Volume 53, Issue 12

Background: More than 40 mathematical indices have been proposed in the hematological literature for discriminating between iron deficiency anemia and thalassemia trait in subjects with microcytic red blood cells (RBCs). None of these discriminant indices is 100% sensitive and specific and also the ranking of the discriminant indices is not consistent. Therefore, we decided to conduct the first meta-analysis of the most frequently used discriminant indices. Methods: An extensive literature search yielded 99 articles dealing with 12 indices that were investigated five or more times. For each discriminant index we calculated the diagnostic odds ratio (DOR) and summary ROC analysis was done for comparing the performance of the indices. Results: The ratio of microcytic to hypochromic RBCs (M/H ratio) showed the best performance, DOR=100.8. This was significantly higher than that of all other indices investigated. The RBC index scored second (DOR=47.0), closely followed by the Sirdah index (DOR=46.7) and the Ehsani index (DOR=44.7). Subsequently, there was a group of four indices with intermediate and three with lower DOR. The lowest performance (DOR=6.8) was found for the RDW (Bessman index). Overall, the indices performed better for adults than for children. Conclusions: The M/H ratio outperformed all other discriminant indices for discriminating between iron deficiency anemia and thalassemia trait. Although its sensitivity and specificity are not high enough for making a definitive diagnosis, it is certainly of value for identifying those subjects with microcytic RBC in whom diagnostic tests for confirming thalassemia are indicated.

A modest amount of cell-free DNA is constantly present in human blood, originating from programmed cell death, apoptosis and rupture of blood cells or pathogens. Acute or chronic cell injury contributes to enhance the pool of circulating nucleic acids, so that their assessment may be regarded as an appealing perspective for diagnosing myocardial ischemia. We performed a search in Medline, Web of Science and Scopus to identify clinical studies that investigated the concentration of cell-free DNA in patients with myocardial ischemia. Overall, eight case-control studies could be detected and reviewed. Although the concentration of cell-free DNA was found to be higher in the diseased than in the healthy population, the scenario was inconclusive due to the fact that the overall number of subjects studied was modest, the populations were unclearly defined, cases and controls were not adequately matched, the methodology for measuring the reference cardiac biomarkers was inadequately described, and the diagnostic performance of cell-free DNA was not benchmarked against highly sensitive troponin immunoassays. Several biological and technical hurdles were also identified in cell-free DNA testing, including the lack of specificity and unsuitable kinetics for early diagnosis of myocardial ischemia, the long turnaround time and low throughput, the need for specialized instrumentation and dedicated personnel, the lack of standardization or harmonization of analytical techniques, the incremental costs and the high vulnerability to preanalytical variables. Hence it seems reasonable to conclude that the analysis of cell-free DNA is not ready for prime time in diagnostics of myocardial ischemia.

The ongoing Ebola virus outbreak in several countries in West Africa was considered by the World Health Organisation (WHO) as a public health emergency of international concern. Healthcare providers must be prepared by organising specific procedures in our hospitals based on recommendations from national and international healthcare organisations. Two aims should be considered: appropriate medical care for patients with suspected or confirmed disease must be ensured, as must measures to prevent transmission to healthcare workers. The clinical laboratory plays an important role and must define and establish its own procedures in accordance with clinicians and integrated into those of the institution, starting with the definition of the organisation model in the laboratory to achieve those goals. In this review we present our experience based on the care of three patients with confirmed cases. We hope it will help other colleagues to plan for Ebola.

Theranos’ Chief Executive Officer recently published a paper in The Wall Street Journal committing to submit all of their tests for FDA approval and renewing her promise that self-testing by the general public will empower people to detect asymptomatic disease early, which will lead to life-saving therapeutic or preventive measures. This opinion paper provides additional information on the benefits and harms of self-testing and self-interpretation of laboratory tests by asymptomatic individuals. We conclude that the health benefit claims of Theranos are hypothetical and they are not supported by evidence. Until such evidence is provided, self-testing of the healthy population should be discouraged.

