Topic Update

Combined metabolomics and genomics approach for the diagnosis of inherited metabolic disorders (IMD)

Combined metabolomics and genomics approach for the diagnosis of inherited metabolic disorders (IMD)

Volume 16, Issue 2, July 2021  (download full article in pdf)

Editorial note:

In this topical update, Dr Eric Law reviews and updates on the current development in metabolomics and genomics and their integrated approach in the study of inherited metabolic disorders (IMD). We welcome any feedback or suggestions. Please direct them to Dr. Sammy Chen of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr Chun-yiu LAW

Consultant, Division of Chemical Pathology, Department of Pathology, Queen Mary Hospital


Inherited metabolic disorders (IMD) refer to a group of heterogeneous biochemical disorders involved in different pathways of metabolism in humans. Metabolism refers to biochemical processes that occur in cells. These are the fundamental chemical reactions related to cell viability, growth, division, etc. Metabolism can be broadly classified into two major processes. One is catabolism, the production of energy from various nutrients, such as glucose, fat, and amino acids. The other process is anabolism, the biosynthesis of new cellular components, such as protein synthesis. A very early description of IMD was detailed by Sir Archibald E. Garrod about alkaptonuria back in 1902, who proposed the conditions to be inheritable and was caused by a specific enzymatic defect [1]. Indeed, IMD is more than just an enzymopathy and the knowledge of IMD and the human metabolome is still expanding in the next 100 years after the discovery of alkaptonuria. A computational analysis of the complete human genome has assigned 2,709 human enzymes to 896 bioreactions [2]. A more recent annotation includes 3,044 human molecular pathways covering 9,022 gene products [3]. According to the latest human metabolome database (HMDB version 4.0), there are 115,398 metabolites that linked with 5,702 different proteins [4].

The human metabolomes

Knowledge of the human metabolomes and their metabolic interactions is important for the understanding of human diseases. For example, it is now recognized that mitochondria are not only a factory for oxidative phosphorylation and energy metabolism, mitochondria also orchestrated with over 1,000 proteins and linked with multiple biochemical processes [5]. Indeed, a disrupted mitochondrial homeostasis had also been observed in some organic acidurias [6, 7]. The interactions of human metabolomes are far more complex than once perceived and involve different cell types, diets, drugs, disease status, microorganisms, and many others (Figure 1). The collective ‘big picture’ can be better studied through exometabolomics [8-10]. For example, acetic acid and trimethylamine (TMA) have been identified as biomarkers of bacterial urinary tract infection (UTI) and Escherichia coli associated UTI, respectively [11, 12]. TMA is one of the examples of mammalian-microbial co-metabolism which the host metabolizes TMA into trimethylamine N-oxide (TMAO) via flavin monooxygenase 3 (FMO3), and in E-coli associated UTI, the endogenous TMAOs are converted back to TMA possibly through the action from bacterial TMAO reductase. Similar mammalian-microbial co-metabolism has been described in aspects of human health that include cardiovascular disease, immunity, gastrointestinal disorders, and cancer [13-16]. The whole metabolomics network is more complex when toxico-metabolomics from drugs, chemicals, and environmental pollutants are taken into account. To-date, 2,280 drug and drug metabolites have been reported in the DrugBank database, and 3,670 toxins and pollutants have been reported in the Toxin and Toxin Target Database [17, 18], and the databank is still expanding.


Figure 1: Simplified diagram to illustrate the structure of a human metabolome which is a complex interplays between host (human) and various factors, e.g. diet, microorganisms, drugs, etc.

Metabolism is the heart of many disease processes. Insights gained from the knowledge of metabolism will inform diagnoses and lead to new treatments. For example, 116 treatable intellectual disability caused by IMD has been described in a 2021 review [19]. In this newsletter, more emphasis will be put on IMD, a heterogeneous condition involving disorders of synthesis, catabolism/anabolism, transport, and storage of metabolites.

Classifications of IMD

The definition for IMD is further refined as described in [20]. Indeed, the presence of an abnormal metabolite is no longer essential for the classification of IMD, but instead includes any condition resulting in the dysfunction of the specific enzymes or biochemical pathways that is intrinsic to the pathomechanism. In individuals, IMD is rare. However, in a population they are collectively “common”. The estimated incidence of IMD is 1 per 4,122 to 4,355 live births [21, 22]. The true prevalence of IMD is difficult to measure due to various factors. It was estimated as 1 in 800 to 2,500 newborns in one study in 2020 [23]. According to the Society for the Study of Inborn Errors of Metabolism (SSIEM), there are over 600 different IMDs, and they are grouped into 15 hierarchical classifications based on the biochemical pathway involved. They are (1) disorders of amino acids and peptide metabolism, (2) disorders of carbohydrate metabolism, (3) disorders of fatty acid and ketone body metabolism, (4) disorders of energy metabolism, (5) disorders of the metabolism of purines, pyrimidines and nucleotides, (6) disorders of the metabolism of sterols, (7) disorders of porphyrin and haem metabolism, (8) disorders of lipid and lipoprotein metabolism, (9) congenital disorders of glycosylation and other disorders of protein modification, (10) lysosomal disorders, (11) peroxisomal disorders, (12) disorders of neurotransmitter metabolism, (13) disorders of the metabolism of vitamins and (non-protein) cofactors, (14) disorders of the metabolism of trace elements and metals, and (15) disorders of and variants in the metabolism of xenobiotics (For more details, please refer to This is a 2012 classification from SSIEM. Knowledge of IMDs is still expanding with the advancements in next-generation sequencing (NGS) which has led to the discovery of more disease-causing genes and disease classes in IMD. For example, new class, such as congenital disorders of autophagy, which cause multiple system involvement have been described in patients with inborn errors of neuro-metabolism [24].

Examples to enhance diagnostic workflow in IMD

Recently, the International Classification of Inborn Metabolic Disorders assigned 1,450 monogenic conditions related to metabolism to 24 categories [25]. These conditions can have overlapping signs and symptoms. Some are rapidly fatal, mainly due to the accumulation of toxic metabolites and/or deprivation of energy; the diagnosis of IMD is clinically vital in this regard since it permits interventions to prevent further metabolic insults and irreversible damages. Unfortunately, there is no simple and single biochemical analysis that encompasses all pathognomonic markers in each IMD. Various methods had been described to decipher human metabolomes [26, 27]. Urine organic acid (UOA) via gas chromatography mass spectroscopy (GC-MS) was introduced in the 1960s and has since been adopted by most clinical laboratories. GC-MS is robust because it generates highly reproducible mass spectra, which allows positive identification using libraries, such as that of the National Institute of Standards and Technology (NIST). Nevertheless, there are several pitfalls of GC-MS. These include low-level metabolites, co-elution, age-dependent variation of metabolite levels, etc. Data interpretation by pathologists is a labour-intensive process. For this reason, an in-house automatic solution was established to address the above pitfalls and assist the UOA reporting process. A checklist composed of almost 100 key metabolites was constructed using over 1,600 sets of UOA GC-MS data and partitioned according to different age groups. Positive identification of metabolites was defined according to their retention times and electron ionisation spectra. The 95th percentile for each compound and in each age group was used as a cut-off to define abnormally high OAs, which would be flagged for in-depth review by chemical pathologists. This algorithm allows: (1) a graphical display of individual UOA levels and comparison with controls according to different age groups, (2) calculation of ratios of metabolites useful in interpreting low-level, but clinically significant, metabolites, (3) pathway analysis by a holistic correlation analysis of all studied OAs, and (4) continual database enrichment. An example of a case of aromatic L-amino acid decarboxylase (AADC) deficiency, a neurotransmitter disorder is shown in figure 2 where a significant increase of vanillactic acid (VLA) is identified. Despite this refinement, the analytical process is time consuming and remains a bottleneck for rapid diagnosis. To further streamline the analytical process for IMDs, we further explored the use of nuclear magnetic resonance (NMR) spectroscopy for IMD diagnosis as an alternative or complementary for GC-MS analysis. This approach for IMD was proposed decades ago in 1999 [28]. To-date, at least 100 IMD conditions can be diagnosed by NMR spectroscopy, as reviewed by Engelke [29] and Moolenaar [30]. In addition, this technique allows for the identification of novel IMDs. Examples include aminoacylase 1 deficiency in patients with metabolic brain diseases [31], beta-ureidopropionase deficiency in patients with movement disorders [32], defective polyol metabolism in patients with leukoencephalopathy [33], and dimethylglycine dehydrogenase in patients with muscle disorders [34].


Figure 2: UOA spectrum by GC-MS from a case of aromatic L-amino acid decarboxylase (AADC) deficiency. The blue arrow points to the diagnostic marker, vanillactic acid (VLA) (Upper). Distribution of VLA levels in different age groups generated from in-house UOA algorithm. The AADC patient shows a marked increase of VLA (red arrow) comparing with age-matched subjects.

A national screening program in Turkey had applied NMR clinically to screen for IEM in newborns [35]. A total of 989 urine samples were collected from neonates and analysed by two laboratories. The results were used to establish a database and routine clinical screening. This NMR-based newborn urine screening has been further extended, covering up to 75 different IEM conditions [36]. The increasing awareness of clinical NMR applications has been further elaborated elsewhere in a 2021 review [37]. In our experience, the diagnostic utility of NMR has been substantiated in various clinical cases, for example, beta-ketothiolase deficiency, beta-ureidopropionase deficiency, citrin deficiency, fructose 1,6 bisphosphatase deficiency, holocarboxylase synthase deficiency, 3-hydroxyisobutyric aciduria, hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome, methylmalonic aciduria (MMA), propionic acid, and succinic semialdehyde dehydrogenase deficiency (SSADHD). The merits of NMR-based urinalysis over GC-MS techniques are the simple sample preparation workflow and a relatively fast analytical time. Sample preparation is a two-step procedure that could be handled in

The choice of metabolic analysis depends on the nature of the pathognomonic metabolites. No single biochemical test that can detect them all. Pathologists have provided input concerning the choice of tests, advice on patient preparation and sample requirements, and clinical interpretations. The many examples include plasma acylcarnitine analysis, plasma/urine/CSF amino acids, urine acylglycines, bile acids, biotinidase activity, chitotriosidase activity, dried blood spot metabolic screening, homocysteine, transferrin isoelectric focusing, glycosaminoglycans analysis, CSF neurotransmitters, urine organic acids, urine sugars, urine guanidinoacetate and creatine analysis, urine oligosaccharides, orotic acids, red blood cells plasmalogens, porphyrins, phytosterols, pristanic and phytanic acids, purine and pyrimidines, very long chain fatty acids, and many more tests. Unfortunately, not all IMD-related tests are available or can be performed in a single centre.

The use of NGS will be a solution which provides additional insight from a genetic dimension, in particular if a biochemical assay is not available or the diagnosis cannot be easily demystified by biochemical tests. Primary coenzyme Q10 deficiency is one of the examples [38]. Affected individuals usually presented with non-specific symptoms and biochemical findings. Indeed, we have reported three cases of COQ4-related mitochondriopathy and identified a hotspot pathogenic variant in this locality using a genomics approach [39]. A plasma COQ10 assay was later developed for this potentially under-recognized condition.

Some IMD conditions are caused by multiple genes, for example, glutaric aciduria type II, methylmalonic aciduria (MMA), maple syrup urine disease (MSUD), propionic academia, phytosterolemia, congenital lactic acidosis, etc. Instead of a conventional gene-after-gene analysis, advances in NGS could allow the detection of the underlying genetic defect through a gene panel approach which effectively saves the time and manpower from managing a huge number of PCR primer bank and gene-specific protocols, not to mention the time spent in their revisions, updates and accreditations.

The diagnostic yield of NGS-based diagnosis for IMD is variable. In one report, NGS diagnosed 59% of the cases with clear clinical and biochemical features and a diagnostic yield of 8% for patients with an unclear phenotype [40]. Another group achieved an overall diagnostic yield of 50% and up to 78% in cases with a clear phenotype [41]. It is technically difficult to compare diagnostic yields of different studies, for reasons that include the scope of the IMD panel used, clinical and analytical aspects that can differ between centres.


With the expanding knowledge of metabolome and genome, more novel metabolites and genes have been discovered. These discoveries are enriching the understanding of IMD. Genomic and metabolomic analyses should be complementary to each other in the study of IMD, particularly in cases with atypical genetic findings or when a particular biochemical assay is not yet available. With the advancement of pharmacological chaperoning, small molecules and gene therapies, etc., more treatment options with improved care will be available in near future. At the same time, there will be increasing role from Pathologists for clinical use of cross-omics approach for disease diagnosis, monitoring and prognostication, with a more accurate and individualized characterization of disease progress.


The author would like to thank the supervision from Prof. Ching-wan Lam, Department of Pathology, The University of Hong Kong for his supervision on the NMR-related works. The author thanks Dr Gary Wong and Dr Jacky Ling, Division of Chemical Pathology, Department of Pathology, Queen Mary Hospital for their works on the in-house automatic solution in UOA analysis.


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Diagnosis of COVID-19

Diagnosis of COVID-19

Volume 16, Issue 1, January 2021  (download full article in pdf)

Editorial note

Coronavirus disease 2019 (COVID-19) is undoubtedly the most topical subject not only in the medical field, but also for humanity globally. In this issue of the Topical Update, Dr. Derek Hung and Prof. Kwok Yung Yuen present an overview on the diagnosis of COVID-19, which underpins effective disease control. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo, Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. Derek HUNG and Prof. Kwok Yung YUEN

Resident, Department of Microbiology, Queen Mary Hospital, Hospital Authority and

Professor, Department of Microbiology, Faculty of Medicine, The University of Hong Kong


Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since December 2019 has infected 54 million population in all six major continents, resulting in over 1.3 million deaths by mid-November 2020. One of the most important aspects in curbing the spread of the virus is rapid yet accurate diagnosis of infection followed by timely isolation and contact tracing. Molecular testing is now the mainstay of diagnosis, supplemented by viral antigen testing. Antibody detection aids in assessment of immunity and disease prevalence in the population. As the disease progresses, there are worldwide efforts in developing a multitude of diagnostic platforms, both in-house and commercial. Studies also endeavour to assess optimal types and timing of specimen collection to enhance diagnostic yield. In this review, we would look at some of the knowledge and practices in making a diagnosis of COVID-19.

Specimen collection

Obtaining the best specimen optimizes the possibility of getting the correct diagnosis based on clinical suspicion. Being a predominantly respiratory pathogen, obtaining respiratory specimens for viral detection remains the primary modality for making a diagnosis of acute infection by SARS-CoV-2. The viral load is highest at or soon after symptom onset, with the viral load in the upper respiratory tract peaking earlier than the lower respiratory tract. The viral load decreases in the respiratory tract at a rate of 1 log10 per week. The World Health Organization (WHO) suggests that testing upper respiratory tract specimens is adequate for early stage infection, especially asymptomatic or mild cases. The Centers for Disease Control and Prevention (CDC) recognizes nasopharyngeal swab, nasopharyngeal wash, nasal wash obtained by health care professionals; nasal mid-turbinate swab, nasal swab obtained by either health care professionals or supervised self-collection on site; and posterior oropharyngeal saliva (POS) by supervised self-collection as valid specimens. Patients with lower respiratory tract symptoms such as productive cough, shortness of breath, or suspicious radiological findings should send sputum to enhance sensitivity. Induced sputum is not recommended due to increased risk of aerosol transmission,. Among different respiratory specimens, broncho-alveolar lavage (BAL) showed the highest positive rate.

For the upper respiratory tract specimen, comparing combined nasal swab/throat swab with nasopharyngeal swab, Vlek et al showed high concordance between these two methods (kappa coefficient 0.95) despite the cycle threshold value (Ct value) obtained from nasopharyngeal swab being lower. Another study suggested nasal swab alone also has good concordance with nasopharyngeal sampling. In contrast, oropharyngeal swab alone has inferior performance. Wang et al showed the sensitivity of oropharyngeal swab was 21.1% and meta-analysis by Bwire et al suggested the positive rate is as low as 7.6% in suspected cases, comparing with 69.6% and 71.3% for nasopharyngeal swab and lower respiratory tract specimen respectively. POS is increasingly studied as an alternative respiratory tract specimen for diagnosis. Theoretically well produced POS can concentrate secretions dripping down from nasopharynx and lower respiratory secretion moved up by ciliary activity of respiratory epithelium. It can be saved by patients themselves with instructions, thus reducing discomfort in specimen collection and minimizing aerosol exposure for health care professionals. The cost of collecting POS could be 2.59-fold lower than nasopharyngeal specimen, which could be significant in resource limited setting. The concordance between POS and nasopharyngeal swab is high, especially in the first 7 days of infection, up to 96.6% positive percent agreement. The sensitivity is comparable with nasopharyngeal swab in properly collected specimen. The sensitivity does not vary much between early morning and at least 2 hours after meal, which provides a convenient option for specimen collection. CDC and Hospital Authority of Hong Kong have adopted POS as an alternative option for upper respiratory specimen collection.

Viral shedding is also found in other specimens with stool being more studied. Meta-analysis showed viral shedding was found in faecal material in 40.5% of patients. The viral shedding in stool is more prevalent in those with gastrointestinal symptoms and may last longer than the shedding in respiratory tract. Viral RNA detected in blood and urine is relatively uncommon, respectively only 1% and 0% in one study with more than 200 patients10. Even without ocular symptoms, the conjunctival secretion may contain a small amount of SARS-CoV-2 RNA in around 8% of patients, warranting appropriate infection control measure in ophthalmological assessment.

Molecular testing

Detection of nucleic acid remains the backbone of diagnosing COVID-19 for treatment and public health purposes. Reverse-transcriptase polymerase chain reaction (RT-PCR) is the most widely used technique. After transcribing the viral RNA into complementary DNA (cDNA) with reverse transcriptase, the cDNA would be amplified and detected by real-time PCR. Potential molecular targets for SARS-CoV-2 include genes encoding structural proteins, e.g. spike (S), envelop (E), helicase (hel), nucleocapsid (N-N1 and N2), transmembrane (M); and non-structural regions, e.g. RNA-dependent RNA polymerase region (RdRp), haemagglutinin-esterase (HE), and open reading frame 1a (ORF1a) and ORF1b. Most scientific institutes and commercial platforms would design primers to target more than one gene, or to target multiple loci of the same gene to enhance diagnostic sensitivity and specificity. Though N gene RNA is shown by nanopore direct RNA sequencing study to be the most abundantly expressed transcript in SARS-CoV-2 infected cells, there is no consensus on which gene confers the best diagnostic performance. Presently, one conserved and one specific target region are recommended to mitigate effect of random mutation or genetic drift while maintaining specificity25. Various regimens for testing are proposed in the literature. Corman et al recommended the Charité protocol, which was to use E gene for screening and RdRp gene for confirmation. CDC used N1 and N2 genes as their diagnostic panel. Chu et al used N gene as screening test and ORF1b as confirmatory assay because the screening N gene assay is 10 times more sensitive than ORF1b. As an alternative confirmatory assay, Chan et al developed a real-time RT-PCR assay locally, targeting RdRp/Hel. This COVID-19-RdRp/Hel assay demonstrated significantly higher sensitivity and specificity for the detection of SARS-CoV-2 RNA than the RdRp-P2 assay in clinical evaluation.

Multiple commercial platforms were developed for molecular SARS-CoV-2 diagnosis for their high throughput, rapid turnaround time and ease of use with automation. Examples are Roche Cobas 6800/8800 system (targets ORF1a and E genes) and Abbott Alinity m SARS-CoV-2 assay (targets RdRp and N genes), where sample preparation, genetic material extraction, target amplification and result reporting are automated inside the system. Molecular point-of-care testing (POCT) refers to diagnostic platform that is portable (often desktop-size), requires minimal sample preparation steps and can provide reliable molecular results within 2 hours. POCT like Cepheid GeneXpert (Xpert Xpress SARS-CoV-2 assay, targets E and N2 genes) enables rapid testing near the site of collection in areas with little laboratory support. Fewer steps in manipulation reduce risk of cross contamination and laboratory error in processing. Many evaluation studies have been published to compare the performance of these commercial platforms against in-house diagnostic tests and for head-to-head comparison between platforms. For example, Cobas system is shown to have high diagnostic agreement with in-house molecular assays,, as well as with other commercial platforms such as Hologic Panther Fusion system and Cepheid GeneXpert. Cepheid GeneXpert reaches an agreement of 100 % compared to three in-house RT-PCRs in a multicentre evaluation in the Netherlands. Among commercial platforms there might be minor discordance between assays at very high Ct values and the viral load of clinical samples used in evaluative studies should be noted in particular.

Another molecular technique is reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) test. Using multiple primers for the genetic target, RT-LAMP amplified nucleic acid by strand displacement in an isothermal condition of around 60- 65oC. It allows synthesis of large amount of genetic material up to 106 to 109 copies of target DNA within 30-60 minutes2. Without the need of thermal cycler as in RT-PCR, RT-LAMP facilitates development of rapid molecular POCT and has an expanding market in commercial diagnostic platform. On the down side, since multiple primers over a relatively small genetic region are needed for amplification, there are constraints in properly designing the primers. Abbott ID NOW is a commercial POCT platform using RT-LAMP, allowing real time detection of SARS-CoV-2 within 15 minutes targeting RdRp gene. Evaluation of ID NOW against other RT-PCR based platforms appears suboptimal in terms of diagnostic sensitivity. Compared to Cobas, ID NOW achieved only 73.9% positive agreement while GeneXpert achieved 98.9% positive agreement. In samples with Ct values greater than 30, positive agreement was 34.3% for ID Now and 97.1% for GeneXpert. A lower sensitivity of ID NOW over GeneXpert was also reported in another evaluation by Basu et al. In contrary, good diagnostic utility has been demonstrated in many other centres including Hong Kong that have designed their own RT-LAMP for COVID-19. Chow et al reported sensitivity of 95% at 60 minutes using RT-LAMP targeting a region across ORF3a/E gene as compared to RT-PCR. Lu et al achieved concordance rate of 93% against RT-PCR using in-house E gene RT-LAMP assay.

In order to improve the diagnostic sensitivity of molecular assays, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been employed by coupling with Cas enzyme. The enzyme would be directed to the target DNA/RNA by a guide RNA complementary to the target sequence. Once bound, the collateral nuclease activity of the Cas enzyme would cleave surrounding reporter fluorophore and lead to signal amplification. DETECTR technology uses Cas12a enzyme to bind target DNA; while SHERLOCK technology uses Cas13a enzymes to bind target RNA. This technology can be incorporated in molecular techniques especially RT-LAMP to enhance the sensitivity and to lower the detection limit.

Next generation sequencing (NGS) enables sequencing of the entire genome in a relatively short period of time. Sharing of genetic data facilitates tracking of disease spread, understanding of disease transmission route, monitoring viral genome evolution and detecting emergence of mutation that may escape detection or enhance virulence. The cost and infrastructure required of NGS and the need of bioinformatics expertise limit its use to larger hospital and research centres.

