Coping skills of olympic developmental soccer athletes.

Athletes at Olympic Developmental Program (ODP) camps experience unusually high levels of expectations and inherent mental and physical challenges within such a short span of time. With the increasing emphasis on talent development, there has been consensus by the ODP staff to more clearly define present levels of coping skills, in order to enhance athletic prediction, maximize training efforts, identify the predisposition to injury, and focus on areas pertinent to successful performance. This study examined athletic and pain coping skills of U. S. ODP soccer athletes not previously investigated. Following written informed consent, 70 males completed the Athletic Coping Skills Inventory and the Sports Inventory for Pain. Data were analyzed by competitive level (U-14, U-15), and skill position (goalkeeper/defense, midfield/foward). MANOVA indicated a significant main effect across competitive level (Wilks' Lambda F(12,57) = 2.27; p = 0.02; n-beta = 0.915) but no significant effect by skill position (Wilks' Lambda F(12,57) = 0.931; p = 0.523; n-beta = 0.457). Post hoc analyses indicated that U-15 athletes scored significantly higher in concentration (p = 0.01) and body awareness (p = 0.03), but lower in avoidance (p = 0.01) than U-14 competitors. In conclusion, older, more experienced athletes revealed more positive athletic and pain coping skills than younger, less experienced athletes, although athletes in skill positions requiring spontaneous decision-making skills and split-second adjustment in a constantly changing sport environment (forwards, midfielders) did not exhibit more positive athletic and pain coping skills than those positions requiring reaction and protection (defenders, goalkeepers).

S-phase modulation by irinotecan: pilot studies in advanced solid tumors.

Two studies of irinotecan (CPT-11) followed 24 h later by an antimetabolite were conducted. The objectives of the studies were: (1) to determine whether the increase in S-phase in tumor cells seen 24 h after CPT-11 administration in animal studies is seen in advanced solid tumors in patients, (2) to determine the dose of CPT-11 required to produce this effect, (3) to compare two methods (immunohistochemistry, IHC, for cyclin A, and DNA flow cytometry, FC) for evaluating S-phase in tumor biopsies from patients, and (4) to establish the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of CPT-11, given 24 h before gemcitabine (GEM, 1000 mg/m(2)). In one study CPT-11 was followed 24 h later by 5-fluorouracil (5-FU), 400 mg/m(2) per week for 4 weeks every 6 weeks. Tumor biopsies were obtained before and 24 h after CPT-11 administration before administration of 5-FU and assayed for S-phase by IHC for cyclin A and by FC. The starting dose of CPT-11 was 80 mg/m(2) per week with subsequent exploration of 40 and 60 mg/m(2) per week to establish the dose-effect relationship of the increase in tumor cells in S-phase. In the second study, CPT-11 was given 24 h before GEM 1000 mg/m(2) per week for 2 weeks every 3 weeks. Doses of 20-80 mg/m(2) were explored to establish the MTD and DLT and to study tumor cell S-phase in selected patients. CPT-11 80 mg/m(2) produced a mean increase in S-phase by IHC for cyclin A of 137%. Lesser increases were seen with 40 and 60 mg/m(2). CPT-11 followed 24 h later by 5-FU 400 mg/m(2) per week for 4 weeks was well tolerated. In the study of CPT-11 followed by GEM 1000 mg/m(2), 60 mg/m(2) of CPT-11 was the MTD.

HLA-DR antigen-negative acute myeloid leukemia.

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter flow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and non-APL cases, and that the morphology of HLA-DR-negative non-APL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular confirmation.

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia.

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40 degrees C for 6-12 h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1beta, MIG). This is in contrast to the stimulatory effects of TNF-alpha or 43 degrees C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the alpha4beta7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.

Acute myeloid leukemia in the setting of low dose weekly methotrexate therapy for rheumatoid arthritis.

Methotrexate is in widespread use as second-line therapy for rheumatoid arthritis. Treatment with methotrexate in this and other settings has not been associated with the development of therapy-related leukemias. Four patients with rheumatoid arthritis are reported who developed acute myeloid leukemia (AML) while receiving low dose weekly methotrexate therapy in the absence of previous or concomitant treatment with known leukemogenic agents. AML in these four patients was of different morphologic subtypes and was associated with heterogeneous cytogenetic abnormalities, cell surface marker expression and multidrug resistance protein expression. None of the recognized features of therapy-related leukemia were present in these four nor in five previously-reported patients. It is likely that the occurrence of AML in patients with rheumatoid arthritis in the setting of methotrexate therapy represents the coincidence of these two diseases, and does not reflect a causal relationship.