Parathyroid hormone (PTH) is a major player in phosphocalcic metabolism and its measurement is very important for the correct diagnosis and treatment of several diseases. PTH determination represents the paradigm of quality in laboratory medicine as many variables in the pre-, intra-, and post-analytical phases strongly affect the value of the clinical information. Analytical determination of PTH has been rendered difficult by the presence, in the circulation, of truncated fragments that can cross-react with the antibodies used for its determination. In addition, pre-analytical phase is complicated by the lack of stability of the peptide and the best sample to use for its determination remains controversial, as well as sample handling and storage. PTH secretion is also affected by circadian and seasonal rhythms and by physical exercise. Finally, from the post-analytical perspective, establishment of reliable reference ranges requires further efforts as the selection criteria for reference subjects should take into consideration new variables such as gender, race and vitamin D levels. Finally, clinical guidelines have recently revised and improved the criteria for a correct interpretation of PTH values.

Quantity quotient (QQ) reporting has been proposed by several authors to improve or support the present situation of presenting quantitative laboratory results. This proposal is based on a concept (symmetrical model) known from the intelligence quotient, which was developed to make intelligence tests comparable. In laboratory medicine, however, most measurands follow a non-symmetrical (skewed) distribution, leading to a compression of the QQ values at the lower end of the reference interval. This effect can be avoided by several alternatives. Three models considering non-symmetrical distributions are compared with the symmetrical model in the present study. The corresponding algorithms can be easily handled on the Excel platform. Graphical presentation of the QQ allows a quick overview of test results if they occur in a large number.

Background: Biobanks are becoming increasingly important for assessment of disease risk as well as identification and validation of new diagnostic biomarkers and druggable targets. The validity of data obtained from biobanks is critically limited by the biomaterial quality of the biological samples. External quality assessment (EQA) programs suitable to comprehensively measure the biomaterial quality in archived materials are currently lacking. We report on quantitative assay designs for the analysis of both structural and functional integrity of DNAs that were applied in a first pilot EQA within the priority program on tumor tissue biobanking funded by the German Cancer Aid. Methods: Participating biobanks isolated DNAs from a standardized set of 10 samples comprising sections of four different formalin-fixed paraffin-embedded tissues using their standard operating procedures. Isolated DNAs and analytical results were returned and analyzed centrally for nucleic acids yield, purity, fragmentation and amplificability at a quantitative level using dedicated assay designs. Results: The amount of extracted DNA varied in isolates ranging between 1.5 μg and 25.8 μg. Quantification of DNA fragmentation and amplificability allowed to highlight considerable discrepancies in DNA quality. Amplicons yielded from the isolates of these identical EQA samples ranged from 105 to 411 bp suggesting differences between residual inhibitors of downstream enzymatic reactions. Conclusions: The quality of extraction of bioanalytes from biomaterial archives is heterogeneous even for stable biomolecules like DNA isolated with highly standardized methods. EQAs are appropriate tools to uncover strengths and weaknesses in biobanks in a systematic fashion. Biomaterial integrity is insufficiently reflected by standard methods, but needs to be assessed to improve biobank interoperability. Finally, our results also point towards the problem of measuring the quality of more delicate biomolecules like proteins or metabolites.

Background: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies. Methods: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). Results: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. Conclusions: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

Background: Fetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR. Methods: Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylated RASSF1A sequences of fetal origin in maternal plasma. Fetal ABO genotypes were determined by SYBR-based real-time PCR with allele-specific primers. The performance of the fetal ABO genotyping was assessed by the blood group serology results of the newborns. Results: The fetal RASSF1A sequences were detectable in all the 73 plasma samples, which confirmed the successful extraction of cffDNA. The diagnostic accuracy of fetal ABO genotyping was 93.2%, in which the accuracy of fetal genotype OO, OA and OB was 100%, 83.3% and 96.8%, respectively. Conclusions: We have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.

Background: Screening for “non-deletion” α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common “non-deletion” α-thalassaemia mutations in populations around the Mediterranean and Middle East. Methods: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes ( HBA1 and HBA2 ) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2 , respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green. Results: All 13 “non-deletion” α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case. Conclusions: The 13 “non-deletion” α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.