Antigen detection

Like other respiratory viruses such as influenza and respiratory syncytial virus (RSV), direct antigen detection from respiratory specimen especially nasopharyngeal sample is another way of making a diagnosis of COVID-19. N protein was found previously to be the predominant structural protein released in large amount in nasopharyngeal aspirate during infection of SARS-CoV, and the same phenomenon is also shown in SARS-CoV-2 where the abundantly expressed N protein is widely used as an antigen detection target in COVID-19. Detection is achieved by capturing viral antigen in clinical specimens by monoclonal antibodies or monospecific polyclonal antibody fixed on a membrane, usually indicated by colour change of the strip in colorimetric lateral flow immunoassay. The assay can be delivered as POCT in an office setting since no complex laboratory support is required and the result can be available within a short period of time, usually <30 minutes. The major setback is the suboptimal sensitivity as compared to molecular diagnosis especially in samples with high Ct values. Evaluation by Lambert-Niclot et al using COVID-19 Ag Respi-Strip CORIS, a nitrocellulose membrane technology with colloidal gold nanoparticles sensitized with monoclonal antibodies directed against SARS-CoV-2 nucleoprotein (NP) antigens, showed sensitivity of only 50% when compared against multiple RT-PCR platforms. For samples with Ct value <25, the sensitivity is higher at 82.2%. In a local evaluation using Biocredit COVID-19 Ag test, the antigen test is 105 fold less sensitive than RT-PCR and it yielded a positive result in 45.7% RT-PCR positive combined nasopharyngeal swab/throat swab specimens only. There are attempts to improve sensitivity of rapid antigen assay. Porte et al evaluated an immune-chromatographic antigen assay using fluorescence signal showing sensitivity of 93% but the Ct value of the sample included in this study is relatively low with mean of 20. Other approaches by concentrating the antigen in specimens before testing with monoclonal antibodies targeting multiple different epitopes of the antigen were also reported. Based on a meta-analysis by Dinnes et al, the average sensitivity is around 56.2% for antigen assay with a high average specificity of 99.5%. Further refinement in antigen detection employs the detection of the change in bioelectric property by antigen binding to the antibody coated membrane. In Seo et al, anti-S antibody binds to SARS-CoV-2 particles to fabricate graphene-based field-effect-transistors (FET) biosensors and can respond down to 16 pfu/mL of virus. One challenge to this advance is the high background noise which can reduce sensitivity of detection. Overall, rapid antigen detection serves only an adjunctive role to molecular assay in making a diagnosis especially in outbreak situation where prevalence is high and molecular assay is not available. WHO has issued interim guidance of use of rapid antigen immunoassays.

Antibody detection

While antibody testing may not be useful in acute setting for COVID-19, it helps establish retrospective diagnosis, predict immunity and understand seroprevalence in a defined community. Commonly employed techniques are lateral flow immunoassay, chemiluminescent immunoassay, immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA). Median seroconversion times following symptom onset are 11 days for total antibodies, 12 days and 14 days for IgM and IgG respectively. Detection rate for IgM ranges from 11-71% in the first 7 days of infection, 36-87% between 8-14 days, and 56-97% after 14 days. For IgG, it ranges from 4-57% in first 7 days, 54-88% between 8-14 days, and 91-100% after 14 days. For SARS-CoV-2, there does not seem to have significant time difference between IgM and IgG response. IgM peaked at around 3 weeks after symptom onset and fell to baseline level at around day 36. The duration of IgG seropositivity remains unknown and longer longitudinal studies are required. Study from Iceland involving over 1200 confirmed patients showed no evidence of antiviral antibody decline by 4 months after diagnosis; and most other studies showed persistently detectable antibodies by 2-3 months after infection60. On the other hand, there are some evidences that the IgG level may decline faster in mild and asymptomatic61 COVID-19 cases.

S protein is an important antigen for neutralizing antibody production. The S1 domain is responsible for receptor binding while the S2 domain is responsible for fusion. The receptor binding domain (RBD) is located at S1. NP, which is a structural component of the helical nucleocapsid, also appears to be an important antigen for the development of serological assays to detect COVID-19. Earlier in the pandemic, using sera collected more than 14 days after symptom onset from 16 patients, To et al showed rates of seropositivity were 94% for anti-NP IgG, 88% for anti-NP IgM, 100% for anti-RBD IgG, and 94% for anti-RBD IgM. Another study compares sensitivity and specificity in testing anti-S and anti-NP IgG for evidence of immunity across multiple platforms, which shows they are comparable by day 37 after infection though seroconversion of anti-NP IgG may precede anti-S IgG by around 2 days (day 9-10 v day 11-12). Caruana et al observed that the decline of anti-NP antibody may be faster than anti-S and thus could be less sensitive longer after infection. Also titre of anti-S antibody may better reflect protection against reinfection67. Multiple commercial platforms were developed for high-throughput antibody testing in clinical laboratory. Automatic platforms such as Abbott SARS-CoV-2 IgG, which is a chemiluminescent micro-particle immunoassay, are also used in public hospital of Hong Kong for a shorter turnaround time.

Neutralization antibody test is important in assessing in vitro the functional capacity of the humoral response of COVID-19 patients to prevent reinfection by the virus. Traditional neutralization assay such as microneutralization and plaque reduction assay require manipulation of live virus and necessitate biosafety level 3 laboratories. As a result, pseudovirus neutralization assay has been developed. Vesicular stomatitis virus (VSV) expressing S protein of SARS-CoV-2, containing the RBD, is used so that the assay can be performed in biosafety level 2 facilities. SARS-CoV-2 neutralizing antibody starts to rise at around 7-10 days after symptom onset and the median peak time is 33 days after symptom onset. The neutralization titres then decline in 93% of the patients and by a median level of 35% over 3 months. Patients with more severe disease requiring ICU admission have accelerated and augmented neutralizing antibody response compared with non-ICU cases. In non-severe cases who have low peak neutralizing antibody titre, neutralizing antibody level might return to baseline within 2 months. Another clinical use of neutralization assay would be to confirm potentially false positive SARS-CoV-2 serology result. Three children with Kawasaki disease without symptoms or epidemiological linkage to COVID-19 were tested positive to anti-RBD and anti-NP antibodies by a microparticle-based immunoassay but were confirmed negative by microneutralization test.

Studies have shown there are serological cross-reactivity between SARS-CoV-2 and SARS-CoV. Testing sera taken from COVID-19 patients by ELISA, cross-reactivity is seen against S protein and RBD of SARS-CoV, though the intensity of cross-reaction against RBD is weaker than S protein. For the full length S protein, the amino acid sequence homology between SARS-CoV-2 and SARS-CoV is around 75%. The homology between them for RBD which is located in S1 domain is around 74%. For the receptor binding motif (RBM) of the RBD where the virus directly binds to angiotensin-converting enzyme 2 (ACE2), the homology is only 50%. The degree of amino acid homology explains the difference in the level of cross-reaction between them on ELISA. Chia et al showed even more significant cross-reactivity between SARS-CoV-2 and SARS-CoV antibody against NP by Luminex assay than antibody against S1 or RBD as the homology between the NP of these 2 viruses is around 90%. Despite some cross-reaction between antibodies against RBD on ELISA, there does not seem to have significant cross neutralization effect73. Only 1 out of 15 COVID-19 sera showed cross neutralization with SARS-CoV at very low titre. Overall the effect of cross-protection in vaccination and whether antibody-dependent enhancement effect would be seen between these 2 closely related viruses remains unknown.

Cross-reactivity against other human coronaviruses in SARS-CoV-2 infection has been investigated in a few trials. In a study by Wölfel et al, using immunofluorescence assay against recombinant S protein, cross-reactivity of SARS-CoV-2 sera is found against human coronaviruses OC43, NL63, HKU1 and 229E on comparing the titres between admission and convalescence samples, especially HKU1 and OC43 which are both betacoronavirus. In Shrock et al, deep serological profiling of sera from SARS-CoV-2 patients and pre-COVID sera are performed. Antibodies against S and NP are the most specific assay to differentiate SARS-CoV-2 and pre-COVID sera. Those with dramatic increase in anti-S antibody after COVID-19 infection also have increase in the intensity of cross-reactivity against other human coronaviruses, especially over more homologous regions of the S protein e.g. at residue 811-830 and 1144-1163. It could be novel antibodies of SARS-CoV-2 that cross-react or boost the anamnestic response against SARS-CoV-2 infection due to existing memory towards other human coronaviruses from past exposure. Moreover, pre-COVID sera also show some cross-reaction towards the homologous region of SARS-CoV-2 S protein and ORF1 in the same study.

Viral culture

Demonstration of live SARS-CoV-2 in cell culture requires biosafety level 3 facilities and are not routinely performed in most of the clinical laboratories. However, live virus isolation is still important for some diagnostic and research purposes so as to determine whether the amount of virus present is infectious to others, to evaluate therapeutic efficacy of potential antiviral compound, to develop viral neutralization assay for testing convalescent sera, to provide positive control for molecular assay development, and to develop vaccine strains. The host cell receptor for SARS-CoV-2 is ACE2. Non-human cell lines such as Vero E6 and Vero CCL-61 which have abundant ACE2 expression are commonly used for isolation. Cytopathic effect is seen by 3 days after inoculation. SARS-CoV-2 also grows in human continuous cell lines such as Calu3 (pulmonary cell line), Caco2 (intestinal cell line), Huh7 (hepatic cell line), and 293T (renal cell line). It grows modestly on U251 (neuronal cell line) which is not seen in SARS-CoV81. Confirmation of SARS-CoV-2 replication in the cell line can be done by molecular testing or immunostaining techniques. Cell lines can be engineered to express a transmembrane serine protease TMPRSS2 for priming of S protein and to facilitate the entry of SARS-CoV-2 into host cell. Organoid systems such as bat and human intestinal organoids are susceptible to SARS-CoV-2 and are developed to better study tissue tropism, the dynamics of infection and testing of therapeutic targets.

Radiological diagnosis and artificial intelligence

There are no pathognomonic radiological features on chest imaging for COVID-19 and the disease should not be ruled in or ruled out based on imaging alone. However, presence of suggestive imaging features can prompt further investigations in suspicious cases, such as lower respiratory tract viral testing for confirmation. Reports in literature have suggested that in some patients, radiological findings may precede the detection of SARS-CoV-2 in clinical specimen,. Chest X-ray (CXR) is a less sensitive modality than computed tomography of the thorax (CT thorax) with a reported CXR sensitivity of 69%85. As in other viral pneumonia, COVID-19 typically presents with multifocal air-space disease, especially with a bilateral lower lung distribution. More specific to COVID-19, it tends to have peripheral lung involvement, seen in 58% of CXR in one study. CT thorax has a higher sensitivity than CXR, quoted at around 60-98%. CT thorax often demonstrates the typical findings of peripheral bilateral ground glass opacities (GGO) with or without consolidation or ‘crazy-paving pattern’. Sometimes the GGO would arrange in a rounded pattern. Isolated lobar or segmental consolidation without GGO, centrilobular shadows, cavitory changes, lymphadenopathy and pleural effusions are rare86. As the disease advances, the opacities might coalesce, affecting central and bilateral upper lobes and may manifest as ‘white lung’ with diffuse infiltrate. The abnormalities usually peak by 2 weeks after symptom onset, replaced by scar tissue with recovery. In the COVID-19 pandemic, artificial intelligence (AI) programme is increasingly studied for screening abnormal radiological result which would be particularly useful for mass screening strategy in outbreak situation. The performance of AI is dependent on the radiological imaging algorithm being fed into the system for deep learning process. So far the result of this research has been promising with reported area under receiver operating characteristic curves greater than 0.9,. However, there are still lots of technical and ethical issue to resolve which include dataset bias, data privacy, and the distribution of ultimate accountability of result.

Detection of host inflammatory reaction

In COVID-19, there are studies to diagnose and predict severe diseases by the host inflammatory response. Apart from direct viral damage, uncontrolled cytokine storm triggered by the virus leads to tissue damage and multiorgan failure. Mean interleukin-6 (IL-6) concentration in serum was found to be 2.9 fold higher in patients with complicated COVID-19 disease than non-complicated disease. It became one of the markers clinicians could use to predict progression into severe disease. Roche Elecsys IL-6 immunoassay received FDA Emergency Use Authorization to help identify patients at high risk of requiring intubation with mechanical ventilation. Molecules targeting IL-6 such as tocilizumab are also studied as therapeutic to prevent disease progress by blocking the inflammatory pathway. It does not show efficacy in preventing intubation or death in moderately ill hospitalized patients in the BACC Bay trial. Elevated CRP is associated with worse outcome, as well as elevated IL-10 which may be related to compensatory anti-inflammatory response and secondary infections. Haematologically, severe disease is associated with higher absolute neutrophil count, D-dimer and LDH but lower absolute lymphocyte101 and platelet count.


Global COVID-19 pandemic stimulates global effort in development of rapid yet accurate diagnostic techniques. Diagnosis is often limited by the low level of viral particles in the specimen and the subtle clinical features in early infection. Though traditional methods like RT-PCR are still the mainstay, we see expanding endeavours to strive for higher speed and lower limit of detection at an earlier time. Molecular techniques such as RT-LAMP, CRISPR/Cas, biosensor technology in antigen detection, AI operating system for image interpretation are pushing the diagnostic ability to the limit. Despite these scientific advances, there are still a lot of gaps to fill especially in understanding the nature and duration of humoral immunity response and its protection against re-infection. All these require continuous global cooperation and information exchange to make them possible.

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Drowning: A Rational Approach to its Diagnosis

Drowning: A Rational Approach to its Diagnosis

Volume 15, Issue 2, July 2020  (download full article in pdf)

Editorial note:

Drowning often presents in various scenarios depending on the circumstances. This Topical Update provides a proper approach to the diagnosis. We welcome any feedback or suggestions. Please direct them to Dr. FOO Ka-chung of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. FOO Ka-chung

Specialty Coordinator (Forensic Pathology), Education Committee

The Hong Kong College of Pathologists


Drowning is referred as “death occurring within 24 hours of a submersion incident”. Definition by World Health Organization is "the process of experiencing respiratory impairment from submersion or immersion in liquid ". [1,2] It is a form of asphyxia with a distinct pathophysiology and mechanism of death. It is also a diagnosis by exclusion, and therefore every piece of information should be regarded as crucial. Pathologists are obliged to work under Coroner’s jurisdiction in interviewing the next-of-kin (if available), reviewing antemortem medical records and preliminary findings provided by investigating officer, performing an autopsy as directed and compiling reports capable of addressing anticipated issues.

As Forensic Pathologists mostly deal with sudden and unexpected deaths, cases of drowning with unsalvageable outcome are often encountered. Hospital Pathologists, on the other hand, are dealing with patients presenting a clinical picture in which death eventually occurred after vigorous cardiopulmonary resuscitation followed by development of various systemic complications, e.g. pneumonia, acute respiratory distress syndrome, multi-organ failure, disseminated intravascular coagulopathy, hypoxic-ischaemic encephalopathy etc.

Manner of Death

Information derived from the Coroner’s Report [3] and the Centre for Health Protection [4] suggested that majority of cases were accidental or suicidal in nature. Only a few were homicides. However, it should be remembered that a body found immersed in water does not necessarily imply a diagnosis of drowning. Nor its manner be automatically presumed basing on the prevalent trend. The deceased can die of natural conditions preceding or during submersion as well as unnatural elements that contributed to the drowning process, explaining the failure of extrication from water versus genuine lethal trauma before or while in water. [5,6]


The mechanism of death is complex involving changes to viscera, biochemical alterations and also at a cellular level. The culprit is the medium imposing hydrostatic and osmotic effect to the lungs. [7] The acute change in intravascular volume with electrolyte imbalance is the consequence. Several stages of drowning present in response to the rising levels of carbon dioxide and decreasing oxygen tension in blood. Voluntary breath holding for about 1 to 2 minutes is followed by a stage of involuntary urge to breath with aspiration of fluid for about 1 to 3 minutes. Tonic-clonic seizures, together with some degree of respiratory activity, will occur in the next 1.5 minutes with eventual involuntary breath holding and terminal gasping before cessation of cardiac activity. [1]

It has even been mentioned that only a few inches of water is sufficient to drown a person, as in the case of sudden incapacitation by onset of acute illness while standing close to a washbasin or bucket. [5,6] It was reported that about 1 mL/kg to 11 mL/kg of water aspirated can result in drowning. [7]

A rare entity underdiagnosed in daily practice, or seldom made by pathologists, is referred as "dry drowning" or "immersion syndrome", with negative autopsy findings of typically drowned lungs due to severe laryngeal spasm, therefore preventing further intake while stimulating the sensitive receptors and subsequently triggering cardio-inhibitory reflexes (Ebbecke reflex, Aschner reflex, Hering reflex). [1,5,6,7,8]

Diagnosis of Drowning

The possibility of drowning should always be considered when a deceased was recovered from a body of fluid or the head was found submerged inside a medium of fluid. The deceased could be found near a body of fluid where it could be washed onto the rocky shore, beach, or riverbank. Domestic environments such as bathtub also house this potential danger affecting all walks of life, especially for those who have chronic illness with sudden unexpected precipitation or the young. While the diagnosis of drowning could be straightforward one, such as a witnessed fall into water with subsequent submersion, it can be extremely difficult when critical information derived from the case is absent or inconclusive. Challenging scenarios can appear with unclear circumstances preventing proper formulation of the manner of death. Moreover, while findings derived from postmortem and ancillary investigations may collaborate with the diagnosis, it can be equally confusing when concomitant conditions are unveiled.

Presence of a natural condition which may contribute to death

Let’s consider the following case:

A 51 year-old female was found collapsed underwater in a public swimming pool of about 1.4 meters deep and was certified dead despite intensive resuscitation. There was no eye witness leading to her collapse. Autopsy revealed severe ischaemic heart disease with no evidence of acute infarction. Both lungs were congested and oedematous but frothy fluid was absent probably due to suction during resuscitation. Cause of death is labelled as drowning as the overall features were compatible with drowning.

The presence of a co-existing medical condition, be it undiagnosed or known to the deceased, has to be evaluated carefully to attribute its extent of contribution to death. A sudden precipitation into cardiac arrhythmia explained the reason why a habitual swimmer is incapacitated and eventually succumbed in the water. From the investigator’s point of view, possible legal issues regarding adequate supervision of the swimming environment may be raised which could lead to possible lawsuit and inquest. As such, the pathologist should be ready to address the extent of contribution of medical condition to the tragic outcome.

Medical background of the deceased has to be thoroughly reviewed including conditions such as asthma, epilepsy, cardiovascular conditions (e.g. Long QT syndrome type 1). Psychiatric history including substance abuse should also be elicited.

Presence of trauma which may be related to death

Another case is presented here:

An 87 year-old female was found floating off shore from a pier. She was known to be a habitual swimmer and there was no known chronic illness. There were multiple lacerations on chest and right upper limb. The thoracic cavity was breached and right lung had collapsed. Tinge of frothy fluid was noticed briefly by paramedics before transportation to mortuary. Autopsy revealed severe coronary stenosis and the left lung was mildly hyperinflated.

The presence of trauma may or may not be related to death as injuries inflicted can be produced ante-mortem or post-mortem. Assessment for vital reactions at the wound margin may be helpful to determine its nature.

All forms of injuries must be explained correlating inanimate objects in the environment. Sliding abrasions may be inflicted upon skidding down a slope while blunt force injuries may be a genuine assault. Self-inflicted injuries may occur in suicide as a back-up technique, for example, a stab to the chest or incised wound on the neck, yet it might at times mimic a homicide.

Dragging effects as a result of contact with river bed or ocean floor propelled by sea waves or tidal current is not uncommon and should be interpreted in light of such movement in water. Abrasions or lacerations may be found on forehead, dorsum, knees and toes. In addition, aquatic animal activity, such as crustaceans, will produce bites and nipping around orifices. The body, on the other hand, may be struck by watercraft or its parts including the propeller, predominantly located below the waist and over the extremities while the subject is maintaining a vertical position. It should be located posteriorly upon floating postmortem. [5,6] At times injuries could be severe enough to hinder the diagnosis by producing serious disruption of the viscera. The presence of postmortem mutilation further complicate the diagnosis, let alone in jeopardizing the facial features and hindering identity as often encountered in mass fatalities.

Healthy adults who can swim rarely drown unless there is an intervening reason such as superimposed injury, fatigue or dangerous environment. The level of fitness, history of risk-taking behaviour, pre-swim activities, swimming ability and experience should be explored.

In the present case, the cause of death is labelled as drowning and suggested an accidental manner with sudden precipitation of undiagnosed cardiac condition, complicated by postmortem propeller injuries by marine traffic, evidenced by lack of blood infiltration at the site of traumatic amputation.

Let’s consider another case:

An 80 year old male, who was an inmate of old aged home with multiple comorbidities confined to a wheelchair, was found submerged underneath river. He was last seen swaying around a footbridge about 3 meters above the river several hours earlier. Probable suicidal intention was identified. Autopsy revealed extensive comminuted fractures of the vault, subarachnoid haemorrhages and cortical contusions. Both lungs did not appear to be waterlogged.

Injuries may also be produced before or upon entering water and their extent have to be assessed. This could be related to subsequent question of survivability. In this case, considering the severity of the head injuries, it would appear that the deceased was unable to survive in water (or at most only a transient period) and succumbed rapidly. The cause of death is therefore attributed to head injuries upon falling with his top of head bumping the river bed.

Another case to ponder:

A 33 year-old female was found submerged about 20 meters off shore. Linear reddish bruising was found on the anterior neck. The face and eyes were congested with petechiae. Small amount of frothy fluid was present. Both lungs were congested and oedematous. Dissection also revealed deep bruising of strap muscles suggestive of pressure applied to neck. Subsequent investigation revealed spouse’s involvement with manual strangulation during a quarrel.

Suspicious injuries should be noticed which may be an act of homicidal drowning. In the present case, the cause of death is a combination of drowning and pressure on neck, with latter being a significant event rendering the deceased unconscious when pressure was applied and succumbed to the effects of immersion.

Presence of drugs which may be related to death

Let’s consider the following case:

A 29 year-old male was found floating in the river reported by local residents. No personal property could be found. No suicide note was present. He was last seen alive by wife 3 days ago and was believed to have quarreled with a female acquaintance, exhibiting violent behavior and soon disappeared afterwards. Wife reported missing to Police the next day and his personal belongings were discovered in a shopping mall. Autopsy revealed features of drowning. Postmortem toxicology analysis showed presence of cocaine and its metabolite benzoylecgonine in blood. It was not known to the family whether he had a history of drug abuse.

Toxicology samples are crucial to exclude conditions that may mimic autopsy features of drowning, such as pulmonary oedema. It may help to exclude an accident, explain for failure to extricate or survival in water, as well as inferring an intention to end one’s life or a deliberate intoxication. In the present case, analysis of hair samples was performed to address the issue whether he was exposed to illicit drug on a chronic basis and therefore exhibiting tolerance.

Presence of decomposition features may obscure the effects of drowning

Let’s consider the following case:

A 32 year-old male was found in the reservoir exhibiting moderate decomposition changes. Suicide note was found in personal property placed neatly on the shore. Autopsy did not reveal any significant trauma or lethal disease conditions. Both lungs were not hyperinflated but huge amount of serosanguinous effusion was present in chest cavities. Police investigation also revealed a strong suicidal intention and third party was not involved.

