​False-positive paracetamol levels in a patient with hyperbilirubinaemia: clinical perspectives

Abstract

​Serum concentrations of paracetamol are measured to investigate the cause of acute hepatitis, monitor the clearance of paracetamol from the body and to determine if supratherapeutic levels warrant treatment with N-acetylcysteine (NAC). ​A 49-year-old man treated for ischaemic colitis developed worsening renal and liver function tests. As part of the investigation of hepatorenal failure, paracetamol levels were requested, which were elevated at 14 mg/L (normal <4 mg/L) resulting in treatment with NAC. Despite treatment, levels of paracetamol remained elevated and the link between hyperbilirubinemia and false-positive paracetamol levels was identified. ​Bilirubin and its by-products have intense absorbance in the ultraviolet and visible regions of the electromagnetic spectrum, causing interference in the enzymatic colorimetric assay most commonly used to measure paracetamol concentration, resulting in false-positive paracetamol levels. Laboratories correct for this interference above a predetermined bilirubin concentration, termed the Icteric Index; however, in our case this interference occurred at a lower level of hyperbilirubinaemia than previously identified as significant. This interaction was found to be more significant at lower bilirubin levels when low or no paracetamol levels were present in the serum, resulting in a change to laboratory practice and development of a ‘Sliding Scale’ approach to analysis. ​Concurrent bilirubin or Icteric Index measurement is recommended for all laboratories that use the enzymatic colorimetric assay for paracetamol measurement. Lower Icteric Index or bilirubin thresholds are required when low or no paracetamol levels are present in the serum to prevent false-positive paracetamol results. We describe a new ‘Sliding Scale’ approach to analysis, and highlight an important interaction for clinicians to be aware of.

Background

Within the UK, drug-induced hepatitis is one of the most common aetiologies of acute liver injury (ALI) or failure, in addition to toxic injury, idiosyncratic reaction to medication, alcoholic hepatitis, viral hepatitis and ischaemic injury.¹ ALI, especially in the early stages, is poorly defined and describes a spectrum of liver dysfunction or damage, which can be marked by significant coagulopathy (defined as an international normalised ratio (INR) ≥1.5) but no discernible hepatic encephalopathy.² Acute liver failure (ALF) is life-threatening liver injury with the development of complications such as hepatic encephalopathy, coagulopathy and impaired protein synthesis within 26 weeks, usually in the absence of any pre-existing liver disease.³ Fulminant liver failure occurs within 8 weeks.¹ For drug-induced hepatitis, paracetamol is the most common causative drug, largely due to the ease of obtaining the medication and Narrow Therapeutic Index in cases of overdose.⁴ Overdose of paracetamol is potentially lethal if not adequately treated with the antidote N-acetylcysteine (NAC).⁵ In cases of ALI or ALF, or suspected paracetamol overdose, serum paracetamol concentrations can be measured to help determine the aetiology of liver dysfunction, and guide treatment with NAC.

Treatment with NAC is generally decided based on time of ingestion and serum concentration of paracetamol. At therapeutic doses, paracetamol is predominantly metabolised in the liver by glucuronide conjugation and sulfate.⁴ A smaller amount, usually less than 5%, is also metabolised via the alternative cytochrome P450 2E1 pathway to the highly reactive and toxic intermediate, N-acetyl-p-benzoquinoneimine (NAPQI), which in turn is reduced to a non-toxic mercaptate or cysteine conjugate by glutathione.^{4 5} In cases of paracetamol overdose, the sulfation and glucuronidation pathways become saturated, resulting in paracetamol metabolisation by the alternative cytochrome P450 2E1 pathway, creating excessive amounts of NAPQI, thus depleting glutathione reserves. The excess NAPQI binds to the hepatocellular membrane causing hepatocyte necrosis and acute drug-induced hepatitis. Treatment with the glutathione precursor NAC replenishes glutathione stores, thereby preventing hepatocellular damage by once again reducing NAPQI to non-toxic metabolites.^4–6

Here, we present a case of ALI of uncertain aetiology, where raised serum paracetamol levels were found despite minimal ingestion at therapeutic levels and ongoing despite cessation of the drug and treatment with NAC. The link between hyperbilirubinaemia and false-positive paracetamol levels is explored, as well as the development of a new ‘Sliding Scale’ technique for analysing samples, which arose from this case.

