For patients with a platelet function disorder, it is often difficult to identify the underlying genetic cause of the bleeding diathesis. However, a study published in Blood Advances found that multiparameter microfluidic testing of thrombus formation can help identify a patient’s platelet disorder.
“While platelets in hemostasis and thrombosis function in an environment of flowing blood, the majority of diagnostic laboratory assays are carried out with citrate-anticoagulated platelet-rich plasma under stasis or slow stirring, i.e., in the absence of wall-shear stress,” explained corresponding author Johan Heemskerk, PhD, of Maastricht University in the Netherlands. “The few whole-blood shear devices that are available (e.g., PFA-100) are considered too insensitive to pick up mild qualitative or quantitative platelet dysfunctions.
“We have developed a flow perfusion assay using three different microspots of platelet-adhesive proteins and providing 48 outcome parameters per flow run. This high-throughput assay was used to phenotype 97 subjects with blood type O, who were fully genotyped, and assay parameters were found to be linked to genetic variance,” said Dr. Heemskerk. “In the present paper, we have applied the high-throughput assay to 16 genetically characterized patients with platelet defects – presenting with bleeding, albinism, or both – as well as 15 mutation-carrying healthy relatives and healthy controls.”
The patients were subjected to genetic screening, which revealed a novel biallelic pathogenic variant in RASGRP2, abrogating CalDAG-GEFI expression, causing a lack of protein expression in platelets; a compound heterozygosity in P2RY12, which was linked to a platelet P2Y12 receptor defect; and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients had confirmed type 1 or 3 Hermansky-Pudlak syndrome. No genetic variant was found in five of the patients.
No participants had used antiplatelet therapy for at least 14 days before undergoing high-content microfluidic blood testing in September 2019. Reference data were obtained from five day-control subjects and the previously published cohort of 97 healthy subjects. Routine laboratory measurements were used to assess platelet functions. Blood samples from all participants and day-control subjects were screened for blood cell counts and microfluidic outcomes on six surfaces.
According to microfluidic data analysis, key parameters of thrombus formation were compromised in the 16 index patients. Separate clusters of patients were identified when compared with heterozygous family members plus control subjects. The clusters segregated further when hematologic values and laboratory measurements were included. An overall impairment in thrombus formation was indicated for the patients who were likely carrying a pathogenic gene variant, but not for the asymptomatic relatives.
“When compared with common diagnostic assays, such as platelet aggregometry, blood cell counts, and flow cytometry, the whole-blood flow assay appeared to be superior in distinguishing between patients on the one hand and healthy relatives and controls on the other hand, because of the high-throughput data generation,” said coauthor Barbara Zieger, MD, of the University of Freiburg in Germany. “The current technique though is laborious with non-replaceable microfluidic chambers and the desktop microscope. Other initiatives have meanwhile been made to adapt the device, obtain single-use microfluidic chips, and automate the image generation and data processing without compromising the high-throughput assay’s sensitivity.”
Compared with previous work, the authors noted their study included multiple patients with newly observed mutations, along with clinically and genetically unaffected family members. However, the small number of patients and relatives was a limitation to the study. Further work is required to determine the additive value of controlled coagulation, the authors suggested.
Any conflicts of interest declared by the authors can be found in the original article.
Reference
Fernández DI, Provenzale I, Canault M, et al. High-throughput microfluidic blood testing to phenotype genetically linked platelet disorders: an aid to diagnosis [published online ahead of print, 2023 Jun 30]. Blood Adv. doi: 10.1182/bloodadvances.2023009860.