Typical findings of drowning are often masked by decomposition changes. In addition, the time of death has to be determined during investigation. For fresh bodies examined at scene, corrective factors should be applied while measuring the core temperature against ambient temperature as the rate of cooling in flowing and still water are different. Casper's dictum refers to the rate of putrefaction after 1 week in air being equivalent to 2 weeks in water and 8 weeks burial in soil. The varying features of decomposition hint to the postmortem interval and is generally slower in cold water than a body discovered on land, but may be accelerated in bacterial laden stagnant water. As micro-organisms continue to disseminate and distribute throughout various body compartments, decomposition will be accelerated upon retrieval.

While the cause of death can remain unascertainable due to decomposition, the pathologist could nonetheless leave a remark stating the overall findings was not inconsistent with that of drowning. This is dependent on the degree of diagnostic certainty dictated by the available circumstances and likelihood of other intervening events, such as injuries (which could also be obscured by decomposition).

Let’s consider another case:

A 69 year-old female with a history of psychotic illness was found floating in the sea, three days after her husband had reported missing to Police. No suicide note was found. Body exhibited early decomposition changes. Postmortem toxicology analysis revealed a toxic level of amisulpride in the blood samples. As there was no concrete evidence about the suicidal intention or actual clinical progress on the psychiatric condition, it remained unclear whether the deceased fell into the water out of her intention.

Destruction of micro-architecture by decomposition permit considerable degree of postmortem redistribution of drugs which possibly account for the elevated levels in the specimen. The cause of death and manner can remain inconclusive.

Mysterious circumstances

Let’s consider a case with apparently suspicious circumstances:

A 32 year-old male with a known history of mood disorder was found floating near a port. His leg was tied to a dumbbell. Suicide note was found at home. He was last seen alive two days ago and reported missing by family another two days later. The body exhibited early decomposition changes but the lungs appeared hyperinflated. Further Police investigation tracked the last whereabouts of the deceased including the use of surveillance camera in the vicinity and revealed no evidence of third party involvement. The shopkeeper selling the dumbbell clearly recalled visit by the deceased on the day of death.

Forensic Pathologists do not interpret a case relying solely on the autopsy findings. Circumstantial information can play a role to hint the pathologist appropriate features that should be looked for during scene and body examination. In the present case, there could be an underlying psychiatric vulnerability suggestive of a suicidal intent. A body with weight affixed to limbs can of course represent an unlawful disposal, but may as well indicate a determination to kill oneself. Examination of the knot tying at the involved body part is crucial.

For suspicious case a detailed investigation into the events before death is expected. The salient areas of such are briefly mentioned here.

Witness account

This is valuable and gives considerable weight to the case. For example, witnessed jumping into the water, signs of mental impairment, activities prior to submersion, the duration of immersion, bystander resuscitation with possibility of repositioning of body, accounts provided by lifeguard and nearby video surveillance, are all hints to the state of mind prior to drowning. [5,6] Homicidal drowning is rare unless one is being incapacitated by alcohol, drugs or physical weakness, or taken by an element of surprise such as being pushed unexpectedly into water. [9]

Scene and environment

Water temperature, current, terrain, water depth, underwater condition, floating objects, marine animal activities or plants, presence of safety and rescue measures are important to consider. A seemingly innocent river with slow volume of flow may harbor strong underwater currents creating significant eddies and vortex sucking the swimmer rapidly, coupled by additional injuries inflicted by submerged rocks and waterfalls, or falling log from trees nearby. [10]

Body floats owing to formation of putrefactive gas producing buoyancy and is affected by lung volume. It could even overcome weights added to the body in concealed homicide. The body will continue to sink as hydrostatic force exert pressure to the chest and abdominal compartments creating negative buoyancy. In extremely cold water with minimal bacterial activity, the body will never resurface and decompose through formation of adipocere. [1,5,6,7,8] Coupled together with witness account about the last seen at the point of immersion, an estimation of current speed and body drop rate (about 1.5 and 2 feet in salt and fresh water respectively) can allow back-calculation of the site of drowning in moving water, i.e. the distance from shore, which is useful for rescue and case reconstruction. [5,6]

For indoor environment a bathroom may present with wet, floor, wet towels and soap scum level in the tub (if water has been drained already). The presence of bucket and mop, and other cleansing material maybe an attempt to disturb the scene. [5,6] A discovery of electrical appliances would call for a proper investigation to the possibility of electrocution.

Location of body

The place where body is discovered does not necessarily indicate the site of drowning. A body can be brought by a receding tide to the shore and there is always a possibility of drowning in another place, such as an indoor environment. [5,6,7] The body maybe disposed into the sea as an act of mimicking suicide. Differentiation between genuine drowning versus other causes; as well as fresh versus salt water immersion would be helpful. The appropriateness of the subject to the location is important. A restricted access may suggest unauthorized entry to the premises and should be investigated.

State of body

The condition of body regarding to its state of dryness or wetness, any attachment by aquatic debris and clothing identified are important. [5,6,7] Minute pieces of evidence pertaining to the identity, drug habit, personal property, weapon and suicide note should not be overlooked. Clothing and status of equipment, especially in diving related fatality should be examined. A naked body may be a deliberate act of hindering proper identification, or could be linked to a sexually motivated homicide. The body composition, water temperature, current action, type of clothing, method of water entry may all affect the presence or absence of clothing on body and should be interpreted with care. [6]

The presence of sand, seaweed or other vegetation should be documented and described, with the possibility of sampling for trace evidence and hinting the location of drowning in doubtful situations. A pair of shriveled and pale hands or feet can be found regardless of whether the individual was alive or not. Commonly referred as "washerwoman's skin”, there is wrinkling and grayish white discoloration of skin at sites devoid of sebaceous glands. Histological features of swelling of epidermis keratinizing squamous epithelium, detachment of horny layer, fraying of keratin lamellae and vacuolation in the basal layer are observed. There are reports in older literature with reference to such histological changes in an attempt to determine the postmortem interval, though subjecting to environmental factors of water type, temperature, movement, pollution and dermal characteristics of the subject. [11]

Hospital Pathologists are familiar with the appearance of hypostasis but such phenomenon would be present on face, upper chest and distal end of extremities. This is explained by dangling position adopted by the body with head and limbs pointing downwards owing its specific gravity while the posterior trunk is floating backup. [1,5,6,7] On the other hand, hypostasis can be minimal when exposed to fast flowing water. [5,6] For bodies lying in bath tub there may be a line of demarcation corresponding to the water level. [7] The importance of visiting a scene cannot be emphasized more.

Clear or blood tinged oedema is usually described as a plume of froth around the nose and mouth. It is non-specific in nature and consists of bronchial mucus, oedematous fluid, air and the drowning medium. The redness is accounted by the ruptured capillaries exuding into the respiratory tract. [7,8,] And most importantly it is transient in nature. In addition, slit, mud, sand, vegetation, algae and shell fragments may be present in bronchi and bronchioles visible both grossly and microscopically. [5,6]

Autopsy Findings

The role of an autopsy is to retrieve relevant findings that support the diagnosis. Not all the features will be present, depending on the nature of drowning process. Interpretation is only meaningful when combined with sufficient circumstantial information.

Emphysema aquosum

A pair of waterlogged lungs is a result of over-distension due to strenuous effort in an attempt to overcome oxygen depletion upon water influx. It is more prominent in the periphery and a combination of both lungs with effusion weighing more than 1000 g is usual. [7,8] There is also overlapping of medial edges in the anterior mediastinum with indentation or imprints by the corresponding ribs. It is distinguished from chronic emphysema by protrusion of sectioned bronchial and vessels at the cut surface for the latter. Histology shows flattened inter-alveolar septa, dilated pulmonary alveoli and compression of septal capillaries. [11] Alveolar macrophages stained CD 68+ (smoker cells) may be washed from the alveoli to heart allowing its detection, as well as stimulation of certain subsets of myelomonocytes in lung tissues [8], though the validity of such remains low from a practical point of view. In addition, aspirated particles such as plant material in the distal bronchioles may be suggestive of ante-mortem aspiration.

Paltauf's spots

These are subpleural haemorrhages located in middle lobe fissure of about the size of a fingernail due to rupture of capillaries by overdistension and haemolysis by fresh water drowning.

Haemorrhage in neck muscles

The strap muscles and posterior occipital muscles may show tiny haemorrhages and altered histological appearance of the myofibrils with fiber degeneration, abnormal clumps of red material and ragged red fibers, owing to anoxic and ischaemic insult secondary to violent convulsive movements. At an ultra-structural level there is myofibrillar disruption and abnormal mitochondria. [12] A prudent approach is to exclude a mechanical cause before ascribing such to the effects of drowning.


A contracted and anaemic spleen due to hypoperfusion and sympathetic stimulation with vasoconstriction is often nonspecific. [8]

Mastoid ear haemorrhages

Haemorrhage into ear compartment occurs as a result of pressure difference subsequent to blockage of Eustachian tube by water. [8,9]

Aspiration of fluid in the sphenoidal sinus:

“Svechnikov's sign” refers to presence of fluid (about 9 ml) in sphenoid and maxillary sinus by water penetration, which could also occur during postmortem. [7,8] It has been studied in literature with recent attempt to quantify and be detected by postmortem CT scan. [13]

Gastric dilatation

“Wydler's sign” refers to swallowing of water with resultant layer of sediment separating into three layers. This is also reported in recent postmortem imaging modalities with a certain degree of diagnostic confidence. [14] Oesophageal mucosal tears can be found occasionally due to distension by water. The presence of superficial radial ruptures of gastric mucosa is referred as "Sehrt's sign".

Ancillary Investigations

These tools can diagnose drowning with a higher degree of confidence, yet their limitations should be observed at the same time.


A differential staining of the intimal of aortic and pulmonary trunk is reported in the literature between saltwater and freshwater drowning. [15]

Immunohistochemical staining

Intrarenal aquaporin-2 (AQP2), intracerebral expression of aquaporin-4, aquaporin-5, HSP70, fibronectin are studied and reported with variable results. Surfactant protein A (SP-A) is produced by type II alveolar cells and showed increased expression with granular pattern in drowning case, despite that these stains could not readily differentiate between fresh and salt water drowning. AQP2, a channel protein for controlling flow of water molecules in the cellular interface, has shown apical expression in the apical membrane of the collecting in salt water drowning. [16] Arginine-vasopressin (AVP) was similarly expressed in the cytoplasm of renal tubules. Both have potentially served as markers to distinguish between salt and fresh water drowning, accounted by the increased binding and expression in a hyperosmolar environment. [17] While differentiation is necessary to exclude unlawful disposal of body, this can occur "naturally" when the body was dragged by sea currents from river in some regions.


There are literatures studying derangement of electrolytes including sodium, chloride, and magnesium between left and right ventricles basing on the effect of hypertonic and hypotonic action of the aspirated water in drowning, referred as the “Getter’s test”. Results were not promising and appeared to be controversial and not adopted for routine use. Strontium was also studied to a certain extent as an indicator of drowning. It has been reported that a difference of 75 µg/L between cardiac chambers could be an indicator of drowning. This test also falls short if the drowning medium has relatively low strontium concentration. [7,8]

Diatom test

This test has often been quoted as a gold standard for some to prove that drowning has occurred. Diatoms are microscopic unicellular algae coated with silica that exist in soil, water and atmosphere. If an individual is drowned in fluid which contains diatoms, they may be identified in the lungs and other organs if circulation is maintained at the time of aspiration. The diatoms can reach various organs such as brain, kidney, liver and bone marrow (femur being the most protected bodily compartment therefore its detection is generally regarding as true positive). The technique in collection of proper bodily samples should be strictly free from environmental contamination. Aided by the oxidizing property of strong acids, detergent or enzyme, the rest of the diatom tissue is consumed leaving a pellet to be centrifuged and then examined microscopically. [1,7,8,11,18] A sample of water must be taken from the suspected site of drowning for comparison. One should notice that a negative result does not rule out drowning as the cause of death.

Its application in cases with advanced decomposition explained why it is often regarded as a gold standard. [19] The confounding factor is often the presence and concentration of diatoms in the environment plus the amount being aspirated. Unfortunately there is scanty environmental data about the species and frequency of their occurrence in local waters. Much data is needed for quantification for the profile of these algae in the environment, before designing an appropriate cut off value and proper positive species identification to achieve a reasonable sensitivity and specificity. Comparison may not be possible when the original site of drowning is unknown.

Postmortem imaging

Postmortem CT scan may show accumulation of aspirated fluid in the maxillary and sphenoidal sinuses (Svechnikov's sign), apart from detection of fluid in trachea and patchy ground glass opacities in the lung parenchyma. In another study, the presence of three layers consisting frothy material, fluid materials and dense component, visualized via different image contrasting features [13,14]. Care should be exercised during transportation as movement of body may result in reshuffling of content.


Despite ever expanding literature on the research about the pathophysiology and findings, as well as validity of ancillary investigations, pathologists are still facing challenges with vague circumstantial information, presence of ante/post-mortem trauma, decomposition changes, as well as non-specific autopsy findings. Nevertheless, as part of the indispensable team in death investigation, pathologists are obliged to take a proactive role in analyzing all available findings which might eventually shed light on any interpretable direction despite circumstantial evidence might still remain unclear. An inquest may be held after careful consideration by the Coroner and this has been the practice adopted to rebut unfounded allegations and refute rumors, when submitted evidence would be intensely examined. It is hoped that evidence presented and testimony of witnesses can address the appropriate issues and allow the next-of-kin to understand the circumstances before the final moment.


  1. Dettmeyer RB, Verhoff MA, Schütz HF. Forensic Medicine: Fundamentals and Perspectives. 1st ed. Springer; 2014. 243-260 p.
  2. Byard RW. Drowning and near drowning-definitions and terminology. Forensic Sci Med Pathol. 2017 Dec;13(4):529-530. doi: 10.1007/s12024-017-9890-5. Epub 2017 Jun 20.
  3. Hong Kong Judiciary. Coroners’ Report 2018. Hong Kong; 2019. 62-73 p. Department of Health, HKSAR. Hong Kong Drowning Report. Hong Kong; 2019. 8-12 p.
  4. Armstrong EJ, Erskine KL. Investigation of Drowning Deaths: A Practical Review. Acad Forensic Pathol. 2018 Mar;8(1):8-43. doi: 10.23907/2018.002. Epub 2018 Mar 7.
  5. Armstrong EJ, Erskine KL. Water-Related Death Investigation Practical Methods and Forensic Applications. 1st edition. CRC Press; 2013. 27-149 p.
  6. Shkrum MJ, Ramsay DA. Forensic Pathology of Trauma: Common Problems for the Pathologist. 1st ed. Humana Press; 2007. 243-293 p.
  7. Lunetta P, Modell JH. Macroscopical, Microscopical and Laboratory Findings in Drowning Victims: A Comprehensive Review. Forensic Pathology Review Volume 3. 1st ed. Humana Press; 2005. 3-77 p.
  8. Leth PM. Homicide by drowning. Forensic Sci Med Pathol. 2019 Jun;15(2):233-238. doi: 10.1007/s12024-018-0065-9. Epub 2019 Jan 5.
  9. Byard RW. Drowning deaths in rivers. Forensic Sci Med Pathol. 2017 Sep;13(3):388-389. doi: 10.1007/s12024-017-9857-6. Epub 2017 Mar 11.
  10. Dettmeyer RB. Forensic Histopathology: Fundamentals and Perspectives. 2nd ed. Springer; 2018. 60-65 p.
  11. Girela-López E, Ruz-Caracuel I, Beltrán C, Jimena I, Leiva-Cepas F, Jiménez-Reina L, Peña J. Histological Changes in Skeletal Muscle During Death by Drowning: An Experimental Study. Am J Forensic Med Pathol. 2016 Jun;37(2):118-26. doi: 10.1097/PAF.0000000000000233.
  12. Lo Re G, Vernuccio F, Galfano MC, Picone D, Milone L, La Tona G, Argo A, Zerbo S, Salerno S, Procaccianti P, Midiri M, Lagalla R. Role of virtopsy in the post-mortem diagnosis of drownig. Radiol Med. 2015 Mar;120(3):304-8. doi: 10.1007/s11547-014-0438-4. Epub 2014 Jul 11.
  13. Gotsmy W, Lombardo P, Jackowski C, Brencicova E, Zech WD. Layering of stomach contents in drowning cases in post-mortem computed tomography compared to forensic autopsy. Int J Legal Med. 2019 Jan;133(1):181-188. doi: 10.1007/s00414-018-1850-4. Epub 2018 Apr 24.
  14. Byard RW. Aortic intimal staining in drowning. Forensic Sci Med Pathol. 2015 Sep; 11(3):442-4. doi: 10.1007/s12024-014-9563-6. Epub 2014 Apr 22.
  15. Barranco R, Castiglioni C, Ventura F, Fracasso T. Immunohistochemical expression of P-selectin, SP-A, HSP70, aquaporin 5, and fibronectin in saltwater drowning and freshwater drowning. Int J Legal Med. 2019 Sep;133(5):1461-1467. doi: 10.1007/s00414-019-02105-1. Epub 2019 Jun 20.
  16. Barranco R, Ventura F, Fracasso T. Immunohistochemical renal expression of aquaporin 2, arginine-vasopressin, vasopressin receptor 2, and renin in saltwater drowning and freshwater drowning. Int J Legal Med. 2020 Apr 2. doi: 10.1007/s00414-020-02274-4. [Epub ahead of print]
  17. Hürlimann J, Feer P, Elber F, Niederberger K, Dirnhofer R, Wyler D. Diatom detection in the diagnosis of death by drowning. Int J Legal Med. 2000;114(1-2):6-14.
  18. Nobuhiro Y, Eiji K, Shuji K. Diatom and Laboratory Tests to Support a Conclusion of Death by Drowning. Essentials of Autopsy Practice Innovations, Updates and Advance in Practice. 1st ed. Springer; 2013. 1-36 p.
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Liver injury associated with immune checkpoint inhibitors - update on clinicopathological features

Liver injury associated with immune checkpoint inhibitors - update on clinicopathological features

Volume 15, Issue 1, January 2020  (download full article in pdf)

Editorial note:

Immune checkpoint inhibitors revolutionize the field of immuno-oncology. They have demonstrated great potential in a wide range of adult cancers by reaching long-lasting objective responses and prolonging survival. Through completed and on-ongoing clinical trials, their indications continue to expand among different cancer types. However, one of their limitations is immune-related adverse events, which are most frequently reported in skin, gastrointestinal tract, and endocrine organs. Immune-related adverse events in liver are less common hepatotoxicity but still reported up to 4 to 10% of patients receiving immune checkpoint inhibitors. This Topical Update provides a concise review on the clinicopathological features of liver injury associated with immune checkpoint inhibitors. We welcome any feedback or suggestions. Please direct them to Dr. Anthony Chan (e-mail: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. Regina Lo

Department of Pathology & State Key Laboratory of Liver Research

The University of Hong Kong

Current applications of immune checkpoint inhibitors

Immune checkpoint inhibitors [ICPI] have been introduced as a form of targeted therapy for human cancers. They exert anti-tumor effects by potentiating T cell functions via removing the inhibitory signals. Programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are receptors located on T cells. Ligand-receptor interactions lead to inhibition of T cell activation, therefore suppressing T cell activity against tumor cells (1). Currently, anti-PD1/PD-1 ligand (PD-L1) and anti-CTLA-4 are the two major forms of ICPI by exploiting an antagonistic approach using specific antibodies that target PD-1 and CTLA-4, respectively. Thus far, several ICPIs were approved by the US Food and Drug Administration for treating cancer (2). Nivolumab and pembrolizumab are FDA approved frontline anti-PD1 agents, while ipilimumab is an anti-CTLA-4 agent. These drugs are given either alone or in combination. Currently there are a number of on-going phase III/IV clinical trials with ICPI for various types of cancers (3).

Clinical features of hepatotoxicity associated with ICPI

Despite the encouraging clinical efficacy, adverse reactions related to ICPI administration have been observed, among which dermatological, gastrointestinal, endocrine manifestations were most frequently reported. These reactions are believed to result from the immune response elicited toward various organs. A meta-analysis of 17 studies revealed an increased risk of all-grade hepatotoxicity with ICPI compared with controls (pooled OR 4.10; PD-1 subgroup 1.94; CTLA-4 5.01) (4). Among all immune-related adverse reactions, hepatotoxicity was observed in a relatively small proportion of cases (up to 4-10%) in most reports (2, 5-9). Susceptibility of adverse reactions in the liver appears to be dependent on the primary cancer, regimen/dose of ICPI, and host factors. It was reported that patients receiving ICPI for HCC were at a higher risk of hepatotoxicity in terms of transaminases levels compared with lung cancer and melanoma (10). Moreover, combination therapy or a higher dose of ICPI was associated with increased risk of hepatic injury (6, 9, 11, 12). Patients may present with fever and jaundice but can also be asymptomatic (13,14). The median time from the first dose to immune-related hepatoxicity was 14.1 weeks (9.4–19.7) for anti-PD-1, 9.9 weeks (6.1–14.7) for anti-CTLA4, and 2.9 weeks for combined therapy (15). The biochemical derangement is usually of a hepatitic or mixed hepatitic/cholestatic pattern. Radiological findings most of the time do not offer additional diagnostic information. In general, hepatotoxicity associated with ICPI is classified according to Common Terminology Criteria for Adverse Events by the National Cancer Institute (CTCAE). This system comprises grades 1-5 (with grade 5 being fatal) based on the serum levels of AST, ALT, ALP, GGT and total bilirubin. Having said that, elevated bilirubin is a less frequent phenomenon than most forms of drug-induced liver injury.

Histological features of liver injury associated with ICPI

The commonest histological features of ICPI-associated hepatotoxicity are lobular hepatitis, portal lymphoid infiltrates and variable degrees of hepatocytic necrosis (16-19). A predominant biliary pattern has been reported but is much less frequently encountered (20, 21). Cholestasis is not commonly seen, with bland cholestasis reported in 1 of 10 cases treated with pembrolizumab (22). Two cases of ICPI-induced hepatitis histologically presenting with fibrin-ring granulomas have also been reported (23). Steatosis is rare. Some histological features may be more readily observed with the use of a specific type of inhibitor. For instance, microgranulomas and central vein endotheliitis were seen in patients who received anti-CTLA4 therapy. With anti-PD1 therapy, more prominent portal tract inflammation was encountered. In contrast to autoimmune hepatitis, plasma cells are usually low in number (24), which is line with the observation that serum IgG level is mostly normal and autoimmune serological markers are negative. Likewise, in a report comparing 7 cases of ICPI-associated hepatitis versus 10 cases of AIH and 10 cases of drug-induced liver injury (DILI) (24), hepatocytic rosettes and emperipolesis were less commonly observed than AIH. When compared with DILI, bile plugs and eosinophils were less readily seen in ICPI-associated hepatitis. On immunohistochemical delineation of the lymphoid cell population in ICPI-associated hepatitis, several reports have consistently demonstrated a predominance of CD8+ lymphocytes (17, 18, 22). This could be distinguishing feature with AIH, in which CD20+ or CD4+ lymphoid cells are frequently encountered.

