Novel Technique for Sickle Cell Anemia Diagnosis

Abstract

Sickle cell anemia (SCA) is a global public health problem that occurs mostly in low‐ and middle‐income countries, and underserved communities. Diagnosis and early treatment are critical to prevent mortality from this disease; however, a large‐scale screening is difficult due to a need for blood collection by trained phlebotomist in designated health centers. The primary goal of this study was to test a new self‐blood collection platform to enable screening in rural and underserved communities. To address this need, a simple, miniaturized device ‐ Noviplex™ card from Novilytic was evaluated. The advantages of this device include the ability to collect 2.7 μL of biofluid as well as the fact that the collected samples are stable at ambient temperature for several weeks and can be mailed to a clinical laboratory for further testing. To obtain the whole blood samples, 1% sodium deoxycholate was added to the blood prior the application or directly to the collection disk of the device. Two mass spectrometry (MS) approaches including MALDI and Multiple Reaction Monitoring (MRM) techniques were developed and validated to 1) detect the normal peptide present in β‐globulin (HBB) from a healthy donor (VHLTPEEK, Mw= 952.50 Da); 2) detect the mutated peptide present in HBB from a SCA donor (VHLTPVEK, Mw= 922.53 Da); and 3) detect a control peptide present in HBB of both healthy and SCA donors (LLVVYPWTQR, Mw= 1,274.72 Da). For MALDI based technique, blood proteins were transferred from the dried collection disc to a PVDF membrane by a semi‐dry blot system, digested with trypsin on the membrane and matrix directly deposited on the top of the blotted sample. Presence or absence of the peptides was determined using a Bruker 7T SolariX Fourier Transform Ion Cyclotron Resonance (FTICR) MS. For MRM, the dried collection disc was transferred to a 96‐well plate, and trypsin was added to cover the collection disc completely. Following digestion, samples were directly analyzed by LC‐MRM for normal, mutated and control peptides. Both techniques were evaluated using the samples from a donor with SCA and from a healthy individual and showed good digestion efficiency and reproducible detection of the peptides of interest. In addition, both methods can be fully automatized for high‐throughput testing in clinical laboratory environment. Sample preparation times for MALDI and MRM techniques were 25 and 3 min, respectively.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.