Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform,three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes

BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.     

A summary of the workshop on passive immunization using monoclonal antibodies for HIV/AIDS, held at the National Institute of Allergy and Infectious Diseases, Bethesda, 10 March 2006

Passive immunization with monoclonal antibodies (MAbs) has been shown to prevent a wide variety of diseases. Currently, there are no MAb products that are licensed for use for immunotherapy or immunoprophylaxis against infection by HIV. However, there are several rational arguments that can be advanced for the use of a passive immunization approaches for counteracting HIV much as for other diseases especially with respect to mother-to-child transmission (MTCT) of HIV and immediate post-exposure situations. Several arguments questioning the feasibility of the approach based on availability of effective drugs, high cost of production and distribution of the MAbs among others, also get raised. It seems that the field now is looking at some promising MAbs as well as several alternate ways to manufacture antibodies and which hopefully may positively affect cost-related issues. This summary of a workshop held to assess the role of MAbs in the treatment and prevention of HIV/AIDS provides a fairly comprehensive analysis of the usefulness of MAb technology for future HIV/AIDS research.     

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.     

Money and microbes: Robert Koch, tuberculin and the Foundation of the Institute for Infectious Diseases in Berlin in 1891

Starting from an assessment of how far Robert Koch's bacteriology had developed in the late 1880s this paper attempts to analyse different aspects of the process that led to the foundation of the Berlin Institute for Infectious Diseases in 1891. With the development of his supposed cure against tuberculosis, tuberculin, Koch attempted to give his research a new direction, earn a fortune with the profits and become more independent of Prussian government officials who, up to that point, had had a major influence on his career. In the period following the presentation of the cure in autumn 1890, however, it became clear that tuberculin's value in treatment was at most dubious. Thus, the failure of tuberculin meant that Koch had to drop his own plans and accommodate those of the Prussian Ministry of Culture. As a result he assumed directorship of the newly founded Institute for Infectious Diseases in Berlin. Even though this was definitely a prestigious position it reaffirmed Koch's dependency on Prussian government officials and was by no means the kind of institution he had aimed for at the outset.     

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.     

Workshop: Potential therapeutic uses of inhibitors of leukotriene generation and function: June 23, 1986, Bethesda,Maryland Sponsored by the National Institute of Allergy and Infections Diseases and the World Health Organization Collaborating Center for Allergic Diseases

In summary, in attempting to assess the clinical efficacy of inhibitors of leukotriene generation and function, it is critically important to sufficiently understand the pharmacology of the drugs being evaluated as well as the disease for which treatment with these agents is being assessed. Failure to address these issues could unfortunately lead to erroneous conclusions regarding a class of potentially important therapeutic agents.     

Diagnosis of unexpected acute myeloid leukemia and chronic lymphocytic leukemia: a case report demonstrating the perils of restricted panels in flow cytometric immunophenotyping

We report on the flow cytometric identification of concomitant acute myeloid leukemia and chronic lymphocytic leukemia in cytology specimens submitted with minimal clinical information. A 64-year-old man presented with fever and progressive dyspnea on exertion. Chest X-ray and computed tomography scan showed a left upper lobe pulmonary mass. Pulmonary capillary pullback specimens were collected to determine infectious verses neoplastic etiology. The pulmonary capillary pullback specimens showed atypical mononuclear cells with enlarged, slightly irregular nuclei; visible nucleoli; and basophilic cytoplasm. Flow cytometric analysis of the specimen for lymphoma was requested. Flow cytometric immunophenotypic studies showed that 78% of the cells were CD34 positive, CD45 dim positive and CD11c positive, consistent with acute myeloid leukemia. About 0. 75% of the cells expressed CD5 as well as dim CD20 and were monoclonal for kappa light chains: consistent with chronic lymphocytic leukemia/small lymphocytic lymphoma. At this time the clinician communicated a history of myelodysplastic syndrome of refractory anemia subtype. Peripheral blood was obtained for further immunophenotyping and the patient was immediately treated for his acute myeloid leukemia. This case demonstrates that a diagnostic antibody panel should allow evaluation of all cell types as per the U.S./Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry (Stewart et al.: Cytometry 30:231-235, 1997). Published 2000 Wiley-Liss, Inc.     

Burden of Infectious Diseases,Substance Use Disorders,and Mental Illness among Ukrainian Prisoners Transitioning to the Community

Background

The epidemics of incarceration, substance use disorders (SUDs), and infectious diseases are inextricably intertwined, especially in the Former Soviet Union (FSU). Few objective data documenting this relationship regionally are available. We therefore conducted a comprehensive, representative country-wide prison health survey in Ukraine, where one of the world’s most volatile HIV epidemics persists, in order to address HIV prevention and treatment needs.