Diagnostic considerations and implications

The diagnosis of ICPI-liver injury can seldom be made by histology alone as there are no pathognomonic features. Before attributing the cause to ICPI, potential etiologies for liver function derangement should be considered. In particular, exclusion of hepatic involvement by tumor and viral hepatitis is needed. According to a recent report, among 491 patients treated with pembrolizumab for melanoma, lung cancer or urothelial cancer, 70 developed liver injury. Among which, a probably drug-related cause was only made in 20 cases after adjudication (25). Liver histology can help to exclude some differential diagnoses and assess the severity of liver tissue injury, which could be useful to guide management plan. The treatment options for adverse reactions would depend on the severity, and include withdrawal/discontinuation of ICPI, corticosteroids (oral or IV) +/- additional immunosuppressant e.g. mycophenolate mofetil (26). The drugs are usually permanently discontinued in cases presenting with Grade 3 or Grade 4 adverse reactions. There are no standard guidelines with reference to reintroducing ICPI after recovery from Grade 1-2 adverse reactions. As far as histology is concerned, it remains an open question whether histological parameters could offer added values in the grading of ICPI-associated hepatotoxicity. Besides, further studies are awaited to better understand the histological features associated different types of ICPI, and to depict the development and progression of fibrosis in this subset of drug-induced liver injury.


  1. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer. 2007;7(2):95-106.
  2. Nadeau A, Fecher LA, Owens SR, Razumilava N. Liver Toxicity with Cancer Checkpoint Inhibitor Therapy. Semin Liver Dis. 2018;38(4):366-78.
  3. Darvin P, Toor SM, Sasidharan Nair V, Elkord E. Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med. 2018;50(12):165.
  4. Wang W, Lie P, Guo M, He J. Risk of hepatotoxicity in cancer patients treated with immune checkpoint inhibitors: A systematic review and meta-analysis of published data. Int J Cancer. 2017;141(5):1018-28.
  5. Suzman DL, Pelosof L, Rosenberg A, Avigan MI. Hepatotoxicity of immune checkpoint inhibitors: An evolving picture of risk associated with a vital class of immunotherapy agents. Liver Int. 2018;38(6):976-87.
  6. Larkin J, Hodi FS, Wolchok JD. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015;373(13):1270-1.
  7. Motzer RJ, Rini BI, McDermott DF, Redman BG, Kuzel TM, Harrison MR, et al. Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J Clin Oncol. 2015;33(13):1430-7.
  8. Weber JS, Kahler KC, Hauschild A. Management of immune-related adverse events and kinetics of response with ipilimumab. J Clin Oncol. 2012;30(21):2691-7.
  9. Cheung V, Gupta T, Payne M, Middleton MR, Collier JD, Simmons A, et al. Immunotherapy-related hepatitis: real-world experience from a tertiary centre. Frontline Gastroenterol. 2019;10(4):364-71.
  10. Brown ZJ, Heinrich B, Steinberg SM, Yu SJ, Greten TF. Safety in treatment of hepatocellular carcinoma with immune checkpoint inhibitors as compared to melanoma and non-small cell lung cancer. J Immunother Cancer. 2017;5(1):93.
  11. Weber JS, Postow M, Lao CD, Schadendorf D. Management of Adverse Events Following Treatment With Anti-Programmed Death-1 Agents. Oncologist. 2016;21(10):1230-40.
  12. Sanjeevaiah A, Kerr T, Beg MS. Approach and management of checkpoint inhibitor-related immune hepatitis. J Gastrointest Oncol. 2018;9(1):220-4
  13. Johnson DB, Chandra S, Sosman JA. Immune Checkpoint Inhibitor Toxicity in 2018. JAMA. 2018;320(16):1702-3.
  14. De Martin E, Michot JM, Papouin B, Champiat S, Mateus C, Lambotte O, et al. Characterization of liver injury induced by cancer immunotherapy using immune checkpoint inhibitors. J Hepatol. 2018;68(6):1181-90.
  15. Gauci ML, Baroudjian B, Zeboulon C, Pages C, Pote N, Roux O, et al. Immune-related hepatitis with immunotherapy: Are corticosteroids always needed? J Hepatol. 2018;69(2):548-50.
  16. Karamchandani DM, Chetty R. Immune checkpoint inhibitor-induced gastrointestinal and hepatic injury: pathologists' perspective. J Clin Pathol 2018;71(8):665-71.
  17. Kleiner DE, Berman D. Pathologic changes in ipilimumab-related hepatitis in patients with metastatic melanoma. Dig Dis Sci. 2012;57(8):2233-40.
  18. Johncilla M, Misdraji J, Pratt DS, Agoston AT, Lauwers GY, Srivastava A, et al. Ipilimumab-associated Hepatitis: Clinicopathologic Characterization in a Series of 11 Cases. Am J Surg Pathol. 2015;39(8):1075-84.
  19. Zen Y, Yeh MM. Checkpoint inhibitor-induced liver injury: A novel form of liver disease emerging in the era of cancer immunotherapy. Semin Diagn Pathol. 2019.
  20. Kim KW, Ramaiya NH, Krajewski KM, Jagannathan JP, Tirumani SH, Srivastava A, et al. Ipilimumab associated hepatitis: imaging and clinicopathologic findings. Invest New Drugs. 2013;31(4):1071-7.
  21. Aivazian K, Long GV, Sinclair EC, Kench JG, McKenzie CA. Histopathology of pembrolizumab-induced hepatitis: a case report. Pathology. 2017;49(7):789-92.
  22. Zen Y, Chen YY, Jeng YM, Tsai HW, Yeh MM. Immune-related adverse reactions in the hepatobiliary system: Second generation checkpoint inhibitors highlight diverse histological changes. Histopathology. 2019.
  23. Everett J, Srivastava A, Misdraji J. Fibrin Ring Granulomas in Checkpoint Inhibitor-induced Hepatitis. Am J Surg Pathol. 2017;41(1):134-7.
  24. Zen Y, Yeh MM. Hepatotoxicity of immune checkpoint inhibitors: a histology study of seven cases in comparison with autoimmune hepatitis and idiosyncratic drug-induced liver injury. Mod Pathol. 2018;31(6):965-73.
  25. Tsung I, Dolan R, Lao CD, Fecher L, Riggenbach K, Yeboah-Korang A, et al. Liver injury is most commonly due to hepatic metastases rather than drug hepatotoxicity during pembrolizumab immunotherapy. Aliment Pharmacol Ther. 2019.
  26. Brahmer JR, Lacchetti C, Schneider BJ, Atkins MB, Brassil KJ, Caterino JM, et al. Management of Immune-Related Adverse Events in Patients Treated With Immune Checkpoint Inhibitor Therapy: American Society of Clinical Oncology Clinical Practice Guideline. J Clin Oncol. 2018;36(17):1714-68.
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Recent Advance in Acute Lymphoblastic Leukaemia

Recent Advance in Acute Lymphoblastic Leukaemia


Volume 14, Issue 2, August 2019  (download full article in pdf)


Editorial note

In this topical update, Dr Albert Sin reviews recent advances in diagnosis and management of acute lymphoblastic leukaemia (ALL).  We welcome any feedback or suggestions. Please direct them to Dr Rock Leung (e-mail: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals and are not necessarily those of the Hong Kong College of Pathologists.


Dr. Albert Sin
Clinical Assistant Professor



Acute lymphoblastic leukaemia (ALL) is an aggressive and highly fatal malignancy resulting from clonal mutations of lymphoid progenitor cells. The incidence of ALL is the most common in childhood and age after 50.18The prognosis of childhood ALL is good with long-term survival rate approaching 90% treated by intensive chemotherapy.12Although the incidence of ALL is less in adolescent, young adult as well as adult, the prognosis of ALL in those people is very poor, with only 30-40% of adult patients able to remit.18 According to the data from US database which registered all patients with diagnosed ALL from 2000 to 2007, the survival rate was 75% at 17 years old, 45% at 20 years old and 15% at 70 years old.14An increasing knowledge of disease biology of ALL transformed into insights for development of novel therapies to improve the treatment outcome of ALL.

One of the reasons of adverse prognosis in adolescent and young adult (AYA) as well as adult patients is that they commonly harbored poor-risk genetic aberrations while less patients carried favorable genetic lesion.14This could explain the sudden drop in survival from 17 years old to 20 years old. 


Ph-like ALL

Ph-like ALL is a newly identified genetic subgroup. The genetic profile of this subgroup of ALL is similar to that of Philadelphia chromosome positive (Ph-positive) ALL but without BCR-ABL1 fusion.17They have a higher frequency of IKZF1 deletion and mutation in genes of lymphoid transcription factors with poor survival.19The incidence of Ph-like ALL increases with ages and approaching 27% of cases of adult B-ALL.14

The nature of genetic aberration is heterogeneous. Despite its complexity, it can be simply classified into five subgroups: 1. CRLF2 rearrangement 2. Rearrangement of ABL-class gene 3. Rearrangement of JAK2 and EPOR 4. Aberrations leading to activation of JAK-STAT or MAPK pathway 5. Other rare kinase alterations.19The distribution of different types of genetic alterations are different among childhood high risk ALL, young adult and adult (Figure 1). CRLF2 rearrangement is the most common type of genetic alteration in Ph-like ALL. CRLF2 gene is responsible for producing lymphopoietin receptor and regulate the process of lymphopoiesis. Common mechanisms of CRLF2 rearrangement include 1. Translocation of CRLF2 gene into IGH gene 2. Fusion between CRLF2 gene and P2RY8 gene. 3. Point mutation F232C at CRLF2 gene. Nearly 50% of CRLF2 rearranged Ph-like ALL have concomitant JAK mutations.19

Figure 1

Figure 1


Diagnosis of Ph-like ALL

Genetic profiling is the gold standard for the diagnosis of Ph-like ALL. However, it is difficult to implement in routine diagnostic laboratory. 

Cytogenetics analysis is a standard test for all cases of ALL which allows a global assessment of chromosomal abnormalities. Some of the recurrent genetic abnormalities, for example t(9;22), hyperdiploidy/hyperdiploidy,   rearrangement involving 11q23, etc, can be detected. However, most of the Ph-like ALL genetic alterations are cryptic, e.g. interstitial deletion of CRLF2, ETV6-RUNX1 fusion, etc and thus they cannot be detected by conventional cytogenetics.2

Fluorescent in-situ hybridization (FISH) can be utilized to detect Ph-like ALL genetic abnormalities. Breakapart probes targeting genes most frequently genes including ABL1, ABL2, PDGFRB, JAK2, CRLF2, and P2RY8 are currently available. Although the positive result upon FISH study needs additional fusion probe for confirmation, it provides a readily available and useful diagnostic tool for establishing the diagnosis of Ph-like ALL. However, some of the important Ph-like ALL genetic rearrangement including intrachromosomal inversions (e.g., inv(9) resulting in PAX5-JAK2 fusion), intra-chromosomal deletions (e.g., del(X)(p22p22)/del(Y)(p11p11) resulting in P2RY8-CRLF2 fusion) are undetectable by FISH technique.2Targeted sequencing by NGS platform is an evolving technique for diagnosis.16

Recently, antibody against CRLF2 is available for flow cytometry study. The expression of CRLF2 as detected by multiparametric flow cytometry is correlated with genetic testing for CRLF2 rearrangement.15This provides a rapid tool for identifying potential cases of Ph-like ALL before the result of genetic tests is available. 

Most of the genetic alterations of Ph-like ALL are targetable kinase lesions, which could be treated by tailored kinase inhibitor therapy (Table 1).19This approach of therapy is current undergoing extensive preclinical studies.9

Table 1


Early T-cell precursor ALL (ETP-ALL)

ETP-ALL is recently characterized subtype of T-ALL. It constitutes around 12% of childhood ALL and 7.4% of adult ALL.11Genetic profiling showed ETP cells are similar to that of haemopoietic stem cells and myeloid progenitor cells.4This subgroup of ALL is characterized by the unique immunophenotype: cytoplasmic CD3+, surface CD3-, CD1a-, CD2-, CD5 dim (<75% positive), CD7 and positive for one or more stem cell and/or myeloid markers including HLA-DR, CD13, CD33, CD34, or CD117.5

While activating mutation of NOTCH1 is a common mutation found in ALL and it account for 50% of cases of childhood ALL, this mutation is less common in ETP-ALL.3ETP-ALL commonly have mutations in FLT3, DNMT3A, IDH1, IDH2, etc.11

ETP-ALL carries a poor prognosis with inferior overall survival when treated with standard chemotherapy regimen comparing with other subtypes of T-ALL.11This subgroup of T-ALL represented a distinct subtype with unique genetic profile and poor prognosis. 


MRD in adult ALL

Minimal residual disease (MRD) describe the very low level of disease burden which cannot be detected by morphology. Measurement of MRD not only pick up a submicroscopic level of disease but also can monitor the disease kinetic during the treatment process of haematological malignancies.10

The following techniques can be used to detect MRD: 

  1. Multiparametric flow cytometry to detect leukaemia-associated immunophenotype (LAIP)
    By using a 4-color or 6-color panel of antibodies, we can identify LAIP in 90% of ALL caes.10Flow cytometry is a quick method and the result of MRD can be generated in a short period of time for clinical decision. The sensitivity of MRD detection by this method is 0.01%. However, in order to define the positive MRD, we need 10-40 cluster of cells and thus higher number of cells are required for assessment which may be difficult for reassessment samples after intensive chemotherapy.7In addition, antigenic shift is commonly occurred in leukaemic cells and normal cells during the therapy. The use of monoclonal antibodies, e.g anti-CD19, anti-CD22 for treatment of ALL will affect the gating strategy used to identify the leukaemic cells.10

  2. Detecting leukaemia-specific fusion transcript by PCR technique
    Quantitative reverse-transcriptase PCR can be employed to detect the amount of leukaemia-specific fusion transcript. The sensitivity is higher compared with flow cytometry (10-4to 10-6).7The test is relatively easy to be performed in standardized diagnostic laboratory. However, only 30-40% of cases of ALL carry leukaemia-specific fusion transcript and thus limited the eligibility of MRD detection by this method. Moreover, the interpretation is challenging for RNA-based test in those cases will poor RNA quality.

  3. Quantitative PCR for immunoglobulin (IG)-T cell receptor (TCR) gene targets
    Quantitative PCR is employed to detect the specific sequence of rearranged IG gene or TCR gene in the sample. The sensitivity of this method is 10-4to 10-5and this method can be applied to all cases of ALL. However, this method of MRD detection requires prior characterization of IG or TCR gene rearrangement by sequencing and designs patient-specific primers for each case for subsequent MRD detection. Extensive standardization and experience are needed for the laboratory to set up this test, which limit the use of this method of MRD detection in diagnostic laboratory. Moreover, the clonal evolution in leukaemic blasts during treatment can make the original rearranged sequence to be lost and thus generate a false negative result. Also, the non-specific primer annealing occurs during the process of marrow regenerative may yield false positive result for the test.7


Application of MRD in treatment of adult ALL

MRD-guided therapy had been gained extensive experience in childhood ALL.8The study group of German Multicenter Study Group for Adult ALL (GMALL) had conducted largest study for the role of MRD in adult Ph-negative ALL. They showed that molecular remission is the only parameters significantly affect the remission duration and survival.6Patients with positive MRD after induction therapy achieved better overall survival after receiving haemopoietic stem cell transplant. Early achievement of MRD negativity after induction chemotherapy is associated with good outcome for adult ALL.10Study showed that MRD level correlates with post-transplant outcome.13Another group found that haemopoietic stem cell transplant benefits the patients with positive MRD at week 6.1These findings may prompt reconsideration of the indications of haemopoietic stem cell transplant for adult patients with ALL, especially those patients achieve MRD negativity after treatment. 


Concluding landmark

The prognosis of acute lymphoblastic leukaemia in young adolescent and adult is poor. The recent discovery of new subtype of acute lymphoblastic leukaemia with characterization of genetic lesions make a breakthrough of understanding of disease biology. Precise disease prognostication can be made. Targeted therapies are being developed for treating those patients. Clinical trials are conducting for evaluating the targeted therapies in those new subtypes of acute lymphoblastic leukaemia. Moreover, the application of MRD monitoring and MRD-adapted therapy in adult ALL can further stratified the patients and select the appropriate candidates of haemopoietic stem cell transplant in order to reduce transplant-related mortality and morbidity. The advances in understanding of molecular mechanism and disease biology of ALL help to improve the risk stratification, rapid development of targeted therapies and hopefully improve the prognosis in young adolescent and adult patients. 





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  7. Jacques J. M. van Dongen., Vincent H. J. van der Velden., Monika Br¨uggemann and Alberto Orfao. (2015) Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies.Blood, 2015;125(26):3996-4009
  8. Marianne Ifversen., Dominik Turkiewicz., Hanne V. Marquart., Jacek Winiarski., Jochen Buechner., Karin Mellgren., Johan Arvidson., Jelena Rascon., Lenne-Triin Ko¨rgvee., Hans O. Madsen., Jonas Abrahamsson., Bendik Lund., Olafur G. Jonsson., Carsten Heilmann., Mats Heyman., Kjeld Schmiegelow., Kim Vettenranta. (2019) Low burden of minimal residual disease prior to transplantation in children with very high risk acute lymphoblastic leukaemia: The NOPHO ALL2008 experience. British Journal of Haematology, 2019(184): 982–993
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Biochemical genetics in the expanded newborn screening era


Biochemical genetics in the expanded newborn screening era


Volume 14, Issue 1 January 2019  (download full article in pdf)


Editorial note

In this topical update, Dr. Calvin Chong reviews and updates on the technological development of biochemical genetics for the three major classes of metabolic disorders, namely aminoacidopathies, organic acidurias and fatty acid oxidation defects. These conditions have increasing local awareness, in particular with the introduction of universal expanded newborn screening. With a rising clinical demand for confirmatory tests in biochemical genetics, the ways to achieve better analytical quality and capacity were discussed. We welcome any feedback or suggestions. Please direct them to Dr. Sammy Chen (email: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Fig 0

Dr. Calvin Yeow-Kuan Chong
Department of Pathology, Princess Margaret Hospital


Biochemical genetics refers to the diagnosis of genetic disorders with biochemical markers. Sir Archibald Garrod first described the biochemical features of alkaptonuria in 19021, and is often named as the founding father of biochemical genetics. Throughout the past century, the practice of biochemical genetics has evolved from spot chemical tests towards the use of chromatography and mass spectrometry2,3. The recent implementation of the pilot study of expanded newborn screening means, on one hand, disorders are diagnosed earlier, and patients with such conditions would fare better, and on the other hand, this represents an increase in the use of confirmatory tests in biochemical genetics which is most acutely felt at the major chemical pathology laboratories.

The screening panel of the government-initiated pilot study included 8 aminoacidopathies, 7 organic acidurias, 6 fatty acid oxidation defects, and 3 other disorders4. Apart from the three disorders which required separate measurements, all three other major classes of disorders (viz. the aminoacidopathies, organic acidurias, and fatty acid oxidation defects) are screened by the use of tandem mass spectrometric measurement of succinylacetone, amino acids and acylcarnitines, all done within a period of around two minutes.

Table 1 lists the confirmatory markers and tests for the screened conditions 5,6: as can be seen, plasma amino acids, urine organic acids, and plasma acylcarnitines represents the main bulk work as confirmatory tests for these conditions. The present article aims therefore to explore the contemporary techniques available for these three major confirmatory tests in biochemical genetics and attempts to identify the approaches a laboratory may seek in order to cater for the increased demands.

Fig t

Table 1. Confirmatory markers and tests for conditions covered in the government-initiated pilot study of expanded newborn screening. Specialized tests not discussed in the present article are italicized.

Amino acids

Quantitative analysis of amino acids in plasma is the first-line confirmatory test for most aminoacidopathies. Amino acids are characterized by the presence of primary amine and carboxylic acid groups in one molecule, though some imino acids, namely proline and hydroxyproline, containing an imino (a functional group containing carbon-nitrogen double bond) as well as a carboxylic acid groups are also considered under the umbrella term amino acids in medical parlance and this use of terminology is adopted in the present article.

The analytical difficulties for amino acids are obvious when one examine their chemical structures: they are amphoteric, very small (glycine has a molecular mass of 75.067 g/mol), and most of them do not possess conjugated double bonds which gives absorbance in the ultraviolet spectra7. The first and second characteristics make separation difficult when reverse phase chromatography is used, whereas the last two give rise to problems in mass spectrometric detection and ultraviolet detection respectively. There are three major methods commonly used in clinical laboratories: amino acid analysers, high performance liquid chromatography (HPLC) with pre-column derivatization, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Irrespective of the methods employed, protein precipitation is necessary before analysis.

Amino acid analysers

Amino acid analysers (AAAs) are standalone machines that employ cation-exchange chromatography using a lithium buffer system, with post-column derivatization with ninhydrin (at some 120-135 degree Celsius) and monitoring at two wavelengths8. It is considered the gold standard method for amino acid analysis. This method overcomes the difficulty in separation of amino acids by utilizing ion-exchange chromatography which relies on the charge of a molecule at a particular pH rather than the hydrophobicity and steric interactions of a molecule and that of detection by formation of purple complexes which absorb strongly at 570 nm for primary amino acids, and yellow complexes which absorb strongly at 440 nm for proline and hydroxyproline9,10. When compared with other methods, AAAs provides higher degree of automation and require less expertise from the laboratory; the major issues with AAAs are the lengthy analytical run (120 minutes is common), the cost of the analytical instruments and the proprietary nature of the reagents. Analytical interference in AAAs are rare but do occur with dipeptides, which occur in prolidase deficiency8,11, and aspartylglucosamine, which occur in aspartylglucosaminuria8, can be spotted by the 570/440 absorbance ratio.

HPLC and LC-MS/MS methods

HPLC coupled with pre-column derivatization is used in the author’s laboratory for PAA determination. The method relied on the pre-column (immediately before injection) derivatization of amino acids by orthophthalaldehyde (OPA) and 3-mercaptopropionic acid (MPA), followed by reversed phase chromatography and ultraviolet detection12-14. In the presence of a thiol reagent, OPA forms a fluorescent derivative with primary amino acids with peak absorbance/excitation at 340 nm and emission wavelength at 450 nm7,8. The major drawback of this method is the inability to detect proline (and hydroxyproline) as OPA does not react with imino acids.

A newer derivatization reagent, AccQ-Tag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), which converts both primary and secondary amino acids into stable fluorescent derivatives, has been used with ultra-high performance liquid chromatography with ultraviolet detection15. This derivatization reagent has the advantages of covering both primary and secondary amino acids and as the derivatization products are stable, advanced autosamplers which can pipet reagent from one vial to another is not required16. Complete chemistry kit-sets that use this derivatization reagent is commercially available17.