Erythropoietin-dependent transformation of myelodysplastic syndrome to acute monoblastic leukemia.

Acute monoblastic leukemia (acute myeloid leukemia [AML], French-American-British type M5a) with leukemia cutis developed in a patient 6 weeks after the initiation of erythropoietin (EPO) therapy for refractory anemia with ringed sideroblasts. AML disappeared from both marrow and skin after the discontinuation of EPO. Multiparameter flow cytometric analysis of bone marrow cells demonstrated coexpression of the EPO receptor with CD45 and CD13 on the surface of blasts. The incubation of marrow cells with EPO, compared to without, resulted in 1.3- and 1.6-fold increases, respectively, in tritiated thymidine incorporation and bromodeoxyuridine incorporation into CD13(+) cells. Clinical and laboratory findings were consistent with the EPO-dependent transformation of myelodysplastic syndrome (MDS) to AML. It is concluded that leukemic transformation in patients with MDS treated with EPO may be EPO-dependent and that management should consist of the discontinuation of EPO followed by observation, if clinically feasible.

High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).

Multiparameter flow cytometry (MFC) has the potential to allow for sensitive and specific monitoring of residual disease (RD) in acute myeloid leukemia (AML). The use of MFC for RD monitoring assumes that AML cells identified by their immunophenotype at diagnosis can be detected during remission and at relapse. AML cells from 136 patients were immunophenotyped by MFC at diagnosis and at first relapse using 9 panels of 3 monoclonal antibodies. Immunophenotype changes occurred in 124 patients (91%); they consisted of gains or losses of discrete leukemia cell populations resolved by MFC (42 patients) and gains or losses of antigens on leukemia cell populations present at both time points (108 patients). Antigen expression defining unusual phenotypes changed frequently: CD13, CD33, and CD34, absent at diagnosis in 3, 33, and 47 cases, respectively, were gained at relapse in 2 (67%), 15 (45%), and 17 (36%); CD56, CD19, and CD14, present at diagnosis in 5, 16, and 20 cases, were lost at relapse in 2 (40%), 6 (38%), and 8 (40%). Leukemia cell gates created in pretreatment samples using each 3-antibody panel allowed identification of relapse AML cells in only 68% to 91% of cases, but use of 8 3-antibody panels, which included antibodies to a total of 16 antigens, allowed identification of relapse AML cells in all cases. Thus, the immunophenotype of AML cells is markedly unstable; nevertheless, despite this instability, MFC has the potential to identify RD in AML if multiple antibody panels are used at all time points. (Blood. 2001;97:3574-3580)

Stereotactic core biopsy breast and blood cell by-products: a source of material for molecular genetics research--initial experience.

Stereotactic core biopsy washings and blood drop samples, routinely discarded by-products, provide satisfactory fresh cellular material for flow cytometry and molecular genetics microsatellite polymerase chain reaction (PCR) analysis for detection of loss of DNA alleles (loss of heterozygosity). Cytokeratin-positive (epithelial) cells from the core biopsy washings were sorted by means of flow cytometry prior to PCR analysis. DNA allele loss was detected in benign breast epithelial cells in three (20%) of 15 patients.

HLA class I antigen cell surface expression is preserved on acute myeloid leukemia blasts at diagnosis and at relapse.

Human leukocyte antigens (HLA) class I molecules restrict the interaction between cytotoxic T cells and target cells. Abnormalities in HLA class I antigen expression and/or function may provide tumor cells with a mechanism for escaping immune surveillance and resisting T cell-based immunotherapies. The potential for applying T cell-based immunotherapy in the treatment of acute myeloid leukemia (AML) has stimulated interest in analyzing HLA class I antigen expression on leukemic blasts in this disease. Little information is available in the literature. We have analyzed HLA class I antigen expression on bone marrow samples from 25 newly diagnosed AML patients by indirect immunofluorescence staining with monoclonal antibodies. Five of these patients were also studied at relapse. Leukemic blasts were resolved from normal lymphocytes by staining with antiCD45 antibody; CD45 expression is dim on leukemia cells, but bright on lymphocytes. HLA class I antigen expression was higher on leukemic blasts than on autologous lymphocytes in all but one case. Moreover, there was no significant change in HLA class I antigen expression at relapse. These results suggest that abnormalities in HLA class I antigens are infrequent in AML and should not represent a major obstacle to the application of T cell-based immunotherapies in this disease.

Multiparameter data acquisition and analysis of leukocytes by flow cytometry.