Case presentation

A 49-year-old man was admitted to a regional hospital with sudden onset abdominal pain, vomiting and diarrhoea associated with rectal bleeding. Medical history included hypertension, ischaemic heart disease (widespread atheroma was seen on coronary angiography in 2015 following an non ST elevation myocardial infarction (NSTEMI)) and post-traumatic paraplegia with neuropathic bladder and right below knee amputation. He was overweight with a body mass index (BMI) of 30 kg/m². His regular medications included bisoprolol, omeprazole, tramadol, lisinopril, atorvastatin, aspirin and long-term over-the-counter ibuprofen and paracetamol use following his injury, always at therapeutic dose or below. He had no known allergies, was a non-drinker and an ex-smoker and had no history of recreational or herbal drug use.

On admission, the patient was hypotensive with syncopal episodes, and had developed stage III anuric acute kidney injury and ALI (see table 1). An initial CT abdomen showed an oedematous and featureless descending colon suspicious of ischaemic colitis. He required admission to the intensive care unit for vasopressor support and renal replacement therapy, and was managed conservatively for ischaemic colitis with intravenous hydration and antibiotics. On day 8, due to ongoing renal and liver failure, he was transferred to our hospital for tertiary hepatology and renal services and further investigation.

Table 1.

Baseline and admission bloods

Blood tests (normal levels)	Baseline bloods (2016)	Baseline bloods (2017)	Admission bloods (2018)
Biochemistry
Sodium (133–146 mmol/L)	138	137	136
Potassium (3.5–5.3 mmol/L)	4.4	5.0	3.4
Urea (2.5–7.8 mmol/L)	7.3	11.1	13.6
Creatinine (59–104 μmol/L)	97	120	220
C-reactive protein (<5 mg/L)	18	–	11
Estimated glomerular filtration rate	>90	>60	21
Paracetamol level (<4 mg/L)			9
Liver function tests
Aspartate aminotransferase (1–45 U/L)	26	110	356
Alanine aminotransferase (0–41 U/L)	–	–	–
Bilirubin (0–21 μmol/L)	8	9	46
Gamma glutamyltransferase (0–71 U/L)	–	–	–
Albumin (35–50 g/L)	39	32	40
Alkaline phosphatase (30–130 U/L)	161	131	199
Haematology
White cell count (4–11×109/L)	10.2	10.4	24.2
Neutrophil count (1.60–7.50×109/L)	6.25	7.92	20.61
Haemoglobin (130–180 g/L)	141	120	155
Mean cell volume (82–98 fL)	88.9	86.3	88.8
Platelets (140–440×109/L)	220	219	118
Coagulation
International national ratio	1.2	1.6	2
Activated partial thromboplastin time (normal 22–30 s)	–	33.5	–

Investigations

Endoscopic investigations were performed for ischaemic colitis and to rule out bowel infarction. Oesophago-gastroduodenoscopy was essentially normal, and flexible sigmoidoscopy and biopsy from the left colon showed non-specific changes (inflamed granulation tissue in keeping with adjacent ulcer, focal acute cryptitis), which were compatible with ischaemic colitis. Echocardiogram showed an ejection fraction of 60% with no acute changes, and no atrial fibrillation or other arrhythmia was detected during admission. Normal sized kidneys were confirmed on ultrasound, and hydronephrosis excluded. The patient remained dialysis dependent, therefore a renal biopsy was performed, which showed only acute tubular necrosis.

By day 10, there was worsening jaundice and further deterioration in liver function tests (LFTs), which had developed a more cholestatic picture. Possible hepatotoxic medications were withheld, including aspirin (due to coagulopathy), atorvastatin and paracetamol. An abdominal ultrasound was performed that showed increased hepatic echogenicity consistent with fatty infiltration, and new-onset hepatosplenomegaly (spleen measured 15 cm). The gallbladder was difficult to visualise, therefore a repeat CT abdomen and pelvis was performed which was reported as no evidence of thrombus in the major abdominal vessels, fatty appearance of the liver, a contracted and thick-walled gallbladder with minimal fat stranding reflecting cholecystitis and no portal vein thrombosis. Doppler ultrasound of the portal vein confirmed no thrombosis. Contrast magnetic resonance cholangiopancreatography showed a mild degree of cholecystitis with no common bile duct or intrahepatic biliary dilatation noted (figure 1).

