Trimethylamine testing at Sheffield Children’s Hospital

Joanne Croft, Clinical Scientist Edwin Smith, Medical Technical Officer

01/03/2018

Sample Reception

pH

Dipstick / indicator paper

Volatile TMA stabilised as HCl salt

Nitrite

Dipstick

Positive result indicates bacterial

contamination

Exclude UTI and repeat sample

Creatinine

Standardisation of results on dilute

/ concentrated urine

Storage: -80C

Initial tests

Analysis

Free Trimethylamine (TMA) Odourous compound

Trimethylamine-N-Oxide

(TMANO) Non-odourous

TMANO not measured directly Converted to TMA

Total TMA measured TMA + TMANO

Subtract free TMA to give

TMANO

What do we measure and how?

Analysis

Alkalinisation Addition of KOH converts

TMA.HCl to volatile TMA

Reduction TiCl3 converts TMANO to

TMA

Followed by alkalinisation

as above

Sample Preparation

Analysis

Headspace GCMS

Analysis

Headspace sampler Samples volatile gas from

“headspace” above liquid phase

Gas Chromatograph Separates compounds using

liquid coated capillary column

Mass Spectrometer Ionises compounds and detects

on basis of mass to charge ratio

Headspace GC-MS

Analysis

Data gives free and total TMA in µmol/L

Divide by creatinine to give results in

µmol/mmol crt

Subtract free from total TMA to give TMANO

Ratio of TMANO to total TMA gives % N-

oxidation

Result calculation

Analysis

Data analysis

TMA

Analysis

Quantitative Free TMA

TMANO

% N-oxidation

Interpretive For a non-specialist doctor

Relies on knowing clinical

details

Reporting

Quality

Quality Covers all aspects of the

laboratory

Assay validation

Internal quality control

Quality Assurance / Sample

exchange

UKAS accreditation

How can we know our result is correct?

Quality

Must be completed before

assay put into routine use

Signed off by manager / HoD

Covers technical performance

of assay Sensitivity

Specificity

Precision

Linearity

Assay Validation

Quality

Reference ranges Assay dependent

From literature as starting point

Masschke et al

TMA 7.7µmol/mmol crt

TMANO 119µmol/mmol crt

N-oxidation 94%

Verified in laboratory

37 samples from unaffected individuals

Mean TMA 0.82µmol/mmol crt (range 0.23 – 3.13)

Mean N-oxidation 98% (range 81 – 100)

Assay Validation

Quality

Sample exchange programme Four participating laboratories worldwide

Children’s Hospital Colorado

LC-MSMS

Radboud UMC Nijmegen

1H-NMR

UniversitätsKlinikum Heidelberg

SPME-GCMS

Sheffield Children’s Hospital

HS-GCMS

Two distributions per year

Five samples per distribution

Scheme begun February 2016

Good performance so far

External Quality Assurance

(EQA)

Quality

United Kingdom Accreditation Service

(UKAS)

ISO15189 – Medical laboratories –

requirements for quality and competence International standards

Cover all aspects of laboratory work

Recent inspection Observed assay being performed

SOPs

Training records / competencies

IQC / EQA

Reporting

Laboratory accreditation

Results

One year on

Trimethylamine testing

Results

Assay running for one year n = 725

Includes repeat samples and monitoring patients,

including those on treatment

147 results below limit of quantitation

Very low levels, unable to provide quantitative result

453 results within ref range for TMA and N-oxidation

94 results outside ref ranges for TMA and N-oxidation

8 results outside ref range for TMA, within ref range

for N-oxidation

23 results within ref range for TMA, outside ref range

for N-oxidation

Mainly known patients on dietary restriction

Feb 2017 – Jan 2018

Results for first year

Interpreting TMA results

Important to note:

Results from the ‘old’ method cannot be directly

compared to those using our ‘new’ method

Results obtained by our method cannot be

interpreted using other laboratories reference ranges

(such as those quoted on scientific papers)

Updated TMAU assay

Values reported and reference ranges:

Reference range Units

Free TMA:creatinine ratio < 7.7 µmol/mmol

creatinine

TMA-N-Oxide:creatinine

ratio

< 119 µmol/mmol

creatinine

% Oxidation > 94 %

Urine creatinine N/A mmol/L

These were originally taken from a paper – we are now confident that these

ranges are correct for our assay.

Summary of reporting

This assay is a ‘screen’

There is much interplay between enzyme activity,

diet, gut flora and hormones

There can be intra-individual variation of results

Clinical details provided are often lacking

For a definitive diagnosis of Primary

Trimethylaminuria genetic testing is required

Summary

Trimethylamine testing requires much expertise

and specialist (expensive!) equipment

Sample exchange scheme set up and showing

good agreement of results between laboratories

Reporting of results of new assay at SCH now

finalised