Identification of Hemoglobinopathies and Thalassemias : A Prospective Study of Up to 10,000 Newborn Samples Thomas Wiesinger (Presenter) ARCHIMED Life Science GmbH Presenter Bio: Thomas Wiesinger has studied chemistry (MSc) at the technical university of Graz and finished his PhD in the field of biocatalysis at the technical university of Vienna in 2018. Already in 2017, he joined the research department of ARCHIMED Life Science GmbH and is mainly working on the development of new diognostic assays by mass spectrometry with focus to rare diseases.

Authors: (1) Thomas Wiesinger,(1) Thomas Mechtler,(1) Markus Schwarz,(2) Xie Xiaolei, (3) Regine Grosse, (4) Zoltan Lukacs, (1) David C. Kasper
(1) ARCHIMED Life Science GmbH, Leberstraße 20, 1110 Vienna; (2) Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose; (3) Pedatric Hematology, Hamburg University Medical Center, Germany; (4) Metabolic Diagnostics Unit, Hamburg University Medical Center, Germany

The early diagnosis of hemoglobin disorders such as sickle cell anemia (SCD) or Thalassemias become more and more important due to increasing carrier-frequencies worldwide (approx. 330.000) as well as in the European Union, especially in urban areas (e.g. Berlin 1 birth in 2.500 newborns).[1] Since the composition of hemoglobin (fetal to adult) is affected by the age of the patient, a reliable strategy, which can handle this challenge of nature, was established and are currently tested in an ongoing study ( 10.000 samples).

Introduction

Over decades, a set of genetic disorders related to hemoglobin (Hb) were characterized and classified into two main groups (i) Hemoglobinopathies and (ii) Thalassemias. These rare diseases mainly contain a mutation in one or both Hb-β chains or a misbalanced ratio between the α/β chains. Besides the identification of Hb S in homozygous form, Hb variants (e.g. Hb C, D, and E) or β-Thalassemia with co-inherited Hb S are the most clinical relevant types. Due to single mutations in the beta chain, these variants differ only by 1 Dalton for the entire protein and that fact leads to resolution issues by mass spectrometry for ion species with high m/z ratios (>15+).[2],[3]

Methods

Since the transition of fetal to adult hemoglobin, within the first weeks after birth, represents an additional biological challenge, a “confirmation strategy” was investigated by extracting Dried Blood Spots (DBS) with 2 different buffer solutions. The obtained solutions of the intact Hb protein and the corresponding tryptic digest are mixed in a defined ratio, to detect all clinical relevant Hb mutations as well as Thalassemia positive patients. The study was approved by the ethic committee Hamburg. Research study, not for diagnostic purposes.

Results

The goal of our ongoing prospective study is to test at least 10.000 samples by our high throughput screening method (2 min/sample) and the observed data will be compared with the state-of-the-art method (BioRad Variant II) and presented. In contrast to this well-established HPLC protocol, our HR/MS method is faster (FIA injection- no chromatographic separation), more robust, and do not require any elaborate sample preparation.

Conclusions & Discussion

Within the first set of patient samples, the observed data have correlated well with the gold standard method (BiaRad Variant II). Additionally, patients that showed different results of HR/MS vs. HPLC method were collected and will be genetically confirmed in the end of our ongoing study.

[1] a) R. Grosse, Pediatr. Blood Cancer 2016, 63, 168-170. b) F. B. Piel, N. Engl. J. Med. 2017, 376, 1561-1573. C.C. Hoppe, Int. Jnl. Lab. Hem. 2013, 35, 297-305

[2] a) R. L. Edwards, Anal. Chem. 2011, 83, 2265-2270. b) I. Zanella-Cleon, Clin. Biochem. 2009, 42, 1807-1817.

[3] Y. A. Daniel, Clin. Chem. 2007, 53, 1448-1454.

ARCHIMED Life Science GmbH is supported by Xiaolei Xie, Claudio DeNardi, and Bradley Hard from Thermo Fisher Scientific.