Figure 1.

Magnetic resonance cholangiopancreatography with contrast: The gallbladder is partially distended with possible sludge. Mild wall thickening of the gallbladder noted in keeping with the degree of inflammation. No evidence of calculi. No intrahepatic biliary radical or common bile duct (CBD) dilation. Unremarkable appearance of the upper abdominal organs. The pancreatic duct is normal in calibre. No focal pancreatic pathology identified. Persistent thickening of the splenic flexure and descending colon noted, which is likely to be infective/ischaemic in nature.

A non-invasive liver screen was essentially normal, besides marginally raised paracetamol levels (see table 2). On initial admission to the regional centre, the patient’s paracetamol levels were checked and found to be 9 mg/L, which although raised (normal <4 mg/L) was in keeping with his regular paracetamol use prior to admission and below the threshold for treatment. His bilirubin at that time was 46 μmol/L (see table 1). Despite subsequent cessation of paracetamol for at least 10 days, when repeated in our tertiary centre, paracetamol levels remained elevated and continued to rise further to 14 mg/L as the bilirubin rose. Due to ongoing liver injury, treatment with NAC was therefore started. Serial paracetamol levels, however, remained raised (see table 3), and following discussion with the clinical biochemistry department, it became apparent that paracetamol levels were likely falsely elevated due to hyperbilirubinaemia. Treatment with NAC was therefore discontinued.

Table 2.

Non-invasive liver screen

Test ordered	Results and laboratory comments
Hepatitis A (IgM), B (surface antigen), C and E (antibody)	Negative
Cytomegalovirus PCR	Negative
Epstein-Barr virus PCR	Negative
Autoimmune liver profile (antibodies to: M2, M2-3E, SP100, PML, GP210, LKM1, LC1, SLA, Ro-52)	Negative
Tissue auto antibodies (reticulin R1, gastric parietal cell, smooth muscle, M2 and non-M2 mitochondria, liver/kidney/microsome, ribosome)	Negative
Extractable nuclear antigen antibodies (Ro, La, Sm, RNP, Jo-1, Scl-70 and CENP-b)	Negative
Alpha-1-antitrypsin level (1.10–2.10)	3.05
Haemochromatosis genotyping	C282Y homozygous related hereditary haemochromatosis is excluded
Antineutrophil cytoplasmic antibodies	Negative
Compliment levels (c3, c4)	Normal
Leptospirosis IgM and DNA	Negative
Immunoglobulins	Mildly elevated IgA and IgG (polyclonal elevation of IgA and IgG associated with drug therapy, chronic mucosal infection, liver disease and connective tissue disease)

Table 3.

Paracetamol levels and liver function tests (LFTs)

Investigation (normal range)	Day 20	Day 23	Day 25
LFTs
Aspartate aminotransferase (1–45 U/L)	155	136	–
Alanine aminotransferase (0–41 U/L)	112	119	130
Gamma-glutamyltransferase (0–71 U/L)	Icterus, at level that renders test inaccurate	Icterus, at level that renders test inaccurate	Icterus, at level that renders test inaccurate
Albumin (35–50 g/L)	22	18	20
ALP	714	779	743
Bilirubin (0–21 μmol/L)	307	321	360
Paracetamol levels
Paracetamol levels (<4 mg/L)	14	10	12
Biochemistry
Sodium (133–146 mmol/L)	130	126	129
Potassium (3.5–5.3 mmol/L)	4.5	4.6	4.6
Urea (2.5–7.8 mmol/L)	17.8	10.6	11.5
Creatinine (59–104 μmol/L)	1078	668	868
Estimated glomerular filtration rate	4	8	6

The patient’s Model For End-Stage Liver Disease Score was calculated at 37, and a fibroScan was reported as showing possible cirrhosis (patient median 13.2 kPa; normal <7 kPa, >14 kPa indicates cirrhosis).⁷ Liver biopsy, however, showed no overt cirrhosis or evidence of hepatocellular damage, but an overall appearance suggestive of intrahepatic and canalicular cholestasis with no signs of obstruction to biliary flow (figure 2).

Figure 2.

