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Assessment of Equine Autoimmune Thrombocytopenia (EAT) by flow cytometry.

Nunez R, Gomes-Keller MA, Schwarzwald C, Feige K - BMC Blood Disord (2001)

Bottom Line: RATIONALE: Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration.Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis.CONCLUSIONS: This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institutes of Virology, University of Zurich, Zurich, Switzerland. nunezr@mskcc.org

ABSTRACT
RATIONALE: Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morphological characteristics paired with detection of platelet-bound IgG. OBJECTIVES: This study is aimed to develop and characterize a single color flow cytometric assay for detection of platelet-bound IgG in horses, in combination with flow cytometric assessment of platelet morphological characteristics. FINDINGS: The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals shows several distinctive features in comparison to the flow cytometric profile of platelets from healthy animals. The thrombocytopenic animals displayed i) increased number of platelets with high FSC and high SSC, ii) a significant number of those gigantic platelets had strong fluorescent signal (IgG bound), iii) very small platelets or platelet derived microparticles were found significantly enhanced in one of the thrombocytopenic horses, iv) significant numbers of these microplatelet/microparticles/platelet-fragments still carry very high fluorescence. CONCLUSIONS: This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

No MeSH data available.


Related in: MedlinePlus

Flow cytometric representation of morphological and fluorescent 1 (FL 1) signal of platelets. (No gate). Density plots of ungated platelets from normal and thrombocytopenic horses. A set of panels displaying the FSC × SSC density plot of platelets derived from three healthy horses are shown in the right panels of figure 1a. The left set of panels of figure 1b represents the FSC × SSC density plots of thrombocytopenic animals. The gates R1, R2 and R3 are showed in each panel.
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Figure 1: Flow cytometric representation of morphological and fluorescent 1 (FL 1) signal of platelets. (No gate). Density plots of ungated platelets from normal and thrombocytopenic horses. A set of panels displaying the FSC × SSC density plot of platelets derived from three healthy horses are shown in the right panels of figure 1a. The left set of panels of figure 1b represents the FSC × SSC density plots of thrombocytopenic animals. The gates R1, R2 and R3 are showed in each panel.

Mentions: The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals showed quite distinctive features in comparison to the FSC and SSC profile of platelets from healthy animals. Most of the platelets from healthy animals were identified in an area located mainly between 101-102 of the FSC axes and 101-102 of the SSC axes. Then, this area was defined as gate R1 (Figure 1 (no gate), right panels). Interestingly, the thrombocytopenic animals showed two additional distinct groups of platelet clouds that prompted us to delineate them by drawing two additional gates. The new gates were labeled R2 and R3. The platelets in R2 displayed high FSC (>102) and High SSC (>102) and could represent either circulating large platelets or megakaryocytes megathrombocytes) (Figure 1 (no gate), left panels). Of note in the case of the megakaryocytes, the maximum size of the cells included in the gate R2 differed by several orders of magnitude to the cells included in the gate R1. Furthermore, The cloud observed in gate R2 for the horse 2 shows two distinctive groups of cells, (i) One with very high FSC; (ii) another with very high SSC, which suggested great internal complexity. Interestingly, horse 1 has in R2 only the cells with high FSC. Further assays including sorting of these cells and staining with antibodies could help to clarify and define the two groups of cells (Figure 1 (no gate), left panels). The platelets in R3 displayed low FSC (<101) and medium SSC (101-102) and represent either very small platelets or platelet derived microparticles (Figure 1 (no gate), left panels). Figure 1 overall shows that the healthy animals (right panels) have the bulk of platelet population mainly located in R1 (> 86%), while the thrombocytopenic animals (left panels) have < 48% (Figure 1 (no gate) and Table 1, row: % of total within R1). In sharp contrast, the healthy ones have only a minute platelet population located in R2 (< 9%), whereas the sick ones have more than 31% of the total platelet counts within the R2 gate (Figure 1 (no gate) and Table 2, row: % of total within R2). Since platelets in R2 are characterized by high FSC and high SSC, and only the thrombocytopenic horses had significant numbers of platelets within this gate, is likely that the presence of circulating large platelets or megakaryocytes will be tightly associated with this disease. The presence of platelet aggregates could be another plausible explanation for the cloud observed in R2. However, only the thrombocytopenic animals have this cloud while the healthy ones do not. Thus, if the cloud were a complex of platelet aggregates, it should be tightly associated to the disease. Further morphological studies after sorting analysis could help to clarify this issue. In addition, one of the thrombocytopenic animals (Horse 1) showed the presence of a significant population of platelets with low FSC and normal SSC that represent either microthrombocytes or platelet fragments. This platelet subset is included within the gate R3 (Figure 1 (no gate), panel: Horse 1).


Assessment of Equine Autoimmune Thrombocytopenia (EAT) by flow cytometry.