Each laboratory has to establish its own experience base and standard operating procedures. The intent of this discussion has been to illustrate the procedures that will lead to good flow cytometry data acquisition and analysis and to illustrate problematic areas. The most important rule of all is to recognize when there is a problem and find the correct solution. It is hoped the information provided herein will be of help in the recognition process.

Titering antibodies.

Flow cytometry using fluorochrome-conjugated antibodies has emerged as a major approach to automated cellular identification. One of the most important issues in immunophenotyping is using the correct amount of antibody. This unit presents techniques for ascertaining the optimal titer for individual, dual, and multiple antibodies used for simultaneous phenotyping, stressing the importance of quality control in making batches of antibody for routine use.

Immunophenotyping.

This unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two-, three-, and four-color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis.

Use of specimen mammography-guided FNA (fine-needle aspirates) for flow cytometric multiple marker analysis and immunophenotyping in breast cancer.

A pilot study of a novel translational research method to simultaneously assay multiple molecular markers and DNA in fine-needle aspirates (FNA) of mammographically detected breast lesions is described. Specimen mammography-guided 20-gauge FNAs obtained from 86 lesions and 22 areas of normal tissue were analyzed by multiparameter flow cytometry for DNA content, her2/neu, transforming growth factor alpha (TGF alpha), and the epithelial marker cytokeratin (CK) simultaneously. Epithelial cell her2/neu positivity was detected in 12 of 44 (27%) of invasive ductal carcinomas and 3 of 9 (33%) ductal carcinoma in situ (DCIS), 10 of 30 (33%) benign lesions, and 4 of 22 (18%) normal tissue aspirates. All lesions and normal tissue showed a similar positive rate for TGFalpha ranging from 61 to 76%. The CK(+)TGF alpha(-)her2/neu(+) immunophenotype was more frequently positive in aneuploid tumors (22%) than all other lesions (7%) (P < 0.05). Specimen mammography-guided FNAs provide fresh cells for flow cytometric multiple marker analysis and immunophenotyping of clinically occult breast lesions and normal tissue.

Immunoreactivity of MIC2 (CD99) in acute myelogenous leukemia and related diseases.

MIC2 is characteristically expressed in lymphoblastic lesions and Ewing's/primitive neuroectodermal tumor sarcomas. Although MIC2 has recently been reported in chloroma and rare terminal deoxynucleotidyl transferase-positive acute myelogenous leukemia (AML), the incidence and the significance of MIC2 (CD99) immunoreactivity in myeloid lesions is not clear. In this study, we evaluated MIC2 positivity in a variety of myeloid diseases and normal marrow to determine its incidence and distribution in myeloid diseases; its correlation with flow cytometric and cytogenetic data in AML; and its association with leukemic transformation, relapse, and chloroma formation. Paraffin sections of 11 chloromas and 94 bone marrow core biopsies from 66 patients were stained with CD99 monoclonal antibody 12E7. Of 94 bone marrow core biopsies, there were 30 AML (fragment antigen binding M0 to M6), 23 remissions, 5 relapses, 12 myeloproliferative disorders, 13 myelodysplastic syndromes, and 11 normal marrows from patients who did not have leukemia. CD99 immunoreactivity was evaluated with light microscopy. MIC2 expression was seen in leukemic blasts in 6 of 11 chloromas (55%) and 13 of 30 AML (43%) but rarely in myeloproliferative disorders, myelodysplastic syndromes, remission, and normal marrow. CD99 tended to be positive in M1-, M3-, and HLA-Dr-negative AML and negative in AML with relapse. MIC2 expression did not correlate with the karyotype independent of French-American-British Cooperative Group classification and the disease remission or occurrence of chloroma in AML. We concluded that MIC2 is commonly expressed in leukemic blasts of AML and is not predictive of leukemic transformation from myeloproliferative disorders and myelodysplastic syndromes or chloroma formation. Caution should be taken when using MIC2 as a marker for Ewing's sarcoma/ primitive neuroectodermal tumor or lymphoblastic lymphoma on paraffin sections of either soft tissue or bone marrow specimens.

Flow cytometric DNA analysis of specimen mammography-guided fine-needle aspirates of ductal carcinoma in situ.