Methods

A nation-wide, multi-site randomly sampled biobehavioral health survey was conducted in four Ukrainian regions in 13 prisons among individuals being released within six months. After consent, participants underwent standardized health assessment surveys and serological testing for HIV, viral hepatitis, and syphilis.

Results

Of the 402 participants (mean age = 31.9 years), 20.1% were female. Prevalence of HIV, HCV, HBV, and syphilis was 19.4% (95% CI = 15.5%–23.3%), 60.2% (95% CI = 55.1%–65.4%), 5.2% (95% CI = 3.3%–7.2%), and 10% (95% CI = 7.4%–13.2%), respectively, with regional differences observed; HIV prevalence in the south was 28.6%. Among the 78 HIV-infected inmates, 50.7% were unaware of their HIV status and 44 (56.4%) had CD4<350 cells/mL, of which only five (11%) antiretroviral-eligible inmates were receiving it. Nearly half of the participants (48.7%) reported pre-incarcertion drug injection, primarily of opioids, yet multiple substance use (31.6%) and alcohol use disorders (56.6%) were common and 40.3% met screening criteria for depression.

Conclusions

This is the only such representative health study of prisoners in the FSU. This study has important implications for regional prevention and treatment because, unlike elsewhere, there is no recent evidence for reduction in HIV incidence and mortality in the region. The prevalence of infectious diseases and SUDs is high among this sample of prisoners transitioning to the community. It is critical to address pre- and post-release prevention and treatment needs with the development of linkage programs for the continuity of care in the community after release.     

Either of the CD45RB and CD45RO isoforms are effective in restoring T cell,but not B cell,development and function in CD45-null mice

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45(+/-) mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.     

Infectious diseases, security and ethics: the case of HIV/AIDS

Securitization of infectious diseases may involve suspension of ordinary human rights and liberties. In the event of an epidemic, therefore, it is important to limit the occasions upon which draconian disease control measures are implemented in the name of security. The term 'security', moreover, should not be used too loosely if it is to retain force and meaning in political discourse. It may be argued that the bar for disease securitization should be set high so that it is limited to contexts involving rapidly spreading pathogens. Such an approach, however, would rule out securitization of more slowly spreading, endemic diseases such as HIV/AIDS. An advantage of characterizing HIV/AIDS as a security threat in developing countries, where the burden of the disease is concentrated, is that this is likely to mobilize resources needed to improve the situation there. That is, if HIV/AIDS is convincingly framed as a security threat, then governments may recognize self-interested reasons to ramp up control measures. Following consideration of arguments for narrow (excluding HIV/AIDS) versus broad (including HIV/AIDS) conceptions of security, we conclude that the legitimacy of 'securitizing' HIV/AIDS ultimately turns on empirical and semantic issues, and we emphasize the importance of distinguishing (1) the nature of the threat posed by HIV/AIDS and (2) the measures required to address that threat.     

Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34.

BACKGROUND: Flow cytometry is potentially suited to the chemosensitivity analysis of peripheral blood or bone marrow subpopulations in patients with leukaemia and myelodysplastic syndromes. METHODS: The use of the fluorescent dye 7-amino-actinomycin (7AAD) on unfixed cells to measure loss of viability at a range of cytosine arabinoside (ara-C) doses was evaluated. A six-tube flow cytometric assay for measuring the sensitivity to ara-C of CD45/side-scatter-gated or of CD34-positive leukemic blasts with 7AAD was established, using fixed stained normal mononuclear cells as an internal standard for quantitation of viable cells following culture. RESULTS: 7AAD dose response curves for 10 patients with acute myeloblastic leukemia (AML) showed a wide range of sensitivities at 2.5-5 microM araC (3.7-97%, mean 54% of control cell viability at 2.5 microM and 4.1-94.6 %, mean 27% at 5 microM). Parallel assays for ATP bioluminescence agreed reasonably well with the 7AAD method, r(s) = 0.78. The chemosensitivity of CD45/SSC-gated blast cells at 2.5 microM araC showed no consistent relationship with the ungated cell populations, such that CD45/SSC-gated blast sensitivity of seven samples ranged from 86% more to 38% less than that of the total population. Similarly, the chemosensitivities of the CD34-gated subpopulations ranged from 51% more to 78% less than those of the total populations. CONCLUSIONS:These results emphasize the necessity of measuring the chemosensitivity of the population of interest rather than of the sample as a whole in heterogeneous clinical material.     