Nunez R, Gomes-Keller MA, Schwarzwald C, Feige K - BMC Blood Disord (2001)

Flow cytometric representation of morphological and fluorescent 1 (FL 1) signal of platelets. (No gate). Density plots of ungated platelets from normal and thrombocytopenic horses. A set of panels displaying the FSC × SSC density plot of platelets derived from three healthy horses are shown in the right panels of figure 1a. The left set of panels of figure 1b represents the FSC × SSC density plots of thrombocytopenic animals. The gates R1, R2 and R3 are showed in each panel.
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Related In: Results  -  Collection

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Figure 1: Flow cytometric representation of morphological and fluorescent 1 (FL 1) signal of platelets. (No gate). Density plots of ungated platelets from normal and thrombocytopenic horses. A set of panels displaying the FSC × SSC density plot of platelets derived from three healthy horses are shown in the right panels of figure 1a. The left set of panels of figure 1b represents the FSC × SSC density plots of thrombocytopenic animals. The gates R1, R2 and R3 are showed in each panel.
Mentions: The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals showed quite distinctive features in comparison to the FSC and SSC profile of platelets from healthy animals. Most of the platelets from healthy animals were identified in an area located mainly between 101-102 of the FSC axes and 101-102 of the SSC axes. Then, this area was defined as gate R1 (Figure 1 (no gate), right panels). Interestingly, the thrombocytopenic animals showed two additional distinct groups of platelet clouds that prompted us to delineate them by drawing two additional gates. The new gates were labeled R2 and R3. The platelets in R2 displayed high FSC (>102) and High SSC (>102) and could represent either circulating large platelets or megakaryocytes megathrombocytes) (Figure 1 (no gate), left panels). Of note in the case of the megakaryocytes, the maximum size of the cells included in the gate R2 differed by several orders of magnitude to the cells included in the gate R1. Furthermore, The cloud observed in gate R2 for the horse 2 shows two distinctive groups of cells, (i) One with very high FSC; (ii) another with very high SSC, which suggested great internal complexity. Interestingly, horse 1 has in R2 only the cells with high FSC. Further assays including sorting of these cells and staining with antibodies could help to clarify and define the two groups of cells (Figure 1 (no gate), left panels). The platelets in R3 displayed low FSC (<101) and medium SSC (101-102) and represent either very small platelets or platelet derived microparticles (Figure 1 (no gate), left panels). Figure 1 overall shows that the healthy animals (right panels) have the bulk of platelet population mainly located in R1 (> 86%), while the thrombocytopenic animals (left panels) have < 48% (Figure 1 (no gate) and Table 1, row: % of total within R1). In sharp contrast, the healthy ones have only a minute platelet population located in R2 (< 9%), whereas the sick ones have more than 31% of the total platelet counts within the R2 gate (Figure 1 (no gate) and Table 2, row: % of total within R2). Since platelets in R2 are characterized by high FSC and high SSC, and only the thrombocytopenic horses had significant numbers of platelets within this gate, is likely that the presence of circulating large platelets or megakaryocytes will be tightly associated with this disease. The presence of platelet aggregates could be another plausible explanation for the cloud observed in R2. However, only the thrombocytopenic animals have this cloud while the healthy ones do not. Thus, if the cloud were a complex of platelet aggregates, it should be tightly associated to the disease. Further morphological studies after sorting analysis could help to clarify this issue. In addition, one of the thrombocytopenic animals (Horse 1) showed the presence of a significant population of platelets with low FSC and normal SSC that represent either microthrombocytes or platelet fragments. This platelet subset is included within the gate R3 (Figure 1 (no gate), panel: Horse 1).

Bottom Line: RATIONALE: Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration.Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis.CONCLUSIONS: This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institutes of Virology, University of Zurich, Zurich, Switzerland. nunezr@mskcc.org

ABSTRACT
RATIONALE: Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i) decreased production; ii) increased utilization; iii) increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT); or iv) platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morphological characteristics paired with detection of platelet-bound IgG. OBJECTIVES: This study is aimed to develop and characterize a single color flow cytometric assay for detection of platelet-bound IgG in horses, in combination with flow cytometric assessment of platelet morphological characteristics. FINDINGS: The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals shows several distinctive features in comparison to the flow cytometric profile of platelets from healthy animals. The thrombocytopenic animals displayed i) increased number of platelets with high FSC and high SSC, ii) a significant number of those gigantic platelets had strong fluorescent signal (IgG bound), iii) very small platelets or platelet derived microparticles were found significantly enhanced in one of the thrombocytopenic horses, iv) significant numbers of these microplatelet/microparticles/platelet-fragments still carry very high fluorescence. CONCLUSIONS: This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

No MeSH data available.


Related in: MedlinePlus