Liquid chromatography coupled with tandem mass spectrometry is also used locally for PAA determination. As these methods are often based on reversed phase chromatography, despite the use of mass spectrometric detector, derivatization is still often employed7. A method based on the proprietary derivatization reagent AccQ-Tag has been recently described in the literature with a run-time of 6 minutes18. Isotopic internal standards are required for LC-MS/MS-based methods and it is important to note that the impracticality of having isotopic internal standards for each and every analyte would mean that the robustness of the assay is considerably weaker than optical-based methods19.

Compared with methods based on optical detection, LC-MS/MS methods had a shorter analytical runtime and better specificity. This is counterweighed by the higher capital and maintenance cost of acquiring an LC-MS/MS (and not to mention the cost of having a backup LC-MS/MS system), as well as the technical expertise necessary to operate and troubleshoot an LC-MS/MS.

Choice of method and the future

The question is different depending on the volume of testing as well as equipment availability. For laboratories that has existing HPLC-DAD equipment employing OPA derivatization there may be little incentive in adopting a new procedure: there is probably little competition for HPLC analyser time, and the inability to detect proline may not be a major issue as hyperprolinaemia type I is benign whereas hyperprolinaemia type II is classically diagnosed by the presence of N-(Pyrrole-2-carboxyl)-glycine in urine organic acids20.

On the other hand, for a laboratory seeking to provide amino acids analysis for the first time, the use of stable derivatization reagent such as AccQ-Tag mentioned above has the advantage of not requiring higher-end liquid chromatographs and the availability of commercial kits means that development time is reduced. For a laboratory anticipating a high workload, a dedicated liquid chromatograph with UV detection appears to be the simplest solution as it is robust and inexpensive. On the other hand, for laboratories with lower service demand, mass spectrometric detection may in fact be more feasible as the notion of spare LC-MS/MS capacity means that the major downside of LC-MS/MS detection, viz capital cost and technical expertise, are sunk cost to the laboratory.

Organic acids

Organic acidurias are diagnosed most commonly by urine organic acid analysis with gas chromatography-mass spectrometry (GC-MS)21, and this technique is used by many local hospitals. Though the term organic acids refers to organic compounds with a carboxylic acid group, a broader spectrum of compounds, such as uracil and xanthine, are detected in practice. Urine organic analysis is usually qualitative though quantitative analyses of some compounds (e.g. orotic acid, methylmalonic acid) are often offered.

GC-MS based urine organic acid analysis

For GC-MS analysis of urine organic acids, a creatinine-corrected amount of urine is subject to liquid-liquid extraction with ethyl acetate after acidification using hydrochloric acid, the organic extract is then dried and derivatized by N,O-bis-tri(methylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)22. The resultant product is injected to the GC-MS operating in scan mode. For reliable detection of ketones, oximation with hydroxylamine hydrochloride can be performed prior to acidic extraction23.

An alternative way of performing urine organic acid analysis is to bypass the extraction step. As no acidic extraction step is done, a limited panel of amino acids, purine, pyrimidines, and mono- and di-saccharides can be detected in the same analytical run. This method has been in-use in the author’s laboratory in the past 2 decades24,25, and allows the detection of a much wider range of metabolites in urine. With no extraction, the chromatograms are extremely complex due to co-elution of analytes and therefore this approach requires expertise in post-analytical data processing to be viable as a routine method.

The interpretation of GC-MS data for urine organic acid analysis is commonly based on examination of the chromatogram (Figure 1), followed by a combination of peak integration in the total ion chromatogram followed by library searching and examination of extracted ion chromatogram at particular retention times for analytes which gives lower responses (Figure 2). The raw analyte response is then compared to locally-established age-specific reference intervals which allow clinical interpretation23.

Fig 1

Figure 1. Total ion chromatogram of a urine sample from a patient with malonic acidemia circulated in the ERNDIM qualitative organic acid program in 2016. The two abnormal peaks are indicated by asterisks: the first peak represents malonic acid (di-TMS derivative) and the second peak represents methylmalonic acid (di-TMS derivative).

Fig 2

Figure 2. Extracted ion chromatogram showing the quantifier ions (m/z 375, for aconitic acid in red; and m/z 254, for orotic acid, in black) and qualifier ions (m/z 285, for aconitic acid in green; and m/z 357, for orotic acid, in blue). They are co-eluting in many GC-MS based urine organic acid assays.

LC-MS based urine organic acid analysis

In the recent years, liquid chromatography-mass spectrometry-based methods have been published for analysis of urine organic acids. The benefit of liquid chromatography-mass spectrometry is clear: there is no need for the cumbersome derivatization step, and heat-labile analytes can be detected26,27. The major drawbacks of LC-MS based methods are the poorer separation of analytes and higher susceptibility towards ion suppression, as electrospray ionization is much less robust against matrix effects compared to electron ionization as used in GC-MS based methods28. The difficulty of developing an in-house LC-MS based method for urine organic acids lies in the procurement of a practically endless list of organic acid standards to establish the retention time and multiple reaction monitoring ratios. At the time of writing, there is at least one commercial kit that has been made available (Zivak Organic Acids LC-MS/MS analysis kit), but this commercial kit did not utilize isotopic internal standards even for critical analytes (such as hexanoylglycine and orotic acid) and one may wish to validate extensively the robustness of such assays against matrix effects.

Choice of method and the future

Out of the three assays discussed in the present article, urine organic acid analysis represents the most difficult of the three to establish in a laboratory. There is first the difficulty in establishing age-specific reference intervals, and then difficulty in acquiring a large number of chemical standards. A good starting point would be obtaining the bi-level quality controls for urine organic acid and special assays for urine from the ERNDIM network which consist of a number of critical analytes. As for choice of internal standards, while traditional choices such as tropic acid and pentadecanoic acid are often employed, deuterated internal standards covering critical analytes are much more available nowadays (e.g. methylmalonic acid-d3 and orotic acid-1,3-15N2) and their addition may improve quantitation of these critical analytes, the former being commonly quantified and the latter being prone to analytical errors29.

For laboratories with existing GC-MS based urine organic acid assay, the question is probably whether to adopt LC-MS based solution. The belief that organic aciduria always results in sky-high level of abnormal metabolites in urine is flawed: the low-excretor phenotype of glutaric aciduria type I can serve as the classical example30. The difficulty may be mitigated somewhat if acylcarnitines and acylglycines are measured by the LC-MS based assays as glutarylcarnitine has been shown to be informative even for low-excretor GA I patients31. Overall, it remains to be seen whether LC-MS based organic acid analysis could stand alone replacing, rather than complementing, GC-MS based assays.

Free carnitine and acylcarnitines

Quantitative analysis of free carnitine and acylcarnitines in plasma represents the first-line confirmatory test for fatty acid oxidation disorders. Analysis of free carnitine with calculation of fraction of excretion can be helpful in the diagnosis of carnitine uptake defect, as is measurement of urine glutarylcarnitine for the diagnosis of glutaric aciduria type I as discussed above31. Carnitine is a quaternary ammonium compound with a carboxylic acid group, as well as a hydroxyl group with which acyl groups are attached to form acylcarnitines; as such, it forms zwitterions under physiological pH. Derivatization to esters by incubation with an alcohol (typically butanol) has been employed32 though locally underivatized analysis at acidic pH is commonly used33.

For underivatized analysis, plasma is first diluted with a mixture of isotopic internal standards, and acidified acetonitrile is slowly added to precipitate plasma proteins. The sample is vortexed, centrifuged, followed by evaporation of the supernatant to dryness and reconstituted for analysis by positive electrospray ionization-tandem mass spectrometry with or without liquid chromatography separation. Data can be collected in multiple reaction monitoring (MRM) mode, or in precursor ion scan with accumulation of ions, commonly known as multichannel acquisition (MCA) mode34 (Figure 3). The American College of Medical Genetics Guideline, published in 2008, suggested the use of precursor ion scan as it permitted the evaluation of the whole acylcarnitine profile, as well as the detection of drug artefacts, interfering compounds and assessment of derivatization35.

Fig 3

Figure 3. Cumulative precursor scan mass spectrum for a blood-spot sample distributed in the ERNDIM Qualitative Acylcarnitine Program in 2014. In this specimen, elevated signals at m/z ratio 260 (C6-carnitine), 288 (C8-carnitine), and 314 (C10:1-carnitine) can be seen. The pattern would be compatible with MCAD deficiency. (Mass spectra courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)

Derivatization and chromatographic separation

In the analysis of carnitine and acylcarnitines, butyl-ester derivatization enhances the formation of positively-charged ion by reacting with carboxylic groups, and causes mass separation of dicarboxylic-carnitines and hydroxy-acylcarnitines (e.g. C4DC-carnitine and C5OH-carnitine), which are isobaric when underivatized36. Derivatization is typically performed at highly acidic conditions (e.g. 3N hydrochloric acid at 65°C for 15 minutes)35. On the other hand, chromatographic separation allows the separate determination of individual isomeric constituents (e.g. C4DC-carnitines include succinylcarnitine and methylmalonylcarnitine; and C5OH-carnitines include 3-hydroxyisovalerylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine) (Figure 4). With meticulous chromatographic separation, most biologically relevant isomeric species could be separately quantified37.

Choice of method and the future

The question for the laboratory is, first, whether to employ the derivatization procedure. The advantage of derivatization is the mass separation of hydroxylacylcarnitines and carnitine derivatives of dicarboxylic acids; the problems associated with derivatization are the partial hydrolysis of acylcarnitines because of the high temperature and strongly acidic condition employed33. The other considerations are whether to employ chromatography and if so, how extensive should it be: it would then be a delicate balance between through-put, diagnostic specificity, and analyser-time that is available. With the improvement of separation capability of liquid chromatographs and sensitivity of modern mass spectrometers, it is suggested that a short UHPLC program combined with both scheduled MRM and precursor ion scan function would be a good compromise.

Fig 4

Figure 4. SRM chromatogram of m/z 262>85 showing chromatographic separation of different species of isobaric (C4DC/C5OH) acylcarnitines (viz. succinylcarnitine at 4.68 minutes, two diastereomeric peaks of methylmalonylcarnitine at 5.2 and 5.35 minutes, and 3-hydroxyisovalerylcarnitine at 5.82 minutes) could be individually identified and quantified with liquid chromatography-tandem mass spectrometry without derivatization. (a) sample with normal amounts of succinylcarnitine; (b) sample with abnormal amounts of methylmalonylcarnitines (Chromatograms courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)



The three assays discussed above represent the bulk of workload for most metabolic laboratories. The planned implementation of universal expanded newborn screening in Hong Kong means that the demand would increase, and the phenotype will be less defined, as tests are requested for patients who may not yet present with features of an inborn error and importantly, patients with only borderline elevation of analytes.

From a Bayesian point of view, this change in pre-test probability would mean that, if the analytical quality and interpretative capacity remains the same (which affect the likelihood ratio of positive results), the post-test probability would suffer from a negative impact. The quest for the metabolic bench of any major pathology laboratory is then to improve both throughput and quality of analysis at the same time, an impossible task, as the Duke of Norfolk wrote in 1538, “a man can not have his cake and eat his cake”38. The technology improvements as reviewed in the present article may aid in the analytical quality but the quest for improved interpretative capacity remains on the training and education of our present and coming generations of pathologists.


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  26. Körver-Keularts IMLW, Wang P, Waterval HWAH, Kluijtmans LAJ, Wevers RA, Langhans C-D, et al. Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism. J Inherit Metab Dis. 2018 May 1;41(3):415–24.

  27. Yassine MM, Dabek-Zlotorzynska E. Application of ultrahigh-performance liquid chromatography–quadrupole time-of-flight mass spectrometry for the characterization of organic aerosol: Searching for naphthenic acids. Journal of Chromatography A. 2017 Aug 25;1512:22–33.

  28. Taylor PJ. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray–tandem mass spectrometry. Clinical biochemistry. 2005;38(4):328–34.

  29. Scott C, Langhans C-D, Roux C. Qualitative Organic Acid. In: ERNDIM Workshop. Manchester, UK; 2017.

  30. Kölker S, Christensen E, Leonard JV, Greenberg CR, Boneh A, Burlina AB, et al. Diagnosis and management of glutaric aciduria type I–revised recommendations. Journal of inherited metabolic disease. 2011;34(3):677.

  31. Tortorelli S, Hahn SH, Cowan TM, Brewster TG, Rinaldo P, Matern D. The urinary excretion of glutarylcarnitine is an informative tool in the biochemical diagnosis of glutaric acidemia type I. Molecular Genetics and Metabolism. 2005 Feb 1;84(2):137–43.

  32. Dhillon KS, Bhandal AS, Aznar CP, Lorey FW, Neogi P. Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process. Clinica Chimica Acta. 2011;412(11–12):873–9.

  33. Ho CS, Cheng BSS, Lam CWK. Rapid Liquid Chromatography–Electrospray Tandem Mass Spectrometry Method for Serum Free and Total Carnitine. Clinical Chemistry. 2003 Jul 1;49(7):1189–91.

  34. Matern D. Acylcarnitines, Including In Vitro Loading Tests. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2018 Nov 30]. p. 171–206. Available here.

  35. Rinaldo P, Cowan TM, Matern D. Acylcarnitine profile analysis. Genetics in Medicine. 2008 Feb;10(2):151–6.

  36. Vreken P, Van Lint AEM, Bootsma AH, Overmars H, Wanders RJA, Van Gennip AH. Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects. Journal of inherited metabolic disease. 1999;22(3):302–6.

  37. Giesbertz P, Ecker J, Haag A, Spanier B, Daniel H. An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues. Journal of lipid research. 2015;jlr. D061721.

  38. Letters and Papers, Foreign and Domestic, Henry VIII, Volume 13 Part 1, January-July 1538 [Internet]. 1892. Available here.


Plasma amino acids (PAA):
Arginine (Arg), Citrulline (Cit), Homocystine (Hcy), Isoleucine (Ileu), Leucine (Leu), Methionine (Met), Ornithine (Orn), Phenylalanine (Phe), Threonine (Thr), Tyrosine (Tyr), Valine (Val).

Urine organic acids (UOA):
ethylmalonic acid (EMA), glutaric acid (GA), 2-hydroxyglutaric acid (2-OHGA), 3-hydroxyglutaric acid (3-OHGA), 3-methylglutaric acid (3-MGA), 3-methylglutaconic acid (3-MGCA), hexanoylglycine (HG), 3-hydroxyisovaleric acid (3-OHIVA), Isovalerylglycine (IVG), 3-methylcrotonylglycine (3-MCG), Methylcitric acid (MCA), Methylmalonic acid (MMA), 3-hydroxypropionic acid (3-OHPA), propionylglycine (PG), phenylpropionylglycine (PPG), Tiglylglycine (TG).

Plasma acylcarnitines (PAC):
Free carnitine (C0).

6-pyruvoyl-tetrahydropterin synthase (PTPS), 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), Carnitine-acylcarnitine translocase (CACT), Carnitine palmitoyltransferase II (CPT-II), Medium-chain acyl-CoA dehydrogenase (MCAD), Very long-chain acyl-coA dehydrogenase (VLCAD).
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Antimicrobial resistance – a global health crisis

Antimicrobial resistance – a global health crisis


Volume 13, Issue 2 July 2018  (download full article in pdf)


Editorial note

With increasing prominence of the threat of antimicrobial resistance both internationally and locally, awareness and knowledge on the problem and prospects are essential, in order for rational application of control measures and monitoring of their effectiveness. In this issue of the Topical Update, Dr. Dominic Tsang and Dr. Christopher Lai present an updated overview of this important subject. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail:, Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Dominic TSANG and Dr. Christopher LAI
Consultant Microbiologist and Associate Consultant, Department of Pathology, Queen Elizabeth Hospital, Hong Kong 


End of modern medicine as we know it

The World Health Organization (WHO) described in 2001 antimicrobial resistance (AMR) as a global problem and an impending crisis. The apocalyptic term “Post-antibiotic era” was mentioned.1 The situation did not improve since. In fact, it deteriorated. In the United Kingdom (UK) government-commissioned “Review of Antimicrobial Resistance” by Lord Jim O’Neill, it was estimated that 700,000 people died from AMR infections in 2016 in the UK alone, and that drug-resistant infections could cause 10 million human deaths annually by 2050, costing the world up to $100 trillion.2 The Chief Medical Officer for England, Dame Professor Sally Davies, has predicted that unless tackled now, AMR could lead to the end of modern medicine as we know it. It could lead to routine operations and even childbirth becoming increasingly dangerous without the required antibiotics. In the UK, over 25,000 deaths a year are attributed to drug resistant infections.3 European Commission estimated the costs associated with AMR infection at €1.5 billion annually.4

The driving force behind emergence and dissemination of AMR is directly related to the use of antibiotics, i.e. the antibiotic selection pressure.5 It is recognized that non-human indiscriminate use of antibiotics in agriculture and animal husbandry to promote growth in animals and the consequential persistence of antibiotics in soil and aquatic environment select for AMR that could be disseminated widely.6 Human overuse nonetheless needs to be controlled by dedicated efforts on strengthening regulations on over-the- counter purchase of prescription only antibiotics, enhancing training in antibiotic prescriptions, monitoring compliance with antibiotic guideline and antibiotic stewardship programme (ASP).7


Concerted effort against AMR

Antibiotics on one hand is an agent required for life saving. But on the other, its usage and the resultant selective pressure have been recognized as the main drivers for AMR. Therefore, its use in human and non-human settings should be balanced against the risk of driving AMR. As such, antibiotic usage data has been linked to AMR surveillance data in the AMR containment strategy.8,9

There are already in place a few surveillance systems on AMR, covering healthy animals, diseased animals, food and humans in countries such as Canada, Denmark, Finland, Germany, Italy, Japan, USA, the Netherlands, Norway, France, and Sweden.10

The UK and China will establish the Global AMR Research Innovation Fund and encourage further investment from other governments and the private sector, helping to address AMR. The new fund will invite bids from industry, academia and other bodies. It aims to create international partnerships to build a global response and support new research to reduce the spread of antibiotic resistance.

The World Health Assembly in May 2015 endorsed a WHO global action plan to tackle antimicrobial resistance to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines.11 The plan sets out five strategic objectives including: to improve awareness and understanding of antimicrobial resistance; to strengthen knowledge through surveillance and research; to reduce the incidence of infection; to optimize the use of antimicrobial agents; and to develop the economic case for sustainable investment that takes account of the needs of all countries, and increase investment in new medicines, diagnostic tools, vaccines and other interventions. Under each of the objectives, specific actions were listed out for member states as well as international partners to implement.


Hong Kong Strategy against Multidrug resistant organisms (MDROs)

The Hong Kong Strategy and Action Plan on Antimicrobial Resistance 2017-202212 was released in July 2017 with the goals to develop a territory-wide network on surveillance on AMR and antimicrobial use, promote appropriate therapeutic use of antimicrobials in human and animals and to promote research on diagnostic and related interventions. Six key areas were targeted in the plan, including strengthening knowledge, optimizing antimicrobial use, reducing infections, improving awareness, promoting research and fostering partnerships among stakeholders.

In terms of AMR surveillance, the overall resistance profiles of MDROs have all along been closely monitored in public hospitals under the Hospital Authority in Hong Kong13 Among the concerned MDROs, Gram positive organisms include methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). Gram negative organisms include extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) and the WHO top priority organisms of carbapenem-resistant Enterobacteriaceae (CRE), carbapenem- resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA).14


Trends in antibiotic resistant organisms

MRSA: In Hong Kong, MRSA bloodstream infection was made a key performance indicator (KPI) to gauge the performance of infection control practices in all public hospitals.15 The corresponding MRSA bacteraemia rate was 0.18- 0.19 per 1,000 acute bed days when the monitoring began in 2008 and declined gradually to 0.144 per 1,000 acute bed days in 2017. At the same time, MRSA constituted 43.1% among S. aureus isolated from local HA hospitals.

VRE: The total number of VRE cases detected remains below 50 a month since 2015, after an upsurge and successful control in the Queen Elizabeth Hospital in 2013 when the peak number of cases detected in a month reached 300.16

CRAB/multi-drug resistant Acinetobacter baumannii (MDRA) is commonly associated with patients with prolonged hospital stay and who required ventilator-assisted ventilation. These patients are also usually put on multiple antibiotics for the treatment of underlying infections.17 Substantial environmental contamination with the resistant bacteria is frequent as a result of nursing care procedures, leading to explosive outbreaks which are difficult to abort unless attention is given to patient segregation and effective disinfection of instruments and environmental surfaces.18,19

ESBL-E began to emerge in late 1980s and peaked in UK in 2006 with 12% E. coli isolated from bacteraemic cases being ESBL positive. ESBL-E has been common locally, at 22% in 2017 compared to 24.3% since 2012. They are especially important as they commonly caused community-acquired as well as hospital-acquired infections. In fact, a local report showed 62% of imported chicken were found to be contaminated with ESBL-E.20 Community carriage rates for ESBL-E are high in East Mediterranean areas and South East Asia, from 30-60%.21,22 For treatment of ESBL-E infections, imipenem has been universally effective since its launch in 1987, and the same applies to meropenem, another carbapenem antibiotic which was launched in 1996.

CPE are members of the Enterobacteriaceae including E. coli and Klebsiella species which possess the gene on plasmids coding for the enzymes such as KPC, NDM and OXA-48 under the Ambler Classification on molecular class A, B or D respectively which enable them to inactivate carbapenem antibiotics and also other beta- lactams. CPE are usually multiply antibiotic resistant, therefore rendering limited options in treatment of their infections.23 The emergence and subsequent spread of CPE followed human movement closely as exemplified by the clustering of KPC-CPE in New York, North-West England, Israel and the spreading of NDM-CPE to Sweden by a returning patient from New Delhi, India.24-26 In Hong Kong, CPE cases were being increasingly detected, from 19 cases in 2011 to 473 cases in 2017. Active bacterial screening (ABS) of patients based on a set of consensus criteria in public hospitals including admission screening of all patients who have stayed in overseas hospitals in the past 12 months, suffering from antibiotic associated diarrhoea, staying in the same cubicle of a known CPE case, etc., helped to pick up an increasing number of CPE. Fortunately, most of the cases (90%) were asymptomatic carriers and did not require specific treatment. Of note, the number of confirmed CPE isolates in England in 2016 was more than 2,500 with KPC, NDM and OXA-48 predominant. Effective treatment options for CPE are few. The activities of ceftazidime and aztreonam with and without avibactam were tested against a large, contemporary, international collection of carbapenemase-producing Gram negative bacilli (CP-GNB) with diverse resistance mechanisms. Aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/litre; ceftazidime- avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-β-lactamase producers. Among the older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.27 Among local CPE isolates, the resistance rate of colistin is around 5.8%.


The “Find and Confine” Control strategy of AMR in HA hospitals

“Find”: This is the single most important control measures aiming to uncover the carriage state of any asymptomatic carrier, especially of VRE and CPE, who could shed the MDRO in the excreta. This is commonly done by ABS in the form of admission screening based on high risk factors such as hospitalization in recent months, a history of exposure to a known case, prolonged hospital stay (e.g. for 14 days or more), development of antibiotic associated diarrhoea etc. The yield of ABS at present is not high, ranging from 0.6% to 1.4% (average 1.0%) based on 2017 CPE ABS data in one of the hospitals, but ABS has undoubtedly provided a substantial impact in mitigating otherwise uninterrupted dissemination of VRE and CPE in the hospital setting. The availability of commercial agar media with the function to differentiate CPEs after overnight incubation has greatly facilitated ABS. Coupled with other rapid enzyme detection and PCR confirmation of CPE, which are also available commercially, the time to detection of CPE carriage in patients has been shortened to one to two days.

