May 2017

ISSN 1650-3414

Volume 28 Number 2

Communications and Publications Division (CPD) of the IFCC
Editor-in-chief : Prof. Gábor L. Kovács, MD, PhD, DSc
Department of Laboratory Medicine, Faculty of Medicine, University of Pecs, Hungary
e-mail: ejifcc@ifcc.org

The
Journal of theInternational
Federation ofClinicalChemistry andLaboratory
MedicineIn this issue

Foreword of the editor
Gábor L. Kovács

98

Advances in the diagnosis of sepsis
Tamás Kőszegi

99Procalcitonin – assisted antibiotic strategy in sepsis
Domonkos Trásy, Zsolt Molnár

104

Advances and pitfalls in using laboratory biomarkers
for the diagnosis and management of sepsis
Dunja Rogić, Gordana Fressl Juroš, Jόzsef Petrik, Ana Lončar Vrančić

114

Nonconventional markers of sepsis
Péter Kustán, Zoltán Horváth-Szalai, Diána Mühl

122

Advances in the diagnosis of sepsis: hydrogen sulfide
as a prognostic marker of septic shock severity
Miha Košir, Matej Podbregar

134

A validation study of after reconstitution stability of diabetes
Shyamali Pal142

A toddler with anasarca caused by congenital nephrotic syndrome
Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay

156

In this issue: Advances in the diagnosis of sepsis

Foreword of the editor

Editor in Chief: Gábor L. Kovács, M.D., Ph.D., DSc

Dr. Tamás Kőszegi presently is a full professor of laboratory medicine at the Departmentof Laboratory Medicine, University of Pécs,
Hungary. He graduated as an MD from the
University of Pécs (1979) and obtained his specialty degree in Medical Laboratory Diagnostics(1984). He wrote his PhD thesis on the releasekinetics of intracellular ATP using different cellular models (1996). His research interest is
wide but in common, he uses mainly thosemethods that are related to luminescence. Oneof his pioneering work on procalcitonin (PCT)
research began in 1999. He published several
papers in collaboration with clinicians on therole of PCT in systemic inflammation (sepsis).
He also proved that neutrophil granulocytes
might be a potential source of PCT release in
septic patients. He is devoted to proteomics
and to find protein biomarkers in systemic diseases with a special emphasis on inflammation. He worked out a method to characterize
perchloric acid soluble serum proteins in systemic diseases related to inflammation (sepsis,

malignancies, autoimmune diseases, Crohn’s
syndrome, etc.). Recently, his interest has been
focusing on serum actin, actin binding proteins(gelsolin and Gc-globulin) and also on urinary
orosomucoid, cystatin C and actin detection insystemic inflammatory conditions. His group
adapted gelsolin, Gc-globulin, urinary orosomucoid and urinary cystatin C to automatedroutine laboratory instruments. These biomarkers may give substantial additional help
for the clinicians at the intensive care unit tomake a quick decision in the treatment of severely ill patients. He also published several
papers on the mode of action, molecular andcellular interactions of mycotoxins with a major
focus on ochratoxin A. His most recent interest
is to capture and characterize circulating tumor
cells and cell-free nucleic acids in breast cancer
patients. Dr. Kőszegi has published more than
300 papers including abstracts, has a cumulative IF 140, and obtained independent citations
close to 500.

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In this issue: Advances in the diagnosis of sepsis

Advances in the diagnosis of sepsis
Guest editor: Tamás Kőszegi1,2
1
2

Department of Laboratory Medicine, University of Pécs Medical School, Pécs, Hungary
János Szentágothai Research Center, Pécs, Hungary

ARTICLE INFOEDITORIAL

Corresponding author:
Dr. Tamás Kőszegi
Department of Laboratory Medicine
University of Pécs
7624 Pécs, Ifjúság u. 13
Hungary
Phone: +36 30 491 7719
Fax: +36 72 536 121
E-mail: tamas.koszegi@aok.pte.hu

Inevitably, sepsis has still remained one of the major challenges at the Intensive Care Units (ICUs)(1). About 30 million cases per year are estimatedworldwide and this tendency is continuously increasing (2). Although sepsis is known for a longtime, its pathomechanism is not completely understood due to the various triggering factors and also
to the altered response of the individuals with different underlying diseases.

Key words:
sepsis, procalcitonin, novel biomarkers,
laboratory management of sepsis
Acknowledgement
The present scientific contribution is dedicatedto the 650th anniversary of the foundationof the University of Pécs, Hungary.
Conflict of interest
The author declares that there is no conflict ofinterest regarding the publication of this article.
.

In sepsis with bacteremia, endotoxins (LPS) of Gram
negative microbes and exotoxins from Gram positive microbes play a major role in the developmentof the symptoms. Currently, it is thought that pathogenesis of sepsis includes microbial interaction withthe host defense system before bacteria can enter
the bloodstream (3-5). Defense mechanisms in tissues differ from those of the intravascular ones. In
the tissues (e.g. first in localized infections) leukocytes are the main antimicrobial factors while inthe bloodstream fight against bacteria is mediatedby humoral factors on the surface of erythrocytes.
Bacteria that can invade the circulatory system possess
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Advances in the diagnosis of sepsis

antioxidant enzymes (SOD, catalase, etc.) protecting them from oxidative injury exerted by
the host. In the bloodstream invading bacteria are attached to the surface of erythrocytes
stimulating oxygen release (from oxyhemoglobin) that might kill bacteria by oxidation. If bacteria escape oxidation they enter erythrocytes
by permeabilizing the membrane. Once inside
the erythrocytes bacteria are most probably to
be killed due to the high concentration of oxygen. On the other hand, bacteria might survive
inside the RBCs at poor oxygenation or whenbacteria are resistant to oxidation. In this way,
RBCs may form a bacterial reservoir where they
can further proliferate (6,7). Inside the erythrocytes bacteria are protected from most of theantibiotics and the antibacterial factors of thehost. Bacterial proliferation damages erythrocytes with a subsequent release of the microbes into the bloodstream (or to other erythrocytes). In case of bacteremia a premature
release of oxygen from erythrocytes and anoxidation resistant infection might occur. As aconsequence, sepsis and in severe cases, septic
shock will develop. Further oxidation of plasma
proteins and lack of proper oxygen content inerythrocytes may cause injury of distant organs
leading to multi-organ failure (MOF) (8,9).
These events are also strongly related to thedevelopment of a misbalance between the
inflammatory and anti-inflammatory cascadeespecially when tissue injury (major surgery,trauma, burns, pancreatitis, etc.) is present.
From the laboratory part, only a few parameters are used routinely for early detection ofsepsis from the more than 200 sepsis related
biomarkers, namely pro-inflammatory andacute-phase proteins (CRP, procalcitonin, interleukines) (10-14), pentraxins (15,16), cytokine/chemokine biomarkers (IL-6, IL-8, IL-10,
TNF- α, etc.) (17,18), macrophage migration inhibitory factor (19,20), high-mobility-group box 1(HMGB1) (21,22), coagulation biomarkers (23,24),

triggering receptor expressed on myeloid cells
1 (TREM-1) (25,26) and midregional pro-adrenomedullin (27). Up to now, no single marker
or a combination of the above markers proved
to be specific and sensitive enough for timely
diagnosis of sepsis. Furthermore, the ultimate
need to predict the outcome of the disease or
to monitor therapeutic efficiency by laboratory
testing has not been fulfilled completely.
The uncertainty regarding both clinical andlaboratory diagnostic criteria has led to the establishment of new sepsis guidelines in 2016.
Among the diverse findings and explanations
in sepsis, the only true fact is, that diagnosis
with proper decision making should be performed within the shortest possible time. The
sooner the antibiotic therapy is begun thehigher chance for the patient to survive. In order to fulfil this requirement both clinical andlaboratory findings (including microbiological
identification) are equally important.
In this issue of the eJIFCC, there are four manuscripts which summarize the present knowledge on the major aspects of diagnosis andtreatment of sepsis with the introduction ofsome unconventional new biomarkers. The
first manuscript of Trásy and Molnár highlights
sepsis management from the point of view ofintensive therapy. The paper is focusing on theimportant aspects of the new sepsis guidelines
and on the pathophysiology of the disease.
The authors describe the body’s immune response to pathogen invasion (pathogen-associated molecular patterns: PAMP and damageassociated molecular patterns: DAMP). The
role of procalcitonin (PCT) in the diagnosis andantibiotic treatment is discussed in details.
Professor Molnár and his group have been involved in the research of diagnostic and prognostic markers of sepsis for more than 15 years
with special emphasis on the clinical usage of
PCT (28,29).

