1. Auditing antenatal diagnoses – what is good practice? Sarah Ball, Tim Hutchin, George Gray, Linda Jenkinson, Mary Anne Preece West Midlands Laboratory for Inherited Metabolic Disorders, Birmingham Children’s Hospital

2. Summary Purpose of antenatal audit Purpose of antenatal audit Practice at West Midlands Inherited Metabolic Disorders Laboratory Practice at West Midlands Inherited Metabolic Disorders Laboratory Audit outcome 1984 – 2005 Audit outcome 1984 – 2005 Details of 2 inconsistent audits and resultant change in practice Details of 2 inconsistent audits and resultant change in practice Suggested system for adoption by other laboratories Suggested system for adoption by other laboratories

3. Purpose of antenatal audit (1) In utero diagnosis of inherited disease: most high risk test for genetic laboratories In utero diagnosis of inherited disease: most high risk test for genetic laboratories Misdiagnosis  Misdiagnosis  loss of a potentially healthy life birth of a child with a painful and crippling disease Huge financial and social costs of misdiagnosis Huge financial and social costs of misdiagnosis

4. Purpose of antenatal audit (2) Laboratory procedures to ensure reliability of test result – minimise pre-analytical, analytical and post-analytical errors Laboratory procedures to ensure reliability of test result – minimise pre-analytical, analytical and post-analytical errors Potential sources of unexpected risks of misdiagnosis: Potential sources of unexpected risks of misdiagnosis:   Genetics of the disease   Expression in utero (biochemical tests)   Technical problems with an established test   Inadequate test validation

5. Purpose of antenatal audit (3) To monitor the quality of our service we routinely attempt audit on ALL our antenatal diagnoses To monitor the quality of our service we routinely attempt audit on ALL our antenatal diagnoses Take action if there are anomalies Take action if there are anomalies

6. Practice in the West Midlands Inherited Metabolic Disorders Laboratory West Midlands population is 5.3million West Midlands population is 5.3million Birth rate of 70000 per year Birth rate of 70000 per year Since 1984 offered a regional service for co-ordinating prenatal tests for IMDs Since 1984 offered a regional service for co-ordinating prenatal tests for IMDs Test may be performed: Test may be performed: In-house (DNA/biochemical) In-house (DNA/biochemical) UK biochemistry/molecular genetics network lab UK biochemistry/molecular genetics network lab Diagnostic lab abroad Diagnostic lab abroad Research lab in UK or abroad Research lab in UK or abroad National/international referral centre for some specialist biochemical and DNA tests National/international referral centre for some specialist biochemical and DNA tests

7. Prenatal test and audit process (1) At time prenatal testing arranged letter to clarify letter to clarify Sample requirement and transport arrangements Sample requirement and transport arrangements Who will dissect sample Who will dissect sample Tests to be done and by whom Tests to be done and by whom Identify who will be giving results to the family Identify who will be giving results to the family Advise that all prenatal tests are audited and recommend early discussion of audit with the family Advise that all prenatal tests are audited and recommend early discussion of audit with the family All prenatal diagnoses are entered in a searchable database (Access) All prenatal diagnoses are entered in a searchable database (Access)

8. Prenatal test and audit process (2) Analytical process Receive sample (CVS or AF – cultured or uncultured) Receive sample (CVS or AF – cultured or uncultured) Perform analysis Perform analysis Results reported and authorised Results reported and authorised

9. Prenatal test and audit process (3) Final report letter = CHASE 1 combines all results (eg MCC exclusion, uncultured and cultured CVS/AF results etc) combines all results (eg MCC exclusion, uncultured and cultured CVS/AF results etc) Requests appropriate audit sample Requests appropriate audit sample TOP: skin/placenta TOP: skin/placenta Continuing pregnancy: blood/biochemistry or clinical report of healthy baby Continuing pregnancy: blood/biochemistry or clinical report of healthy baby Date of first chase letter recorded on database Date of first chase letter recorded on database

