U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: selection of antibody combinations.
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Biomedical subjects
Publications and source records attributed to J L Carey.
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BACKGROUND: Anticardiolipin antibodies (aCL) have been associated with an increased risk of stroke and thrombo-occlusive events. Little is known about the influence of aCL on recurrent thrombo-occlusive events. METHODS: Consecutively identified patients (n = 132) with focal cerebral ischemia [stroke = 112, transient ischemic attack (TIA) = 20] harboring aCL of at least 10 GPL units at the time of their index event were prospectively followed to estimate the effect of aCL titer on time to and risk of subsequent thrombo-occlusive events (stroke, TIA, deep venous thrombosis, pulmonary embolism, myocardial infarction) and death. On the basis of prior literature, we divided patients into those with aCL < or = 40 GPL (n = 111; mean age, 63 +/- 14 years; mean follow-up, 1.95 years) and those with aCL > 40 GPL (n = 21; mean age, 54 +/- 20 years; mean follow-up, 1.50 years). RESULTS: There was no difference between groups for prevalence of hypertension, diabetes mellitus, cigarette smoking, atrial fibrillation, prior TIA, or sex. The GPL > 40 group was younger (54 +/- 20 versus 63 +/- 14 years; P = .055), had more prior strokes [9/21 (48%) versus 27/111 (20%); P = .030], more frequent subsequent thrombo-occlusive events and death [15/21 (71%) versus 51/111 (48%); P = .030], and a shorter median time (years) to event (0.15 versus 0.61, log rank P = .005). The risk ratio for recurrent event and death with GPL > 40 obtained from Cox proportional hazards models, adjusted for prior strokes, prior TIAs, hypertension, diabetes mellitus, atrial fibrillation, and cigarette smoking was 1.9 (95% confidence interval, 1.0 to 3.5; P = .051). CONCLUSIONS: Our data suggest that subsequent thrombo-occlusive events and death after focal cerebral ischemia associated with IgG aCL may occur sooner and more frequently with GPL > 40.
To evaluate the potential advantages and disadvantages of the EANA, the immunopathology laboratory of the Henry Ford Health System compared the qualitative and quantitative results from several EANA assays to those from a well-standardized FANA assay and then correlated them with the clinical data.
An algorithm (directed thyrotropin [TSH], directed thyroid testing algorithm [DRTSH]) for the initial evaluation and monitoring of thyroid function was established in our institution in 1990. The algorithm begins with measurement of TSH by a sensitive immunoassay. If TSH is either < 0.4 mU/L or > 5.5 mU/L, a free thyroid index (T4 x Resin Uptake Ratio (RU)) is automatically performed on the same sample on the same day. In the setting of a large, predominately outpatient, prepaid health care population, the algorithm reduces unnecessary testing and focuses resources on the patients who need it. Three years after its introduction, physician acceptance of this approach is high ( > 90%), test utilization is reduced, test turnaround time is reduced, and significant cost-savings can be demonstrated.
Idiopathic CD4+ T-lymphocytopenia (ICL) in HIV-seronegative patients is a newly described, rare entity. The common underlying abnormality is a usually stable depletion in CD4+ lymphocytes in patients, some of which have unexplained opportunistic infections. We present a previously unreported condition of an asymptomatic individual with CD4+ T-lymphocytopenia and a selective IgA deficiency. The subject is a 35-year-old healthy white male with a documented 5-year history of low CD4+ T cell counts. He has been repeatedly HIV seronegative and has no risk factors for HIV infection. Data were obtained from several laboratories over a 5-year period and include standard WBC differentials, HIV testing, serum immunoglobulin quantitation, mitogen stimulation assays, diphtheria and tetanus antitoxin titers, and flow cytometric immunophenotyping. The composite results show a subject with a normal white blood cell count, an absolute lymphopenia, a slight granulocytosis, and a selective IgA deficiency. Leukocyte subset analyses show essentially normal B but significantly altered T cell phenotypes. The normal CD4:CD8 ratio shows extreme inversion, primarily due to CD4 T-lymphocytopenia.
