High-Throughput identification of hemoglobinopathiasand thalassemias by HRAM/MS

Z. Lukacs1, R. Grosse1, R. Santer1, S. Murko1, T. Wiesinger2, D. Kasper2

1 UKE, Hamburg, Germany2 ArchimedLife Science GmbH, Wien, Austria

Newborn Screening and Metabolic Diagnostics Unit

Research Institute

Lukacs et al. Hb screening with HRMS and HPLC 2

Disclosures

- Travel and research grants from Genzyme B.V., NL

- Travel and research grants from BioMarin Ltd, UK

- Research grant from Alexion Pharma GmbH, Germany

- Research grant from Ultragenyx, Germany

- Consultant to Archimed LifeScience GmbH, Austria

Sickle Cell Disease and Thalassemias

Lukacs et al. Hb screening with HRMS and HPLC 3

• Piel et al, N Engl J Med, 376, 2017, 1561

• UCSF, Oakland, USA

Hemoglobin subunit change with age

Lukacs et al. Hb screening with HRMS and HPLC 4

Source: UCSF Benioff Children’s Hospital Oakland

Type Characteristics

Alpha-thalassemia Absent or decreased production of alpha globin subunits

Beta-thalassemia Absent or decreased production of beta globin subunits

Rare thalassemia Affecting the production of delta or gamma globin subunits

Lukacs et al. Hb screening with HRMS and HPLC 5

Pilot Study for Hemoglobinopathias in Hamburg

- 17 018 newborns have been included in the study- all children born in the greater Hamburg area- no bias- anonymized samples- 321 samples have been excluded because of poor sample quality- First-tier testing : HPLC-analysis (BioRad)- second-tier testing : molecular genetic testing (same sample)

Lukacs et al. Hb screening with HRMS and HPLC 6

Pilot Study for Hemoglobinopathias in Hamburg

Grosse, R., Lukacs, Z., Nieves Cobos, P. et al. Pediatr Blood Cancer 2016: 168-170

Lukacs et al. Hb screening with HRMS and HPLC 7

HPLC Analysis with the BioRad VariantNBS

Lukacs et al. Hb screening with HRMS and HPLC 8

HPLC Analysis with the BioRad VariantNBS

S

A

F

A

FAST E/A2

• Problem : to reliably detect thalassemias

Lukacs et al. Hb screening with HRMS and HPLC 9

New Approach : HR/MS- Q Exactive Focus; LC: TLX 2

Supported by:

Lukacs et al. Hb screening with HRMS and HPLC 10

“Confirmation Strategy“

• Dual easy and cheap sample preparation of dried blood spots (DBS)

• Intact chains and tryptic digest (trypsine porcine)

• Hb S/C, Hb S are selectively detected in 2 min

• Duplex mode up to 720 sample/day

Tryptic peptidesIntact chains

Intact Hb chains Tryptic peptides

Thomas WiesingerResearch Institute

Lukacs et al. Hb screening with HRMS and HPLC 11

Identification of hetero- or homozygt. forms

Genetically confirmed positive patient sampleswere used to define threshholds

Full scan data to calculated different ratio´s Alpha/(beta+gamma)

Calculated Ratio´s clear indication for Hb and Thalpositive patients

Tryptic Digest “Confirmation Strategy“Ratio of

Intact Hb

NORMAL

Co-inherited Hb S with C, D

or E

Thalassemia

Homozygot. Hb S (Sicklecell disease)

Hb S

Hb A

Hb B

Hb C

Hb D

Hb E

Area

cou

nts

HB SS

Hb S

Hb A

Hb B

Hb C

Hb D

Hb E

Area

cou

nts

Hb SC

Lukacs et al. Hb screening with HRMS and HPLC 12

HR/MS may pick up thalassemiasby using the intact protein chains in a more precise way 𝜷𝜷 Thalassemia (minor) should give a α/β- ratio = 2

By comparing the intact protein chains, an unselective trypsin digest of the alpha or beta chains can be avoided

Calculated α/β- ratio = 2for 𝜷𝜷 Thalassemia

(minor)

α αβ

βα αβ

Only 1 β chain is expressed

Lukacs et al. Hb screening with HRMS and HPLC 13

Hb S/Hb beta T1

Hb C/Hb beta T1

Hb D/Hb beta T13

Hb E/ Hb beta T3

Variant II HRMS

0% 2% 87% 0% Hb FAD Hb FAD11127% 20708% 1% 0% Hb FSC Hb FSC

89% 0% 0% 0% Hb FAS Hb FAS2466% 2% 9% 0% Hb FAS Hb FAS

0% 129% 1% 0% Hb FAC Hb FAC8303% 16110% 2% 0% Hb FSC Hb FSC

61% 0% 1% 0% Hb FAS Hb FAS18944% 26% 2% 0% Hb SS Hb SS

1% 135% 1% 0% Hb FAC Hb FAC88% 0% 0% 0% Hb FAS Hb FAS

7250% 1% 2% 0% Hb FAS Hb FAS115% 0% 3% 0% Hb FAS Hb FAS

0% 0% 101% 0% Hb FAD Hb FAD81% 0% 7% 0% Hb FAS Hb FAS

105% 0% 2% 0% Hb FAS Hb FAS0% 0% 0% 175% Hb FAES Hb FAES

Comparison of positive samples

Verified the workflow by using 37 positive donor samples confirmed by HPLC and genetic tests

