A systematic review of the diagnostic performance of serum markers in identifying liver fibrosis on biopsy in patients with ALD using standard methodology found 15 primary studies. The evaluations used 13 different markers, for single markers most commonly HA (n = 7), and 10 marker panels. Serum markers were able to identify those people with severe fibrosis/cirrhosis with reasonable diagnostic accuracy (based on AUROCs). HA as a single marker performed well in identifying cirrhosis, as do some panels of markers. The performance of the serum markers was poorer at identifying lower grades of fibrosis, although few studies evaluated this. The paucity of the literature precluded further conclusions and summative analysis was not possible due to study heterogeneity.
The evidence base for serum markers in ALD lags behind that of Hepatitis C and non alcoholic fatty liver disease. The studies are fewer in number, have fewer participants, vary considerably in inclusion criteria, and have a higher prevalence of cirrhosis/severe fibrosis than in similar studies in Hepatitis C and NAFLD. They also tend to be older studies than other liver disease aetiologies, being less informed by recent advances in the rigour and standardisation required from design and reporting of diagnostic studies . More recent studies have evaluated panels (two of which were external validation studies). Panels varied in their individual constituents, and in the number of components. Generally the values of AUROCs of panel tests in patients with ALD in predicting cirrhosis /sever fibrosis are comparable with those in NAFLD or Hepatitis C. For example in a metaanalysis of Fibrotest in Hepatitis C the mean AUROC for predicting significant fibrosis was reported as 0.77 (95% CI 0.75, 0.79) and in NAFLD 0.81 (95% CI 0.74 0.86) , and a summary AUROC for cirrhosis 0.82 . Certain panels such as APRI seem to perform less well in ALD than in Hepatitis C. Summary AUROC for significant fibrosis was reported as 0.76 (95% CI 0.74 0.79) and for cirrhosis 0.82 (95% CI 0.79 0.86) [33, 34].
There have been reports in the literature of the effect of current heavy alcohol consumption on circulating serum markers which may limit their performance in identifying the chronic effect of alcohol on fibrosis in patients who may be current drinkers. The mode of action of alcohol on the markers is unclear. Animal models have shown that alcohol may have an effect on serum markers such as HA in several ways- by alteration of communication between liver cells thereby affecting HA clearance and by direct effect on induction of hepatic sinusoidal endothelial cell dysfunction [35, 36], Studies have shown that some markers are more susceptible to influences of acute consumption but results are not consistent. One study reported that some markers are affected (tenascin, laminin), some are unaffected (PIIINP, TIMP1), and some very variable (HA) . One small study reported that mean levels of PIIINP but not TIMP1 rise with abstinence . This confirmed the results from an earlier study which showed similar effect of alcohol on PIIINP  Direct studies of effects of alcohol on serum markers in clinical studies involve very small numbers and few studies have reported in the last 5 years. Most alcohol status (were reported ) is self report with some studies using collateral evidence when available. The included studies in this review did not all report current drinking status in detail. In 4 studies included patients were in-patients for alcohol withdrawal /rehabilitation, in 2 studies the patients were not abstinent. More data from large robust studies are needed to properly evaluate the influence of current alcohol intake (ideally quantified with objective measures/triangulated evidence) on markers, reporting results in terms of level of alcohol consumption and time of abstinence.
A major concern in drawing overall conclusions from this review is the considerable heterogeneity of the study populations. Whilst all included studies recruited patients from specialist clinics in secondary or tertiary settings (there were no studies set in primary care), there was variation in the population characteristics, such as level of alcohol consumption, and differences in the prevalence of severe fibrosis. This may lead to spectrum bias influencing diagnostic performance and additionally, affect generalisability. Design of the studies differed with variation in recruitment methods and inclusion criteria. All patients had to have had a biopsy (from inclusion criteria) which could introduce verification bias compared to those patients with excess alcohol consumption not selected for biopsy having a different disease severity than those who were selected. Only four studies reported any parameters by which biopsy quality could be judged, and half of these reported findings stratified by biopsy quality. Even when the tests were similar between studies, the thresholds used were different or not reported. Direct comparison between studies was made more difficult by the use of a range of fibrosis staging systems, largely locally generated. There was heterogeneity and lack of standardization of analytical methods used for the markers measurements and as these different assays may not be well correlated, external validity may be reduced and the determination of a single generalisable threshold remains problematic for those markers assayed locally. Access and availability of serum markers using commercial automated platforms may address this issue. There was incomplete reporting of co-morbidities and diagnostic test results, making appraisal and summative assessment difficult. The paucity of studies which looked at direct comparisons between panels, and between single marker and panels make it difficult to say one panel is more accurate than another. It is clear from this systematic review that the current serum markers are promising, improving and may provide additional diagnostic information in the identification and management of people with ALD.