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In the next paper Rogić and her co-authors, besides the classical CRP and PCT markers highlight the potential use of presepsin as a recent
laboratory parameter for early detection of sepsis. Presepsin is a 13 kDa soluble form of CD14
cluster surface glycoprotein derived mainly
from membrane bound CD14 on the surfaceof monocytes (mCD14). Presepsin enables thebinding of LPS and the LPS-binding protein(LBP) complex to toll-like receptors (TLRs), augmenting the inflammatory response. Even if theclinical usefulness of presepsin has not beenverified in every detail yet, the major advantageof this test lies in the very early rise of presepsinin sepsis (within 1 hour). Another advantage ofthe test is that measurement of presepsin canbe done at the bedside with a POC method.
Professor Rogić’s basic fields of research andprofessional activities are evidence-based laboratory medicine, organization and managementof medical biochemistry laboratory, point-ofcare testing, and organization and managementof laboratory parameters of renal diseases. The
next review of Kustán et al. deals with unconventional biomarkers with potential clinical usefulness at the ICU. A challenging observation insepsis and septic shock is the release of large
amounts of a physiological intracellular protein,
actin into the circulation. Once freed from thecells, excess actin is toxic and enhances therisk for respiratory distress syndrome, formingof micro emboli and development of multiple
organ dysfunction syndrome (MODS). Excessive
actin release into the bloodstream decreases
the level of the actin scavenger proteins gelsolin and Gc globulin. In septic patients, especiallywith acute kidney injury (AKI) urinary actin levelis strongly associated with kidney status. In critically ill patients, urinary alpha-1-acid glycoprotein or orosomucoid (u-ORM) as an inflammatory marker is extremely elevated and may beconsidered as a non-invasive marker for diagnosis of sepsis. Kustán and his co-authors haveworked out an automated immune turbidimetric assay for measuring of u-ORM and that ofgelsolin is under development (30). Finally, themanuscript of Miha Košir and Matej Podbregar
are discussing the function and clinical usage ofa less known gaseous transmitter, hydrogen sulfide. Besides NO and CO, hydrogen sulfide (H2S)
is the third known gasotransmitter molecule
influencing many physiological processes suchas maintenance of vascular tone, modulating
the inflammatory response, scavenging reactive oxygen species, etc. Its plasma concentration has a predictive value for the outcome ofsepsis. Interestingly, too high or too low plasma
H2S levels exert unfavorable effects predicting the severity of the disease and indicatinga worse outcome. Professor Podbregar and his
team are attempting to place successful basic
research into clinical context including interestin pathophysiology of shock, hemodynamic stabilization, prediction of severity of shock, cytokine removal techniques/modulation of inflammation and bioactive gases (NO, H2S). They arealso interested in development of point of careprediction tools.
In conclusion, the successful diagnosis andtreatment of sepsis is based on the correct interpretation of clinical signs and symptoms andalso on the availability of laboratory tests withhigh specificity and sensitivity. Measurementof one lab parameter is never enough andmonitoring of key markers such as procalcitonin is essential. The tendency (rising or falling)of the biomarker is usually more important
than the absolute values. Further evaluation ofpresepsin in comparison with well-establishedmarkers (PCT, CRP) and with possible interfering factors (kidney failure) is of utmost importance. The introduction of non-commerciallyavailable tests as u-ORM, gelsolin, Gc globulin
and H2S into the routine laboratory palette inevitably would give valuable complementary
data for sepsis management.

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remains the dominant challenge in the care of critically illpatients. Crit Care Resusc 2005;7:32–9.
2. Fleischmann C, Scherag A, Adhikari NK, Hartog CS, Tsaganos T, Schlattmann P, et al. International forum of acutecare trialists. Assessment of global incidence and mortality of hospital-treated sepsis. Current estimates and limitations. Am J Respir Crit Care Med 2016;193(3):259–72.
3. Cohen J, Vincent J-L, Adhikari NKJ, Machado FR, Angus
DC, Calandra T, et al. The Lancet infectious diseases commission: sepsis: a roadmap for future research. Lancet
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6. Minasyan H. Erythrocyte: bacteria killer and bacteria
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surgical and medical patients with septic shock. Crit Care
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16. Bottazzi B, Garlanda C, Cotena A, Moalli F, Jaillon S,
Deban L, et al. The long pentraxin PTX3 as a prototypic
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17. Tsalik EL, Jaggers LB, Glickman SW, Langley RJ, van Velkinburgh JC, Park LP, et al. Woods CW discriminative valueof inflammatory biomarkers for suspected sepsis. J Emerg
Med 2012;43(1):97–106.
18. Andaluz-Ojeda D, Bobillo F, Iglesias V, Almansa R, Rico
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19. Calandra T, Roger T. Macrophage migration inhibitory
factor: a regulator of innate immunity. Nat Rev Immunol
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7. Minasyan H. Erythrocyte and leukocyte: two partners
in bacteria killing. Int Rev Immunol 2014;33(6):490–7.

20. Bozza FA, Gomes RN, Japiassú AM, Soares M, Castro-Faria-Neto HC, Bozza PT, et al. Macrophage migration inhibitory factor levels correlate with fatal outcome
in sepsis. Shock 2004;22(4):309–13.

8. Shacter E. Quantification and significance of protein oxidation in biological samples. Drug Metab Rev
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21. Wang H, Yang H, Tracey KJ. Extracellular role of
HMGB1 in inflammation and sepsis. J Intern Med 2004;
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9. Hovorka SW, Hong J, Cleland JL, Schöneich Ch. Metalcatalyzed oxidation of human growth hormone: modulation by solvent-induced changes of protein conformation.
J Pharm Sci 2001(January);90(1):58–69.

22. Karlsson S, Pettilä V, Tenhunen J, Laru-Sompa R, HynninenM, Ruokonen E. HMGB1 as a predictor of organ
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10. Tschaikowsky K, Hedwig-Geissing M, Braun GG,
Radespiel-Troeger M. Predictive value of procalcitonin, interleukin-6, and C-reactive protein for survival inpostoperative patients with severe sepsis. J Crit Care
2011;26(1):54–64.

23. Sakr Y, Reinhart K, Hagel S, Kientopf M, Brunkhorst F.
Antithrombin levels, morbidity, and mortality in a surgicalintensive care unit. Anesth Analg 2007;105(3): 715–23.

11. Ho KM, Lee KY, Dobb GJ, Webb SA. C-reactive protein
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12. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med
1999;340(6):448–54.
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ES, Snider Jr RH, Becker KL. Procalcitonin has bioactivity at
calcitonin receptor family complexes: potential mediator
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14. Clec’h C, Fosse JP, Karoubi P, Vincent F, Chouahi I, Hamza L, et al. Differential diagnostic value of procalcitonin in

24. Dhainaut JF, Shorr AF, MaciasWL, Kollef MJ, Levi M,
Reinhart K, et al. Dynamic evolution of coagulopathy inthe first day of severe sepsis: relationship with mortality
and organ failure. Crit Care Med 2005;33(2):341–8.
25. Bouchon A, Facchetti F, Weigand MA, Colonna M.
TREM-1 amplifies inflammation and is a crucial mediatorof septic shock. Nature 2001;410(6832):1103–7.
26. Cohen J. TREM-1 in sepsis. Lancet 2001; 358(9284):
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27. Struck J, Tao C, Morgenthaler NG, Bergmann A. Identification of an Adrenomedullin precursor fragment inplasma of sepsis patients. Peptides 2004;25(8): 1369–72.
28. Molnar Z, Szakmany T, Koszegi T. Prophylactic N-acetylcysteine decreases serum CRP but not PCT levels andPage 102

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microalbuminuria following major abdominal surgery.
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29. Trásy D, Tánczos K, Németh M, Hankovszky P, Lovas
A, Mikor A, Hajdú E, Osztroluczki A, Fazakas J, Molnár Z.
Delta Procalcitonin Is a Better Indicator of Infection Than

Absolute Procalcitonin Values in Critically Ill Patients: A
Prospective Observational Study. J Immunol Res. 2016;
Epub 2016 Aug 15
30. Kustán P, Szirmay B, Horváth-Szalai Z, Ludány A, Kovács
GL, Miseta A, Kőszegi T, Mühl D. Urinary orosomucoid: a
novel, early biomarker of sepsis with promising diagnostic
performance. Clin Chem Lab Med. 2017;55(2):299-307.

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In this issue: Advances in the diagnosis of sepsis

Procalcitonin – assisted antibiotic strategy
in sepsis
Domonkos Trásy, Zsolt Molnár

Department of Anaesthesiology and Intensive Therapy, Faculty of Medicine, University of Szeged, Hungary

ARTICLE INFOABSTRACT

Corresponding author:
Zsolt Molnár
Department of Anaesthesiology
and Intensive Therapy
Faculty of Medicine
University of Szeged
6 Semmelweisst
6725 Szeged, Hungary
Phone: +36 62 545168
Fax: +36 62 545593
E-mail: zsoltmolna@gmail.com

Sepsis is one of the biggest challenges in critical care
nowadays. Defining sepsis is a difficult task on its
own and its diagnosis and treatment requires welltrained, devoted personnel with interdisciplinary
collaboration in order to provide the patients thebest chance for survival. Immediate resuscitation,
early adequate antimicrobial therapy, source control
and highly sophisticated organ support on the intensive care units are all inevitable necessities for successful recovery.