Background: Medicine is a highly professionalized endeavour, by tradition centred on the authority of physicians. Better education and the advent of the information age cater for increased demands on society in general and on health care in particular to enable people to make informed decisions regarding themselves. Participation in medical decisions requires informed knowledge which is hard to obtain without substantial and time consuming professional help. Methods: We performed a survey amongst the member organizations of European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) in order to investigate the recognition and preparedness of providing help to patients in interpreting their laboratory results. Results: Out of 40 EFLM Member Societies, 27 sent their responses to the survey. In most cases the first line delivery of laboratory results to physicians is by computer link (63%). Patients receive their laboratory results on demand from their physician in 60% of cases. However, 34% of laboratory specialists showed a negative attitude for delivering laboratory results to patients. Yet, in 48% of countries 1–5 patients per day ask a laboratory specialist about the significance of laboratory results outside the reference range. When patients are informed about the purpose of laboratory testing, they seek information primarily from their physician, followed by the internet and the Specialist in Laboratory Medicine. Conclusions: Changing practices increasingly enabling patient access to their records are on the increase facilitated by recent innovations in information technologies. Successful transfer of some of the responsibilities of physicians, demands a mutual triangular dialogue between the patient, their physician and laboratory medicine.

Background: Ebola virus, an enveloped virus, is the cause of the largest and most complex Ebola virus disease (EVD) outbreak in West Africa. Blood or body fluids of an infected person may represent a biohazard to laboratory workers. Laboratory tests of virus containing specimens should be conducted in referral centres at biosafety level 4, but based on the severity of clinical symptoms, basic laboratories might be required to execute urgent tests for patients suspected of EVD. The aim of this work was to compare the analytical performances of laboratory tests when Triton X-100, a chemical agent able to inactivate other enveloped viruses, was added to specimens. Methods: Results of clinical chemistry, coagulation and haematology parameters on samples before and after the addition of 0.1% (final concentration) of Triton X-100 and 1 h of incubation at room temperature were compared. Results: Overall, results showed very good agreement by all statistical analyses. Triton X-100 at 0.1% did not significantly affect the results for the majority of the analytes tested. Conclusions: Triton X-100 at 0.1% can be used to reduce the biohazard in performing laboratory tests on samples from patients with EVD without affecting clinical decisions.

Background: Gestational age-specific reference values are essential for the accurate interpretation of haemostatic tests during pregnancy. Methods: Our 1-year prospective study included 40 healthy pregnant women with a median age of 30 (range 22–40) years; the subjects were followed in order to establish the gestational age dependent values for endogenous thrombin potential (ETP), D-dimer and protein S (activity and free). Results: During the first trimester 50% of studied women had ETP >100% (reference values out of pregnancy); in the second trimester an ETP over 100% was observed in all women; ETP values remained unchanged during the third trimester. In the first trimester, the median D-dimer concentration of 0.30 mg/L, in the second 0.91 mg/L and in the third of 1.45 mg/L were observed. During the first trimester 14/40 subjects had protein S activity below reference range (<59%, out of pregnancy); the median value of 61.35; interquartile range (IQR) 20.38; in the second 21/37; the median value of 53.1 (IQR 15.65); in the third trimester 28/37 had low level of protein S activity with the median value of 49.0 (IQR 18.8). Free protein S showed a slight decrease from the first trimester; it remained almost stable during the rest of pregnancy, with the equal number of pregnant women with reduced free protein S. Conclusions: Related to the gestational age, a significant increase of ETP and D-dimer, from the second trimester was observed; the decrease of protein S was observed already from the early pregnancy, with more pronounced variability of protein S activity.