“Confine”: It is an important part of standard precautions (SP) plus contact precautions (CP) in the care of a known MDRO case. A room with en- suite is the preferred placement especially for those who suffer from diarrhoea and therefore at a higher risk of transmission of MDROs. This is widely practiced in developed countries. When en-suite rooms are not available, segregation of MDRO carriers by “cohorting” patients carrying the same MDRO is a pragmatic alternative.

Hand hygiene (HH): Healthcare workers (HCWs) strictly observing proper HH, e.g. WHO 5 moments, is undoubtedly the most important and effective measure in aborting the dissemination of MDROs in healthcare setting. The use of alcohol handrub in place of hand washing in situations where soiling is minimal has greatly improved HH compliance. However, HH is an action governed by human behavior which unfortunately also suffers from all factors that affect our behavior, such as physical fatigue, forgetfulness, motivation, persistence, peer influence, etc. For the same reason, the reliability of HH compliance monitoring by direct observation is also limited by the well-known “Hawthorn effect” in that, as humans, we tend to perform when we are aware of being observed.28 Further, in periods of high bed occupancy like influenza seasons, it is very hard to expect HCWs to fully comply with HH and as a consequence, these periods are more prone to cross transmission and outbreaks of MDROs. Apart from HCWs, patients and visitors are equally important in observing HH in order to avoid cross transmission. Similar moments of HH are being promoted for patients in local hospitals. In the long run, the development of a positive and motivated infection control culture and HH habit would not only maintain the cross transmission of MDROs at a low level but also mitigate the risk of other cross infections.

Environmental hygiene: The inanimate environment plays an important role inperpetuating the dissemination of MDROs in the hospital settings, especially VRE and MDRA.29,30 High-touch (frequently touched) surfaces such as patients’ privacy curtain, bed rails, door knobs, nursing trolleys, drip stands are often contaminated with the MDROs during outbreaks.31 The conventional cleansing and disinfection by using diluted sodium hypochlorite solution, although effective, is labour intensive. Also, to ensure the cleaning procedure is meticulously performed, it is commonly monitored by the use of a surrogate marker (e.g. UV-fluorescent marker) to ensure satisfactory performance.32 To circumvent such drawback, there are now plenty of effective new products on environmental disinfection, ranging from self- disinfecting surface coating sprays, hydrogen peroxide vapor, UV-C device, 2-in-1 disinfectant wipes to antimicrobial privacy curtains that could achieve effective decontamination with less labour. In a multi-centre study, 42.7% of standard hospital curtains were contaminated with MRSA and 42.3% with CRAB.33 The use of disposable antibacterial privacy curtains, e.g. nanoparticle silver or quaternary ammonium impregnated curtains, have been shown to prolong the time to contamination and reduce the bio-burden even after extended usage in acute care setting.34

Reducing overuse of antibiotics: It is imperative to maintain the use of antibiotic at the minimum essential level in order to prevent the emergence of resistance.34 Education and training at an early stage of the medical curriculum is critical in establishing the concept and skills in prudent antibiotic use. Guideline such as the local IMPACT guideline on antibiotic use is indispensable in providing the guidance on the right indications, choice, dose, route, and duration of antibiotic use. In addition, antibiotic stewardship program (ASP) has been widely practiced and proven to be effective in ensuring appropriate use of antibiotics.35 Locally, a multi- disciplinary team of clinical microbiologists, physicians, pharmacists and infection control nurses has been put in place in all HA hospitals to provide concurrent feedback on the use of targeted “big-gun” antibiotics. The percentage of appropriate use stays at above 80% in general and feedbacks on antibiotic use are welcomed in most of the cases.

Another very important aspect in the control of AMR is diagnostic support in infection.36 The rapid isolation and identification of an aetiological agent lends strong support in the continuation of antibiotic treatment, or its discontinuation in the absence of any evidence of infection. This is made possible with the introduction of molecular platforms such as 16S ribosomal RNA PCR and metagenomic studies.37

Surrogate biomarker of infection, in particular procalcitonin (PCT) which exhibits greater specificity than other proinflammatory markers such as C reactive protein (CRP) helps in identifying patients with sepsis and can be used for diagnosing infections, especially ventilator- associated pneumonia (VAP).38,39 The short half- life (25-30 hours in plasma) of PCT and its absence in healthy state make it the preferred biomarker for bacterial infections. PCT levels


The outlook of the challenges from AMR

The outcome in our battle against MDROs depends on how successful we are in preserving the efficacy of our existing antibiotics for treatment of infections and the result of our search for new antibiotics. Both require resources, efforts and dedication. While preserving existing effective antibiotics demands the aforementioned One Health approach, research breakthroughs arguably provide us the only hope in winning the battle against MDROs and to ensure effective treatment of infections for the continual practice of modern medicine.

Not long ago, researchers have identified from a soil sample a new cell wall inhibitor, teixobactin, from a previously unknown Gram-negative bacterium that lives in soil but which cannot be cultured in the laboratory using standard technique.40 The researchers used the “Ichip”, an isolation chamber, in which a soil sample is diluted with agar and a single bacterial cell in a chamber is then placed in soil where the bacteria could access to nutrients and growth factors. The teixobactin identified has excellent activity against Gram-positive pathogens including MRSA, Clostridium difficile, Bacillus anthracis and Mycobacterium tuberculosis. Another recent breakthrough was reported41 on accessing hidden natural products (NP) made by bacteria not by culturing but by sequencing, bioinformatics analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Such technique has led to the discovery of malacidins, a distinctive class of antibiotics which are active against MRSA infections without selection for resistance under the laboratory conditions. These discoveries are definitely good news given the great potential for more to be discovered by using these innovative technologies. Of course, there are still tests to be done before they could become clinically useful, but at least these discoveries through our human creative and innovative minds are holding promise in the battle against AMR. Meanwhile, we must not be distracted away from the momentum in tackling the rapidly deteriorating AMR situation.



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  39. Schuetz P, Wirz Y, Sager R, Christ-Crain M, Stolz D, Tamm M, Bouadma L, Luyt CE, Wolff M, Chastre J, Tubach F, Kristoffersen KB, Burkhardt O, Welte T, Schroeder S, Nobre V, Wei L, Bucher HC, Bhatnagar N, Annane D, Reinhart K, Branche A, Damas P, Nijsten M, de Lange DW, Deliberato RO, Lima SS, Maravic-Stojkovic V, Verduri A, Cao B, Shehabi Y, Beishuizen A, Jensen JS, Corti C, Van Oers JA, Falsey AR, de Jong E, Oliveira CF, Beghe B, Briel M, Mueller B. Procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections. Cochrane Database Syst Rev [Internet]. 2017 [cited 2017 10 12];10CD007498. In: Ovid MEDLINE(R) In- Process & Other Non-Indexed Citations [Internet].

  40. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schaberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A new antibiotic kills pathogens without detectable resistance. Nature [Internet]. 2015 [cited 2015 Jan 22];517(7535):455-9. In: Ovid MEDLINE(R) [Internet].

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An overview of NMO Spectrum Disorder and the diagnostic utility of anti-NMO antibodies

An overview of NMO Spectrum Disorder and the diagnostic utility of anti-NMO antibodies


Volume 13, Issue 1 December 2017  (download full article in pdf)


Editorial note

NMOSD is an immune mediated demyelinating disease. Though its clinical presentation may overlap with multiple sclerosis, distinguishing these two entities is important in view of differences in treatment. Anti-NMO antibodies play a crucial role in the workup and diagnosis of NMOSD. In this review, Dr Elaine Au provided an overview of the NMOSD condition and the use of anti-NMO antibody assays. We welcome any feedback or suggestions. Please direct them to Dr Elaine Au (email: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr Au Yuen Ling Elaine
Associate Consultant, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital 


NMO is an idiopathic immune mediated demyelinating disease that predominantly affects optic nerves and spinal cord. The prevalence range from 0.3 to 4.4 per 100000 people, with Asian and African-American more affected than Caucasian, where multiple sclerosis is more common in the white population (1-5). The condition has been named as Devic’s disease in the past (6), which described a monophasic disorder presenting with simultaneous bilateral optic neuritis and transverse myelitis. With the availability of specific serological marker, antibodies that targeted the water channel aquaporin-4 (AQP4), the clinical and neuroimaging spectrum of NMO disease is broadened. Instead of being a monophasic disorder, NMO antibodies positive patients with recurrent attacks are not uncommonly found. Moreover, the clinical presentations are more variable than the traditional Devic disease. There is no pathognomonic clinical feature of spectrum disorder (NMOSD), though certain clinical presentations are particularly suggestive of the disorder. These include simultaneously bilateral optic neuritis, complete spinal cord syndrome and area postrema clinical syndrome.


Multiple sclerosis is an important differential diagnosis of this condition in view of the overlapping clinical features of these two conditions. In NMOSD, optic neuritis tends to be more severe, more often with simultaneous bilateral involvement or sequential in rapid succession, compare to multiple sclerosis. Complete spinal cord syndrome, with longitudinally extensive transverse myelitis in MRI, is more suggestive of NMOSD than multiple sclerosis. Differentiating NMOSD from other demyelinating disease, i.e. multiple sclerosis is important since the treatment is different. Indeed, some multiple sclerosis therapies may aggravate NMO disorders (7-10).


Diagnostic Criteria

In 2006, the serological marker was first incorporated into the revised NMO diagnostic criteria. In 2007, NMOSD was introduced to include seropositive patients who do not follow the classical monophasic bilateral optic neuritis and transverse myelitis. Lately, the International Panel for NMO Diagnosis (IPND) has further revised the diagnostic criteria (11). For anti-NMO antibodies positive cases, presenting at least one core clinical characteristic of the disease is required for diagnosis. Core clinical characteristics include optic neuritis, acute myelitis, area postrema syndrome (episode of otherwise unexplained hiccups or nausea and vomiting), acute brainstem syndrome, narcolepsy or acute diencephalic syndrome with typical Magnetic Resonance Imaging (MRI) lesions and symptomatic cerebral syndrome with typical MRI lesions. On the other hand, more stringent clinical criteria, with additional neuroimaging findings, are required in seronegative patients to fulfill the diagnostic criteria (See appendix). 

For the workup of the disease, MRI, cerebral spinal fluid (CSF) exam and serological test for anti-NMO antibodies are important. In some patients, CSF pleocytosis, usually in the form of monocytosis and lymphocytosis, are present. Increased CSF protein levels are noted in 46-75% of cases (12, 13). Nevertheless, presence of CSF oligoclonal bands is uncommon in NMOSD, and at most transient, in contrast to the presence of persistent CSF oligoclonal bands in the case of multiple sclerosis. Finally, visual evoked potentials, somatosensory evoked potentials and brainstem acoustic evoked potentials examination may also be helpful in the workup.


Clinical course and prognosis

In NMOSD syndromes affecting regions other than optic nerve and spinal cord, not uncommonly patients will relapse with more classical involvement in subsequent attacks. Majority of NMOSD patients suffer from recurrent attacks (80- 90%), less frequently monophasic attack (10-20%) (14), while gradual progressive course with neurological deterioration is very rare (15). Relapses usually occur in clusters, but unpredictable intervals. In the Mayo Clinic series, the second relapse occurred within 1 year in 60% of cases, and within 3 years in 90% of cases (16). Repeated NMO attacks not uncommonly lead to accumulation of neurological impairment. NMO is associated with more adverse outcome than MS in general(14).

NMOSD has been shown to be frequently associated with other autoimmune disorders, including lupus, Sjogren’s syndrome, etc (17-19). On contrary, NMOSD is not common in rheumatic disease patients.


Anti-NMO antibodies

Among the different investigations, NMO antibodies test is central to the workup. Anti-NMO antibodies are pathogenic. The third extracellular loop of AQP4 is the major epitope for the anti- NMO antibodies. Biopsy and autopsy tissue obtained from seropositive patients demonstrate loss of AQP4 immunoreactivity. Perivascular complement activation in actively demyelinating lesions is also happened. In the central nervous system (CNS), AQP4 is expressed at the foot processes of astrocytes, near the basement membranes, in the optic nerve, in a subpopulation of ependymal cells, in hypothalamic nuclei and in the subfornical organ (20, 21). Truncated astrocyte processes or cell loss were found in demyelinating lesions (11). In rat models, passive transfer of the antibodies leaded to the development of disease (22, 23). These pathological findings distinguish NMOSD from multiple sclerosis.

The Anti-NMO antibodies can be detected by indirect immunofluorescence staining on tissue slide using mouse cerebellum tissue section, cell- based assays, radioimmunoprecipitation assays and enzyme-linked immunosorbent assays (ELISA). Overall, cell-based assay is preferred in view of better assay sensitivity and specificity compared to other methods. Ideally, confirmatory testing with one or more techniques is suggested (11), especially in cases with atypical presentation or borderline results are obtained.

In tissue based indirect immunofluorescence test, NMO antibodies positive case is characterized by the binding to structures adjacent to microvasculature, the Virchow-Robin spaces (VRS) and pia mater (Fig.1).


Fig 1

Fig. 1. Mouse cerebellum tissue section stained with anti-NMO antibodies.


This assay allows the detection of any coexisting anti-neuronal antibodies, which may be important as differential diagnosis and workup. However, the method is observer dependent and subjective. The interpretation of antibody staining may easily be affected by non-specific background staining on the tissue. In some rare occasions, antibodies other than anti-NMO may mimic the staining pattern and lead to false positive results. Moreover, indirect tissue based immunofluorescence test has relative low sensitivity (63-64%) (24-27).

Cell-based assay utilize cell lines such as human embryonic kidney 293 (HEK293) cells or Chinese hamster ovary (CHO) cells that have been transfected with AQP4 gene expression vector, so that expressing much higher level of antigen comparing to normal tissues. Cell lines from different units may use different ratio of the two isoform of AQP4: M1 and M23 in order to obtain optimal antigen presentation. Cell-based assay can be assessed by indirect immunofluorescence staining or flow cytometry. For indirect immunofluorescence cell- based assay, slide with fixed AQP-4 gene transfected cells and non- transfected cells growing on different biochips are placed side by side for comparison. Therefore, false positivity is minimized with the inclusion of control non-transfected cells. A higher expression of antigen in the transfected cells also enhance the assay sensitivity compared to tissue-based indirect immunofluorescence testing. (Fig.2)


Fig 1

Fig.2. HEK 293 cells transfected with AQP-4 gene expression vector and stained positive with anti- NMO antibodies.


Overall, cell-based assay is the recommended assay in view of good sensitivity and specificity (mean sensitivity 76.7% in a pooled analysis; 0.1% false positivity in a multiple sclerosis cohort) (24- 27). Commercial kits for indirect immunofluorescence cell-based assay are available, which facilitate the assay setup in service laboratories. Nevertheless, indirect immunofluorescence method is semi-quantitative and observer dependent.

Protein-based assays, like ELISA and radioimmunoprecipitation assays, in general, have lower sensitivity compared to cell based assays. In addition, ELISA, in particular at low titer, may yield nonspecific results. However, these assays provide quantitative results which may potentially be used for serial monitoring (24).

Though NMO antibody testing in serum is well- established, the diagnostic role of testing the antibody in CSF is controversial. Most of the cases reported in literature are diagnosed by serum test, though there have been rare cases reported that were CSF positive but serum test negative (28, 29). When studying paired CSF and serum samples with antibody indices calculated, intrathecal production of the NMO antibody is rare (24). NMO antibodies can be present in patients few years before and after the disease presentations. Lately, there is increasing evidence that the antibody titre may reflect disease activity. Elevated antibody levels at relapse and decrease in titre after immunosuppressant treatment has been reported in literature (30-34). Therefore, serial monitoring may possibly facilitate management and medication adjustment. However, there is no general threshold value for clinical relapse and the absolute level varies with individual patients. Rising level may not predict relapse in all cases. In addition, some methodologies, like indirect immunofluorescence test, only provide semi-quantitative results, and inter-run reproducibility is another issue. Other factors including the frequency of test necessary to achieve meaningful disease status monitoring and the cost involved are also important consideration. Therefore, it remains to be determined whether the marker should be serially monitored for treatment response and disease activity monitoring.



The treatment for classical Devic’s disease presentation and relapsing NMOSD presentation is no different. High dose steroid is commonly employed as first- line of treatment in acute presentation. Plasma exchange may be considered in treatment refractory cases. Immunomodulatory treatment with interferon β, which is a treatment option in multiple sclerosis, may exacerbate NMOSD disorders. Therefore, differentiating between these two conditions is important. Options of steroid sparing immunosuppressants to consider in NMOSD include azathioprine, methotrexate, mycophenolate mofetil, rituximab, etc (14).



NMOSD is a rare but increasingly recognized condition, which present as an inflammatory and demyelinating autoimmune disorder affecting the central nervous system. With the availability of a serological marker, anti-NMO antibody, the diagnosis and differentiating from related conditions is facilitated. Timely diagnosis and treatment is important for the management of these patients.



  1. Kira J. Multiple sclerosis in the Japanese population. The Lancet Neurology. 2003;2(2):117- 27.

  2. abre P, Signate A, Olindo S, Merle H, Caparros-Lefebvre D, Bera O, et al. Role of return migration in the emergence of multiple sclerosis in the French West Indies. Brain : a journal of neurology. 2005;128(Pt 12):2899-910.

  3. Asgari N, Lillevang ST, Skejoe HP, Falah M, Stenager E, Kyvik KO. A population-based study of neuromyelitis optica in Caucasians. Neurology. 2011;76(18):1589-95.

  4. Cossburn M, Tackley G, Baker K, Ingram G, Burtonwood M, Malik G, et al. The prevalence of neuromyelitis optica in South East Wales. European journal of neurology. 2012;19(4):655-9.

  5. Mealy MA, Wingerchuk DM, Greenberg BM, Levy M. Epidemiology of neuromyelitis optica in the United States: a multicenter analysis. Archives of neurology. 2012;69(9):1176-80.

  6. Jarius S, Wildemann B. The history of neuromyelitis optica. Journal of neuroinflammation. 2013;10:8.

  7. Kimbrough DJ, Fujihara K, Jacob A, Lana- Peixoto MA, Leite MI, Levy M, et al. Treatment of Neuromyelitis Optica: Review and Recommendations. Multiple sclerosis and related disorders. 2012;1(4):180-7.

  8. Kleiter I, Hellwig K, Berthele A, Kumpfel T, Linker RA, Hartung HP, et al. Failure of natalizumab to prevent relapses in neuromyelitis optica. Archives of neurology. 2012;69(2):239-45.

  9. Min JH, Kim BJ, Lee KH. Development of extensive brain lesions following fingolimod (FTY720) treatment in a patient with neuromyelitis optica spectrum disorder. Multiple sclerosis. 2012;18(1):113-5.

  10. Papeix C, Vidal JS, de Seze J,Pierrot- Deseilligny C, Tourbah A, Stankoff B, et al. Immunosuppressive therapy is more effective than interferon in neuromyelitis optica. Multiple sclerosis. 2007;13(2):256-9.

  11. Wingerchuk DM, Banwell B, Bennett JL, Cabre P, Carroll W, Chitnis T, et al. International consensus diagnostic criteria for neuromyelitis optica spectrum disorders. Neurology. 2015;85(2):177-89.

  12. O'Riordan JI, Gallagher HL, Thompson AJ, Howard RS, Kingsley DP, Thompson EJ, et al. Clinical, CSF, and MRI findings in Devic's neuromyelitis optica. Journal of neurology, neurosurgery, and psychiatry. 1996;60(4):382-7.

  13. de Seze J, Stojkovic T, Ferriby D, Gauvrit JY, Montagne C, Mounier-Vehier F, et al. Devic's neuromyelitis optica: clinical, laboratory, MRI and outcome profile. Journal of the neurological sciences. 2002;197(1-2):57-61.

  14. Sellner J, Boggild M, Clanet M, Hintzen RQ, Illes Z, Montalban X, et al. EFNS guidelines on diagnosis and management of neuromyelitis optica. European journal of neurology. 2010;17(8):1019-32.

  15. Wingerchuk DM, Pittock SJ, Lucchinetti CF, Lennon VA, Weinshenker BG. A secondary progressive clinical course is uncommon in neuromyelitis optica. Neurology. 2007;68(8):603-5.

  16. Wingerchuk DM, Hogancamp WF, O'Brien PC, Weinshenker BG. The clinical course of neuromyelitis optica (Devic's syndrome). Neurology. 1999;53(5):1107-14.

  17. Jarius S, Jacobi C, de Seze J, Zephir H, Paul F, Franciotta D, et al. Frequency and syndrome specificity of antibodies to aquaporin-4 in neurological patients with rheumatic disorders. Multiple sclerosis. 2011;17(9):1067-73.

  18. Pittock SJ, Lennon VA, de Seze J, Vermersch P, Homburger HA, Wingerchuk DM, et al. Neuromyelitis optica and non organ-specific autoimmunity. Archives of neurology. 2008;65(1):78-83.

  19. Wandinger KP, Stangel M, Witte T, Venables P, Charles P, Jarius S, et al. Autoantibodies against aquaporin-4 in patients with neuropsychiatric systemic lupus erythematosus and primary Sjogren's syndrome. Arthritis and rheumatism. 2010;62(4):1198-200.

  20. Graber DJ, Levy M, Kerr D, Wade WF. Neuromyelitis optica pathogenesis and aquaporin 4. Journal of neuroinflammation. 2008;5:22.

  21. Tait MJ, Saadoun S, Bell BA, Papadopoulos MC. Water movements in the brain: role of aquaporins. Trends in neurosciences. 2008;31(1):37-43.

  22. Kinoshita M, Nakatsuji Y, Moriya M, Okuno T, Kumanogoh A, Nakano M, et al. Astrocytic necrosis is induced by anti-aquaporin-4 antibody- positive serum. Neuroreport. 2009;20(5):508-12.

  23. Kinoshita M, Nakatsuji Y, Kimura T, Moriya M, Takata K, Okuno T, et al. Neuromyelitis optica: Passive transfer to rats by human immunoglobulin. Biochemical and biophysical research communications. 2009;386(4):623-7.

  24. Jarius S, Wildemann B. Aquaporin-4 antibodies (NMO-IgG) as a serological marker of neuromyelitis optica: a critical review of the literature. Brain pathology. 2013;23(6):661-83.

  25. Waters PJ, McKeon A, Leite MI, Rajasekharan S, Lennon VA, Villalobos A, et al. Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays. Neurology. 2012;78(9):665-71; discussion 9.

  26. Sato DK, Nakashima I, Takahashi T, Misu T, Waters P, Kuroda H, et al. Aquaporin-4 antibody- positive cases beyond current diagnostic criteria for NMO spectrum disorders. Neurology. 2013;80(24):2210-6.