Key words:
sepsis, septic shock, procalcitonin,
antibiotic, biomarkers
Author’s disclosures:
Zsolt Molnar receives lecture honoraria
from ThermoFisher Scientific.

To help fast and accurate diagnosis biomarkers havebeen measured for decades. Procalcitonin (PCT) is
one of the most studied, but the results are conflicting. Sepsis means a very loose cohort of a large heterogeneous patient population, hence defining certain cut off values for PCT to differentiate between
different severities of the disease is almost impossible. Clinicians first have to understand the pathophysiological background of sepsis to be able to interpret correctly the PCT results.
Nevertheless, PCT has been shown to have the best
sensitivity and specificity to indicate infection, antibiotic appropriateness and stopping therapy.
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Procalcitonin – assisted antibiotic strategy in sepsis

In this article we will focus on some important
aspects of pathophysiology and advice on how to
implement that in the everyday clinical practice.
We believe that this multimodal evaluation of
the clinical picture together with PCT results canbe a useful tool to make the most out of the PCT
results, and do the best for patients on the ICU.
INTRODUCTION
One of the most challenging tasks in critical care
medicine is the treatment of serious infection
related multiple organ dysfunction, termed ingeneral as sepsis, and septic shock. Early detection of infection and the immediate start ofresuscitation parallel with adequate antimicrobial therapy undoubtedly give the best possible
chance for survival and received strong recommendation by the Surviving Sepsis Campaign
guidelines [1]. However, while recognizing organ failure via objective signs is relatively easy,
diagnosing infection as the possible underlying
cause remains a challenge. Due to the non-specific properties of conventional signs of infection, such as body temperature and white cellcount (WCC), biomarkers have been utilized
to aid diagnosis for decades. One of the moststudied biomarkers is procalcitonin (PCT) [2]. Itsrole in assisting antibiotic (AB) therapy has beenstudied extensively, with contradicting results.
There are positive studies [3, 4] showing that a
PCT-guided patient management reduced antibiotic exposure and length of antibiotic therapy
without affecting patient outcomes. There arealso negative studies, which could not show thisbenefit [5-7]. However, to understand the values and limitations of inflammatory biomarkers
it is inevitable to understand the immunologicalbackground of critical illness determined mainlyby the host response. Moreover, putting the results of these studies in context, based on new
insights of the pathomechanism of sepsis andsystemic inflammation generated mainly by theindividuals’ host response, may explain the differences between the reported results and help
the clinician to interpret PCT data with more confidence at the bedside.
SEPSIS SYNDROME AS A DISEASE
In most surgical and medical specialties we diagnose definitive diseases, which would indicate
definitive treatment. However, defining, hence
diagnosing sepsis is not that simple.
The term “sepsis syndrome” was invented during the designing of the protocol of one of thefirst prospective randomized trials in sepsis,
performed by a group of scientists led by thelate Roger Bone in Las Vegas in 1980 [8]. Several
years later a statement paper was publishedby the same authors titled “Sepsis syndrome: avalid clinical entity” [9], after which the medical society started to deal with sepsis as with adefinitive disease, which created false expectations:1) physicians wanted one single test withhigh sensitivity and specificity to diagnose sepsis, and 2) there was an urge to find an “antisepsis magic bullet”. Neither of these wishes
have and will never ever come true.
Regarding the definition and diagnosis of sepsis,the classical signs of the “sepsis syndrome” suchas fever/hypothermia, leukocytosis/leukopenia,
tachycardia and hypotension, meant a very large
and non-specific cohort of patients. For this reason, a consensus conference was brought together which defined the so called “consensus
criteria” of sepsis, which has been used for decades in research and clinical practice alike [10].
However, the uncertainty about sepsis definitions lingered on that resulted the recently published new definitions as “Sepsis 3” [11]. In this,sepsis is defined as a “life- threatening organ
dysfunction caused by a dysregulated host response to infection”. As categories only sepsis,
septic shock, and organ dysfunction remained.

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Procalcitonin – assisted antibiotic strategy in sepsis

These efforts clearly show that finding the appropriate definition of sepsis has been a continuous
challenge for more than 30 years. The difficulty
in defining sepsis originates from its complex
pathophysiology, which is affected by numerous individual variations of the host response.
Furthermore, in most specialties diagnostic laboratory or radiological tests have very high sensitivity and specificity often reaching almost 95100% [12]. However, in the case of sepsis, it isdifferent, which makes not just the diagnosis, but
the interpretation of the results of clinical trials
and also epidemiological data very difficult.
THE IMMUNE RESPONSE FOR AN INSULTThe immune system is a complex network andthe immune response to pathogens relies onboth innate and adaptive components, dynamically defined as the pro-, and anti-inflammatory
forces. The innate immune system (including
the complement system, sentinel phagocyte
Figure 1

and natural killer cells), is responsible for theeradication of the invaders, while the adaptive
immune system’s role is to control the process
and keep it localized to the site of the insult[13]. Under normal circumstances these mechanisms remain in balance. The innate systemacts by broad recognition of antigens, mainly
by triggering “pathogen-associated molecularpatterns” (PAMP) of lipopolysaccharide elements of the surfaces of invading pathogens.
When there is an imbalance due to the dysregulation of the pro-, and anti-inflammatory
forces, the local response escalates into a systemic host response also termed as “cytokine
storm” [14]. It was a surprising finding, thatafter trauma, burns, ischemia-reperfusion, pancreatitis, major surgery, etc., same or similarmolecules are released mainly from the mitochondria of the injured or stressed cells that arefound during PAMPs, and can also cause a cytokine storm. This process accompanying tissue

The molecular responses for damage (DAMP)
and pathogen (PAMP) type insults

For further details, see text.

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Procalcitonin – assisted antibiotic strategy in sepsis

injury is called “damage-associated molecularpatterns” (DAMP). In the case of bacterial infection this similarity is due to the fact that thebacteria and the mitochondria (which is moreor-less an encapsulated bacterium) share very
similar genetic background. This explains why
tissue injury induced DAMP and bacterial infection induced PAMP manifest in similar host
responses and clinical manifestations [15]. Thissimilar pathophysiological response is summarized in Figure 1. This indicates that in addition
to PAMP, DAMP can also cause the induction of
the production of similar cytokines, hormokines
and also PCT. This on the one hand provides thepotential benefit of PCT in diagnosing infection(PAMP) but also limits its accuracy as levels may
increase in scenarios without infection (DAMP).
This is the reason, why unexpected PCT values(high or low) are often interpreted as “false negative” or “false positive”. However, understanding the nature of PCT production helps a lot in
the interpretation of PCT values at the bedside.

critical illness and suspecting infection and eventhe source of infection, but they cannot proveit on their own. Conventional indicators suchas fever/hypothermia, leukocytosis/leukopenia, tachypnea, tachycardia, hypotension, takenfrom the classical “sepsis-syndrome” criteria
are non-specific, and in fact poor indicatorsof infection. To fill this gap inflammatory biomarker measurements have been developed[2]. Every biomarker has its own merit andlimitations, but there is no “ideal” biomarker,
and there may never be one. Biomarkers can
support decision making but they will never
be able to differentiate between inflammatory
response for infection from host response for
non-infectious insults with a 100% sensitivity
and specificity due to the complex, overlapping
pathomechanism of PAMP and DAMP. This is in
sharp contrast with the diagnostic power of certain biomarkers used in the world of “definitive”
diseases, where several laboratory parameters
have this ability. Furthermore, learning how to
use biomarkers is not easy either.

THE ROLE OF PCT
IN DIAGNOSING INFECTION

The two most commonly used markers in infection/sepsis diagnostics and for guiding therapeutic interventions are PCT and CRP [2]. Oneof the main limitations of CRP is that it moves“slowly”, and after a certain insult it reaches its
maximum value usually 48 hours later. This is in
general unacceptable on the ICU, as every hour
delay in starting for example appropriate antibiotic treatment can affect mortality as indicatedby the study of Kumar et al. [16]. Furthermore,levels are generally elevated in most ICU patients, making interpretation of CRP very difficult [17].

The so called “sepsis biomarkers” do not recognize sepsis per se, but inflammation. The reasons have been explained in the previous paragraphs, namely that both damage and pathogen
related insults can provoke a very similar inflammatory host response. Therefore, in this context, the right question is: whether the criticallyill condition is due to infection or not? Because
if it is, we should start anti-microbials or othersource control. But if it isn’t, then anti-microbial
therapy should not be commenced, due to its
several undesired effects. Therefore, it is not“sepsis” what we treat, but organ dysfunction
and infection.
Diagnosing infection on the ICU is not easy andrequires a multimodal approach. Clinical signs
are obviously the most important in recognizing

Procalcitonin is detectable in the serum within afew (4-6) hours after its induction, which is mostoften bacterial infection. During the “normal”
course of an infection it reaches its peak within
24 hours and then starts its decline in the caseof adequate treatment with levels reducing by
roughly 50% daily according to its half-life [18].