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.     

纳米抗体在传染病的预防、诊断和治疗中的应用 *

传染病是一种由致病性微生物引起,能够影响人类身体健康甚至引发严重社会危机的传播性疾病。近年来,新冠、埃博拉等传染病的恶性暴发促使人们寻找更为高效便捷的防治手段以遏制疾病的进程。抗体在传染病防治中的应用引起了广泛关注,palivizumab是目前唯一被批准应用于呼吸道合胞病毒在免疫力低下人群的预防的单克隆抗体。纳米抗体(nano-antibody, Nb)是目前已知的能与抗原稳定结合的最小功能性单域抗体,具有稳定性高、亲水性强、易于表达和改造等优势。独特的分子特性使其在病毒、细菌、寄生虫等引发的传染病的预防、诊断和治疗中展现出良好的应用前景,相关研究显示纳米抗体对艾滋、流感、新型冠状病毒等都有很好的治疗效果。重点叙述纳米抗体的结构特点及其在传染性疾病中的研究进展。     

Comparison of Methods for Correction of Mortality Estimates for Loss to Follow-Up after ART Initiation: A Case of the Infectious Diseases Institute,Uganda

Background

In sub-Saharan Africa, a large proportion of HIV positive patients on antiretroviral therapy (ART) are lost to follow-up, some of whom are dead. The objective of this study was to validate methods used to correct mortality estimates for loss-to-follow-up using a cohort with complete death ascertainment.

Methods

Routinely collected data from HIV patients initiating first line antiretroviral therapy (ART) at the Infectious Diseases Institute (IDI) (Routine Cohort) was used. Three methods to estimate mortality after initiation were: 1) standard Kaplan-Meier estimation (uncorrected method) that uses passively observed data; 2) double-sampling methods by Frangakis and Rubin (F&R) where deaths obtained from patient tracing studies are given a higher weight than those passively ascertained; 3) Nomogram proposed by Egger et al. Corrected mortality estimates in the Routine Cohort, were compared with the estimates from the IDI research observational cohort (Research Cohort), which was used as the “gold-standard”.

Results

We included 5,633 patients from the Routine Cohort and 559 from the Research Cohort. Uncorrected mortality estimates (95% confidence interval [1]) in the Routine Cohort at 1, 2 and 3 years were 5.5% (4.9%–6.3%), 6.6% (5.9%–7.5%) and 7.4% (6.5%–8.5%), respectively. The F&R corrected estimates at 1, 2 and 3 years were 11.2% (5.8%–21.2%), 15.8% (9.9%–24.8%) and 18.5% (12.3% –27.2%) respectively. The estimates obtained from the Research Cohort were 15.6% (12.8%–18.9%), 17.5% (14.6%–21.0%) and 19.0% (15.3%–21.9%) at 1, 2 and 3 years respectively. Using the nomogram method in the Routine Cohort, the corrected programme-level mortality estimate in year 1 was 11.9% (8.0%–15.7%).

Conclusion

Mortality adjustments provided by the F&R and nomogram methods are adequate and should be employed to correct mortality for loss-to-follow-up in large HIV care centres in Sub-Saharan Africa.     

Morphological and flow cytometric characterization of leukocytes from the notothenioid teleosts Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni

To date, the adaptive immune response of the notothenioid fishes of the Sub- and High-Antarctic has received little attention. Here we characterize leukocyte preparations derived from head (pronephric) kidney, spleen, and intestine of three notothenioid species, Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni. Cells were collected from head kidney, spleen, and intestine, and were fixed in paraformaldehyde and analyzed by flow cytometry and optical microscopy. The three species displayed typical leukocyte populations composed of lymphocytes, granulocytes, monocytes/macrophages and thrombocytes with a peculiar organ distribution and peculiar flow cytometric patterns. This work represents the first characterization of cells involved in immune reactions in the investigated species.     

Commuter Mobility and the Spread of Infectious Diseases: Application to Influenza in France

Commuting data is increasingly used to describe population mobility in epidemic models. However, there is little evidence that the spatial spread of observed epidemics agrees with commuting. Here, using data from 25 epidemics for influenza-like illness in France (ILI) as seen by the Sentinelles network, we show that commuting volume is highly correlated with the spread of ILI. Next, we provide a systematic analysis of the spread of epidemics using commuting data in a mathematical model. We extract typical paths in the initial spread, related to the organization of the commuting network. These findings suggest that an alternative geographic distribution of GP accross France to the current one could be proposed. Finally, we show that change in commuting according to age (school or work commuting) impacts epidemic spread, and should be taken into account in realistic models.