Liver biopsy results and ligand for photomicrographs: (A) These are H&E-stained sections, showing different magnifications. Portal zones (PZ) contain an occasional bile ductule (arrow). Areas of pigment retention (cholestasis) are present (marked C). (B) Special stains show mild fibrous expansion of portal zones (green areas in Masson trichrome). Normal liver plate architecture in reticulin-stained section. Iron is seen in Kupffer cells and cholestasis noted.

Differential diagnosis

The exact cause of ALI and severe, cholestatic jaundice was never fully determined, despite liver biopsy. Given the splenomegaly on imaging, the bilirubin ratio was calculated as roughly 0.8 (direct or conjugated blirubin/total bilirubin) ruling out prehepatic causes of jaundice (ie, haemolysis), which was also excluded due to a stable haemoglobin. The initial liver injury was suggested to be secondary to a period of ischaemia and hypotension, and the cholestasis related to medications (in particular antibiotics given for ischaemic colitis) and ductopenia. Important to note, however, was that despite significantly deranged LFTs, the patient showed no other signs of decompensation or ALF, such as hepatic encephalopathy, ascites or hyperammonaemia. He did, however, have an ongoing coagulopathy in terms of both INR (range 1.2–1.7), prothrombin time (13.8 s) and activated partial thromboplastin time (32 s). This was thought to be a combination of both liver dysfunction, dietary vitamin K deficiency and the use of routine low molecular weight heparin during haemodialysis to prevent clotting of the dialysis circuit.

The ischaemic colitis resolved with conservative management with antibiotics and intravenous fluids. The only positive microbiology was a stool screening sample for vancomycin resistant enterococcus, which required barrier nursing but no further specific treatment. As with the ALI, the underlying cause for ischaemic colitis in a young patient was never fully ascertained. He had several risk factors for vascular disease including hypertension, raised BMI and the presence of atheroma on coronary angiography; however, no definitive trigger was found either through history or investigations. Of note, there was no history of herbal or illicit drug abuse, which may have exacerbated mesenteric vasoconstriction or contributed to liver injury.

Outcome and follow-up

At the time of writing, the patient remained dialysis dependent, and due to ongoing liver dysfuction was undergoing further investigation by the liver transplant unit, as well as nutritional support.

Discussion

Several interesting points are raised by this case. First, the association between hyperbilirubinaemia and false-positive paracetamol levels was not well known among clinicians outside of the biochemistry department, and should be highlighted as an important consideration for all physicians practicing acute medicine.

Second, even though hyperbilirubinaemia causing false-positive paracetamol levels is well recognised within biochemistry, it is usually at higher bilirubin levels and automatically corrected for during analysis. Paracetamol levels can be measured through a variety of methods;4–6 8–10 however, the most common, which is also used by our laboratory, is the spectrophotometric method of the enzymatic colorimetric assay, which uses light absorption as part of the technique. Although enzyme-based assays are quick, more convenient and cheaper compared with other techniques, they are generally more prone to interference from biological molecules, such as bilirubin and its by-products or haemolysis.4–6 8–10 Bilirubin has considerable potential for interfering with spectrophotometric measurements because of its broad and intense absorbance in the ultraviolet and visible regions of the electromagnetic spectrum. Paracetamol levels are measured by the change in absorbance noted at 600 nm, which is directly proportional to the quantitative paracetamol concentration in serum. An increase in the background absorbance at 600 nm caused by the presence of bilirubin and its by-products, however, may therefore be falsely interpreted as the presence of paracetamol in the serum.^{5 6 11} To adjust for this, the laboratory will use an Icteric Index to quantify hyperbilirubinaemia; a figure that is determined by comparing the colour of the test serum against a set of colour standards. An Icteric Index of 15 or above indicates active jaundice, and at an Icteric Index of more than or equal to 25, the laboratory will automatically adjust for and report on interference if paracetamol levels are requested by the clinician. An Icteric Index of 25 equates to a bilirubin of approximately 427 μmol/L and is the cut-off given by the analyser manufacturer. However, in our case the maximum bilirubin concentration measured peaked at 307 μmol/L, which was below the threshold to trigger adjustment, and paracetamol levels were subsequently reported high (range 10–14, normal value <4 mg/L).