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Procalcitonin – assisted antibiotic strategy in sepsis

Procalcitonin differentiates bacterial infections
from systemic inflammatory response of otheretiologies with higher sensitivity and specificity as compared to CRP [19], and also have agood prognostic value regarding survival [20].
However, interpreting PCT values on admission
or after the onset of an acute insult, let it be infectious or not, is not simple. But this holds true
for any biomarker, as they show a large scatter
between patients with a seemingly similar clinical condition, hence single absolute values are
difficult to interpret.
There are many studies reporting that PCT values correlate with severity and differ significantly in patients with SIRS, sepsis, severe sepsis
and septic shock [21]. Clec’h et al., found thatpatients with septic shock had more than 10times higher median PCT levels as compared tothose admitted with shock of non-septic origin[22]. However, looking at the data carefully reveals that although there was a remarkable andstatistically significant difference, but there isalso a huge scatter and overlap of the PCT data
between the groups (septic shock: 14 [0.3-767]
vs. non-septic shock: 1 [0.15-36] ng/ml, respectively), which makes individual interpretation ofa single measurement very difficult - a finding,which is generally true for every biomarker of
inflammation. This has been reinforced by thesame group in a subsequent study, in which
they found that the median PCT value in medical vs. surgical patients differed both in SIRS:
0.3 (0.1-1.0) vs. 5.7 (2.7-8.3) ng/ml, and in septic shock: 8.4 (3.6-76.0) vs. 34.0 (7.1-76.0) ng/
ml, respectively [23]. These differences and thelarge overlap can be explained by the PAMP and
DAMP based host response. In certain cases
there is a single PAMP or DAMP, but they canalso occur in combination as PAMP+DAMP. The
latter is bound to have a pronounced inflammatory response reflected in several times higher
PCT values. Therefore, it has become clear thatthe same PCT value, in other words a given“normal” value, cannot be used in every condition. Medical patients with infection in general
should have lower PCT values (single insult of
PAMP) as compared to surgical patients with infection, where DAMP and PAMP are present at
the same time. Moreover, it is also importantto acknowledge, that any cellular injury, let it
be direct tissue or ischemia-reperfusion injury
without infection can result in elevation of PCT
induced by a single DAMP type insult.
Although PCT absolute values have the above
mentioned limitations, but there is overwhelming evidence that in most cases high PCT values
indicate bacterial infection. The shortcomingsof PCT absolute values might be compensatedwhen the kinetics of PCT is taken into accountto indicate infection.
PCT-ASSISTED ANTIBIOTIC THERAPY
There are three fundamental questions to beanswered during our ward rounds when treating patients with suspected or proven infections on the ICU: 1) is there infection, in otherwords should we start empirical antibiotic therapy; 2) is the commenced antibiotic effective;
and finally 3) when should we stop antibiotic
treatment?
In this article we are giving some aspects to answer these questions referring to the result ofprevious studies which were performed at our
department in the last few years in the field of
procalcitonin and antibiotic therapy [24, 25, 26].
1. Is there infection?
It has been explained earlier that either PAMP
or DAMD can induce PCT production. Serum levels of any biomarker show large scatter even ina seemingly homogenous patient population.
That is why it is so difficult, almost impossible, todefine an exact PCT value that indicates bacterial
infection. Indeed, even the most accurate studies
can only show 75-85% sensitivity and specificity.

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Unfortunately, most clinicians tend to interpret
sepsis as a definitive disease, therefore they
have false expectations from the role of thebiomarkers in the diagnosis of infection, andthey become “biomarker sceptic”, when theyfind high levels of PCT after a non-infectious
insult, such as surgery, trauma, or after cardiopulmonary resuscitation. But those who know
the patophysiology of inflammation (mechanism of DAMP, PAMP) are not surprised by thisphenomenon, because they understand that
this is due to the etiology and heterogeneity ofpatients, more precisely due to the individual
immune response after a particular insult.
Our recently published results showed that PCT
kinetics could give a much more reliable help
to the clinicians’ decision making than absolutevalues. As it has a half-life of less than 24 hours,
we hypothesized that kinetics may produce different pattern in those who receive adequate
treatment as compared to those who don’t. In
the EProK („Early Procalcitonin Kinetics”) study
patients were enrolled who were thought tohave infection [24]. From the enrolled 209 patients in 114 cases PCT was available from theprevious day before the infection was suspected [25]. Throughout this 24 hours we foundthat PCT elevation was approximately twice
higher in patients who turned out to have infection versus those who did not. So a more than
88% PCT elevation in 24 hours refers to infection with 75% (65-84) sensitivity and 79% (6092) specificity (AUC 77%). It can be an absolutevalue independent indicator of infection.
It is important to note that if the patient is hemodynamically unstable and infection is likely,
by definition he/she has septic shock or at least
one cannot exclude it, hence antibiotic therapy
shouldn’t be delayed but has to be commenced
immediately, regardless of the PCT or any biomarker value [16]. However, if the patient is stable hemodynamically, and PCT is “low” or decreasing then we can wait, observe the patient

and reassess later. What “low” means as anexact value is difficult to define, as it depends
on the etiology and the patient. Therefore, wehave to admit, that diagnosing infection with orwithout PCT remains a challenge.
2. Evaluating antibiotic appropriatenessAfter commencing empirical antibiotic therapy,
it is indispensable to confirm appropriatenessto correct treatment if needed as soon as possible because it is upmost vital. In septic shock
every hour delay in starting adequate antibiotic
therapy could have serious effect on survival[16]. But unnecessary overuse of antibiotics canalso cause increased bacterial resistance, invasive fungal infections, side effects and increased
costs [27]. Despite international guidelines areavailable to help in choosing the right medication with the best possible chance, unfortunately it seems that inappropriate empiricalantibiotic therapy can be as high as 25-30% on
the ICU [28, 29]. The gold standard for proving
appropriateness of antibiotic therapy is the microbiological confirmation of the bacteria andits susceptibility. However, these results may
come far too late, in reality days after the specimen had been sent, but treatment cannot bedelayed. At present there is very little to help
the clinicians at the early stage of patient care
to confirm appropriate antibiotic treatment.
The above mentioned EProK study showed that
there was a significant difference in the earlykinetics of PCT between patients receiving appropriate as compared to those getting inappropriate antibiotic therapy (24). Serum PCTlevels were measured right after ABs were commenced then 8 hourly in the first day. In thosepatients who received effective AB therapy PCT
reached the highest level at 16 hours and started to decline at the end of the first day while
those whose therapy turned out to be inadequate the PCT level continued to increase during
the first day (Figure 2).These data suggest that

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early response of PCT within the first 24 hours
of commencing empirical antibiotics in criticallyill patients may help the clinician to evaluate theappropriateness of therapy.
Measuring PCT when antibiotics were commenced, then 12-16 and 24 hours later can reveal a certain kinetics. In the case of continuous
increase (red arrows), it is likely that the empirical ABs are inappropriate. However, a “roof-top”
type PCT pattern, indicating, the levels peakedsomewhere around 12-16 hours can indicate appropriate antibiotic therapy.
Once empirical antibiotics were commenced,
daily re-evaluation of the situation is needed. In
addition to the clinical picture and PCT levels,
microbiology should also be taken into account.
Microbiological data usually becomes available
2-3 days after specimen were sent, and then it’stime to re-evaluate the situation. The courseof the clinical condition, combined with boththe microbiological and PCT results, what weterm as a multimodal approach, can assist us
whether to continue, reconsider or change antibiotics and/or reassess organ support and mostFigure 2

importantly, to stop antibiotics even on day 3 ifthey are considered unnecessary.
Despite no clinical improvement a decrease in
PCT still may indicate that the infection is under control, but the patient needs more time
to gain benefit from treatment. Therefore, ABs
should be continued. On the contrary, if PCT is
not decreasing or even increasing, these can beimportant signs that infection is not under control, hence source of infection and antibiotics(type, dose) should be reassessed. If antibiotics
are appropriate, depending on PCT changes ABs
should either be continued or other sources ofinfection should be looked for. In case of inappropriate ABs and no clinical improvement, regardless of the PCT, therapy should be changed.
If there is clinical improvement but no proof of
infection (micro: negative), based on PCT changes (↓ or ↑) infection may be excluded and ABs
stopped, or continued. Similar algorithms canbe applied if ABs are appropriate. If ABs are inappropriate and PCT decreases, then one may
consider the microbiology as false positive andstop ABs, because it is highly unlikely that there