Leukocytes may have an important role in the pathogenesis of brain injury after ischemia. Expression of adhesion molecules on leukocytes and/or endothelia is needed for leukocytes to adhere to endothelia and infiltrate into the injured brain. The purpose of the present pilot study is to delineate whether the expression of leukocyte adhesion molecules, CD11a and CD18, are upregulated in patients with ischemic stroke and transient ischemic attack. Ten patients with ischemic stroke, 6 with transient ischemic attack (TIA), and 11 age and risk factor matched controls were studied. Using immunofluorescence phenotyping and flow cytometry, leukocyte membrane expression of CD11a and CD18 were measured within 72 h after onset of ischemia. Follow-up measurements were performed at 5-7 days after ictus in 6 patients with stroke, and at 3-5 days after ictus in 3 patients with TIA. CD11a immunofluorescence (IF) was significantly increased within 72 h after onset of symptoms in patients with stroke as well as TIA compared with the control group (p < 0.017). IF of CD18 also increased in both patient groups, but significance was reached only in the TIA group (p < 0.05). No difference of CD11a and CD18 IF was detected between stroke and TIA groups. Follow-up measurement of CD11a and CD18 showed a trend of decrease, but CD11a IF remained significantly elevated compared with the control group (p < 0.017). Expression of leukocyte adhesion molecules CD11a, and CD18 are upregulated in patients with ischemic stroke and TIA. Although these data are preliminary, our data suggest that these molecules are associated with cerebrovascular disorders including ischemic stroke and TIA.
Previous reports have suggested that nodular lymphocyte predominance Hodgkin's disease (NLPHD) is a germinal center-derived B-cell lymphoma that is distinct from other types of Hodgkin's Disease. A relationship between NLPHD and simultaneous or subsequent development of large-cell (LC) non-Hodgkin's lymphoma (NHL) has been established. Both Reed-Sternberg cell variants in NLPHD and NHL cells in these cases express B-cell-associated antigens, and in some cases the B-cell lineage of the NHL has been confirmed by immunoglobulin gene rearrangement studies. The B-cell phenotype and the indolent course of both lymphomas suggest histologic progression of NLPHD to B-cell NHL, rather than a de novo LCNHL unrelated to Hodgkin's Disease. We report a unique case of T-large-cell lymphoma (TLCL) following successful chemotherapy of NLPHD. A 54-year-old male was treated with seven cycles of mechlorethamine, vincristine, procarbazine, prednisone chemotherapy for NLPHD and 4 years later developed recurrent adenopathy. Lymph node biopsy showed a diffuse LCNHL. Frozen section immunotyping and gene rearrangement studies confirmed the diagnosis of TLCL. To our knowledge, this case represents only the second report of TLCL associated with NLPHD and is of significance in that: (1) it demonstrates that T-cell neoplasia can occur in the setting of NLPHD; (2) this case does not appear to represent histologic progression of NLPHD and most likely represents de novo disease that may be secondary to chemotherapy; and (3) the clinical course may differ from the favorable prognosis seen in NLPHD associated with B-cell NHL.
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Megakaryoblastic termination of myeloproliferative disorders is rare. The morphology of megakaryoblastic transformation can be subtle and is often mistaken for myeloid or lymphoid proliferations. Previously reported observations suggest a relatively poor prognosis for this category of patients, making precise diagnosis imperative. A multifaceted approach using morphology, ultrastructure, cytochemistry, and immunological membrane analysis may be helpful. We present two cases of myeloproliferative disorder with aggressive megakaryoblastic phases (myelofibrosis with agnogenic myeloid metaplasia and chronic myeloid leukemia with blast crisis). The clinical course is described and the results of the morphological, cytochemical, ultrastructural, and cytogenetic studies of both cases are presented. In addition, immunochemical studies (flow cytometry) and platelet function studies (aggregation, beta-thromboglobulin, and platelet factor IV release) were done for one of these patients.
Flow cytometric analyses have become commonplace in the clinical laboratory for determining cell lineage and for quantitation of cells bearing a given phenotype. Because these assays are being conducted to support diagnoses or assist in determining therapy, it is crucial to ensure that these tests are highly accurate and reproducible within a laboratory and among laboratories involved in similar endeavors. This quality assurance has been slow evolving in clinical flow cytometry for a variety of reasons: the exquisite sensitivity and delicacy of the instrumentation that recognize previously undetectable variations in staining; the constant improvement of the hardware and software; the rapid development of new techniques and reagents of clinical interest; and the failure of any existing specialty or subspecialty to encompass all aspects of flow cytometry. This article provides an overview of quality assurance necessary for the flow cytometric analysis of cell surface markers. Practical experience, published studies, and suggested guidelines from accreditation agencies have been combined to develop the text.