  27. Pittock SJ, Lennon VA, Bakshi N, Shen L, McKeon A, Quach H, et al. Seroprevalence of aquaporin-4-IgG in a northern California population representative cohort of multiple sclerosis. JAMA neurology. 2014;71(11):1433-6.

  28. Klawiter EC, Alvarez E, 3rd, Xu J, Paciorkowski AR, Zhu L, Parks BJ, et al. NMO-IgG detected in CSF in seronegative neuromyelitis optica. Neurology. 2009;72(12):1101-3.

  29. Long Y, Qiu W, Lu Z, Peng F, Hu X. Clinical features of Chinese patients with multiple sclerosis with aquaporin-4 antibodies in cerebrospinal fluid but not serum. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 2013;20(2):233-7.

  30. Jarius S, Aboul-Enein F, Waters P, Kuenz B, Hauser A, Berger T, et al. Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain : a journal of neurology. 2008;131(Pt 11):3072-80.

  31. Jarius S, Franciotta D, Paul F, Ruprecht K, Bergamaschi R, Rommer PS, et al. Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance. Journal of neuroinflammation. 2010;7:52.

  32. Jarius S, Franciotta D, Paul F, Bergamaschi R, Rommer PS, Ruprecht K, et al. Testing for antibodies to human aquaporin-4 by ELISA: sensitivity, specificity, and direct comparison with immunohistochemistry. Journal of the neurological sciences. 2012;320(1-2):32-7.

  33. Kim W, Lee JE, Li XF, Kim SH, Han BG, Lee BI, et al. Quantitative measurement of anti- aquaporin-4 antibodies by enzyme-linked immunosorbent assay using purified recombinant human aquaporin-4. Multiple sclerosis. 2012;18(5):578-86.

  34. Takahashi T, Fujihara K, Nakashima I, Misu T, Miyazawa I, Nakamura M, et al. Anti-aquaporin- 4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain : a journal of neurology. 2007;130(Pt 5):1235-43.



NMOSD diagnostic criteria for adult patients

Diagnostic criteria for NMOSD with NMO-IgG

  1. At least 1 core clinical characteristic

  2. Positive test for NMO-IgG using best available detection method (cell-based assay strongly recommended)

  3. Exclusion of alternative diagnoses

Diagnostic criteria for NMOSD without NMO-IgG or NMOSD with unknown NMO-IgG status

  1. At least 2 core clinical characteristics occurring as a result of one or more clinical attacks and meeting all of the following requirements:

    - At least 1 core clinical characteristic must be optic neuritis , acute myelitis with LETM, or area postrema syndrome

    - Dissemination in space (2 or more different core clinical characteristics)

    - Fulfillment of additional MRI requirements, as applicable

  2. Negative tests for NMO-IgG using best available detection method, or testing unavailable

  3. Exclusion of alternative diagnoses

Core clinical characteristics

  1. Optic neuritis

  2. Acute myelitis

  3. Area postrema syndrome: episode of otherwise unexplained hiccups or nausea and vomiting

  4. Acute brainstem syndrome

  5. Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions

  6. Symptomatic cerebral syndrome with NMOSD-typical brain lesions

Additional MRI requirements for NMOSD without NMO-IgG and NMOSD with unknown NMO-Ig status

  1. Acute optic neuritis: require brain MRI showing (a) normal findings or only nonspecific white matter lesions, OR (b) optic nerve MRI with T2-hyperintense lesion or T1-weighted gadolinium-enhancing lesion extending over >1/2 optic nerve length or involving optic chiasm

  2. Acute myelitis: requires associated intramedullary MRI lesion extending over >=3 contiguous segments (LETM) OR >=3 contiguous segments of focal spinal cord atrophy in patients with history compatible with acute myelitis

  3. Area postrema syndrome: requires associated dorsal medulla/ area postrema lesions 4. Acute brainstem syndrome: requires associated periependymal brainstem lesions

Neurology 2015;85:177-189


Clinical features and laboratory findings atypical for NMOSD, that need to consider alternative diagnoses

  1. Progressive overall clinical course (neurologic deterioration unrelated to attacks: Consider MS)

  2. Atypical time to attack nadir: less than 4 hours (consider cord ischemia/ infarction); continual worsening for more than 4 weeks from attack onset (consider sarcoidosis or neoplasm)

  3. Partial transverse myelitis, especially when not associated with LETM MRI lesion (consider MS)

  4. Presence of CSF oligoclonal bands (oligoclonal bands occur in < 20% of cases of NMO vs > 80% of MS

Comorbidities associated with neurologic syndromes that mimic NMOSD

  1. Sarcoidosis, established or suggestive clinical, radiologic or laboratory findings thereof (e.g. mediastinal adenopathy, fever and night sweats, elevated serum angiotensin converting enzyme or interleukin-2 receptor level)

  2. Cancer, established or with suggestive clinical, radiologic or laboratory findings thereof; consider lymphoma or paraneoplastic disease ( e.g. collapsing response mediator protein-5 associated optic neuropathy and myelopathy or anti-Ma- associated diencephalic syndrome)

  3. Chronic infection, established or with suggestive clinical radiologic, or laboratory findings thereof ( e.g. HIV, syphilis)

Neurology 2015;85:177-189

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Molecular alterations of gastrointestinal stromal tumor - Prognostic and therapeutic implications

Molecular alterations of gastrointestinal stromal tumor - Prognostic and therapeutic implications 


Volume 12, Issue 2 August 2017  (download full article in pdf)


Editorial note:

Gastrointestinal stromal tumor is the commonest mesenchymal tumor in the digestive system. It is a genetically heterogeneous disease with various mutations apart from classical activation mutations in KIT  and PDGFRA genes. In the topical update, Dr. Anthony Chan provided an overview of molecular alterations of gastrointestinal stromal tumor with emphasis on their prognostic and therapeutic significance. We welcome any feedback or suggestions. Please direct them to Dr. Anthony Chan (e-mail: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists. 

Dr. Anthony W.H. Chan
Clinical Associate Professor
Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong 


The gist of GIST

Gastrointestinal stromal tumor (GIST) is a rare tumor with the annual incidence rate of 10- 15/1,000,000, but it is the commonest mesenchymal tumor in the digestive system. It affects both sexes equally and presents at any age from children to elderly with the median age of mid 60s. Stomach (55.6%) is the most frequent primary tumor site followed by small intestine (31.8%), large intestine (6.0%) and esophagus (0.7%). Other uncommon primary sites, such as omentum, mesentery and liver, accounts for 5.5% of all GISTs.(1) Important milestones of GIST in diagnostic, prognostic and therapeutic aspects are briefly summarized in this section. 

In the past, GIST was regarded as leiomyoma, leiomyoblastoma or leiomyosarcoma before the era of wide availability of immunohistochemistry. In 1983, Mazur and Clark first applied the term "stromal tumor" to describe a group of gastric mesenchymal tumor lacking ultrastructural features of smooth muscle or schwann cells.(2) In 1989, a short-lived term, gastrointestinal autonomic nerve tumor (GANT), was used to describe a small subset of GIST featured by small intestinal location, epithelioid appearance and focal immunoreactivity towards neural markers (S100, neurofilament and synaptophysin).(3) In 1995, CD34 was found to be the first useful diagnostic immunohistochemical marker to differentiate GIST from leiomyoma and schwannoma although only 60-70% of all GISTs are immunoreactive to CD34.(4) In 1998, the hallmark constitutive activation mutation of KIT gene and overexpression of KIT/CD117 protein in GIST were discovered by Hirota et al.(5) This finding also suggested that GIST may be originated from interstitial cells of Cajal, pacemaker cells of intestine, which express KIT and CD34. However, activation mutation of KIT gene and overexpression of KIT are not consistently correlated. A subset of KIT positive GISTs was found to lack KIT mutation and this observation led to the subsequent discovery of gain-of-function mutation of platelet-derived growth factor receptor alpha (PDGFRA) gene in 2003.(6, 7) KIT and PDGFRA mutations are mutually exclusive. About 5-10% of GISTs, particularly those with PDGFRA mutation do not express KIT. In 2004, West et al. identified a novel gene, DOG1 (discovered on GIST-1), through cDNA microarray, and showed DOG1 protein was highly expressed in GISTs (97.8%), including those KIT negative GISTs.(8) KIT and/or DOG1 become crucial diagnostic immunohistochemical markers for GIST. A small subgroup of GISTs with immunoreactivity of KIT/DOG1 lack neither KIT or PDGFRA mutation was first designated as wild-type GISTs in the same year.(9) Wild-type GISTs are later shown to be a heterogeneous group with various mutations.(10- 13)

Prognosis of patients with GIST is shown to be correlated with tumor size and mitosis. The first consensus risk stratification was proposed by investigators in National Institutes of Health (NIH) in 2002 (Table 1).(14) Anatomical location of GIST is also an important prognostic factor and firstly integrated to the Armed Forces Institute of Pathology system in 2006 (Table 2)(15). Gastric GIST behaves more indolent than small and large bowel GIST with similar size and mitosis. Tumor rupture is an additional prognosticator for GIST patients and incorporated into the modified NIH system in 2008 (Table 3).(16) Finally, the most widely adopted tumor staging system, American Joint Committee on Cancer (AJCC), include GIST risk stratification composed of tumor size, mitosis, anatomical location, nodal and distant metastases in the 7th edition in 2010, which remains unchanged in the recently released 8th edition (Table 4 and 5).

Surgical resection remains the mainstay of curative therapy for GIST but a substantial portion of GIST patients present in advanced stage beyond surgical intervention. Imatinib, a multi-targeted tyrosine kinase inhibitor specific for c-abl, c-kit and PDGFR, was first used in a patient with metastatic GIST in 2001.(17) The dramatic clinical response from this patient and the subsequent successful phase II clinical trial in 2002 secured the first-line role of imatinib for patients with inoperable GIST and pioneered molecular targeted therapy for sarcoma.(18) Primary and acquired resistance to imatinib among GIST patients led to development of newer targeted agents. Two hallmark phase III randomized controlled trials on sunitinib (NCT00075218) and regorafenib (NCT01271712) for GIST were completed in 2006 and 2013, respectively.(19, 20) Sunitinib and regorafenib are indicated for patients with advanced GIST resistant or intolerant to imatinib. 


Table 1: NIH risk stratification for GIST (14) 




Very low risk <2 ≤5
Low risk 2-5 ≤5
Intermediate risk <5 6-10
5-10 ≤5
High risk >5 >5
>10 Any
Any >10


Table 2: AFIP risk stratification for GIST (15) 

1 ≤2 ≤5 None None None None
2 >2-5 ≤5 Very low Low Low Low
3a >5-10 ≤5 Low Moderate - -
3b >10 ≤5 Moderate High High High
4 ≤2 >5 None High - High
5 >2-5 >5 Moderate High High High
6a >5-10 >5 High High - -
6b >10 >5 High High High High


Table 3: Modified NIH risk stratification for GIST (16) 

Very low risk ≤2 ≤5 Any
Low risk >2-5 ≤5 Any
Intermediate Risk >2-5 >5 Gastric
≤5 6-10 Any
>5-10 ≤5 Gastric
High risk >5 >5 Any
>10 Any Any
Any >10 Any
Any Any Tumor rupture
>5-10 ≤5 Non-gastric


Table 4: AJCC staging system for gastric and omental GIST 

IA ≤5 0 0 ≤5
IB >5-10 0 0 ≤5
II ≤5 0 0 >5
>10 0 0 ≤5
IIIA >5-10 0 0 >5
IIIB >10 0 0 >5
IV Any 1 0 Any
Any Any 1 Any


Table 5: AJCC staging system for small/large bowel, esophageal, mesenteric and peritoneal GIST 

I ≤5 0 0 ≤5
II >5-10 0 0 ≤5
IIIA ≤2 0 0 >5
>10 0 0 ≤5
IIIB >2 0 0 >5
IV Any 1 0 Any
Any Any 1 Any



Mutational landscape of GIST

KIT and PDGFRA mutations are major driver mutations in GIST tumorigenesis. Both genes encode type III receptor tyrosine kinases with similar structures: extracellular ligand binding domain and dimerization domain, a transmembrane sequence, a juxtamembrane domain and intracellular kinase domain (Figure 1). Binding of corresponding ligands, stem cell factor and PDGFA, to c-kit and PDGFRA receptor, respectively, dimerizes and activates receptor tyrosine kinases. In GIST, activation mutations in KIT and PDGFRA lead to uncontrolled ligand- independent receptor activation. Mutation hotspots of KIT gene are located at exons 9, 11, 13 and 17, whereas those of PDGFRA gene are situated at exons 12, 14 and 18. Mutation of extracellular domain of KIT encoded by exon 9 facilitate receptor dimerization. Mutations in the juxtamembrane domain, which is encoded by exon 11 of KIT and exon 12 of PDGFRA, allow dimerization of receptor without binding of ligands. Mutations of ATP binding region of kinase domain (encoded by exon 13 of KIT and exon 14 of PDGFRA) enhance kinase activity, while mutations of activation loop (encoded by exon 17 of KIT and exon 18 of PDGFRA) promote active conformation of kinase.(21) Table 6 and Figure 2 summarize the mutational landscape of GIST based on the data from population-based studies and clinical trials.(22-29) Frequencies of PDGFRA mutations are significantly lower among patients in clinical trials (mean 1.7%) than those in population-based studies (mean 14.9%) because GIST patients with PDGFRA mutations are associated with better prognosis and earlier stage and hence do not require systemic therapy.(9, 22, 23, 29)

KIT mutation accounts for 71.5% (64.8-89.1%) of mutations in GISTs.(24, 25, 27-29) Exon 11 mutation is the commonest mutation (61.1%, range: 56.1-77.1%). Deletion, substitution and duplication contribute to 23-28%, 2-20% and 2-7%, respectively. Deletion in exon 11 is associated with younger age, larger tumor size, higher mitotic count and poor prognosis, whereas duplication is associated with female and stomach predilection and better prognosis. Exon 9 mutation is found in 7.1-10.9% of GISTs, particularly in those arising from small and large intestine, and associated with poor prognosis. Exon 13 and exon 17 are rare mutation hotspots (<1-2%) in GISTs, which are almost exclusively spindle in morphology and more frequently developed in small intestine. GISTs with exon 13 and 17 mutants are associated good and intermediate prognosis, respectively.

PDGFRA mutation accounts for 14.9% (4.7-21.1%) of mutations in GISTs.(24, 25, 27-29) About 30- 40% of GISTs without immunoreactivity of KIT/CD117 harbour PDGFRA mutation. GISTs with PDGFRA mutation generally show predilection to gastric location (>90%) and epithelioid/mixed morphology, and favourable prognosis (except non-D842V exon 18 mutation).

Wild-type GIST, which express immunoreactivity of KIT/DOG1 but lack neither KIT or PDGFRA mutation, contributes to 13-18% of adult GISTs and 85% of pediatric GIST.(10-12) As previously mentioned, it is a genetically heterogeneous group (Figure 3). Wild-type GIST can be further stratified by using succinate dehydrogenase B (SDHB) immunohistochemistry and familial syndromes. On one hand, SDHB deficient wild-type GISTs accounts for about 5% of all GISTs, and can be sporadic or related to Carney triad and Carney- Stratakis syndrome. Carney triad is a constellation of GIST, paraganglioma and pulmonary chondroma with undetermined germline mutation, whereas Carney-Stratakis syndrome is an autosomal dominant disease with dyad of GIST and paraganglioma, and germline mutations in SDHB, SDHC or SDHD genes.(30) SDHB deficient wild-type GISTs are featured by female predominance (except for Carney-Stratakis syndrome), exclusive location in stomach, multifocality, epithelioid/mixed morphology, unpredictable clinical outcome by histology, indolent clinical course despite frequent nodal metastasis, and mutation id SDH subunits (except for Carney triad). On the other hand, SDHB proficient wild-type GISTs make up 10.5% of all GISTs, and are either sporadic (9%) or syndromic (1.5%). Syndromic SDHB proficient wild-type GISTs are associated with neurofibromatosis type 1, absence of sex/age predilection, small intestine in location, multifocality, spindle morphology, and favorable prognosis. Sporadic SDHB proficient wild-type GISTs can be further classified according to BRAF mutation. Sporadic SDHB proficient wild-type GISTs with BRAF mutation usually occur in 6th decade of age and small intestine with spindle morphology. Prognosis of this subgroup is inconclusive.(10, 29, 31, 32) Sporadic SDHB proficient wild-type GISTs without BRAF mutation are also known as quadruple wild-type GISTs without any mutation in KIT, PDGFRA, SDH and genes in RAS pathway (BRAF/NF1).(12, 13) They represent the commonest subgroup (7%) of wild-type GISTs and a genetically heterogeneous subgroup harboring ETV6-NTRK3 translocation, FGFR1-TACC1 translocation, mutation of MEN1 and MAX, and overexpression of COL22A1 and CALCRL.(12, 29, 33, 34) Due to complex genetic heterogeneity, clinicopathological features of this subgroup have not been well characterized.


Figure 1: Schematic diagram of the structures of KIT and PDGFRA receptor tyrosine kinases 

Fig 1


Figure 2: Mutational landscape of GIST 

Fig 2


Figure 3: Classification of wild-type GIST 

Fig 3


Table 6: Mutational landscape of GIST

Study Region n KIT exon PDGFRA exon Wild type
9 11 13 17 12 14h 18
Wozniak 2012 (24) Poland 427 7.3% 61.1% 0.5% 0.5% 0.2% 0.7% 11.9% 17.8%
Wozniak 2014 (28) Europe 1056 7.4% 61.4% 1.8% 0.6% 0.9% 0.3% 12.8% 14.9%
Künstlinger 2013 (25) Germany 1366 9.2% 59.3% 1.8% 0.8% 1.8% 0.6% 13.8% 12.7%
Wang 2014 (27) China 275 10.9% 77.1% 1.1% 0.0% 1.1% 0.0% 3.6% 6.2%
Rossi 2015 (29) Italy 451 7.1% 56.1% 0.9% 0.7% 2.2% 1.6% 17.3% 14.2%
ACOSOG Z9001 (26) 507 6.9% 67.3% 1.8% 0.2% NA NA NA 12.8%
CALGB 150105 (23) 378 8.2% 72.8% 0.8% 1.1% 0.0% 0.0% 1.6% 15.3%
EORTC 62005 (22) 377 15.4% 65.8% 1.6% 0.8% 0.8% 0.0% 1.9% 13.8%



Clinical implications of mutations in GIST

Different mutations in GIST have their own characteristic prognostic and therapeutic implications. Prognostic significance of individual mutations have been described by various investigators and briefly mentioned in the previous section. Rossi et al. recently systemically analyzed the prognostic impact of mutations among 451 patients with primary localized treatment-naive GISTs.(29) By multivariable Cox regression, mutational status was an independent prognosticator in addition to patient's age, tumor location, tumor size and mitotic count. Three molecular risk groups with prognostic significance were identified: Group 1 with the most favorable outcome is composed of mutations in KIT exon 13, PDGFRA exon 12 and BRAF; Group 2 with the intermediate outcome (hazard ratio 3.06) consists of KIT/PDGFRA/BRAF triple negative, and mutations in KIT exon 17, PDGFA exon 14 and 18 (D842V); and Group 3 with the most unfavorable outcome comprises mutations in KIT exon 9 and 11, and PDGFRA exon 18 (non-D842V).

Clinical response toward imatinib among GIST patients is closely related to tumor genotype. In a phase III clinical trial (SWOG S0033/CALGB 150105), the investigators demonstrated that patients with KIT exon 11 mutation (complete response [CR]/partial response [PR] 71.7%) had better response to imatinib than those with KIT exon 9 mutation (CR/PR 44.4%) and wild-type KIT (CR/PR 44.6%).(23) They also showed that doubling the dose of imatinib (from 400 mg to 800 mg) improved response rates for patients with exon 9-mutant tumors (CR/PR 17% vs. 67%). Double dose of imatinib did not offer any better response rate among patients with exon 11 mutant or wild- type KIT. A subsequent meta-analysis of 1,640 patients with advanced GIST receiving imatinib confirmed that double dose of imatinib improved progression-free survival and objective response rate, but not overall survival, among patients with KIT exon 9-mutant GIST.(35) PDGFA exon 18 (D842V) mutation and KIT/PDGFRA wild-type are responsible for primary resistance to imatinib.(36) Among patients with advanced GIST receiving imatinib, a substantial proportion of initial responders will develop acquired resistance. Secondary mutations in exon 11 (L576P and V559A), exon 13 (V654A), exon 14 (T670I), exon 17 and exon 18 (A829P) of KIT, and exon 18 of PDGFRA are related to acquired resistance to imatinib.(36)

Clinical response to sunitinib, the second line targeted therapy after imatinib failure, is also considerably affected by primary and acquired mutations of KIT. Patients with primary KIT exon 9 mutation or wild-type KIT had better overall and progression-free survival than those with KIT exon 11 mutation, whereas patients with acquired KIT exons 13 or 14 mutations had better outcome than those with KIT exon 17 or 18 mutations.(37) Similarly, clinical response to regorafenib, the third line therapy after imatinib and sunitinib failure, is significantly influenced by tumor genotype. Regorafenib provided better clinical outcome among patients with primary KIT exon 11 mutation and SDHB deficient GIST, (38) as well as those with secondary mutation of KIT exon 17, which are resistant to both imatinib and sunitinib.(39)



GIST is a genetically heterogeneous tumor. Genotypes and phenotypes are closely interrelated. Specific mutations have their characteristic clinicopathological features, prognostication and therapeutic implications. Genetic analyses KIT and PDGFRA are highly recommended especially among patients with advanced diseases undergoing targeted therapy. Wild-type GISTs are recommended to be further analysed by SDHB immunohistochemistry and BRAF mutation test. 



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9. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol. 2004;22(18):3813-25.

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11. Janeway KA, Kim SY, Lodish M, Nose V, Rustin P, Gaal J, et al. Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations. Proc Natl Acad Sci U S A. 2011;108(1):314-8.

12. Nannini M, Astolfi A, Urbini M, Indio V, Santini D, Heinrich MC, et al. Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST). BMC Cancer. 2014;14:685.

13. Pantaleo MA, Urbini M, Indio V, Ravegnini G, Nannini M, De Luca M, et al. Genome-Wide Analysis Identifies MEN1 and MAX Mutations and a Neuroendocrine-Like Molecular Heterogeneity in Quadruple WT GIST. Mol Cancer Res. 2017;15(5):553-62.

14. Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ, et al. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol. 2002;33(5):459-65.

15. Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23(2):70-83.

16. Joensuu H. Risk stratification of patients diagnosed with gastrointestinal stromal tumor. Hum Pathol. 2008;39(10):1411-9.

17. Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med. 2001;344(14):1052-6.

18. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347(7):472-80.

19. Demetri GD, van Oosterom AT, Garrett CR, Blackstein ME, Shah MH, Verweij J, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet. 2006;368(9544):1329-38. 

20. Demetri GD, Reichardt P, Kang YK, Blay JY, Rutkowski P, Gelderblom H, et al. Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381(9863):295-302.

21. Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer. 2011;11(12):865-78.