Lukacs et al. Hb screening with HRMS and HPLC 14

For the identification of Hb S, C, D, E a ratio is calculated by using the equivalent digest sequence of the „healthy Hb b“ chain.(Similar STRATEGY was published Daniel Y.A British Journal of Haematology, 130, 635–643; SpotON a British spin off provides a Hb kit for MSMS)

HR/MS

Hb S/Hb beta T1

Hb C/Hb beta T1

Hb D/Hb beta T13

Hb E/ Hb beta T3

Hb Oarab/Hb beta T13

Hb Alpha intact /Hb beta intact (total*)

0% 0% 7% 0% 0% 0,680% 0% 11% 0% 0% 0,510% 6% 11% 0% 0% 0,420% 0% 10% 0% 0% 0,495% 0% 3% 0% 0% 0,63

65% 0% 15% 0% 0% 1,010% 0% 11% 0% 0% 0,94

26% 0% 63% 0% 0% 1,080% 0% 51% 0% 0% 0,850% 4% 14% 0% 0% 0,960% 1% 24% 0% 0% 1,176% 3% 15% 0% 0% 2,30

* Hb beta intact total = Sum of all related Hb beta chain proteins (β + γ)

Lukacs et al. Hb screening with HRMS and HPLC 15

Summary of the prospective study

In total 5336 samples were measured on both systems Hb Presbyterian detected only by HRMS 5 genetic tests are required and will be performed in Hamburg

Variant II HRMS HRMS2nd Tier Testing

Genetic Testing

FAS 35 35 - n.p.

FAC 9 9 - n.p.

FAD 8 8 - n.p.

FAE 4 1 - *

β Thal 0 4 1 *

Hb Presbytarien 0 1 1 *

Hb unknown 0 1 0 *

Lukacs et al. Hb screening with HRMS and HPLC 16

Several known beta Thal patient samplesfrom the hematological unit (UKE Hamburg) as well as prospectively identified patientshave been analyzed

The calculated cutoff was confirmed(around a/b= 2)

To avoid false negative results we set thecutoff of at 1.6

These samples were applied to a 2nd tiertesting protocol, where a single DBS spot isextracted (without tryptic digest)

496

239237

228

41171182311 321 1 1 1

0

62

124

186

248

310

372

434

496

-1.25-0.5 0.25 1 1.75 2.5 3.25 4 4.75 5.5 6.25 7 7.75 8.5

Freq

uenc

y

Hb Alpha intact/Hb Beta intact (total)

Calculated α/β- ratio = 2for 𝜷𝜷 Thalassemia

(minor)

α αβ

βα αβ

Only 1 β chain is expressed

Lukacs et al. Hb screening with HRMS and HPLC 17

# HPLC (BioRad Variant II) Primary Screening (HRMS)

2nd Tier Testing (HRMS)

Genetic confirmation

1 beta Thal/ FAS beta Thal/ FAS FAS pending

2 Beta Thal/FAS Beta Thal/FAS Beta Thal/FAS n.p.

3 - Hb Presbyterian Hb Presbyterian pending

4 FAS suspected Hb Presbyterian/ HbGhanaien

Hb Presbyterian/ HbGhanaien

Hb Presbyterian/ HbGhanaien

5 beta Thal - - pending

6 FAE or Beta Thal beta Thal - pending

Some selected results

Lukacs et al. Hb screening with HRMS and HPLC 18

# HPLC (BioRad Variant II) Primary Screening (HRMS)

2nd Tier Testing (HRMS)

Genetic confirmation

1 beta Thal/ FAS beta Thal/ FAS FAS pending

2 Beta Thal/FAS Beta Thal/FAS Beta Thal/FAS n.p.

3 - Hb Presbyterian Hb Presbyterian pending

4 FAS suspected Hb Presbyterian/ HbGhanaian

Hb Presbyterian/ HbGhanaian

Hb Presbyterian/ HbGhanaian

5 beta Thal - - pending

6 FAE or Beta Thal beta Thal - pending

Some selected results

Lukacs et al. Hb screening with HRMS and HPLC 19

Conclusion HRMS

496

239237

228

41171182311 321 1 1 1

0

62

124

186

248

310

372

434

496

-1.25-0.5 0.25 1 1.75 2.5 3.25 4 4.75 5.5 6.25 7 7.75 8.5

Freq

uenc

y

Hb Alpha intact/

Calculated α/β- ratio = 2for 𝜷𝜷 Thalassemia

(minor)

α αβ

βα αβ

Only 1 β chain is expressed

α thalassemia

Limited to HbH and severely affected patientsdue to the phenomenon that no quantitativedifference is observed for protein amounts incases of α−thalassemia minor.

Rare Hb mutations/β-thalassemia

Led to the identification of Hb Presbyterianβ-thalassemia more reliably detectable

Hb S homo or heterozygote

Mass shift of 30 Da can be detected

Lukacs et al. Hb screening with HRMS and HPLC 20

Conclusion

• Generally data compares well between both methods• All data can be reprocessed later to assess further variants• HRMS shows robust performance (rare cleaning, ca. 2000 samples)• β-thalassemia more reliably detectable by HRMS• Further confirmed patient samples will be run on both devicesbut• BioRad may be easier to use for HbS only screening programs

Thank you !

The Hb team at Hamburg University