The application of flow cytometry to cancer diagnosis and the prediction of tumor behavior continues to expand. The ability of flow cytometry to examine multiple parameters of large numbers of individual cells is being exploited increasingly to characterize neoplastic processes better. This will allow closer examination of tumor heterogeneity and identification of subpopulations with different behavioral patterns. Flow cytometry is being used with greater frequency in attempts to predict tumor behavior and response to therapy. Flow cytometry may have the most to offer in this area. Diagnosis of cancer by routine histopathological examination will not be replaced by flow cytometry; flow cytometry will be used in conjunction with morphological descriptions. The detection of proliferation antigens and oncogene products, as well as cell cycling, provides information on neoplastic progression that is not readily obtainable by other methods. In addition, flow cytometry will undoubtedly be used to measure other features of neoplastic cells, such as enzyme levels and ion fluxes, which may better characterize the behavior of the tumor. Advances in flow cytometry instrumentation, light sources, fluorochrome development, and basic aspects of cellular and molecular biology will continue to permit this technology to define neoplastic cells and their behavior better, resulting in both improved patient care and a better understanding of tumor biology.
To date, the detection of oncogene products in human neoplasms has relied primarily upon immunoblot analysis of specimen homogenates. Herein are reported the results of a study using flow cytometry to evaluate the expression of the ras p21 gene product in both fresh and cryopreserved specimens of human acute leukemias. Cell lines known to express ras p21 were used as positive controls and normal peripheral blood was used as a negative control. Intensity of staining for ras p21 (Ip21) was expressed as the ratio of the peak channel numbers of the peak generated by staining with anti-ras p21 to the peak obtained by staining with an isotype control. Using this method, 21 out of 32 clinical specimens of acute leukemia were found to express ras p21 in elevated amounts compared to normal peripheral blood. Flow cytometry appears to be a practical method for routine screening of clinical specimens for the expression of oncogene products on individual cells rather than cell homogenates.
Achieving a vigorous secretory immunoglobulin A (IgA) response in intestinal secretions usually requires multiple doses of antigen given orally, while systemic immunity is more easily attained by parenteral immunization. This study examines the role of combined parenteral and oral immunizations to enhance the early mucosal immune response to an enteropathogen. We have used a chronically isolated intestinal-loop model in rabbits as a probe to monitor kinetically the initial (primary) local immune response to shigella lipopolysaccharide (LPS) following combinations of parenteral immunization intramuscularly (i.m.) and oral stimulation with shigellae. Predictably, effective stimulation of systemic immunity was elicited when heat-killed preparations of Shigella sp. strain X16 were given i.m., as shown by strong serum IgG and weak intestinal IgA activity to shigella LPS. A single oral dose of live Shigella sp. strain X16 given to unprimed rabbits elicited only a typical weak IgA response in intestinal secretions. However, when an i.m. dose of heat-killed shigellae was followed 1 day later by an oral dose of live Shigella sp. strain X16, a hyperstimulation of the early secretory IgA response was elicited, and the response reached levels found previously only after multiple oral administrations of live shigellae. This stimulation did not require the use of an adjuvant. At the same time, the animals receiving this combined oral and i.m. regimen had a lower IgG antishigella LPS activity in serum compared with their response after receiving parenteral antigen in adjuvant alone. These findings indicate that while a dichotomy exists between the systemic and mucosal immune responses, careful orchestration of the stimulatory events can promote a vigorous early local IgA response.
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rho proteins isolated from strains of Escherichia coli with the suA1, suA100, and suA120 polarity suppressor mutant alleles of the rho gene are shown to be defective in termination of T7 DNA transcription in vitro. These mutant rho factors are found to be less active than normal rho in catalyzing the hydrolysis of ATP with isolated T7 RNA as cofactor, and their lower ATPase activities correlate well with their decreased abilities to mediate release of RNA from isolated T7 DNA transcription complexes. The mutant rho factors also have lower ATPase activities than wild type rho with certain synthetic RNA polymers, but they have full activity with poly(C). Because the mutant rho factors have normal ATPase activity with some RNAs, we conclude that their defects are in their abilities to interact with certain RNA cofactors and not in their catalytic capacities. Binding studies indicate that their defects do not appear to involve the primary interaction between rho and RNA. When poly(dC) is used to saturate the primary site for RNA on rho, the Km values for (Cp)7C as a secondary site activator of rho ATPase are 3.4, 80, and 15 microM for rho from the strains with the wild type, suA1, and suA100 alleles, respectively. These results suggest that the defects in the rho factors isolated from strains with the polarity suppressor alleles suA1 and suA100 involve their abilities to interact with RNA at their secondary sites.
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