9. Judith Timms HIV screening incidents

HIV screening: what can go wrong?
Learning from screening safety incidents
Judith Timms
Consultant Virologist and IDPS laboratory
advisor

FALSE POSITIVE HIV RESULTS
2 HIV screening: what can go wrong? Learning from screening safety incidents
What does this mean?
o For this talk ‘false positive’ refers to the wrong result on a patient
rather than a low level positive that is not confirmed by further tests
on the same sample.
o Due to a breakdown somewhere in the pathway between the
patient being bled and the result being issued.

What are the causes?

o Wrong blood in tube
o Laboratory error

o Laboratory error
Which is the commonest cause?

o Laboratory error
o Laboratory error
Incidents from 2014 to early 2017 reviewed: the error originated in the
laboratory in 8 of 9 cases.

o Laboratory error
o Laboratory error
Observation: when a repeat blood test on a patient fails to confirm the
results of the first sample laboratories tend to assume that there has
been a mix up during the taking and labelling of the sample.

o Laboratory error
o Laboratory error
Observation: when a repeat blood test on a patient fails to confirm the
results of the first sample laboratories tend to assume that there has
been a mix up during the taking and labelling of the sample. This can
delay appropriate laboratory investigation.

EVIDENCE EXAMINED
o All false positive HIV results reported via STEIS and DATIX from
2014 to early 2017.
o Data examined included: -
• the initial Screening Incident Assessment Form (SIAF).
• the more detailed root cause analysis report (if available).
o Looking for any common themes or lessons that should be shared
with all IDPS providers.

IMPLICATIONS OF FALSE POSITIVE RESULTS
o Some pregnant women suffered the distress of being told that they were HIV
positive only to have that diagnosis reversed.
o The anxiety and distress that a diagnosis of HIV can cause should not be
underestimated.
o In some instances the error was picked up before the woman was given the
incorrect diagnosis.
o The work required to thoroughly investigate the cause of a false positive
result is considerable.
o It is therefore important that all those who provide screening services should
examine their own processes to see if there is anything they can improve
upon that reduces the risk of generating false positive results.
o Incidents will happen: sharing what we learned will help future investigations.

WRONG BLOOD IN TUBE: THE ONLY INCIDENT
o A booking blood was reported as HIV positive: the sample was
taken from a known HIV positive patient.
o But both form and sample were labelled with the labels from a
different patient with the same name who was HIV negative.
o No discrepancy noticed by laboratory as form and sample matched.
o The sample was booked in and subsequently reported on the wrong
patient, who had not actually had a blood test that day.
How did it happen?
o The midwife asked the clinic receptionist for labels but did not check
that 3 details on the labels matched the patient before taking blood.
o The results were not given to the patient as a discrepancy in the full
details was noticed prior to patient interview.

LABORATORY ERROR: CASE 1
o The initial HIV screening test on a booking blood was positive.
o This was communicated to the screening team and the patient before the
confirmatory results had been performed at a second laboratory.
o The sample did not confirm in further assays, nor did a repeat sample.
o Screen positive results should not be communicated until confirmatory tests
are completed on the screening sample.
o This incident was a failure to follow the guidelines.
o It is often not possible to give a definitive negative result on a sample that
has tested positive in one assay. However, if the full results are available
and two out of three tests are negative then the conversation with the
patient is rather different.

o Are there ever any acceptable exceptions to the guidelines regarding
completion of confirmatory testing prior to communication of results?

o Are there ever any acceptable exceptions to the guidelines regarding
completion of confirmatory testing prior to communication of results?
o When the test is urgent rather than routine because the patient is unbooked
and in labour or has just delivered.
o Notifying urgent results on the basis of a single test is most likely to happen
• Out of hours - even large laboratories with OOH service may not have
confirmatory tests available 24/7.
• Smaller laboratories without in-house confirmatory assays.
o The balance of risks is between acting on a single test or delaying post-
exposure prophylaxis for the baby.

o A booking blood was reported as being positive for HIV but repeat samples
failed to confirm this result.
o The possibility of mislabelling the sample by the midwife was excluded,
hence contamination was likely to have occurred within the laboratory.
o Samples known to be HIV positive were processed in the same batch,
raising the possibility of cross contamination.
o The lab conducted titration tests to determine what level of contamination
could be responsible for the positive result.
o Less than 2 microlitres, possibly even as little as 0.2 microlitres of a positive
sample produced a positive result when added to a seronegative sample.