The limitations of this review include lack of data to perform summative analyses and a focus on the ability of diagnostic tests to identify fibrosis alone. Detection of inflammation has not been addressed. Issues of spectrum bias which may have an impact on performance characteristics of the tests making direct comparisons between studies problematic, and this has not been directly addressed in this review. This is due to several main problems in accounting for such as bias. The first is a lack of a universally accepted system of dealing with this issue, especially in this group of patients with ALD. There have been some methodological suggestions published by one group in chronic Hepatitis C , who have used this method in a study in ALD patients . Authors used standard population of same prevalence for all fibrosis stages and currently it is unclear if this has external validity or international acceptance by professionals working in this field. In addition the studies included in this review are older, use different classification systems for histology and have inconsistent and incomplete reporting of the individual stages of study participants. All of this makes accounting for spectrum bias problematic, complex and of questionable validity in this review. However it is an important issue and should be borne in mind when looking at results between studies.
For preventing and managing ALD it is important to identify those patients who are drinking hazardously and have clinically silent severe fibrosis/cirrhosis in order to focus interventions, to begin to screen for varices and Hepatocellular carcinoma or to prepare for possible liver transplant. Data presented in this review suggest that marker panels could be used effectively in this situation. It would be clinically useful to patients and clinicians to identify the proportion of hazardous drinkers who have developed liver disease to monitor disease progress more closely and to offer an opportunity for strategies aimed at reduction/abstention. Repeated serum marker measurement showing rise or decline in results may have an impact on lifestyle choices again allowing scope for reduction in alcohol consumption. These are speculative ideas and require further research. This group of patients often has erratic attendance at outpatient and biopsy appointments and may present in settings where invasive tests are inappropriate/difficult (e g prison). Access to non-invasive tests of liver fibrosis would be useful in the management of such patients.
Large studies of patients with ALD need to be designed which can directly compare and validate in external populations, performance of existing markers, the identification of new markers or enhancement of existing tests to identify any, mild or moderate fibrosis. For example, methods such as proteomics and metabonomics may identify markers that can be incorporated into existing or new panels of markers, either in isolation or in combination with quantitative imaging techniques (such as elastography). This process might be facilitated by establishing an international reference library and quality assurance scheme. The evaluation of diagnostic performance should be accompanied by parallel evaluation of test performance for properties such as reproducibility, stability and linearity. Further work is needed to ascertain the diagnostic performance of markers in primary care setting. The limitations of liver biopsy may create a glass ceiling for potential non-invasive tests, and future studies should consider use of clinical outcomes as the reference standard. The few studies that have been reported in the literature on performance of serum markers in ALD predicting clinical outcomes rather than fibrosis have shown good performance for some panels of serum markers . Fibrotest, Hepascore and Fibrometer A has been shown to be able to predict liver related mortality at 5 years and 10 years (AUC = 0.79 (95% CI 0.68,0.86) 0.77(95% CI 0.69,0.85) 0.80(95% CI 0.71,0.87) respectively, at least as well as biopsy (AUC 0.77 (95% CI 0.70,0.83). Forns index, APRI and FIB 4 had lower performance in predicting liver related mortality -AUCs 0.40 (95% CI 0.30,0.49), 0.60 (95% CI 0.50,0.69), 0.65 (95% CI 0.54 0.74 respectively. In a smaller population of patients with ALD the predictive performance of the ELF test has also shown AUC 0.80 (95% CI 0.70, 0.89) for liver related morbidity/mortality at 7 years (personal communication with Authors). Additional larger studies that can evaluate and compare performance of non invasive methods in predicting clinical outcomes in patients with ALD are needed.
In summary, none of the serum markers reported so far in the literature appear to have a very good performance for fibrosis severity less than moderate/severe fibrosis/cirrhosis. In general, performance decreases as severity of fibrosis being identified/ruled out decreases. HA shows some promise as a single marker in ruling out cirrhosis and to an extent severe fibrosis, but it is hard to know what threshold to use. Other single markers have less good performance when used alone. Some Panels (Fibrometer, Fibrotest Hepascore, and ELF) show promise in diagnosing cirrhosis/severe fibrosis but studies in ALD have small numbers.