10. Prenatal test and audit process (4) Audit sample Receive audit sample for testing and enter result on data base Receive audit sample for testing and enter result on data base Or receive clinical report (acceptable where condition manifest in neonatal period) Or receive clinical report (acceptable where condition manifest in neonatal period) Result of audit sample testing or clinical report entered on database Result of audit sample testing or clinical report entered on database CHASE 2 CHASE 2 If no audit sample received 3 months after the edd write to request appropriate samples If no audit sample received 3 months after the edd write to request appropriate samples

11. Prenatal test and audit process (5) Unaudited prenatal test If no response after 3 months of second chase letter then prenatal test recorded as unaudited If no response after 3 months of second chase letter then prenatal test recorded as unaudited

12. Results of antenatal audit Total number of auditable antenatal diagnoses 1984 - 2005 340 Completed audits 278 (82%) Uncompleted audits 62(18%)

13. Completed Audits Total completed audits 1984 – 2005 278 Misdiagnoses 2 (0.07%)

14. Uncompleted audits Total uncompleted audits 62 No reply to audit request 27 (44%) Parents refused testing 11 (18%) Samples received but tests failed 4 (6%) No sample received on TOP or foetal loss 20 (32%) TOP when predicted affected 9 (45%) TOP for other abnormality when predicted unaffected 2 (10%) TOP for social reasons when predicted unaffected 1 (5%) Miscarriage subsequent to testing 8 (40%) No sample received:-

15. Inconsistent case 1 Index case Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Prenatal test (1995) VLCFA quantification in uncultured CVS VLCFA quantification in uncultured CVS Predicted unaffected male foetus Predicted unaffected male foetusAudit Neonatal plasma VLCFA indicated affected status Neonatal plasma VLCFA indicated affected status Audit trail on u-CVS – no evidence for sample swap Audit trail on u-CVS – no evidence for sample swap Reanalysis of c-CVS – variability of VLCFA levels with passage Reanalysis of c-CVS – variability of VLCFA levels with passageConclusion Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses

16. Inconsistent case 1 Index case Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Prenatal test (1995) VLCFA quantification in uncultured CVS VLCFA quantification in uncultured CVS Predicted unaffected male foetus Predicted unaffected male foetusAudit Neonatal plasma VLCFA indicated affected status Neonatal plasma VLCFA indicated affected status Audit trail on u-CVS – no evidence for sample swap Audit trail on u-CVS – no evidence for sample swap Reanalysis of c-CVS – variability of VLCFA levels with passage Reanalysis of c-CVS – variability of VLCFA levels with passageConclusion Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses

17. Inconsistent case 1 Index case Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Prenatal test (1995) VLCFA quantification in uncultured CVS VLCFA quantification in uncultured CVS Predicted unaffected male foetus Predicted unaffected male foetusAudit Neonatal plasma VLCFA indicated affected status Neonatal plasma VLCFA indicated affected status Audit trail on u-CVS – no evidence for sample swap Audit trail on u-CVS – no evidence for sample swap Reanalysis of c-CVS – variability of VLCFA levels with passage Reanalysis of c-CVS – variability of VLCFA levels with passageConclusion Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses

18. Inconsistent case 1 Index case Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Baby boy with X-linked adrenoleukodystrophy (X-ALD) by plasma very long chain fatty acid quantification Prenatal test (1995) VLCFA quantification in uncultured CVS VLCFA quantification in uncultured CVS Predicted unaffected male foetus Predicted unaffected male foetusAudit Neonatal plasma VLCFA indicated affected status Neonatal plasma VLCFA indicated affected status Audit trail on u-CVS – no evidence for sample swap Audit trail on u-CVS – no evidence for sample swap Reanalysis of c-CVS – variability of VLCFA levels with passage Reanalysis of c-CVS – variability of VLCFA levels with passageConclusion Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Result published and change of practice worldwide: VLCFA quantification AND oxidation of VLCFAs Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses Mutation analysis of ABCD1 gene – now preferred procedure for X-ALD prenatal diagnoses