o Two possibilities for contamination identified relating to the liquid handling
device: -
• A drip falling from the tip of the liquid handling device as it passed over
the tubes to load the plates;
• An airborne droplet created in a tube containing HIV positive blood
which then splashed into an adjacent tube.
o A potential contributing factor was the use of a relatively old (15 years)
piece of equipment that was difficult to maintain properly due to the
shortage of replacement parts.

o A booking blood reported as being positive for HIV but a repeat sample
o The first sample gave what was regarded as a ‘high’ positive result which
was repeatable and confirmed at a second laboratory.
o There was limited investigation into processes in the laboratory and the
incident appears to have been attributed to mislabelling of the sample either
in phlebotomy or in the laboratory.
o Contamination does not appear to have been considered, perhaps because
the initial result was ‘high’ although the assay is not actually quantitative.

o A booking blood reported as being positive for HIV but a subsequent point
of care test in GUM and repeat testing in the screening laboratory and a 2nd
laboratory failed to confirm this result.
o The initial assumption was that this was a ‘wrong blood in tube’ incident.
o This was on the basis that the result was ‘high’ and the assumption that any
carry-over of a positive sample would have produced a ‘low’ positive result.
o The process was highly automated and used a tracked system with a
random-access analyser.
o However, investigation of the procedures undertaken by the midwife
suggested ‘wrong blood in tube’ was highly unlikely.
o It was concluded that contamination within the laboratory was the only
possible explanation, although the laboratory staff were not able to identify
a definite cause or any issues with the automated system.

o In this case the patient’s booking blood was reported as positive for both
HIV and Hepatitis B.
o A repeat sample was negative for both viruses.
o The initial sample was positive for both hepatitis B surface antigen and ‘e’
antigen and gave a viral load of 1.3 x 105 copies/ml (perhaps a little low for
an ‘e’ antigen positive sample).
o The confirmatory HIV tests were also positive (4th and 3rd generation
assays) although an aliquot of the original sample sent to Colindale for
typing was too weakly positive to give a typing result.
o Repeat samples from other women who had booking bloods taken on the
same day were negative.

o Microsatellite analysis of free-circulating DNA showed that the original
sample and 2 subsequent samples were from the same woman.
o ie there had been no mislabelling/sample switch in the antenatal clinic or
laboratory, hence the original sample must have been contaminated.
o The laboratory equipment had been replaced less than a year before and
compromised a highly automated platform but the pre-analytic robot had
broken down and was not in operation.
o The sample did not pass through the centrifugation, decapping and track
modules but instead was centrifuged ‘offline’, manually de-capped and then
front-loaded on to the analyser.
o It was concluded that the contamination event took place during the manual
decapping and tube handling operation.
o None of the other front loaded samples had a dual infection and none of the
positive samples were in proximity to the original antenatal tube.

o The laboratory undertook two simulations which demonstrated: -
• When the track system was used without manual intervention it was
highly unlikely to cause contamination.
• The manual handling to uncap tubes involved in front loading posed a
risk of tube to tube contamination from glove contamination.

o A booking blood reported as being positive for HIV but two repeat samples
o For the initial sample the screening HIV test and one of the confirmatory
HIV tests were positive whilst the second confirmatory test was
indeterminate. The tests were repeated as they were unexpected (negative
3 years previously) and the same results obtained.
o The assay values consistent with a recent, primary infection – which in the
context of a patient who was previously HIV negative was a real possibility
– but would also be consistent with contamination.
o The possibility of contamination was ruled out by the laboratory as all the
antenatal samples were tested from the primary tube and on a dedicated
analyser and there were no other HIV positives tested on that day and the
results were regarded as a biological false positive.

Biological false positive?
o The interval between the booking blood and the repeat samples is not given
but is unlikely to have been more than 2 weeks.
o Unusual for a patient’s sample to give a false positive result in 2 different
assays and indeterminate in one assay that then resolved completely.
o The vast majority of biological false positives are in one assay only (which is
why positive results in additional assays are required before saying a
patient is positive) and do not resolve so quickly.

