scholarly journals Analysis of Antibody Hybridization and Autofluorescence in Touch Samples by Flow Cytometry: Implications for Front End Separation of Trace Mixture Evidence

2016 ◽  
Author(s):  
M. Katherine Philpott ◽  
Cristina E. Stanciu ◽  
Ye Jin Kwon ◽  
Eduardo Bustamante ◽  
Susan Greenspoon ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Human Leukocyte ◽  
Cell Populations ◽  
Str Typing ◽  
Leukocyte Antigen ◽  
Palmar Surface ◽  
Allele Specific ◽  
A Cell ◽  
Biochemical Variation ◽  
Hla Antibody

AbstractThe goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to differentially label and then sort cell populations from separate contributors in a trace biological mixture. Cell characterizations initially focused on two different protein systems, Human Leukocyte Antigen (HLA) complex and cytokeratin (CK) filaments. Hybridization experiments using pan and allele-specific HLA antibody probes showed that surface antigens on cells transferred from the palmar surface of volunteers are largely unreactive, suggesting that they cannot be used to differentiate cell populations in a touch mixture. Touch samples were also hybridized with the pan-CK probe AE1, which targets CK proteins 10, 14, 15, 16 and 19. Fluorescence levels of AE1 hybridized cells were observed to vary across donors, although these differences were not consistent across all sampling days. We then investigated variations in red autofluorescence profiles (650-670nm) as a potential signature for distinguishing contributor cell populations. Although distinct differences in red autofluorescence profiles were observed ‐‐ with one donor consistently exhibiting higher levels of fluorescence than others ‐‐ some variation was also observed in touch samples collected from the same individual on different days. While this suggests that contributor touch samples cannot be defined by a discrete level of autofluorescence, this attribute may still be a useful means of isolating contributors to some touch mixtures. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two person touch mixture was separated into two fractions via Fluorescence Activated Cell Sorting (FACS) using gating criteria based on intensity of 650-670nm emissions, and then subjected to DNA analysis. STR typing of the sorted fractions provided partial profiles that were consistent with separation of individual contributors from the mixture.

scholarly journals Development of a humanized mouse model to analyze antibodies specific for human leukocyte antigen (HLA)

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0236614
Author(s):  
Senichiro Yanagawa ◽  
Hiroyuki Tahara ◽  
Takayuki Shirouzu ◽  
Shintaro Kawai ◽  
Yuka Tanaka ◽  
...  
Keyword(s):  
Mouse Model ◽  
Human Leukocyte Antigen ◽  
B Cell ◽  
Human Leukocyte ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Leukocyte Antigen ◽  
Antibody Mediated Rejection ◽  
Hla Antibody ◽  
Human B Cell

In organ transplantation, human leukocyte antigen (HLA)-mismatch grafts not only induce the activation of cellular mediated immune response but also the development of chronic antibody-mediated rejection due to the donor-specific anti-HLA antibody (DSA) produced by B cells and plasma cells interacting with the graft endothelium. Significant improvement in long-term survival after transplantation can be expected if antibody-mediated rejection due to the DSA can be overcome. However, the mechanism of producing or controlling the DSA remains to be elucidated. In recent decades, “humanized” mouse models have been widely used for the basic research of human immune systems, but a humanized mouse model to analyze the mechanism of DSA production has not been established yet. Thus, we aimed to create a humanized mouse using a severe immunodeficiency mouse (NSG mouse) administered with human peripheral blood mononuclear cells (PBMCs). Initially, we detected a very low level of human total-IgG and no anti-HLA antibodies (Abs) in these mice. In our next attempt, we mixed PBMCs of various HLA antigenic combinations with or without regulatory T cells and preconditioned them by culturing on feeder cells stably transfected with human CD40 ligand (h-CD40L) alone or with h-CD40L and human B cell activating factor (h-BAFF). They were subsequently co-cultured with the corresponding irradiated stimulator PBMCs, and all cells were administered into naïve NSG mice. Although all three humanized models had sufficient human total-IgG and anti-HLA antibody production, allospecific anti-HLA Ab production was prominently suppressed whereas non-specific anti-HLA Abs were sufficiently detected. Therefore, this novel humanized mouse model might be useful for analyzing the mechanism of anti-allogeneic human B cell tolerance induction.

scholarly journals Development of a humanized mouse model to analyze antibodies specific for human leukocyte antigen (HLA)

2020 ◽  
Author(s):  
Senichiro Yanagawa ◽  
Hiroyuki Tahara ◽  
Takayuki Shirouzu ◽  
Shintaro Kawai ◽  
Yuka Tanaka ◽  
...  
Keyword(s):  
Mouse Model ◽  
Human Leukocyte Antigen ◽  
B Cell ◽  
Human Leukocyte ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Term Survival ◽  
Leukocyte Antigen ◽  
Antibody Mediated Rejection ◽  
Hla Antibody