Background: The non-vitamin K antagonist oral anticoagulants (NOACs) apixaban, dabigatran, and rivaroxaban are being administered in fixed doses without routine monitoring of anticoagulant activities. Despite this key advantage over vitamin K antagonists (VKAs), assessment of anticoagulant intensities is required in various clinical circumstances. We developed a multi-analyte approach for mass spectrometric analysis of NOACs in human plasma. Methods: Plasma samples were precipitated with acetonitrile. Separation was achieved by liquid chromatography using a C18 column and a gradient elution within a run time of 2.5 min. Positive electrospray ionization was used and ion transitions monitored by a triple quadrupole mass spectrometer. Stable-isotope-labeled analogues of analytes were employed as internal standards for quantitative analysis. Certified external quality control samples were obtained for external validation. Results: For all analytes, linearity could be demonstrated over the concentration range of 1–500 μg/L (R 2 >0.999), and the calculated limits of quantification were <1 μg/L. Results for inter- and intra-day assay precision and trueness were obtained using internal quality control samples and remained within the acceptance criterion of ±15%. External quality control samples were measured at the specified nominal values with inter- and intra-day precisions <14%. Matrix effects were fully compensated by co-eluting internal standards, which in turn did not relevantly influence ionization efficiency. Conclusions: The method enables rapid and reliable simultaneous determination of NOAC concentrations in human plasma. It was successfully introduced into clinical practice; a case with rivaroxaban overdose is presented to exemplify the method’s applicability.

Background: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. Methods: ANA and autoantibodies (autoAbs) to double-stranded DNA (dsDNA), CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis thereof by second generation ANA analysis in patients with systemic lupus erythematosus (n=174), systemic sclerosis (n=103), Sjögren’s syndrome (n=46), rheumatoid arthritis (n=36), mixed and undetermined connective tissue diseases (n=13), myositis (n=21), infectious disease (n=21), autoimmune liver disease (n=93), inflammatory bowel disease (n=78), paraproteinemia (n=11), and blood donors (n=101). Results: There was very good agreement of second generation ANA testing with classical one by IIF and ELISA regarding testing for ANA and autoAbs to dsDNA, CENP-B, SS-B, RNP-Sm, Scl-70, SS-A/Ro52, and SS-A/Ro60 (Cohen’s κ>0.8). The agreement for anti-Sm autoAb was good (κ=0.77). The differences of both approaches were not significant for autoAbs to SS-B/La, RNP-Sm, Scl-70, SS-A/Ro60, and SS-A/Ro52 (McNemar’s test, p>0.05, respectively). Conclusions: Second generation ANA testing can replace the two-tier analysis by combining IIF screening with multiplex confirmative testing. This addresses shortcomings of classical ANA analysis like false-negative ANA findings and lack of laboratory efficiency and standardization.

Background: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. Methods: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 °C for 24 h or 48 h. Results: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 10 3 –10 6 WBC/100 mg faeces. The lower limit of detection was 4 WBC/μL and the lower limit of quantification was 5 WBC/μL. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)=4.28+0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. Conclusions: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease.

Background: Cerebral folate deficiency (CFD) may be underdiagnosed, as it manifests with various non-specific neurological symptoms. The diagnosis of CFD requires a determination of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF), which is available in a limited number of specialized laboratories. In clinical biochemistry laboratories, total folate (TF) determination in serum or plasma is routinely performed by automated analyzers. The aim of this study is to determine whether the automated assay of CSF TF is a helpful screening tool for CFD. Methods: We analyzed CSF samples collected from 73 pediatric patients. We measured CSF TF, serum TF, and CSF 5MTHF in 73, 70, and 48 patients, respectively. The assay of 5MTHF was conducted by a newly developed system utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). We investigated the correlation between TF and 5MTHF in the CSF. Results: There was a strong positive correlation between CSF TF and 5MTHF (ρ=0.930, p<0.0001, n=48). Age was negatively correlated with CSF TF (ρ=−0.557, p<0.0001, n=51), serum TF (ρ=−0.457, p=0.0008, n=51), and CSF 5MTHF (ρ=−0.387, p=0.0263, n=33), but not with the CSF/serum TF ratio. Conclusions: The automated assay of CSF TF is helpful to estimate CSF 5MTHF. The CSF TF assay may have a significant impact on the early diagnosis of CFD, because clinicians have better access to it than the 5MTHF assay.