Misinterpretation of forensic tests?
o It was concluded that contamination could not have occurred because
forensic DNA tests on the original sample and a follow-up sample showed
the samples to have come from the same patient and no contaminating
DNA was detected.
o This conclusion is unreliable since forensic tests would not be expected to
pick up a very low level of contaminating DNA.

o The initial HIV screening test on a booking blood was positive.
o A second blood sample was requested whilst confirmatory tests were being
performed at a reference laboratory. This does not follow national policy
whereby testing should be completed before results are communicated.
o The patient was not given the initial result but was told that there were
‘technical difficulties’ with the previous sample.
o The first sample was positive in 2 confirmatory assays but the repeat
sample was negative in the initial screening assay and subsequent assays.
o Forensic testing revealed identical DNA profiles in the clot and serum from
the index case and the repeat sample.
o No additional DNA was detected in the index sample. This suggests
contamination by a small amount of HIV positive sample, too small to be
detected in DNA studies but sufficient to produce a positive HIV test.

o The laboratory used a highly automated track for testing antenatal samples.
o In this instance there was insufficient sample to facilitate automated
pipetting on the pre-analytics although there was sufficient for testing (and
for confirmation) if this step was by-passed by manual intervention.
o In this situation the sample is usually described as a ‘short-sample’.
o This meant that sample required manual intervention by way of pipetting the
serum and transferring it to a ‘hanging cup’ (which reduces the dead
volume) for re-testing on the track.
o It was not possible to determine the exact circumstances of the
contamination at as there was no record of when, where and by whom the
sample was transferred to the hanging cup.

o A booking blood was reported as being positive for HIV but repeat sample
o Investigation indicated that the primary tube had tested positive but an
aliquot tested negative.
o Despite the discrepancy the primary tube result was regarded as correct
and that sample was sent away for confirmation at a 2nd laboratory.
o It appears that the primary sample was contaminated after aliquoting.
o Splashing of samples had been observed with the analyser so it was
possible this was how the contamination had occurred.
o It is good laboratory practice to confirm from the primary tube when an
aliquot is tested or from an aliquot when the primary tube is tested but
although this happened the discrepant result was disregarded.

CONCLUSIONS
o The majority of false positive HIV results were due to laboratory error.
o This includes both manual and automated steps in the testing process and
also decision making in dealing with results.
o The ability of low level contamination to produce strongly positive results
should be more widely recognised.
o Some incident investigations have tended to assume that low volume
contamination could only have produced low level positive results.
o Incident investigation would be more thorough and wide-ranging if this was
better known.

CONCLUSIONS
o Forensic tests (microsatellite analysis of free-circulating DNA) are useful in
demonstrating whether or not samples came from the same patient.
o Use forensic tests early - before recalling large numbers of women for
retesting - the results will usually mean that such recall is unnecessary.
o The limitations of these tests must also be recognised: they are useful for
demonstrating whether or not samples came from the same patient (wrong
blood in tube?) but NOT for excluding low levels of contamination.
o The degree of contamination needs to be of the order of 5-10% to be
reliably detected and would certainly not detect the human DNA
contamination from a splash, drip or reused pastette.

LESSONS
1. False positive HIV results are usually generated by errors within the
laboratory and are rarely due to ‘wrong blood in tube’.
2. The HIV screening assays are highly sensitive and a very small amount of
contamination can cause what appears to be a strongly positive result.
3. The early use of forensic tests can be useful to exclude ‘wrong blood in
tube’ and prevent unnecessary recall of patients.
4. Track systems which include pre-analytic modules are very safe and reduce
the risk of contamination.
5. If the pre-analytics module is not operational consideration should be given
to delaying testing of all but the most urgent samples until the track is fully-
functional since manual work-arounds increase contamination risk.
6. Laboratories and screening services should consider whether to reject short
samples which are insufficient for automated testing since this would avoid
resorting to manual intervention which increases the risk of contamination.

ACKNOWLEDGEMENTS
o The obstetrics services and screening laboratories who declared their
incidents and shared their investigations what went wrong.
o Without this openness we would have been unable to analyse the overall
picture or to share the learning from these incidents.

9. Judith Timms HIV screening incidents

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