The raised paracetamol levels resulted in treatment with NAC for 3 days, which was tolerated well and resulted in no adverse effects to the patient. NAC is an extremely safe medication to administer; however, side effects can be experienced. Gastrointestinal effects, for example vomiting, are most common, and although older regimes reported an anaphylactoid reaction in up to 11%–13% of patients (defined as patients who either had NAC treatment interrupted, an intervention with an antiallergy drug, or both), newer treatment regimes have reduced this to 2%.^{11 12} Although in our case treatment with NAC was not indicated, in cases of confirmed or suspected paracetamol poisoning, the potential for side effects should not delay treatment with NAC which can be lifesaving. Furthermore, in our case, treatment with NAC did not delay further investigation and treatment proceeding, as this was continued contemporaneously while NAC was being administered. This is important to acknowledge, as due to false-positive paracetamol levels treating physicians may wrongly assume the cause for ALI, making them less likely to pursue other aetiologies. Patients are therefore at risk of missing vital treatment for a diagnosis that was never found.^4–6

This case reveals a disadvantage of the most commonly used method of the colorimetric enzyme assay, in that despite current protocol and parameters being followed a false-positive result still occurred. The interaction between hyperbilirubinaemia and paracetamol measurement was therefore discovered to be more complex than previously thought, and it is now evident that the apparent level of serum paracetamol detected not only depends on the concentration of serum bilirubin, but also on the true paracetamol concentration itself.^{5 10} As shown in the previous literature, during extreme hyperbilirubinaemia, when there are also true high levels of paracetamol in the serum, an Icteric Index of 25 (bilirubin 427 μmol/L) is an accurate threshold at which to adjust for interference.^{5 10} However, when there are truly no, or low, levels of paracetamol in the blood, a lower concentration of hyperbilirubinaemia is significant to cause an interaction.¹⁰ In our case, no paracetamol had been administered in the upcoming days to when levels were measured; therefore, a bilirubin of 307 μmol/L (or lower) was significant enough to cause a false reading as no paracetamol was truly present in the serum. This has prompted a change in our laboratory practice, and a ‘Sliding Scale’ method of paracetamol level analysis has been developed, whereby lower Icteric Index thresholds are used when lower levels of paracetamol are detected. All medical laboratories should be aware of this complex interference, and ensure that mechanisms are in place to recognise and mitigate against it.

Finally, in clinical practice, paracetamol levels are most commonly requested in the context of acute paracetamol overdose, where the patient may present earlier and liver damage and subsequent hyperbilirubinaemia has not yet occurred.¹⁰ Therefore, the interaction during analysis may not be applicable. However, in cases of staggered paracetamol overdose or delayed presentation, liver damage may already be present, and clinicians and laboratories need to be aware of the potential for interaction with results. In our case, the history, timeline and pattern of results and medication use did not fit with paracetamol-induced ALI. Interesting to note is that his initial paracetamol levels were measured at 9 mg/L, which was in keeping with his therapeutic use prior to admission. Bilirubin at that time was only 46 μmol/L, and therefore would not have contributed to the paracetamol level being raised. However, despite cessation of paracetamol, his levels continued to rise and remained elevated as his bilirubin rose, in keeping with interference. This case reinforces the importance of not only interpreting investigation results in the context of history and clinical situation, but also being judicious in the selection of investigations. Similarly, physicians need to remain vigilant for outlier results, and through open interspecialty discussion work with other teams to identify and improve changes to practice, if needed.

Learning points.

• Caution is necessary when interpreting paracetamol results for jaundiced patients, and consultation with the laboratory may be needed—especially in the context of unexpected slight elevations in paracetamol level.

• This case report reveals a disadvantage of the most commonly used enzymatic colorimetric assay due to interference from bilirubin and its by-products in the serum.

• When little or no paracetamol present, interference with the enzymatic assay method becomes significant at lower levels of hyperbilirubinaemia—a new ‘Sliding Scale’ protocol for analysis has therefore been developed.

• Bilirubin measurement is recommended for all samples in laboratories that use the enzymatic colorimetric assay for paracetamol measurement. Questionable paracetamol results in icteric serum can be confirmed by dilution studies.

• Due to false-positive paracetamol levels, treating physicians can wrongly assume that the cause for acute liver injury is found, resulting in wrong diagnosis and missed management.