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is clinical improvement and decreasing PCT if an
infection is not under control due to inappropriate ABs. This scenario happens when there
are pathogens (colonization for example), but
no infection. Finally, in case of inappropriate
ABs and unfavourable PCT changes, consultation with infectologists and microbiologists is
recommended.
This multimodal evaluation could help to individualize suspected infection management in
the early course of sepsis on the ICU.
3. Stopping antibiotic therapy
Procalcitonin, mainly due to its favourable kinetic
profile can potentially be a useful biomarker foralso the cessation of antibiotic treatment [30]. In
the first trial on 600 ICU patients, the PRORATA
study [4], PCT-guided antibiotic management
was tested. Antibiotics were encouraged in caseof elevated PCT levels, and discouraged whenlevels were low. The novelty of this trial wasthat investigators were encouraged to discontinue antibiotics when PCT concentration wasless than 80% of the peak value or when absolute concentration of less than 0.5 ng/ml wasreached. The same protocol was repeated in alarge recent study on 1500 patients by de Jonget al., in a multicenter prospective trial [31]. The
results were similar just like the previous one
applying this approach shortened the durationof antibiotic treatment and the daily dose antibiotic consumption, in addition the mortality
in this group was significantly lower in the PCTgroup as compared to conventionally treatedpatients. In spite of the reinfection rate being
higher in the PCT guided group the cumulative
cost of antibiotics per patient was significantly
lower. Despite the significantly shorter antibiotic therapy, they were unable to show any
difference in outcome between the groups, inother words patients did not suffer harm fromnot receiving antibiotics for the length of time
recommended by guidelines.

CONCLUSION
In this deadly battle of fighting the burden of serious infections on the ICU, we often keep missing the point. Although sepsis exists, just likecritical illness, but precisely defining it is probably impossible due to its diversity in etiology,
pathomechanism and clinical manifestation.
Therefore, interpreting the results of sepsis studies is a daunting task. Procalcitonin is definitely
one of the most reliable inflammatory markers
in the critically ill to date, and there is also convincing evidence that its use to guide antibiotic
therapy can rationalize starting, escalating andstopping antibiotic therapy. Furthermore, when
the concept, highlighted in this paper is applied,
PCT may also become cost effective, by not starting at all, or stopping antibiotic therapy early.
However, starting or stopping antibiotic treatment is more complex than just treating one
single figure or even the kinetics of PCT values.
A multimodal, individualized concept, consistingof a) recognizing organ dysfunction, b) identifying the possible source, c) following the clinicalpicture and d) taking PCT and PCT-kinetics into
account, is necessary to make the most out ofyour PCT and to do the best for your patients inyour everyday practice. Indeed, it requires welltrained, devoted, thinking physicians who dial in
all information such as the results of physical examination, laboratory data, and physiologicmeasurements and make the decisions. Therefore,
PCT is not the answer, but it can certainly help,
considering that we understand what’s going on
in our patients.
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In this issue: Advances in the diagnosis of sepsis

Advances and pitfalls
in using laboratory biomarkers
for the diagnosis and management of sepsis

Dunja Rogić1,3, Gordana Fressl Juroš2, Jόzsef Petrik3, Ana Lončar Vrančić1
Department of Laboratory Diagnostics, University Hospital Center Zagreb, Croatia
Children’s Hospital Srebrnjak, Zagreb, Croatia
3
Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry,
University of Zagreb, Croatia
1
2

ARTICLE INFOABSTRACT

Corresponding author:
Dunja Rogić
University Hospital Center
Kišpatićeva 12
10 000 Zagreb
Croatia
Phone: +385-1-2367288

Sepsis is a critical patient condition with high mortality rate caused by a complex and inadequate host
response to infection. Since early identification andstart of antibiotic therapy in the first few hours after
sepsis development dramatically improves outcomes,
it is of utter importance to offer fast, reliable and specific early laboratory biomarkers to help clinicians insepsis recognition. On the other hand, the biomarkers should also be helpful in excluding sepsis and/or
confirming therapy effectiveness, and thus prevent
overprescribing of antibiotics. In this paper, we discuss the significance and relative merits of three currently available protein biomarkers: C-reactive protein, procalcitonin and presepsin. Although useful,none of these biomarkers has been shown to completely fulfill the roles mentioned above.

Key words:
management of sepsis, C-reactive protein,procalcitonin, presepsin

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Advances and pitfalls in using laboratory biomarkers for the diagnosis and management of sepsis

INTRODUCTION

C-REACTIVE PROTEIN

The 2016 Guidelines of the Surviving Sepsis
Campaign (SSC) classify sepsis and septic shock
as medical emergencies and therefore treatment and resuscitation must begin immediately(1). Sticking to the shortest possible time framefor diagnosis and treatment is crucial since
it often means life or death for the patient.
However, it is –especially in the early stages–
not easy to be sure if a patient is septic or not.
As a consequence, to be on the safe side, antibiotics are often overprescribed, which generates
further problems. As sepsis represents one of
the major problems in intensive care units, it isessential for clinicians to have fast, accurate andreliable biomarkers that can help them to makea quick diagnosis and appropriately manage or
exclude this life-threatening condition. An ideal
biomarker should have all of the following characteristics: fast and specific increase in sepsis,
rapid decrease after effective therapy, short halflife and fast and widely available and reliable
method of determination. None of the current
biomarkers exhibits all of these specifications
in full, but the best currently used biomarker
available both as a point of care test (POCT) andas a part of several major in vitro diagnostics(IVD) manufacturers’ portfolio is procalcitonin(PCT) (2). However, since CRP is still uniformly
and inevitably used worldwide, its importancein sepsis diagnosis and management will bebriefly discussed. The focus of attention will beplaced on the new and rapidly advancing biomarker presepsin whose role is still uncertain,
but which might represent a step towards earlier and better sepsis recognition by laboratory
means (3). Of course, the golden standard forsepsis confirmation remains within the scope of
microbiology laboratory, but the time to result
even with the recent advancement of Matrix
Assisted Laser Description/Ionization - Time of
Flight (MALDI TOF) technology is still inferior to
the above mentioned surrogate biomarkers.

C-reactive protein (CRP) belongs to the pentraxin family of calcium-dependent, ligand-binding
proteins. Human CRP molecule has a discoid
shape and consists of five identical nonglycosylated polypeptide subunits, each containing 206
amino acid residues (4). The CRP gene is locatedon the first chromosome (1q21q23).
CRP was isolated from the sera of patients infected with Streptococcus pneumoniae, and was
first described by Tillett and Frances in 1930 (5).
The CRP, named for its capacity to precipitate
the somatic polysaccharide-F of Streptococcus
pneumoniae, was the first acute-phase protein
to be described and is an exquisitely sensitivemarker of systemic inflammation and tissue
damage (4).
CRP is synthesized primarily in the liver, andat lower levels in adipocytes as a response tointerleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α). CRP is an acutephase protein. During acute-phase response toinfection, inflammation or tissue damage, theconcentration of CRP increases several thousand times within 48 hours. Authors Pepys andHirschfield stated that the median concentration of CRP is 0.8 mg/L in healthy young adultvolunteer blood donors, the 90th percentile is
3.0 mg/L, and the 99th percentile is 10 mg/L
(1) but, following an acute-phase stimulus, thevalues may increase from less than 50 μg/L to
more than 500 mg/L, that is, 10,000-fold (6).
The function of CRP is manifold. The CRP can bindto specific ligands and activate a complement on
the classical pathway and thereby participate innon-specific defences against infection and prevent the development of autoimmune diseases.
The main ligand for CRP is phosphocholine which
is present in the cells of most pathogens, including bacteria and fungi. Furthermore, it is considered that the CRP interacts with damaged endothelial cells or the apoptotic and necrotic cells.

