Porphyrin methodology Jackie Woolf Senior BMS Cardiff Porphyria - - PowerPoint PPT Presentation

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Porphyrin methodology Jackie Woolf Senior BMS Cardiff Porphyria - - PowerPoint PPT Presentation

Jun 24, 2023 307 likes •889 views

Porphyrin methodology Jackie Woolf Senior BMS Cardiff Porphyria Service Introduction The right choice General principles for sample handling Methods - Aminolaevulinic acid - Porphobilinogen - Total urine porphyrin - Plasma

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SLIDE 1

Porphyrin methodology

Jackie Woolf Senior BMS Cardiff Porphyria Service

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SLIDE 2

Introduction

  • The right choice
  • General principles for sample handling
  • Methods
  • Aminolaevulinic acid
  • Porphobilinogen
  • Total urine porphyrin
  • Plasma porphyrin screening
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SLIDE 3

Is this investigation appropriate?

  • Acute vs. cutaneous - protocols
  • Patient age
  • Childhood onset (EPP, CEP, ALAD,

homozygous porphyrias )

  • Adult onset (AIP, VP, HC)
  • Red herrings (Tyrosinaemia, Pb poisoning,

secondary coproporphyria)

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SLIDE 4

Sample choice

  • Photosensitivity (EPP) EDTA blood
  • Skin fragility (early onset) Urine + EDTA
  • Skin fragility (VP, HC, PCT) Urine + EDTA

+ faeces

  • Acute (Child) Urine
  • Acute (Adult) Urine + EDTA
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SLIDE 5

Sample handling

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SLIDE 6

Urine and faecal samples should be stored frozen unless they are to be assayed immediately. Do not freeze whole blood samples.

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SLIDE 7

A random urine sample, protected from light, is the preferred specimen for urinary porphyrin analysis.

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SLIDE 8

Arguments against 24 hr urine collections

  • Unnecessary delay
  • Incomplete collections
  • Bacterial action
  • Inconvenience to patients
  • Expense of transport
  • Exposure to light
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SLIDE 9

Protection from light

  • Pre-analytically
  • Throughout analysis
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SLIDE 10

Urine samples must NOT be centrifuged

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SLIDE 11

Care should be taken when interpreting the results from very dilute urine samples

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SLIDE 12

High risk samples

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Clinical details Genetic consent

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Methods - generally

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SLIDE 15

Methods for porphyrin screening should fulfil the following criteria :

  • Robust methodology
  • Internal quality control
  • External quality assurance
  • Realistic turn-around times
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SLIDE 16

Methods – acute presentation

  • ALA
  • PBG – screening vs quantitation
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All laboratories should be able to offer a rapid, sensitive and proven method for urinary porphobilinogen analysis.

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SLIDE 18

PBG methodology

  • Acute attack = increased urine PBG
  • Requirement to detect ALL urines with

clinically significant PBG

  • Detection limit
  • Interference
  • Speed
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SLIDE 19

PBG methodology (cont)

  • Watson-Schwartz
  • Trace kit
  • Mauzerall & Granick (modified)
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SLIDE 20

Watson - Schwartz

  • Qualitative
  • Quickest
  • Urine + Ehrlich’s (DMAB)
  • Organic solvent
  • False +ve vs false –ve
  • Validated protocols
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SLIDE 21

1ml QC or urine

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SLIDE 22

+ 1ml Ehrlichs reagent

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SLIDE 23

+ 2ml saturated sodium acetate

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SLIDE 24

+1ml amyl alcohol

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SLIDE 25

Re-extract with amyl alcohol

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SLIDE 26

Clear upper layer

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SLIDE 27

Trace kit

  • Resin filled syringes + filters
  • Comparison with artificial standards
  • Quick
  • Symptomatic initial screening
  • ? Asymptomatic screening - unsuitable
  • ? More sensitive & specific
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SLIDE 28

Mauzerall & Granick

  • Time consuming
  • Gold standard (reliable & specific)
  • Few interferences
  • Follow-up to +ve screen or continuing

clinical suspicion.

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SLIDE 29

Basic ingredients

100g Resin 200ml H2O Working resin

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SLIDE 30

Loading 2ml resin

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SLIDE 31

Column volume resin wash (H2O)

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SLIDE 32

Ehrlichs reagent

DMAB Glacial acetic acid 70% perchloric acid

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SLIDE 33

Commercial QC and samples

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1ml QC or urine sample

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H2O column wash

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Elute PBG – 1st stage

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SLIDE 37

Elute PBG – 2nd stage

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Volume adjustment

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Mix well

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1ml eluate + 1ml Ehrlichs

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Mix well

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15 minute incubation

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Incubation complete

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Spectrophotometer (553nm)

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Increased PBG vs. Normal PBG

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Methods - Photosensitivity

  • Plasma fluorescence emission

spectroscopy

  • Calibration of fluorimeters for porphyrin

analysis is instrument specific

  • A red-sensitive photomultiplier is essential

for porphyrin analysis

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SLIDE 47

50 100 150 200 250 590 600 610 620 630 640 650 Wavelength (nm) Fluorescence Units

Plasma porphyrin fluorescence

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SLIDE 48

Methods – skin fragility

  • The soret band
  • Porphyrin carboxylic acids and their methyl

esters in organic solvents have a characteristic absorbance peak at 400-410nm

  • The wavelength and relative intensity vary

according to the β-substituents present.

  • Absorbance at soret band maximum in acid

solution widely used to determine conc.

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SLIDE 49

Absorption spectrum of an acidified urine sample

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SLIDE 50

Methods - fractionation

  • HPLC
  • C18 column with guard
  • Gradient elution
  • 50ul sample volume
  • 25min run time/sample
  • Fluorimetric detector (red-sensitive PMT)
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SLIDE 51

Advantages of HPLC

  • Ease of separation + rapid
  • Can use products from quantitative assays
  • Good resolution
  • Quantitative
  • Less labour intensive
  • BUT - costs more
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SLIDE 52

Unaffected patient

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SLIDE 53

CEP

4 6 9 11 13 15 18 20 FU

4 6 8 10 12 14 16 18 20 FU

Urine

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Enzymes - to measure or not to measure

  • Cytosolic (PBGD) - OK
  • Mitochondrial (ALA synthase, COPPOX,

PPOX, Ferrochelatase,) - small sample, lymphocytes, fibroblasts + low activities. Technically difficult

  • Overlapping ranges
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SLIDE 55

Tandem MS

  • Complete isomer separation (providing all

have different molecular weights)

  • Sensitivity (pmol)
  • Speed
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Summary

  • Minimum service
  • Collaboration
  • Encourage appropriate investigation
  • Maintain analytical and interpretative

expertise

  • Use satisfactory methods – sensitive and

specific

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SLIDE 57

Summary continued

  • Use appropriate IQC
  • Participate in available EQA schemes
  • Seek advice from specialist centres.