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. 2024;16(19-20):1025-1032.
doi: 10.1080/17576180.2024.2395706. Epub 2024 Sep 4.

Assessing prognosis by quantifying FcγRIIa on fixed platelets

David J Schneider  1 , Heidi S Taatjes-Sommer  1 , Peter M DiBattiste  2 , Kanwal S Palla  3 , Tyler Shovah  3 , Subhanip Biswas  3 , Jeanne Ohrnberger  2
Affiliations

Assessing prognosis by quantifying FcγRIIa on fixed platelets

David J Schneider et al. Bioanalysis. 2024.

Abstract

Introduction: FcγRIIa amplifies platelet activation and higher platelet FcγRIIa identifies patients at greater risk of subsequent cardiovascular events. We report the accuracy and precision of a modified test to quantify FcγRIIa on previously fixed platelets (pFCG test).Methods & results: An antibody clone (5G1) was developed after exposure of mice to formaldehyde treated FcγRIIa. Accuracy and precision of the modified test was evaluated with biologic specimens (platelets) and engineered synthetic cells conjugated with FcγRIIa (Slingshot Biosciences). The modified pFCG test on fixed platelets (using 5G1) consistently identified modestly more (∼300 molecules) of FcγRIIa on platelets compared with the pFCG test on nonfixed platelets (using clone FL18.26). With biologic specimens, the intra-assay coefficient of variation (CV) was 2.1 ± 0.1% (standard error of the mean, n = 750). The interassay CV was assessed intraday (4.5 ± 1%) and interday (up to 5 days after fixation, 6.5 ± 0.4%, n = 50). The pFCG test performed on Slingshot Synthetic cells conjugated with FcγRIIa demonstrated accuracy, linearity (R2 = 0.984) and similar interassay CV both intraday (2% ± 0.6%) and interday (20 nonconsecutive days, 9.9% ± 2.1%).Conclusion: In summary, modification of the pFCG test to be performed on fixed platelets allows accurate quantification of pFCG with high precision.

Keywords: accuracy; biomarker; platelet; precision; prognosis.

Plain language summary

[Box: see text].

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Conflict of interest statement

DJ Schneider is named inventor on a patent (10,502,737, 11,747,335 B2) that propose the use of FcγRIIa for assaying platelet reactivity and treatment selection and on a pending application (application 63/371,636) that describes quantification of FcγRIIa on fixed platelets. DJ Schneider is co-founder of Prolocor Inc.; PM DiBattiste is co-founder of Prolocor, Inc, J Ohrnberger is employed by Prolocor Inc. KS Pallah, T Shovah and S Biswas are employed by Slingshot Biosciences. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed.

Figures

Figure 1.
Figure 1.
The pFCG test was performed with the addition of clone FL18.26 to nonfixed platelets and the addition of clone 5G1 to fixed platelets (n = 100 patients with MI). Patients with MI were chosen for this analysis because the range of expression (∼400->5000 molecules/platelet) is greater than that seen in healthy subjects (<1000 molecules/platelet). The graph on the left shows a comparison of the two methods across a broad range of FcγRIIa expression. The graph in the middle shows the absolute difference between results obtained with the two methods (results with 5G1 minus results with FL18.26) over a broad range of expression. The image on the right shows results from western blot analysis. Lane 1 shows results obtained with probing of the lysate from CHO cells (that do not express FCγRIIa). Lane 2 shows results from CHO cells that have been stably transfected to express FCγRIIa. Lane 3 shows results from a platelet lysate. Similar specificity of 5G1 and FL18.26 is demonstrated. The greater intensity associated with the 5G1 clone is consistent with greater affinity. CHO: Chines Hamster Ovary; MI: Myocardial infarction; pFCG: Platelet FcγRIIa.
Figure 2.
Figure 2.
FcγRIIa was conjugated to Slingshot synthetic cells (TruCytes™). The pFCG test was performed and results were compared with the expected results (left). Linearity of the pFCG test (right) was assessed with the use of Slingshot synthetic cells (TruCytes™). The pFCG test was performed (n = 765 determinations) on samples that ranged from 591 to 7640 molecules of FcγRIIa/bead. This range encompasses values seen in healthy subjects and patients with MI. Linear regression demonstrated R2 = 0.984. MI: Myocardial infarction; pFCG: Platelet FcγRIIa.
Figure 3.
Figure 3.
The intra-assay CV was assessed during the performance of the assay on biological specimens (platelets from 750 patients). CV: Coefficient of variation.
Figure 4.
Figure 4.
The interassay CV was assessed with biological samples (platelets from 50 patients). Intraday samples were performed on the same day. The second pFCG test was performed at least 2 h after the first test. interday samples were performed on days 1–5 after fixation of samples. CV: Coefficient of variation.
Figure 5.
Figure 5.
The interassay CV was assessed with TruCytes™ synthetic cells conjugated with FcγRIIa. Intraday samples were performed on the same day. The second pFCG test was performed at least 2 h after the first test. interday samples were performed on 20 nonconsecutive days. CV: Coefficient of variation.
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