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Advances and pitfalls in using laboratory biomarkers for the diagnosis and management of sepsis

The major CRP drawback in sepsis lies in its lackof specificity. Elevated CRP concentrations in
the circulation may indicate inflammation and/or tissue damage of any origin, such as bacterial
and viral infections, mycoses, allergic complications of infection, various inflammatory reactions, necrosis, trauma, or malignancies (7-13).
The particular problem is a common postoperative CRP elevation with values that might easily
overlap with the septic ones, particularly in theinitial stages when early recognition is of utter
importance.
The CRP values are not affected by diurnal variations, food intake and most medications.
Recent evidence suggests that aging has a significant effect on inflammatory response and theimmune system of elderly people, which is to beconsidered when interpreting CRP values (14).
Acute phase proteins such as CRP, PCT, serum
amyloid A, IL-6, and hepcidin have been investigated in multiple studies related to neonatal
sepsis. CRP is usually used as an indicator ofbacterial sepsis in newborns since PCT values
may not be easily interpreted in the first few
days of life. However, the determination of
CRP has a few drawbacks; it is not useful as amarker for the early phase of infection because
it can only be detected about 12 hours after the
onset of clinical symptoms, it reaches its maximum after 20 to 72 h (15) and does not show
satisfactory specificity (16). However, due to its
universal availability and fairly straightforward
interpretation of values, CRP is still the mostcommon marker used worldwide in all hospitalized patients, including those with high risk ofsepsis development.
PROCALCITONIN
PCT is a calcitonin precursor prohormone which
consists of 116 amino acids and is normally expressed by neuroendocrine cells of the thyroid
gland, lungs and pancreas. In healthy people

PCT values are low, less than 0.046 μg/L (95thpercentile) (17,18). PCT values in various nonseptic infections are usually lower than 0.5 μg/L
(18). This fact, i.e. exclusively bacteria-related
increase represents one of the crucial PCT
advantages when compared to CRP. In severe
septic shock PCT can rise up to 1000-fold. The
pathomechanism of blood PCT elevation is areaction to various exogenous and endogenous
stimuli, such as inflammatory interleukins,
membrane elements, bacteria lipopolysaccharides or peptidoglycans as well as bacterial endotoxins. In sepsis, PCT is synthesized mostly inliver, but also in other parenchymal organs (19).
Increase in PCT level in patient’s blood can bedetected approximately 2 to 4 hours after the
onset of sepsis (19), which represents anothermajor advantage making it both more suitable
and specific than CRP in this clinical context. It is
important to know that PCT has a plasma halflife of 20-24 hours (20) Therefore, according tocurrent recommendations, the minimum time
before testing needs to be repeated (minimum
retesting interval) should be 24 hours (21).
PCT can serve as an aid for clinicians in assessing the risk category for their patient of developing sepsis or septic shock, according to theclassification (18) in Table 1.
After initial measurement, PCT can be used tomonitor progression of the disease and therapy
effectiveness, taking into account minimum retesting interval for PCT of 24 hours.
PCT has also proved to be useful in guiding
antibiotic therapy. This approach was mainly
evaluated in patients with respiratory tract infections; however, it can also be used in critically ill patients with sepsis or severe sepsis ofvarious origins (21,22). In those patients, daily
measurement of PCT is indicated and discontinuation of antibiotic therapy should be considered when PCT levels decrease to less than
80% of the peak value or below 0.5 μg/L (23).

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Table 1

Risk categories according to PCT values

PCT value (μg/L)

Risk category< 0.5

low risk of systemic bacterial or fungal infection

0.5 - 2.0high risk of systemic bacterial or fungal infection

2.0 – 10high risk of sepsis and progression to septic shock> 10

high risk of septic shock

This approach has been shown to significantly reduce the antibiotics use without compromising the patient outcome, which mightprove beneficial towards the goal of minimising
the antibiotic usage both for the sake of patients and hospital resources. It is important tonote that current recommendations state thatmeasurement of PCT levels can be used to support shortening the duration of antimicrobial
therapy in sepsis patients; however, as yet thisapproach is not based on strong evidence (1).
Similar strength of recommendations pertains
to the fact that PCT levels can be used to support the discontinuation of empiric antibiotics
in patients who initially appeared to have sepsis, but have subsequently been proven to have
limited clinical evidence of infection (1).
In conclusion, PCT has proven to be a helpful
biomarker in early diagnosing of sepsis in emergency departments and intensive care units. It
has also been recommended for monitoring effectiveness and modulating duration of antibiotic therapy. It is now widely available both as alaboratory and as a point-of-care test, whereas
laboratory methods are preferred due to semiquantitative nature of results on most POCT devices. When ordering PCT, it should be essentialthat the result provides the clinician with an answer that could not have been resolved by other laboratory tests combined with clinical signs
and symptoms, such as complete blood countor CRP measurement. It is important to notethat PCT and all other biomarkers can provide
only supportive and supplemental data to clinical assessment. Decisions on initiating, altering,or discontinuing antimicrobial therapy should
never be made solely on the basis of changes
in any biomarker, including PCT (1). The following questions therefore cannot as yet unequivocally be answered exclusively either by PCT or
any other available biomarker: Does my patient
have sepsis? Is this antibiotic therapy effective
for my patient? Can I now safely discontinue
antibiotic therapy? PCT seems to be of help in
answering those questions in many clinical situations; however, it cannot fulfill this role by itself and should therefore be ordered rationally.
PRESEPSIN
Presepsin is a newly investigated sepsis marker
that has been shown to have potential as an
early marker of sepsis recognition, for antimicrobial therapy monitoring and as a prognostic
marker. Presepsin (sCD14-ST) is a peptide sized
13 kDa that is generated by proteolytic cleavage of soluble forms of CD14 cluster (sCD14).
CD14 is a cell surface glycoprotein with molecular mass of 53-55 kDa that is anchored by glycosyIphosphatidylinositol (GPI) to cell membraneand represents a membrane form of CD14(mCD14). The mCD14 as a co-receptor mediates the binding of the bacterial endotoxin,

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lipopolysccharides (LPS) and complex LPSlipopolysaccharide binding protein (LBP) totoll-like receptors (TLRs), causing the activation of inflammatory responses: cell activation,
fagocytosis and cytokine production defending
host against pathogen (24-26). After the sTLR
activation, the mCD14 undergoes the proteolysis, producing two soluble forms (sCD14) ofdifferent sizes. The smaller sCD14 is produced
by protease cleaving of mCD14 and the bigger
one is produced intracellularly and is directly
released from the cell in the protease-independent manner (27-29). CD14 is present mostly
on monocytes, macrophages, neutrophils,
B-lymphocytes and also on chondrocytes, dendritic cells and human epithelial intestinal cells.
Hepatocytes can also express CD14, especiallyduring endotoxemia (30). Recent investigationof presepsin kinetics has shown that, when thepolymorphonuclear and monocytic cells were
exposed to LPS, presepsin could be detected
as early as one hour after the exposure, withmaximum concentration in the third hour (31).
This finding may confirm that presepsin can bea useful marker of host response to bacteria
and can be both a specific marker of infection
as well as an early indicator, compared to current markers.

and non-bacterial infective disease. Presepsin
at the cut-off value of 600 ng/L has diagnostic
sensitivity and specificity of 87.8% and 81.4%,
respectively, which is comparable to PCT at the
cut-off of 0.5 µg/L. A study showed that there
was no statistically significant difference in presepsin levels between patients with localized
and systemic infection, which might preclude
its usefulness in sepsis (33).

Many studies have investigated accuracy of presepsin in diagnosis of system immune response
syndrome (SIRS), sepsis and septic shock in different clinical conditions. The systematic reviews and meta-analysis showed that presepsin
as a diagnostic marker of sepsis has diagnostic
sensitivity and specificity of 0.83 and 0.78, respectively, diagnostic accuracy (expressed as
ROC AUC, receiver operating characteristic,
area under the curve) of 0.88, and positive andnegative likelihood ratios of 3.9 and 0.21, respectively (32).

Another study has shown that presepsin levels
are elevated at an early stage of sepsis and increase with its progression. The plasma presepsin levels reached the highest level in septic
shock. ROC analysis of presepsin to differentiate SIRS (systemic inflammatory response syndrome) and sepsis revealed that, at the cut-offof 581 ng/L, sensitivity and specificity were 65%
and 100%, respectively, AUC was 0.830. Whenpresepsin is combined with the MEDS (Mortality
in Emergency Department Sepsis) scoring system, the AUC was significantly higher - 0.95,
with the sensitivity and specificity of 85% and100%, respectively (36). A similar investigationof the diagnostics of sepsis has shown AUC for

Multicenter prospective studies have demonstrated a statistically significant difference inpresepsin levels between patients with bacterial

A multicenter randomized clinical trial whichinvestigated the clinical role of presepsin assay
for monitoring disease in relation to the development of complications (34) found that higher
presepsin levels on the first day were closely associated with higher incidence of subsequentorgan failures (SOFA score). It also studied presepsin role in monitoring the host response to
antimicrobial therapy and appropriateness oftherapy – the trial confirmed that patients withincreasing presepsin concentrations during thefirst 7 days were less likely to have received
early appropriate antibiotic therapy. The study
also stated prognostic accuracy of presepsin forearly and long-term outcomes; early presepsin
was higher in non-survivors than in survivors.
Patients with lung infection had lower baseline
presepsin levels than patients with abdominal
and urinary tract infections (34,35).