Background: Red blood cell distribution width (RDW) was recently shown to be age-dependent when using Sysmex XE-2100 hematology analyzers. As measuring RDW is subject to technology, we have investigated if this relation also exists when using a different hematology analyzer, Abbott CELL-DYN Sapphire. In addition, as RDW is generally expressed relative to mean red blood cell volume (MCV), we have explored how MCV influences the age-dependency of RDW. Methods: We measured RDW and MCV in a large cohort and calculated RDW-SD (the “absolute” RDW), which does not contain MCV. For establishing reference intervals we used Bhattacharya statistics. Results: In our study cohort of 8089 individuals we found a strong association between RDW and age, but not with gender. Also MCV showed an age-related increase over the entire age range. The conventional RDW increased by 6% from the youngest to oldest age class, whereas RDW-SD increased by nearly 15%. This difference was caused by a mean age-related increase in MCV of 6.6%. Age-dependent reference intervals were established for RDW, RDW-SD and MCV. Conclusions: The age-dependency of RDW seems to be a universal biological feature rather than related with a single type of hematology analyzer. As not only RDW, but also MCV increases with age, we propose that future studies on the prognostic significance of RDW take its age-dependency into account and focus on RDW-SD as a potential marker of adverse events in many clinical conditions.

Background: ITPA polymorphisms have been associated with protection against ribavirin-induced anemia in chronic hepatitis C (HCV) patients. Here we determined the association of inosine triphosphate pyrophosphohydrolase (inosine triphosphatase or ITPase) enzyme activity with ITPA genotype in predicting ribavirin-induced anemia. Methods: In a cohort of 106 HCV patients, hemoglobin (Hb) values were evaluated after 4 weeks (T 4 ) and at the time of lowest Hb value (T nadir ). ITPase activity was measured and ITPA genotype determined. Single-nucleotide polymorphisms (SNPs) tested were c.124+21A>C and c.94C>A. ITPase activity ≥1.11 mU/mol Hb was considered as normal. Results: After 4 weeks of treatment, 78% of the patients with normal ITPase activity were anemic and 21% of the patients with low ITPase activity (p<0.001). Stratified by genotype, the percentages of anemic patients were: wt/wt 76%, wt/c.124+21A>C 46% (p=0.068), and wt/c.94C>A 29% (p=0.021). At T nadir , virtually all patients with normal ITPase activity were anemic, compared to only 64% of the patients with low activity (p=0.02). Thirteen patients had wt/c.124+241A>C genotype. Within this group all five patients with normal ITPase activity and only four of eight with decreased activity developed anemia. Presence of HCV RNA did not influence ITPase activity. Conclusions: This study is the first to report that ITPase activity predicts the development of anemia during ribavirin treatment. ITPase activity and ITPA genotype have high positive predictive values for development of ribavirin-induced anemia at any time during treatment, but ITPase activity predicts ribavirin-induced anemia more accurately.

Background: Diagnosis of spontaneous bacterial peritonitis (SBP) is based on a differential ascites leukocyte count which does not provide prognostic information. We performed a pilot study to assess calprotectin in ascites as an alternative diagnostic and prognostic marker. Methods: We collected ascites from patients with liver cirrhosis from March 2012 to July 2013. Routine clinical and laboratory data of the patients were recorded. Ascites calprotectin levels were determined by ELISA. Results: Overall, we collected 120 ascites samples from 100 patients with liver cirrhosis and from eight patients with malignant peritoneal effusion as disease control. Samples without infection had significantly lower calprotectin levels (median 34 ng/mL, range 5–795) than SBP samples (median 928 ng/mL, range 21–110,480; p<0.001) and malignant effusions (median 401, range 47–2596 ng/mL; p<0.001). In non-infected ascites, calprotectin levels were higher in Child-Pugh stage B versus C (median 57 ng/mL vs. 17 ng/mL; p<0.001) and in alcoholic versus viral cirrhosis (median 37 ng/mL vs. 10 ng/mL; p=0.015). The ratio of ascites calprotectin to total protein was a better marker for SBP than calprotectin alone (AUROC=0.93; p<0.001; sensitivity 93%, specificity 79%; positive predictive value 60%; negative predictive value 97%). In addition, high levels of the ratio to total protein were associated with poor 30-day survival (p=0.042). Conclusions: The ratio of ascites calprotectin to total protein may be a promising new diagnostic and prognostic marker in patients with liver cirrhosis and SBP and should be evaluated further.