19. Inconsistent test 2 Index case Neonatal hypoglycaemia, lactic acidosis Neonatal hypoglycaemia, lactic acidosis then hypotonia, liver failure, nystagmus and died then hypotonia, liver failure, nystagmus and died Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Prenatal test PEPCK very rare no experience of prenatal testing worldwide PEPCK very rare no experience of prenatal testing worldwide Testing lab agreed to test c-AF cells on research basis only Testing lab agreed to test c-AF cells on research basis only Parents counselled that experimental procedure Parents counselled that experimental procedure C-AF cell PEPCK within normal limits C-AF cell PEPCK within normal limitsAudit Baby born with same symptoms as index case Baby born with same symptoms as index case Fibroblast PEPCK within normal limits Fibroblast PEPCK within normal limits Further studies suggested a possible mtDNA depletion disorder Further studies suggested a possible mtDNA depletion disorderConclusion Involvement of PEPCK remains unknown Involvement of PEPCK remains unknown Availability of information on reliability of test crucial for informed genetic counselling Availability of information on reliability of test crucial for informed genetic counselling

20. Inconsistent test 2 Index case Neonatal hypoglycaemia, lactic acidosis Neonatal hypoglycaemia, lactic acidosis then hypotonia, liver failure, nystagmus and died then hypotonia, liver failure, nystagmus and died Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Prenatal test PEPCK very rare no experience of prenatal testing worldwide PEPCK very rare no experience of prenatal testing worldwide Testing lab agreed to test c-AF cells on research basis only Testing lab agreed to test c-AF cells on research basis only Parents counselled that experimental procedure Parents counselled that experimental procedure C-AF cell PEPCK within normal limits C-AF cell PEPCK within normal limitsAudit Baby born with same symptoms as index case Baby born with same symptoms as index case Fibroblast PEPCK within normal limits Fibroblast PEPCK within normal limits Further studies suggested a possible mtDNA depletion disorder Further studies suggested a possible mtDNA depletion disorderConclusion Involvement of PEPCK remains unknown Involvement of PEPCK remains unknown Availability of information on reliability of test crucial for informed genetic counselling Availability of information on reliability of test crucial for informed genetic counselling

21. Inconsistent test 2 Index case Neonatal hypoglycaemia, lactic acidosis Neonatal hypoglycaemia, lactic acidosis then hypotonia, liver failure, nystagmus and died then hypotonia, liver failure, nystagmus and died Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Prenatal test PEPCK very rare no experience of prenatal testing worldwide PEPCK very rare no experience of prenatal testing worldwide Testing lab agreed to test c-AF cells on research basis only Testing lab agreed to test c-AF cells on research basis only Parents counselled that experimental procedure Parents counselled that experimental procedure C-AF cell PEPCK within normal limits C-AF cell PEPCK within normal limitsAudit Baby born with same symptoms as index case Baby born with same symptoms as index case Fibroblast PEPCK within normal limits Fibroblast PEPCK within normal limits Further studies suggested a possible mtDNA depletion disorder Further studies suggested a possible mtDNA depletion disorderConclusion Involvement of PEPCK remains unknown Involvement of PEPCK remains unknown Availability of information on reliability of test crucial for informed genetic counselling Availability of information on reliability of test crucial for informed genetic counselling

22. Inconsistent test 2 Index case Neonatal hypoglycaemia, lactic acidosis Neonatal hypoglycaemia, lactic acidosis then hypotonia, liver failure, nystagmus and died then hypotonia, liver failure, nystagmus and died Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Fibroblast phosphoenolpyruvate carboxylasekinase (PEPCK) showed marked deficiency Prenatal test PEPCK very rare no experience of prenatal testing worldwide PEPCK very rare no experience of prenatal testing worldwide Testing lab agreed to test c-AF cells on research basis only Testing lab agreed to test c-AF cells on research basis only Parents counselled that experimental procedure Parents counselled that experimental procedure C-AF cell PEPCK within normal limits C-AF cell PEPCK within normal limitsAudit Baby born with same symptoms as index case Baby born with same symptoms as index case Fibroblast PEPCK within normal limits Fibroblast PEPCK within normal limits Further studies suggested a possible mtDNA depletion disorder Further studies suggested a possible mtDNA depletion disorderConclusion Involvement of PEPCK remains unknown Involvement of PEPCK remains unknown Availability of information on reliability of test crucial for informed genetic counselling Availability of information on reliability of test crucial for informed genetic counselling