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presepsin to be 0.82. For predicting severe sepsis in sepsis patients, the AUC was 0.840 and,with the combination of presepsin and MEDS
score the AUC was 0.875 (35).
A very important piece of information about
presepsin should be emphasized. As presepsin
is a small protein (13 kDa) and is filtered by kidneys, its level is strongly dependent on kidney
function. The decreased glomerular filtration
causes elevated levels of presepsin in circulationand thus, presepsin levels above the cut-off value in patients with renal failure have to be interpreted with caution (37,38). A study showed nostatistical difference in presepsin concentration
between septic and non septic oliguric patients(stage failure in RIFLE criteria; RIFLE, concensus
classification criteria for acute kidney injury:
Risk, Injury, Failure, Loss, End-stage kidney disease), which might preclude its use in patientswith renal failure (38). In this regard, however,
future studies are necessary to confirm thisfinding. There is also a recent study (39) which
deemed presepsin as a valuable biomarker for
diagnosis of infection and sepsis; however, its
diagnostic accuracy has not shown any superiority compared to PCT. Therefore, the authors
question its validity for introduction into clinical practice. Similarly, its added value has beenquestioned in patients with pyelonephritis (40).
Despite the somewhat controversial findings
about its validity, presepsin might be an effective biomarker for the timely diagnosis of sepsis,
particularly due to its early elevation. Besides
diagnosis, it might prove useful for monitoring
therapy effectiveness and it might serve as an
aid in prognosis. It is important to note that its
measurement can be performed quickly andeasily (41) as a POC test; however, as it is still
not widely available on common laboratory
platforms, its utility might be hampered. Alongwith, or –as a better scenario for healthcare resources– instead of other diagnostic inflammatory markers, presepsin might therefore get its

place in the septic patient care as a routine laboratory marker used to facilitate current diagnostic strategies, particularly for early recognitionof sepsis within the first few hours, which nowadays still represents a diagnostic challenge.
CONCLUSION
So far in clinical practice no single, optimal biochemical marker is available to confirm or exclude the diagnosis of severe infection within
the clinically required time frame. Therefore thediagnosis has to include consideration of all important signs of infection. The quest for an ideal
sepsis biomarker is still going on. The most reliable one according to current knowledge is still
PCT, while the emerging and promising markers
such as presepsin still lay in waiting to be unequivocally proven as more useful and effective.
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34. Masson S, Caironi P, Spanuth E, Thomae R, Panigada
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In this issue: Advances in the diagnosis of sepsis

Nonconventional markers of sepsis
Péter Kustán1,2, Zoltán Horváth-Szalai2,3, Diána Mühl1

Department of Anaesthesiology and Intensive Therapy, University of Pécs Medical School, Pécs, Hungary
Department of Laboratory Medicine, University of Pécs Medical School, Pécs, Hungary
3
János Szentágothai Research Center, Pécs, Hungary
1
2

ARTICLE INFOABSTRACT

Corresponding author:
Dr. Péter Kustán
Department of Laboratory Medicine
University of Pécs
7624 Pécs, Ifjúság u. 13
Hungary
Phone: +36 30 248 3289
Fax: +36 72 536 121
E-mail: peter.kustan89@gmail.com

Sepsis still remains a challenging healthcare problem
with high mortality rate. To improve outcome, early
diagnosis and monitoring of sepsis is of utmost importance. In this process objective laboratory parameters are the most helpful.

Key words:
sepsis, presepsin, actin, gelsolin,
Gc-globulin, orosomucoid, urine
Acknowledgement
The present scientific contribution is dedicatedto the 650th anniversary of the foundation of
the University of Pécs, Hungary. The presented
work was funded by NKFI-EPR K/115394/2015/
HU and GINOP-2.3.2-15-2016-00021 grants.
Hereby we express our special thanks
for the invaluable help of our nurses,
assistance and colleagues.
Conflict of interest
The authors declare that there is no conflict ofinterest regarding the publication of this article.

Procalcitonin and C-reactive protein are the mostcommonly used and recommended markers of sepsis however, more than 200 sepsis biomarkers havealready been published. This mini review focuses on
nonconventional novel possibilities for the recognition of sepsis severity. Presepsin, actin and actin scavenger proteins (gelsolin and Gc-globulin) and orosomucoid are discussed. Besides serum parameters,
the urinary levels of these markers are also elaborated, since urinary biomarkers of sepsis provide new
diagnostic implications and are helpful for monitoring both the kidney function and the septic process.
Increasing serum actin levels and decreasing levelsof actin binding proteins seem to be associated withsepsis severity and outcome. Actin can be detectedin the urine samples of septic patients as well, andstrongly elevated levels of it were found in sepsisrelated acute kidney injury. Both serum and urinary
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Nonconventional markers of sepsis

orosomucoid might be able to indicate sepsis,
however urinary orosomucoid is a more sensitive inflammatory marker.
Novel laboratory tests can provide rapid help forclinical decision making because the key point insuccessful treatment lies in the early diagnosisof sepsis.


INTRODUCTION
Although sepsis is one of the oldest syndromes
in medicine it is a challenging healthcare problem
even nowadays. In spite of the era of modern antibiotics and intensive therapy sepsis is still one of
the leading causes of morbidity and mortality (1).
Sepsis is a heterogeneous and complex syndrome
with various etiology, severity and prognosis. To
our present knowledge the inflammatory response is the key role in the pathophysiologyof sepsis however; a kind of uncertainty exists
regarding the factors most likely to lead to increased lethality. In spite of the uncertainties
one fact is obvious: the earlier the diagnosis ofsepsis is raised, the more favorable outcome
may be predicted (2, 3).

Microbiological identification of pathogens isessential for efficient therapy of sepsis, because
the clinical signs are nonspecific. Gold standard
microbiological culturing methods require quite
a long time (days), but new molecular biological
techniques, polymerase chain reaction and mass
spectrometric methods can shorten pathogen
identification in the bloodstream (5). However,these methods can not differentiate between
colonization and infection, moreover they needa well trained and equipped laboratory.
The diagnosis and monitoring of sepsis is of utmost importance, in this regard objective laboratory tests may provide rapid information forproper decision making. Up to now, more than
200 sepsis biomarkers have already been studied, most of them belonging to the inflammatory mediators’ family (acute phase proteins,
cytokines, chemokines, CD markers, adhesion
molecules, etc.) (6, 7).
This mini review discusses classical sepsis biomarkers as well but the major focus will be onsome of novel interesting nonconventional markers of sepsis.
CONVENTIONAL SEPSIS MARKERS:SERUM PCT AND CRP

Based on the novel results and advances ofpathobiology, management and epidemiologyof sepsis, the definitions of the syndrome havebeen changed recently. Sepsis-3 consensus defines sepsis as a life-threatening organ dysfunction caused by a dysregulated host response toinfection (4).

The diagnostic and therapeutic guidelines ofsepsis management recommend the use of procalcitonin (PCT) and C-reactive protein (CRP)
measurements for early recognition of the syndrome (2, 8).

The diagnosis of sepsis is most often not easy
especially in newborns or in patients whose immune response is not adequate. Therefore, it isof utmost importance to introduce diagnostic
biomarkers which can predict or verify systemic
inflammation as early as possible. These tests
should also be applicable for monitoring of thedisease progression and efficacy of therapy as
well.

Blood levels of PCT rise 4-6 hours after the onsetof systemic infection and PCT’s half-life is about
one day. Procalcitonin concentrations showed
good correlation with the severity of sepsis,
higher PCT levels correlated with higher risk ofmortality (9). Massive tissue damage could also
provoke elevated serum PCT values without infection, but fungal and viral infections do notelevate the PCT concentrations (10).

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Monitoring of PCT kinetics is recommended because delta PCT is a better marker of infection
than absolute levels and furthermore, early PCT
kinetics could indicate the efficacy of antibiotic
therapy (11, 12).
CRP is a non-specific inflammatory marker,
therefore it increases in many acute and chronic
diseases (tissue injury, autoimmune disorders,malignancies), however in sepsis management,
CRP could supplement PCT measurements.
After infections serum CRP reaches its maximum within 48-72 hours. Strongly elevated CRP
levels were found to be severity and mortality
predictors in sepsis (13). The measurement ofhigh sensitivity CRP (hsCRP) is recommended.
Since both biomarkers have some limitations,promising other possibilities should be searched
for and in fact, are available nowadays.
PRESEPSIN
CD14 molecule is a pattern recognition receptor
existing in two forms: as a membrane-boundtype (mCD14) and a soluble form (sCD14). Bothforms play a role in recognition of LPS and in cell
activation. Soluble CD14 subtype (sCD14-ST)
also called as presepsin elevates significantly
during inflammation and seems to be usable indifferentiating between bacterial and nonbacterial infections (14).
Presepsin is normally present in very low concentrations in the serum of healthy individuals.
In response to bacterial infections, its concentration increases within 2 hours, according to
the severity of the disease (15). Studies havebeen reported with various diagnostic cut-off
levels for sepsis between 400–600 pg/ml (16,17). Preliminary studies showed that plasmapresepsin is a highly sensitive and specific marker of sepsis, and its concentration significantly
correlates with the severity of the disorder andin-hospital mortality of patients suffering fromsevere sepsis and septic shock (18). A novel

point of care test is available on the market for
rapid presepsin determination, which can help
clinicians in rapid decision making.
Due to its 13 kDa molecular weight, presepsin
is filtered through the glomeruli, then reabsorbed, and catabolized within proximal tubular cells (19). There is increasing evidence, that
presepsin levels are affected by kidney function.
Elevated presepsin levels were found in patientswith decreased renal function and inverse correlation was described between presepsin and
GFR as well (19, 20).Therefore, presepsin levels
should be interpreted more attentively in patients with kidney disease.
ACTIN
Actin is a multifunctional 43 kDa protein which
is present in all eukaryotic cells in monomeric/
globular (G-actin) and in polymeric/filamentous(F-actin) form (Figure 1). The two forms dynamically change due to the very rapid polymerization and depolymerization of the molecule.
Actin takes a pivotal part in many cellular processes (building up microfilamental cytoskeleton, motility, moving, division, junctions) andin muscle contraction, too (22).
As actin is one the most abundant intracellular
protein, during massive cell injury and catabolic
conditions high amounts of actin can release
into the circulation. Free extracellular actin has
toxic effects, since actin filaments are thought
to increase blood viscosity, to activate platelets, and cause endothelial cell damage andsmall blood vessel obstructions. Therefore, highamounts of extracellular actin may contributeto the development of multiple organ failure(23, 24).
The so called actin scavenger system is responsible for the protection of the body from actin
toxicity; however the capacity of this defense
system can be overwhelmed by massive tissue
injury (25).

