CD133- ones, and that CD133+ AML progenitors might contribute to resistance to chemotherapy through preferential activation of PI3K/AKT, MAPK and Bcl-2 survival response. Disclosures:No relevant conflicts of interest to declare.

ROR1 Is Expressed on the Surface of Non-Neoplastic Human B-Lymphocyte Precursors (Hematogones) and on a Subset of B Acute Lymphoblastic Leukemia

AbstractAbstract 2735ROR1 is a putative receptor tyrosine kinase that is expressed during embryogenesis, on chronic lymphocytic leukemia (CLL) cells, but not on normal mature lymphocytes, including normal CD5+ B cells (Fukuda et al PNAS 2008; 105:3047). ROR1 binds Wnt5a, and this ligand enhances the survival of CLL cells in vitro, suggesting that ROR1 functionally contributes to leukemogenesis. Additional evidence for the importance of ROR1 function in leukemia is the noted decrease in survival of B acute lymphoblastic leukemia (B-ALL) cells in vitro after RNAi targeting of ROR1 (Tyner et al. PNAS 2009; 106:8695). To further explore the significance of ROR1 in B-ALL, we analyzed 30 cases of B-ALL for surface expression of ROR1 via multiparameter flow cytometry using a fluorochrome-conjugated mAb specific for ROR1. Only two of 30 cases of B-ALL showed uniform surface ROR1 staining with a mean fluorescence intensity (MFI) that was 6.2 or 3.6 times greater than that of B-ALL cells stained with a control mAb. The other 28 B-ALL cases showed a mean MFI ratio of 1.4 (S.D. = 0.4). The two ROR1+ cases shared distinctive immunophenotypic and cytogenetic changes consistent with a more mature B-ALL subtype. We also evaluated marrow from patients recovering from chemotherapy or hematopoietic stem-cell transplantation and found that non-neoplastic B-lymphocyte precursors (BLP, hematogones) also expressed surface ROR1. These BLP were positive for CD10, CD19, CD38, and dim CD45 with variable expression of CD20, CD34, TdT, and cytoplasmic immunoglobulin. While the surface antigen phenotype of B-ALL is similar to BLP, flow cytometry can distinguish B-ALL from BLP because B-ALL shows more uniform antigen expression, lacking the continuum of maturation stages present in BLP. Also, B-ALL usually has aberrant antigen-expression intensity or aberrant antigen co-expression such as terminal deoxynucleotidyl transferase (TdT) plus CD20 on the same cell. Of 22 marrow samples tested with 1–20% hematogones, all had CD10+, CD19+, dim CD45+ cells that expressed ROR1 with a mean MFI ratio of 4.9 (S.D. 2.0). Further study found ROR1 was restricted to a subset of CD10, CD19, and low-level CD45 cells that lack expression of CD34 or TdT. This subset is consistent with an intermediate stage of B-cell maturation, possibly associated with B-lymphoid positive and negative selection. In contrast, CD10+, CD19+ germinal center B cells from reactive lymph nodes were negative for surface ROR1 (N=4). This pattern of ROR1 expression on BLP and a subset of distinctive B-ALL suggests that surface expression of ROR1 occurs during a discrete stage of B-cell maturation in the marrow that may be expanded during recovery from myeloablative therapy and/or hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.