AbstractIn organ transplantation, human leukocyte antigen (HLA)-mismatch grafts not only induce the activation of cellular mediated immune response but also the development of chronic antibody-mediated rejection due to the donor-specific anti-HLA antibody (DSA) produced by B cells and plasma cells interacting with the graft endothelium.Significant improvement in long-term survival after transplantation can be expected if antibody-mediated rejection due to the DSA can be overcome. However, the mechanism of producing or controlling the DSA remains to be elucidated.In recent decades, “humanized mouse model” have been widely used for the basic research of human immune systems, but a humanized mouse model to analyze the mechanism of DSA production has not been established yet. Thus, we aimed to create a humanized mouse using a severe immunodeficiency mouse (NSG mouse) administered with human peripheral blood mononuclear cells (PBMCs). Initially, we detected very low level of human total-IgG and no anti-HLA antibodies (Abs) in these mice. The responder PBMCs with antibody-producing B cell activating factors added or regulatory T cells depleted were subsequently co-cultured with the irradiated stimulator PBMCs in vitro, and these whole cells were administered into naïve NSG mice. The humanized model with sufficient human total-IgG and anti-HLA antibody production was consequently established. Interestingly, in all these mouse models, allo-specific anti-HLA Abs production was prominently suppressed, whereas non-allo-specific anti-HLA Abs were sufficiently detectable.Therefore, this novel humanized mouse model might be useful for analyzing the mechanism of anti-allogeneic human B cell tolerance induction.

scholarly journals The fraction of sensitization among lung transplant recipients in a transplant center in Japan

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Sakiko Kumata ◽  
Takashi Hirama ◽  
Yui Watanabe ◽  
Hisashi Oishi ◽  
Hiromichi Niikawa ◽  
...  
Keyword(s):  
Japanese Population ◽  
Lung Transplant ◽  
Human Leukocyte ◽  
Cross Sectional Study ◽  
University Hospital ◽  
Antibody Testing ◽  
Cross Sectional ◽  
Leukocyte Antigen ◽  
Lung Transplant Recipients ◽  
Hla Antibody

Abstract Background Anti-human leukocyte antigen (HLA) antibody testing was approved by the Japanese government in 2018. As such, there was no longitudinal data regarding the HLA-sensitization of lung transplant (LTX) patients in Japan. We therefore set out to measure anti-HLA antibodies from all our LTX patients during their annual follow-up to characterize the sensitization status in the Japanese population. Methods The cross-sectional study was conducted for consecutive LTX recipients who underwent transplantation from January 2000 to January 2020 at Tohoku University Hospital (TUH). The serum from the recipients was screened for anti-HLA antibody with the panel-reactive assay (PRA) and the donor-specific antibodies (DSA). Results Sensitization was reviewed in 93 LTX recipients, showing 23 positive (24.7%) and 70 negative (75.3%) PRA. More sensitized recipients were found in recent transplantations (60.9% (14/23), ≤5 years post LTX) than in older transplantations (17.4% (4/23), 5–10 years or 21.7% (5/23), ≥10 years post LTX) (p = 0.04). Even fewer recipients had DSA (5.4%, 5/93), among whom 4/5 (80%) were recently transplanted. Conclusion The rate of PRA positive LTX recipients in our population was lower compared with those in previous reports from US and Europe. More sensitized LTRs were found in recent transplantations than the older cohort, and DSA was identified primarily in the recent recipients. Due to several limitations, it is still unclear whether the sensitization would be related the development of CLAD or survival, yet this study would be fundamental to the future anti-HLA body study in Japanese population.

scholarly journals Human Leukocyte Antigen-C and Haplotypes: Associations with Resistance and Susceptibility to HIV-1 Infection among Serodiscordant Couples in Nigeria

2021 ◽  
Author(s):  
Ngozi Mirabel Otuonye ◽  
Ma Luo ◽  
Onaiwu Idahosa Enabulele ◽  
Ayorinde James ◽  
Felix Emele ◽  
...  
Keyword(s):  
High Resolution ◽  
Human Leukocyte ◽  
Hiv Positive ◽  
Serodiscordant Couples ◽  
Leukocyte Antigen ◽  
Class 1 ◽  
Allele Specific ◽  
Hla Class 1 ◽  
Susceptibility And Resistance ◽  
Hiv 1