23. A system for antenatal audit Database in place for audit Database in place for audit Facilitates running work lists regularly to chase audit gaps Facilitates running work lists regularly to chase audit gaps Add data: Add data: Before prenatal test Before prenatal test Final result of prenatal test: date of first chase Final result of prenatal test: date of first chase Date of second chase (if necessary) Date of second chase (if necessary) Result of audit Result of audit

24. Minimum data required for audit database Before prenatal test Mother’s name Mother’s name Disease Disease Date of sampling procedure Date of sampling procedure Sample type Sample type

25. Minimum data required for audit database After prenatal test Result of diagnostic test Result of diagnostic test Date of first chase (i.e. final report letter) Date of first chase (i.e. final report letter) Pregnancy continuing or termination of pregnancy Pregnancy continuing or termination of pregnancy

26. Minimum data required for audit database Audit test If termination of pregnancy: If termination of pregnancy: Foetal skin/placenta Foetal skin/placenta Result consistent or inconsistent Result consistent or inconsistent If pregnancy continuing: If pregnancy continuing: Await appropriate sample from baby Await appropriate sample from baby Result of biochemistry or DNA analysis on neonatal sample Result of biochemistry or DNA analysis on neonatal sample Result consistent or inconsistent Result consistent or inconsistent

27. Minimum data required for audit database Second chase 3 months after live birth expected 3 months after live birth expected If no audit sample or appropriate clinical report If no audit sample or appropriate clinical report Repeat request for audit sample Repeat request for audit sample If still no reply or no sample after a further 3 months record as unaudited If still no reply or no sample after a further 3 months record as unaudited

28. Conclusions (1) Essential to ensure awareness of audit in clinical team and counsel family before prenatal test performed Essential to ensure awareness of audit in clinical team and counsel family before prenatal test performed Audit is routine Audit is routine Attempt to audit all prenatal tests Attempt to audit all prenatal tests Parents may decline audit specimen Parents may decline audit specimen Parents may chose not to be informed of the audit result if TOP Parents may chose not to be informed of the audit result if TOP

29. Conclusions (2) Clinical and lab audit is essential to good practice Clinical and lab audit is essential to good practice Our results show value of antenatal audit Our results show value of antenatal audit Systems are easily set up – high success rate of audit completion Systems are easily set up – high success rate of audit completion Audit permits evaluation of new methods and checking performance of established methods Audit permits evaluation of new methods and checking performance of established methods Failure to detect technical error (e.g. atypical enzyme deficiency or anomalous mutation test) would mean the same misdiagnosis could be repeated in subsequent pregnancies Failure to detect technical error (e.g. atypical enzyme deficiency or anomalous mutation test) would mean the same misdiagnosis could be repeated in subsequent pregnancies Antenatal screening committee document on “Prenatal screening standards and protocols” April 2008 will have a standard on audit of amniocentesis and CVS diagnostic procedures and outcomes of pregnancy Antenatal screening committee document on “Prenatal screening standards and protocols” April 2008 will have a standard on audit of amniocentesis and CVS diagnostic procedures and outcomes of pregnancy

30. What is done in other labs? What is the current practice in ACC and CMGS laboratories? What is the current practice in ACC and CMGS laboratories? Are audits attempted on all antenatal diagnoses, some or none? Are audits attempted on all antenatal diagnoses, some or none? If only some, how are they selected? If only some, how are they selected?