Flow cytometric studies of screening mammography detected ductal carcinoma in situ (DCIS) are limited by the lack of fresh cell samples. We have performed flow cytometric DNA analyses of specimen mammography-guided fine-needle aspirates of 50 consecutive DCIS lesions detected by screening mammography. The comedo histologic subtype had an aneuploidy rate of 39% (9 of 23); noncomedo subtypes had an aneuploidy rate of 19% (5 of 27), p=ns. Noncomedo subtypes were more likely to have low (less than 2.2%) S-phase percentages, 59% (16 of 27) as compared to comedo, 9% (2 of 23), p<0.05. High and intermediate nuclear grade DCIS lesions had an insignificantly greater rate of aneuploidy, 35% (9 of 26) and 33% (4 of 12) respectively, as compared to low nuclear grade lesions, 8% (1 of 12), p=ns. Low and intermediate nuclear grade DCIS lesions had low S-phase percentage rates of 67% and 50% respectively, as compared to the high nuclear grade lesions low S-phase percentage rate, 15%, p=ns. Aneuploidy and lesser rates of low S-phase percentages were significantly associated with necrosis and apoptosis. Our data suggest that flow cytometric DNA analysis of mammographic lesion-specific, fresh cell samples obtained by fine-needle aspiration under specimen mammographic guidance can assess mammography-detected DCIS lesions when gross fresh tissue procurement is not possible.

Aberrant antigen expression detected by multiparameter three color flow cytometry in intermediate and high grade B-cell lymphomas.

The aberrant expression of antigens (Ag) in lymphoproliferative disorders may cause a diagnostic problem when single parameter immunohistochemical assays are performed on frozen or paraffin sections because coexpression by relevant cells is not determined. This aberrant expression also raises the question as to whether mixed lineage (biphenotypic) lymphoid proliferations exist. Marrow (6) and extramedullary (20) tissues from 26 patients with diffuse, intermediate and high grade, B-cell lymphomas (IWF E=1, F=1, G=19, H=1 and J=4) were analyzed with 19 markers using 3-color flow cytometry. The percentages (%) of patients with double Ag coexpression in at least 20% of the CD19+ or CD20+ lymphoma cells were: stem cell (SC) Ag: CD10 = 58 and CD34 = 15; T-cell Ag: CD2 = 38, CD5 = 19 and CD7 = 19; myeloid (My) Ag: CD13 = 19 and CD33 = 8. The corresponding % with unusual triple Ag coexpression in at least 10% of the CD19+ B-cells were SC+T+ Ag: CD10CD2 = 50, CD10CD5 = 27, CD10CD7 = 38, CD34CD2 = 31, CD34CD5 = 19 and CD34CD7 = 27; T+T+ Ag: CD2CD5 = 35, CD2CD7 = 42 and CD5CD7 = 31; T+My+ Ag: CD2CD13 = 35 and CD2CD33 = 12; and My+My+ Ag: CD13CD33 = 12. Ten of 12 lymphomas tested showed clonal immunoglobulin (Ig) heavy chain gene rearrangements in the absence of clonal T-cell receptor (TCR) gene rearrangements. None (0%) of the My Ag positive cases showed immunoreactivity for myeloperoxidase. We conclude that the anomalous T and My Ag expression seen in the above B-cell lymphomas is not indicative of mixed lineage proliferation but represents the aberrant expression of these antigens by the malignant cells.

Four color compensation.

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate.

Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry.

BACKGROUND: The combination of in situ PCR and cell phenotyping by antibody labeling (ISPCR/Flow) allows for the identification of cell subsets carrying a particular genetic sequence. ISPCR utilizes thermal cycling for genetic amplification, which can reduce the effectiveness of surface antibody labeling. This study explored and characterized the effects of thermal exposure on antibody labeling using CD4 and CD45. METHODS: Single temperature incubations and thermal cycling exposures were performed on leukocytes labeled with either direct antibody conjugates or with biotinylated antibodies and PE-streptavidin. RESULTS: Fluorescence emission decreased above 70 degrees ( )C when cells were stained with directly conjugated antibodies or a biotinylated antibody and PE-streptavidin prior to high heat exposure. If counter stained with PE-streptavidin after heat, fluorochrome fluorescence was detectable. We tested a second CD4 clone, that provided poor results under similar labeling conditions, suggesting the combination of fixation and heat may have an epitope specific effect for the same cellular antigen. CONCLUSIONS: Immunophenotyping can be combined with ISPCR, but each antibody must be tested to determine its efficacy. The denaturation of protein above 70 degrees C appears to be the main reason for loss of fluorescence. The best procedure is to first stain cells with a biotinylated antibody to an epitope that survives fixation and thermocycling. The cells are then subjected to the desired PCR procedure. Finally they are stained with a fluorochrome conjugated streptavidin.