ABSTRACTINTRODUCTIONThe Human Leucocyte Antigen (HLA) class-1 is known to play a significant role in mediating resistance or susceptibility to HIV infection in the clinical course of AIDS. Recent studies have identified HLA-C as a key molecule that affects HIV disease progression. However, the role of HLA class 1 in heterosexual HIV-1 susceptibility or resistance in serodiscordant couples is not known in Nigeria. Therefore, this study evaluated the association between HLA-C susceptibility and resistance in HIV-1 transmission amongst heterosexual serodiscordant couples in Nigeria.METHODSA total of 271 serodiscordant, concordant HIV positive and negative couples who gave informed consent were enrolled into this study. Extracted genomic DNA was sequenced for high resolution HLA-C class 1 genotypes using allele-specific primers (on exons 2 and 3) for HLA-C sequencing and typing.RESULTSThe highest frequency distribution of high-resolution HLA-C alleles observed in the HIV positive subjects were: HLA-C*040101 178 (35.0%) followed by C*0701 124 (24.9%) compared with HIV negative subjects: C*040101 108(39.0%) followed by C*0701 64(24.7%). Alleles C*070201 (OR = 4.19, P<0.05) and C*0804 (OR = 3, P<0.045) were found to be independently associated with HIV-1 susceptibility in the cohort of serodiscordant couples. HLA-C*0802 (OR=0.5. P<0.005) and C*0304 (OR=0.34. P<0.002) were significantly associated with HIV-1 resistance to HIV-1 infection among the cohort.CONCLUSIONThe result has contributed to the importance of how host HLA-C genetic factors can influence HIV-1 disease susceptibility (HLA-C*070201; C*0804) and resistance (HLA-C*0802; C*0304) in serodiscordant couples. This information may contribute to the development of future effective HIV vaccine in Nigeria.

Survival of transfused donor white blood cells in HIV-infected recipients

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 272-279 ◽  
Author(s):  
Margot S. Kruskall ◽  
Tzong-Hae Lee ◽  
Susan F. Assmann ◽  
Megan Laycock ◽  
Leslie A. Kalish ◽  
...  
Keyword(s):  
Blood Cells ◽  
White Blood Cells ◽  
Human Leukocyte ◽  
Pcr Primers ◽  
Leukocyte Antigen ◽  
Specific Pcr ◽  
Before And After ◽  
Allele Specific ◽  
Hla Haplotypes ◽  
To Receive

The appearance and expansion of donor white blood cells in a recipient after transfusion has many potential biologic ramifications. Although patients with HIV infection are ostensibly at high risk for microchimerism, transfusion-associated graft-versus-host disease (TA-GVHD) is rare. The purpose of this study was to search for sustained microchimerism in such patients. Blood samples were collected from 93 HIV-infected women (a subset from the Viral Activation Transfusion Study, an NHLBI multicenter randomized trial comparing leukoreduced versus unmodified red blood cell [RBC] transfusions) before and after transfusions from male donors. Donor lymphocytes were detected in posttransfusion specimens using a quantitative Y-chromosome–specific polymerase chain reaction (PCR) assay, and donor-specific human leukocyte antigen (HLA) alleles were identified with allele-specific PCR primers and probes. Five of 47 subjects randomized to receive nonleukoreduced RBCs had detectable male lymphocytes 1 to 2 weeks after transfusion, but no subject had detectable male cells more than 4 weeks after a transfusion. In 4 subjects studied, donor-specific HLA haplotypes were detected in posttransfusion specimens, consistent with one or more donors' cells. None of 46 subjects randomized to receive leukoreduced RBCs had detectable male lymphocytes in the month after transfusion. Development of sustained microchimerism after transfusion in HIV-infected patients is rare; HIV-infected patients do not appear to be at risk for TA-GVHD.

Identification by flow cytometry of the prostaglandin-producing cell populations of term human decidua

1991 ◽  
Vol 131 (2) ◽  
pp. 327-334 ◽  
Author(s):  
E. R. Norwitz ◽  
P. M. Starkey ◽  
A. López Bernal ◽  
A. C. Turnbull
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Human Leukocyte ◽  
Cell Types ◽  
Cell Populations ◽  
Decidual Cell ◽  
Leukocyte Antigen ◽  
Hla Dr ◽  
Pure Cell

ABSTRACT Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0·25% bovine serum albumin at 37 °C. PGF2α and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2α (fmol/106 cells per 3 h) exceeded that of PGE2 at 273 (108–322) 322) versus 97 (38–127) respectively (median (range)). The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2α and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2α and PGE2 production rates in human term decidua in vitro. Journal of Endocrinology (1991) 131, 327–334

scholarly journals Flow cytometry dataset for cells collected from touched surfaces

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 390
Author(s):  
Ye Jin Kwon ◽  
Cristina E. Stanciu ◽  
M. Katherine Philpott ◽  
Christopher J. Ehrhardt
Keyword(s):  
Flow Cytometry ◽  
Optical Properties ◽  
Genetic Analysis ◽  
Human Leukocyte ◽  
Cell Populations ◽  
Single Source ◽  
Forward Scatter ◽  
Physical Separation ◽  
Leukocyte Antigen ◽  
Genetic Profiles

‘Touch’ or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures.  This dataset comprises the results from antibody hybridization surveys using Human Leukocyte Antigen (HLA) and Cytokeratin (CK) probes, as well as surveys of optical properties of deposited cells, including forward scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel.  All analyses were performed on “touch” samples deposited by several different contributors on multiple days to assess inter- and intra-contributor variability.

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