Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti‐human platelet antibodies

Hayashi, Tetsusuke; Amakishi, Etsuko; Matsuyama, Nobuki; Yasui, Kazuta; Furuta, Rie; Hori, Yuji; Tanaka, Shigenori; Fukumori, Yasuo; Hirayama, Fumiya; Inoue, Masayasu

doi:10.1111/j.1365-3148.2010.01064.x

Cited by 20 publications

(16 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The MAIPA system, similar to MAIGA, is the gold standard for detecting HPA Abs [22], and we have shown that the cell line-based MAIPA (HP-MAIPA) system is also useful, because the preparation of a typed panel of platelets can be avoided [23]. In a previous study, the use of cell lines instead of panel platelets meant that MoAbs was not required, avoiding the risk of epitope competition between the MoAbs and human Abs for detection [24].…”

Section: Discussionmentioning

confidence: 97%

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

et al. 2015

View full text Add to dashboard Cite

Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

show abstract

Section: Discussionmentioning

confidence: 97%

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Overall, the cell stabilization methods optimized and evaluated in this study may facilitate the distribution and use of transfected cells expressing low-incidence antigens, which could supplement and improve RBC-based antibody identification assays. Likewise, these methods may potentially simplify the detection of alloantibodies against human neutrophil antigens (HNA) [42, 43] or human platelet antigens (HPA) [44, 45] by means of HNA and HPA transfected cells, also available.…”

Section: Discussionmentioning

confidence: 99%

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells

et al. 2016

View full text Add to dashboard Cite

BackgroundThe identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.MethodsWe generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.ResultsTransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.ConclusionsBoth stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

show abstract

“…Immunoprecipitation and Western blot analysis was carried out according to a previously described method [31,33] using M280 magnetic beads (Invitrogen).…”

Section: Immunoprecipitation and Western Blottingmentioning

confidence: 99%

“…Transfections to produce K562 cells expressing HPA-4b were performed according to a previously described method [31,33] using retroviral particle carrying both GPIIb and GPIIIa containing HPA-4b genes. The cells were cloned by a limiting dilution method in the presence of 1.0 mg/mL puromycin and 0.8 mg/mL G418.…”

Section: Gene Transductionmentioning

confidence: 99%

Establishment of a cell line panel for the detection of antibodies against human platelet antigen 4b

Hayashi

Amakishi²,

Inoue

et al. 2011

Int J Hematol

View full text Add to dashboard Cite

Antibodies against human platelet antigens (HPAs) play important roles in thrombocytopenia. In Japan, HPA-4b antibody is frequently responsible for HPA-related neonatal alloimmune thrombocytopenia. A highly sensitive assay using platelets has been developed for the detection of antibodies against HPAs. However, it is difficult to obtain the platelets expressing specific HPAs required for the assay. Therefore, an alternative method not requiring platelets would be helpful to detect antibodies against HPAs. Glycoprotein IIIa (GPIIIa) cDNA encoding HPA-4b was individually co-transduced with that of wild-type GPIIb in K562 cells, which is a non-adherent cell line, using a retroviral vector. The expression of transgene products in cultured cells were observed for over 6 months. Next, to evaluating the sensitivity and specificity of this cell line panel, we performed monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay with a serum previously identified by another method. All HPA-4b antibodies in serum samples were positive, and all serum samples, including normal serum and serum containing HLA antibodies were negative. No difference was observed in the specificity and sensitivity between our method and conventional MAIPA using platelets. The present results indicate that this established cell line panel permits highly sensitive detection of specific antibodies against HPA-4b.

show abstract

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti‐human platelet antibodies

Cited by 20 publications

References 22 publications

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells

Establishment of a cell line panel for the detection of antibodies against human platelet antigen 4b

Contact Info

Product

Resources

About