How Mass Spectrometry Revolutionised New Born Screening

A big part of my interest in mass spectrometry as a diagnostic tool and belief in it’s potential comes from an under recognized contribution to humanity. Not potential, but real world impact of it. Every year, over 10 million babies worldwide are tested using the triple quadrupole mass spectrometer via a simple heel prick blood spot card within 5 – 8 days of birth. This is an incredible achievement, and I’ve attached some links that I would urge you to read. Have a browse of these journal articles about David S. Millington and the triple quadrupole by Yost and Enke.

“They now test about 10 million babies around the world each year, and this instrument is the only way you can really do it,” Yost said. “We probably save 10,000 babies from early death. It’s certainly the application that makes my wife the proudest.” “Now it’s the most common mass spec in the world, and it sells over $1 billion a year, which is fairly mind-boggling for something that was a stupid idea that was never going to work.”

This article will go into depth in the analytical chemistry and how it could potentially work in future with portable mass spectrometers.

The Problem

Worldwide roughly 1 out of every 1,500 to 2,500 babies are born with inborn errors of metabolism. These are simply invisible at birth but will cause irreversible brain damage, organ failure or sudden death if left untreated. There are many neonatal screening programmes, roughly 30 - 40 metabolic markers are screened by mass spectrometry. One of the diseases that is targeted is Phenylketonuria (PKU).

PKU is a rare, inherited autosomal recessive inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). This defect prevents the conversion of the amino acid phenylalanine into tyrosine, leading to toxic accumulation in the blood and brain. Untreated PKU is associated with an abnormal phenotype which includes growth failure, poor skin pigmentation, microcephaly, seizures, global developmental delay and severe intellectual impairment. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives.

As PKU sufferers have a deficiency in their PAH enzyme, the blood concentration of phenylalanine (Phe) are far higher than normal. PAH deficiency can be classified into classic PKU (Phe >1200 μmol/L), mild PKU (Phe = 600-1200 μmol/L) and mild HPA, where blood Phe is elevated above upper reference limit, but <600 μmol/L.

How does the early diagnosis work?

This information comes from NHS England’s “Newborn blood spot screening: laboratory guide for IMDs” Note, none of these are 1 complete workflow, unfortunately I’ve only been able to find different parts of the workflow from google. But nonetheless it will be an accurate but not perfect representation of the entire process.

Dried Blood Spots

The initial screening tests use blood collected on the standard newborn screening blood sample collection card. The routine sample is collected on day 5, where the day of birth is day 0. Consent is obtained and recorded. The card has to contain the relevant baby and maternal identifiers and sampling information. NHS guidance recommends obtaining four good-quality blood spots. A heel prick allows for a drop of blood onto a filter paper. The filter paper spots are stable for many years, and the PKU screening tests have been reported to have a low error rate. The laboratory checks the card for things like insufficient blood, uneven saturation, layered blood, contamination, poor drying, transfusion problems.

Blood spot discs are punched (3.2 mm in diameter) into a multi-well plate, and the analytes are extracted using a solvent based extraction solution ~125 µL. The extraction solution contains internal standard of stable isotopes of Phe, Tyr, Leu, Met, C8, C10, C5, C5Dc and SUAC (succinylacetone). Following appropriate incubation and the transfer to a fresh plate if required according to kit instruction, the plate can be sealed and loaded onto a tandem mass spectrometer.

UPLC-Quadrupole, Single Reaction Monitoring (link)

For separation, traditionally liquid chromatography is used. This is a separation technique that separated molecules based on physiochemical properties of the analyte, stationary phase and mobile phase. For the analyte it is properties such as size, charge, polarity and volatility. For the stationary phase it’s polarity, charge and viscosity and for the mobile phases it is the interaction with the analyte and stationary phases. However more modern high-throughput newborn screening for companies like SCIEX has moved to Flow injection analysis (FIA) tandem mass spectrometry.

After the dried blood spot punch, into NeoBase 2 extraction solvent 125 µL which is 74.0 % methanol and 25.9% water. This is shaken and incubated at 45 °C for 30 minutes, then 100 µL is transferred to a fresh microplate, sample is held for 1 hr before undergoing FIA-MS/MS. FIA is flow injection analysis where samples are directly injected into a mass spectrometer as a continuous mobile phase stream without prior chromatographic separation. This process takes roughly 2 minutes injection to injection.

Fig 1. Assay procedure

It then undergoes Electrospray ionisation (ESI). ESI is an ionisation technique using a capillary with a high electric potential put across it. This causes the solvent to evaporate leaving charged ions which enter the detector.

 A spectrometer separates charged gaseous molecules based on their mass to charge ratio. It works by creating ions which can be manipulated by an electric or magnetic field separating them, allowing for identification and quantitation.

The quadrupole is a type of mass analyser used for this purpose. Single quadrupole instruments are among the most compact, robust and cost effective mass spectrometers available. It is comprised of 4 parallel metal rods connected pairwise to a combination of radio frequency and DC voltages. Ions will travel through the quadrupole in the z axis towards the detector. In order to do this, the ion must experience an electric field that gives it a stable trajectory. The equations that describe whether or not an ion of a particular m/z will experience a stable or unstable trajectory are called Matthieu equations. As RF and DC voltages are controllable, you can decide either to set DC voltages to 0 and have only RF voltages this allows all ions to be transmitted. Otherwise, you can select for a particular ion of interest.

The way triple quadrupole works is that 3 quadrupoles are set up in series. For single reaction monitoring, the first quadrupole scans for a particular m/z, the second undergoes collision induced dissociation. This is where an increase in DC voltage, increases the kinetic energy of the ions and their internal energy causing them to fragment. The last quadrupole scans for a particular product ion which is analysed by the detector.

Quantitation

Finally, the analyte signal is compared to the isotopically labelled internal standard signal giving an estimated whole blood concentration.

Sources of Error

The NHS document contains many sources for potentials for false positives. With LC there is no way to distinguish isomers and isobars.

 The largest source of uncertainty is the dried blood spot itself. It cannot correct for haematocrit-dependent spreading this is where the volume fraction of red blood cells in blood alters its viscosity and physical movement when soaking into porous materials like diagnostic filter paper. Uneven spot formation, too small spots, blood layered onto blood, differences between the centre and edge of the spot, contamination before punching.

The full workflow

Pre-analytical

Baby → heel → DBS → drying → shipping → accession → spot-quality assessment → punching.

Sample preparation

DBS disc → stable-isotope extraction solution → incubation → SUAC reaction where required → extract transfer → QC checks.

Analytical

Autosampler → FIA carrier → ESI → Q1 → collision cell → Q3 → detector → analyte/IS ratio → concentration.

Post-analytical

QC acceptance → screening algorithms → cut-offs → repeat abnormal sample in duplicate → ratios/second-tier analysis → clinical scientist review → urgent referral if screen positive → separate diagnostic testing.

Fig 2. Example workflow

Conclusion

Via this process many inborn errors in metabolism are found early allowing for treatment before it causes death. As Richard Yost mentioned earlier, it screens over 10 million babies a year saving 10,000 each year from early death. That is a huge legacy to leave, and certainly something to be proud of.