22. Debiec-Rychter M, Sciot R, Le Cesne A, Schlemmer M, Hohenberger P, van Oosterom AT, et al. KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours. Eur J Cancer. 2006;42(8):1093- 103.

23. Heinrich MC, Owzar K, Corless CL, Hollis D, Borden EC, Fletcher CD, et al. Correlation of kinase genotype and clinical outcome in the North American Intergroup Phase III Trial of imatinib mesylate for treatment of advanced gastrointestinal stromal tumor: CALGB 150105 Study by Cancer and Leukemia Group B and Southwest Oncology Group. J Clin Oncol. 2008;26(33):5360-7.

24. Wozniak A, Rutkowski P, Piskorz A, Ciwoniuk M, Osuch C, Bylina E, et al. Prognostic value of KIT/PDGFRA mutations in gastrointestinal stromal tumours (GIST): Polish Clinical GIST Registry experience. Ann Oncol. 2012;23(2):353-60.

25. Kunstlinger H, Huss S, Merkelbach-Bruse S, Binot E, Kleine MA, Loeser H, et al. Gastrointestinal stromal tumors with KIT exon 9 mutations: Update on genotype-phenotype correlation and validation of a high-resolution melting assay for mutational testing. Am J Surg Pathol. 2013;37(11):1648-59.

26. Corless CL, Ballman KV, Antonescu CR, Kolesnikova V, Maki RG, Pisters PW, et al. Pathologic and molecular features correlate with long-term outcome after adjuvant therapy of resected primary GI stromal tumor: the ACOSOG Z9001 trial. J Clin Oncol. 2014;32(15):1563-70.

27. Wang M, Xu J, Zhao W, Tu L, Qiu W, Wang C, et al. Prognostic value of mutational characteristics in gastrointestinal stromal tumors: a single-center experience in 275 cases. Med Oncol. 2014;31(1):819.

28. Wozniak A, Rutkowski P, Schoffski P, Ray-Coquard I, Hostein I, Schildhaus HU, et al. Tumor genotype is an independent prognostic factor in primary gastrointestinal stromal tumors of gastric origin: a european multicenter analysis based on ConticaGIST. Clin Cancer Res. 2014;20(23):6105-16.

29. Rossi S, Gasparotto D, Miceli R, Toffolatti L, Gallina G, Scaramel E, et al. KIT, PDGFRA, and BRAF mutational spectrum impacts on the natural history of imatinib-naive localized GIST: a population-based study. Am J Surg Pathol. 2015;39(7):922-30.

30. Stratakis CA, Carney JA. The triad of paragangliomas, gastric stromal tumours and pulmonary chondromas (Carney triad), and the dyad of paragangliomas and gastric stromal sarcomas (Carney-Stratakis syndrome): molecular genetics and clinical implications. J Intern Med. 2009;266(1):43-52.

31. Hostein I, Faur N, Primois C, Boury F, Denard J, Emile JF, et al. BRAF mutation status in gastrointestinal stromal tumors. Am J Clin Pathol. 2010;133(1):141-8.

32. Huss S, Pasternack H, Ihle MA, Merkelbach-Bruse S, Heitkotter B, Hartmann W, et al. Clinicopathological and molecular features of a large cohort of gastrointestinal stromal tumors (GISTs) and review of the literature: BRAF mutations in KIT/PDGFRA wild-type GISTs are rare events. Hum Pathol. 2017;62:206-14.

33. Brenca M, Rossi S, Polano M, Gasparotto D, Zanatta L, Racanelli D, et al. Transcriptome sequencing identifies ETV6-NTRK3 as a gene fusion involved in GIST. J Pathol. 2016;238(4):543-9.

34. Shi E, Chmielecki J, Tang CM, Wang K, Heinrich MC, Kang G, et al. FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors. J Transl Med. 2016;14(1):339.

35. Gastrointestinal Stromal Tumor Meta- Analysis G. Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: a meta-analysis of 1,640 patients. J Clin Oncol. 2010;28(7):1247-53.

36. Sankhala KK. Clinical development landscape in GIST: from novel agents that target accessory pathways to revisiting non-targeted therapies. Expert Opin Investig Drugs. 2017;26(4):427-43.

37. Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D, et al. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. J Clin Oncol. 2008;26(33):5352-9.

38. Ben-Ami E, Barysauskas CM, von Mehren M, Heinrich MC, Corless CL, Butrynski JE, et al. Long-term follow-up results of the multicenter phase II trial of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of standard tyrosine kinase inhibitor therapy. Ann Oncol. 2016;27(9):1794-9.

39. Yeh CN, Chen MH, Chen YY, Yang CY, Yen CC, Tzen CY, et al. A phase II trial of regorafenib in patients with metastatic and/or a unresectable gastrointestinal stromal tumor harboring secondary mutations of exon 17. Oncotarget. 2017;8(27):44121-30. 

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Laboratory testing for Direct Oral Anticoagulants (DOACs): Are we ready?

Laboratory testing for Direct Oral Anticoagulants (DOACs):

Are we ready?


Volume 12, Issue 1 January 2017  (download full article in pdf)


Editorial note:

In this topical update, Dr Rock Leung reviews the testing strategy and quality assurance issues on laboratory testing for direct oral anticoagulant (DOACs). We welcome any feedback or suggestions. Please direct them to Dr Rock Leung (e-mail: of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.


Dr. Rock LEUNG
Associate Consultant, Division of Haematology, Department of Pathology and Clinical Biochemistry
Queen Mary Hospital, Hong Kong



DOACs Direct oral anticoagulants
FIIa Thrombin
PK Pharmacokinetics
PD Pharmacodynamics
PT Prothromhin time
APTT Activated partial thromboplastin time
TT Thrombin time
dTT Diluted thrombin time
ECT Ecarin clotting time
DRVVT Diluted Russell’s viper venom time



The newly available Food and Drug Administration (FDA) -approved oral anticoagulants, namely dabigatran extexilate, rivaroxaban, apixaban and edoxaban, have been more commonly used nowadays for treatment and prophylaxis of venous thromboembolism, as well as for prevention of stroke in non-valvular atrial fibrillation. This new class of anticoagulants has been referred as novel oral anticoagulants (NOACs), target-specific oral anticoagulants (TOACs), or direct oral anticoagulants (DOACs). For the sake of standardization, the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SCC) for the control of anticoagulation recommends the term DOACs. DOACs have been shown to be at least as effective as warfarin in various clinical trials. Moreover, there was reduced incidence of intracranial haemorrhage reported in some studies when compared with warfarin [1]. Unlike warfarin, DOACs do not need routine therapeutic monitoring given their predictable pharmacokinetics (PK), pharmacodynamics (PD) and wide therapeutic windows. There are, however, clinical conditions that measurement of anticoagulation activity of DOACs is necessary or potentially useful, e.g. before invasive procedures, during adverse events like break-through bleeding or thrombosis, and pre- and post-administration of reversal therapy for patients with DOACs overdose. Thus, there is a role for laboratory, by testing for DOACs, to help clinicians on patient management. In addition, it is the responsibility of the laboratory to acknowledge the interferences of DOACs on conventional and special coagulation tests as part of the laboratory quality assurance in the era of gaining popularity of DOACs usage.


Mechanisms of actions of DOACs

In contrast with heparin that can only inhibit free protease, DOACs are rapidly-acting, target-specific anticoagulants that inhibit both the free and bound activated serine protease [2]. The fact that DOACs can inactivate bound serine protease explains their more robust action than warfarin or heparin. Dabigatran is a direct thrombin (IIa) inhibitor while rivaroxaban, apixaban and edoxaban are direct inhibitors of activated factor X (Xa). Most of the DOACs are cleared by liver and kidney, with the exception of dabigatran being almost exclusively excreted by kidney. DOACs reach peak plasma levels within approximately two hours and plasma trough levels within 12 hours or 24 hours depending on their frequency of administration [3]. The DOACs can be withhold a few days before elective surgery or invasive procedures due to their short half-lives and favourable pharmacokinetics.


To test or not to test?

Routine monitoring of DOACs is not required. Testing on patients on DOACs is generally indicated in certain clinical circumstances, including acute bleeding, suspected DOACs overdose, drug interaction, in patients with impaired renal function, before surgery or invasive procedure in patients who have taken the drug beyond 24 hours and with creatinine clearance of <50 mL/min or with extreme body weight [4]. Recently, more pharmacokinetics and pharmacodynamics data on indications of clinical testing came up. Currently it is recommended that checking of drug-specific peak and trough levels for DOACs should be performed for patients with body mass index (BMI) of >40 kg m^2 or weighted over 120 kg [5]. There is currently no consensus on when to test for DOACs activities when these drugs are to be used in women with childbearing potential. One should however note that animals studies have shown teratogenic effect of dabigatran, edoxaban and rivaroxaban, these drugs were assigned by the FDA as pregnancy category C, reflecting their potential teratogenicity. Whereas no teratogenicity has been demonstrated in animals for apixaban as of today, it was categorized as pregnancy catergory B by FDA [6]. On the other hand, the use of DOACs is considered an off-label clinical application for paediatric thromboemobolic diseases [7]. It is not unreasonable to obtain information about anticoagulation activity by laboratory assay for this special group of patients, as in the case of low-molecular-weight heparin (LMWH) usage in select paediatric patients.

Given the predictable pharmacokinetics of DOACs, it was proposed that a pharmacokinetic strategy by stopping the drug for a time frame adequate for washout of drug effect is safe before surgery or invasive procedures. This approach can only be applied for planned surgery or invasive procedures, with available information regarding patient’s renal function as well as the dose and timing of the last DOAC administration. For emergent or unplanned procedures in patients with renal insufficiency or unplanned procedures when the timing of the last DOAC administration is uncertain, measurement of residual drug level will be valuable to assist clinical decisions, including the assessment of bleeding risk and the need for antidote for prompt reversal of DOAC effect before surgery. In life-threatening bleeding associated with the use of DOACs, the measurement of drug level can supplement clinical information to determine whether the bleeding is contributed by the anticoagulation effect of DOACs and whether the administration of DOAC-specific antidotes is required. If antidote is applied, laboratory test can monitor the extent of reversal.


What tests to do?

The ideal test for DOACs shall be accurate, readily available on a 24-hour basis in order to accommodate emergency clinical situations, and with a reasonably short turnaround time (TAT).
Gold standard method using ultra-performance liquid chromatography – tandem mass spectrometry (UPLI-MS/MS) provides the most accurate information about the drug levels for patients on DOACs. However, the test is not readily available in most of the laboratories.

Routine coagulation screening tests, i.e., prothrombin time (PT), activated partial thromboplastin time (APTT) or thrombin time (TT), have been suggested as screening tests for DOACs. For routine coagulation screening tests to be useful and suitable for testing for DOACs, linearity and adequacy of test response to increasing dosage and amenability to standardization are prerequisites [8]. For dabigatran, TT is readily available in most laboratories and prolongation of clotting time is linearly and dose-dependently related to dabigatran concentrations. However, responsiveness is excessive. Therefore, a normal TT should rule out a dabigatran anticoagulant effect but the degree of prolongation poorly reflects drug concentration. Dilute TT (dTT), i.e., testing of TT on diluted plasma, is adequately responsive to dabigatran and suitable for assessment of dabigatran activity. Ecarin clotting time (ECT), using ecarin for the conversion of FII to meizothrombin, to assess anticoagulant effect of dabigatran was also shown to have satisfactory linearity and responsiveness to increasing dabigatran concentrations. APTT, though being demonstrated to have satisfactory responsiveness to dabigatran, lacks linearity upon increasing drug concentration and there is significant inter-reagent variability [9]. PT is insensitive to dabigatran and not suitable for testing.

Rivaroxaban prolongs the PT in a concentration-dependent manner, but the correlation is generally weak and became weaker with increasing drug concentration. Significant reagent-dependent differences in assay sensitivity are noted in multiple studies, limiting its use for assessment of rivaroxaban activity if the in-house thromboplastin reagent for routine coagulation screening is insensitive to rivaroxaban [10]. APTT is insensitive to rivaroxaban and shall not be used for assessment of rivaroxaban activity. For apixaban, both PT and APTT are insensitive to increasing drug concentrations and for edoxaban, PT performance is similar to that observed for rivaroxaban and APTT is insensitive [11].
Therefore, routine coagulation screening tests PT, APTT and TT cannot provide a reliable measurement of DOAC anticoagulant effect in most circumstances. One exception being a normal TT excludes significant residual effect of dabigatran in patients. Moreover, PT and APTT are either insensitive or show variably sensitivity to the on-therapy range of DOACs and limit their use in determining whether the drug concentration is in subtherapeutic or supratherapeutic ranges. Furthermore, these coagulation screening tests are potentially affected by the presence of lupus anticoagulants and conditions resulting in factor deficiency as in liver disease or dilutional coagulopathy. Thus, the sensitivity & specificity in reflecting the anticoagulant effect of DOACs is limited.

Anti-Xa assay is a chromogenic assay based on the measurement of residual FXa with synthetic substrates upon mixing of plasma with FXa. Although one study showed the feasibility of using of anti-Xa assay for LMWH to assess the presence of rivaroxaban [12], it is recommended to use drug-specific calibrator rather than adopting the anti-Xa assay for measurement of heparin activity due to the following reasons: 1) assays to measure indirect Xa inhibitors, e.g., LMWH, are measured in IU/ml and direct Xa DOACs are measured in ng/mL and there is no direct relationship between these two units of measure, 2) there is significant variability in measured drug concentration, as demonstrated by rivaroxaban, between various anti-Xa kits and 3) the therapeutic range, at least for apixaban and rivaroxaban, far exceeds the typical calibration range for UFH and LMWH (in the 5-9 IU/ml range) and 4) the assay is not specific for anti-Xa DOACs and will detect all anti-Xa anticoagulants2.

Commercially available drug-specific coagulation assays for testing of DOACs use calibrators and controls specific for the DOAC being measured [13-15], enabling the reporting of a drug concentration upon testing of patient’s plasma sample. Multiple calibrators and test plasma dilutions are employed to ensure the test sample responses are within the range of the calibration curve and also to allow for assessment of linearity and parallelism [16]. It was recommended that anti-Xa assay and diluted TT shall be employed when carrying out the drug-specific coagulation assay for anti-Xa inhibitor and anti-IIa inhibitor respectively, given their linear relationship and good correlation with drug concentration as measured by mass spectrometry [11]. Although an ecarin chromogenic assay (ECA) for direct IIa inhibitor and a DRVVT-based assay for both direct IIa and direct Xa inhibitors have been calibrated for testing of DOACs, ECA was shown to have suboptimal accuracy when compared UPLC-MS/MS and DRVVT-based assay would give false positive result in the presence of lupus anticoagulant [17,18]. Studies have shown that various drug-specific coagulation assays differ significantly in quantitation of the DOAC being measured when compared with UPLC-MS/MS in terms of precision and accuracy [17].


What is the meaning of drug concentration?

Drug-specific assay is by no means a direct measurement of drug concentration for DOACs. Instead it is an extrapolation of drug concentration by its anticoagulation activity measured by clot-based or chromogenic assay.
Therapeutic ranges of DOACs have not been validated by the manufacturing pharmaceutical companies. Moreover, there is no established range of concentrations associated with bleeding. In clinical use, expected trough and peak concentrations as predicated on prescribed dose and frequency are often taken as a reference during result interpretation of drug levels [17] (Table 1).


There is no consensus on whether trough level is superior to peak level when interpreting the findings during monitoring of DOACs. The sample for DOAC level is often collected at a random time during emergency clinical situations. A meaningful interpretation of drug level requires the knowledge of the time of last dose of DOAC, the drug dosage and patient’s renal and liver functions so that the trend of drug concentration over time can be better predicted.
With increasing use of DOAC assay, it is expected that DOAC plasma concentration shall be a standard study parameter in future clinical trials. This will allow the identification of drug concentration threshold associated with bleeding, the establishment of a therapeutic range for different kinds of DOACs and better definition of DOAC-induced bleeding complications.


Antidote for reversal of DOACs

Non-specific reversal agents like prothrombin complex concentrates, “bypassing agent” like factor eight inhibitor bypass activity (FIBA) and activated FVIIa were used for the correction of DOAC effect. They only had a general antagonizing action on the anticoagulation effect of DOAC without targeting the specific DOACs themselves. Three antidotes for the DOACs are now under various stages of development. Idarucizumab (Praxbind®), the antidote for dabigatran, is now licensed in the United States and recommended for licensing by the European Medicines Agency. Andexanet alfa, the antidote for the oral anti-Xa inhibitors, is undergoing phase III study. Ciraparantag (PER977), an agent reported to reverse the anticoagulant effects of all of the DOACs is at an earlier stage of development [19]. In life-threatening bleeding, administration of antidote or reversal agent before emergency operations shall not be delayed until the availability of test results. Otherwise, the decision on whether antidote is indicated can be guided by suitable laboratory assay as mentioned in the previous section. Drug-specific assay is considered the most suitable candidate given its superior sensitivity and probably better specificity than conventional coagulation assay and better accessibility and faster turnaround compared with mass spectrometry. Measurement of drug activity shall guide the antidote treatment and allow more effective use of this costly medicine. The importance is highlighted by one study on idaruxizumab for dabigatran reversal in which dTT was normal on study entry in nearly one quarter of the study population, indicating little or no circulating anticoagulant in this group of patients, whom benefit from the administration of idaruxizuman was minimal [20]. Although DOAC concentrations warranting the administration of antidote were recommended (e.g., a drug concentration over 50 ng/mL in serious bleeding and 30 ng/mL in patients requiring urgent intervention) [19], these actionable limits have not been validated in clinical studies.


Quality assurance issues on DOACs testing

Laboratories should develop customized algorithms on DOACs testing strategy for DOACs based on their need. The relative sensitivity of routine coagulation screening test, especially APTT and TT for dabigatran and PT for rivaroxaban, apixaban and edoxaban shall be validated by calibrated materials. Most published algorithms [21, 22] assume patient’s coagulation status is solely under the effect of DOACs and may not be applicable for patients with massive transfusion, disseminated intravascular coagulopathy (DIC) or presence of lupus anticoagulant that may have contributed to the abnormal coagulation screening results. Moreover, it is not practical to change the service PT and APTT reagents solely for DOACs detection.
The set up of clot-based or chromogenic drug-specific assay needs careful literature review on the performances of different commercially available assays. For example, one study reported overestimation of rivaroxaban levels with an anti- Xa assay utilizing exogenous antithrombin [23] and ISTH [4] recommended against its use. Nevertheless, the choice of commercially available assay may be limited by its compatibility with the automated coagulometers in service.
There are currently no standards or guidelines on the validation of drug-specific coagulation assays. Same principles on validation for clot-based or chromogenic coagulation assay shall follow, including testing for accuracy, within-run & between-run precisions and lower limit of quantification (LOQ). The testing of accuracy may be limited by accessibility to mass spectrometry. This can be resolved by testing accuracy against different lot of calibrators. Precision at low drug concentration is important to determine any significant residual DOAC effect in emergency setting. Assay kit with incorporation of low-level calibrators is favored over those with calibrators only covering the usual on-therapy concentration ranges. For the same reason, LOQ validation is important and the report shall report results as “less than” numerical LOQ value (ng/mL). Testing on plasma collected from normal subjects not taking DOACs shall be carried out to determine the intrinsic anti-Xa or anti-IIa activity from natural anticoagulant, e.g., antithrombin, which may also affect the lowest reportable limit of the assay.

As part of the quality assurance, PT, APTT, TT and fibrinogen activity shall be assessed for samples sent for quantitation of DOACs. When TT is prolonged, heparin contamination shall be excluded by carrying out protamine neutralization test. Before reporting the drug concentration, linearity of the calibrator curves shall be verified. Calibrator curve shall be acquired for every patient sample instead using stored calibrator curves as a control of lot-to-lot variation of calibrators for this relatively infrequent test. Results shall be reported in ng/mL, and there should be an accompanied comment about the appropriate range of results (peak or trough levels) based on publication. Drug level shall be interpreted in light of the time since last dose of DOAC intake as well as the dosage of DOAC taken. It is critical to have continuous surveillance of test performance over time. This can be achieved through enrollment in External Quality Assurance Programme (EQAP) (e.g., College of American Pathologist).

Drug-specific coagulation assay can be performed by automated coagulometers with pre-set dilution and analysis protocols with a low to moderate level on skill requirement and hence amenable to the organization of a laboratory-wide staff training programme to cater for the development of a routine 24-hour DOAC laboratory testing service. Interval refreshment training shall be organized to upkeep staff competence. Clinical pathologists shall be involved in communication with clinicians during emergency management of patients requiring DOAC testing to ensure efficient delivery of accurate information to facilitate patient management.


Impact of DOACs on special coagulation assay

It is important for laboratories that carry out special coagulation assay to acknowledge the interferences of DOACs on special coagulation assay. These include clot-based and chromogenic assay. ELISA-based and molecular assays are essentially not affected by DOACs (Table 2) [2, 17].



DOACs are more commonly used nowadays. While clinical indications for laboratory testing are more available, there is a pivotal role of laboratories to formulate a testing strategy for DOACs. Routine coagulation screening tests are not informative in most cases. Development of drug-specific assay for DOACs testing is needed. The interpretation of drug level generated by drug-specific assays needs to be facilitated by more data on the association between drug concentrations and bleeding risks expected in future studies.


Table 1. 5th – 95th percentile of peak and trough concentrations of DOACs obtained from pharmacokinetic and pharmacodynamics studies on patients prescribed with fixed dose and frequency of DOACs.

2.5 mg twice daily
10 mg twice daily


150 mg twice daily


30 mg once daily
60 mg once daily


10 mg once daily
20mg once daily




Table 2. Impact of DOACs on select special coagulation assays

Assay Anti-FIIa DOAC Anti-FXa DOAC
Clauss fibriongen May be falsely decreased No effect
One-stage APTT-based factor assays May demonstrate false decrease in factor activity May demonstrate false decrease in factor activity
One-stage PT-based factor assays May demonstrate false decrease in factor activity May demonstrate false decrease in factor activity
Chromogenic FVIII activity No effect May demonstrate false decrease in factor activity
Bethesda assay False inhibitor present False inhibitor present
AT activity: thrombin substrate May demonstrate false increase in AT activity; may mask AT deficiency No effect
AT activity: FXa substrate No effect May demonstrate false increase in AT activity; may mask AT deficiency
PC activity: clot based May demonstrate false increase in PC activity; may mask PC deficiency May demonstrate false increase in PC activity; may mask PC deficiency
PC activity: chromogenic No effect No effect
PS activity: clot-based May demonstrate false increase in PS activity; may mask PS deficiency May demonstrate false increase in PS activity; may mask PS deficiency
PS activity: chromogenic No effect No effect
PS activity: ELSA-based or LIA-based No effect No effect
LA testing Possible to misclassify as LA present Possible to misclassify as LA present
Activated PC resistance Falsely increased ratio; possible to misclassify as FV Leiden mutation absent Falsely increased ratio; possible to misclassify as FV Leiden mutation
AT, antithrombin; PC, protein C; PS, protein S, LA, lupus anticoagulant; LIA, latex immunoassay


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