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Figure 1

The structure of native G-actin

G-actin contains 4 subdomains, and includes one ATP molecule and one Ca2+ ion located in the nucleotide cleft, which
exists in a closed state (21).

In healthy individuals the major source of extracellular actin is most probably the skeletal muscle with its large mass and high actin content.
Circulating actin levels might provide clinically
relevant information on disease severity, serum
actin (se-ACT) levels were found to be higher inseptic patients (3.5 (1.6-6.1) mg/L) than in controls (3.0 (2.1-3.7) mg/L) however did not meetcriteria for statistical significance (Figure 2A).
The cause of increased se-ACT levels in systemic
inflammation and in sepsis might be the extensive tissue injury and detritus of blood cells (26).
There is only scarce data on urinary appearanceof actin, however due to its molecular weight
free actin could be filtrated through the glomeruli. Recently, our research group has observed
the presence of actin in urine samples of septic
patients in contrast, actin could not be detectedin urine specimens from healthy individuals(27). Urinary actin (u-ACT) levels were determined by quantitative western blot, as in serum.
Significantly higher urinary actin was measured
in samples of patients with sepsis-related acutekidney injury (AKI, Figure 2B) compared to nonAKI patients (8.17 (2.09-45.53) ng/mL vs. 4.03
(091-10.21) ng/mL). Dialyzed patients showed
extremely high u-ACT levels (36.02 (4.7-176.56)
ng/ml). U-ACT correlated significantly (p<0.01)
with kidney function markers (serum creatinine:
0.315, urinary albumin: 0.704) but no correlation was found with se-ACT levels (27).
Previously Kwon et al. found increased u-ACT
levels as predictors of kidney failure after ischemic injury in renal allografts (u-ACT/u-Cr were1095.6 ± 729.6 ng/mg in cadavers with sustained
acute renal failure and 355.0 ± 247.0 ng/mg in

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Figure 2

Serum and urinary actin levels in septic patients

Based on (26), (27).

cadavers recovering from acute renal failure;p<0.05) (28). However the appearance of actin
in urine has not been clarified, u-ACT excretion
may reflect overall cellular damage in the kidneys, thus it might provide novel possibility forearly diagnosis of AKI, which is the most severe
complication of sepsis.
ACTIN-BINDING PROTEINSIn order to protect the body from overwhelming
actin toxicity, there are two major extracellularactin-binding proteins called gelsolin (GSN)and Gc-globulin (group specific component,
also called vitamin D-binding protein) (Figure3). Both plasma proteins are essential actin
scavengers working in concert. GSN severs anddepolymerizes actin filaments originating from
disrupted cells, and Gc-globulin frees GSN fromactin monomers and sequesters them. The
bound actin filaments and monomers are finally
cleared from the circulation by the reticuloendothelial system (31). Furthermore, both GSN
and Gc-globulin could modulate inflammatory
processes. Under physiological conditions, theconcentration of actin in the blood is far lessthan that of actin binding proteins. Interestingly,
in case of severe systemic inflammation, due toexcessive tissue injury the excessive amount of
extracellular actin and the pro-inflammatory
mediators exceed the binding capacity of the
scavenger proteins, so the plasma concentrationof these drops significantly (25). Both actinbinding proteins are cleared from the circulationby the reticulo-endothelial system, however
urinary levels of them are also studied (31).
GELSOLIN
Gelsolin is a ubiquitous, multifunctional protein.
Three different isoforms exist in humans, twocytoplasmic forms and one circulatory isoform(32). Circulatory GSN is mainly secreted by muscle tissue (26). Circulatory GSN is a 93 kDa Ca2+dependent protein and its plasma values range
between 190-300mg/L (but these are highly
method-dependent) (31,32). Besides actin, plasma GSN may also be able to bind to bioactive
molecules (lysophosphatidic acid, sphingosine
1-phosphate, fibronectin and platelet activating

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Nonconventional markers of sepsis

Figure 3

Crystal structures of calcium-free human gelsolin
and that of uncomplexed Gc-globulin

A: GSN consists of six domains (G1-G6) indicated by different colors. In the Ca-free, inactive form of GSN, the six similarly
folded domains adopt a compact globular structure held together by extensive noncovalent interactions of G2 with bothG6 and the C-terminal tail (29).
B: Gc-globulin is built up of 3 three homologous α-helical domains. Domains I and II can be subdivided further into two
structurally related subdomains (30).

factor), pro-inflammatory mediators and bacterial wall components (lipoteichoic acid andlipopolysaccharides).

of GSN have been proven by administration of exogenous gelsolin to rodents with septicemia andsevere injury yielding reduction in mortality (37).

In follow-up studies, first-day GSN levels wereproven to have a significant distinguishing ability regarding the septic and the non-septic statesfurthermore, GSN also predicted the outcome ofsepsis (26, 34-36). Non-survivor septic patients
showed lower levels of serum GSN (Figure 4).
Recently, our research group introduced a newpromising marker besides GSN, the serum actin/
GSN ratio (derived from the same patients’ actin and GSN levels) which had similar prognostic
value as APACHE II clinical scores regarding intensive care unit mortality (26). One limiting factor isthe lack of a rapid detection method for actin and
GSN, which is the current focus of our research.

Studies regarding urinary GSN (u-GSN) levels insepsis have been scarcely performed. Ferreia
et al. (38) described u-GSN as a discriminating
protein regarding cisplatin- and gentamicininduced AKI in rats. Another study of Maddens
et al. (39) reported increased u-GSN levels in
septic mice. Both of these observations basedon Western blot analyses indicated that u-GSN
originates from the blood by glomerular filtration. In addition, u-GSN seems to be a possible
diagnostic marker in patients suffering fromtype I diabetes mellitus (40). Interestingly, decreased u-GSN levels were found in rheumatoid
arthritis patients (41), however they did notoffer any predictive value. So far, all studies regarding u-GSN are promising starting points andshould be further validated.

Higher plasma GSN levels seem to have good prognostic value in sepsis, moreover the protective role

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Nonconventional markers of sepsis

Figure 4

A: First-day serum GSN levels in septic survivor and non-survivor patients
based on 7-day mortality
B: Receiver operating characteristic curve of serum GSN for predicting
7-day mortality of sepsis

AUC: 0.74, cut-off value: 11.38 mg/L (sensitivity: 76.2%, specificity: 72.7%). Based on (26).

GC-GLOBULIN
Plasma Gc-globulin (52 - 59 kDa) is a memberof the albuminoid superfamily. Gc-globulin ismainly produced by the liver (serum level: 300600 mg/L) owning 3 major isoforms (Gc1f, Gc1s,
Gc2) (42). Gc-globulin seems to act as an acutephase protein after injury. Also, important function of Gc-globulin is binding and transporting
25-OH-D and 1,25-(OH)2D3 vitamin metabolites.
Furthermore it enhances neutrophil chemotaxis and could modulate T cell responses (42).
Admission plasma concentration of Gc-globulin
below 134 mg/L (determined by immune nephelometry) was found to be associated with organ
dysfunction (hematologic or respiratory failure)and sepsis after traumatic injury (43). Jeng et al.
found an association between critical illness andlower 25-OH-D and Gc-globulin levels in critically illpatients when compared to healthy controls (44).

Gc-globulin is filtered freely through the glomeruli because of its low molecular weight. In thekidney, Gc-globulin is involved in the vitamin
D biosynthesis process. Under normal circumstances, Gc-globulin is reabsorbed and catabolized by proximal tubular epithelial cells resulting
only in a trace urinary excretion (42). Therefore,
acute tubular injury is expected to result in exaggerated urinary Gc-globulin excretion. Recently,
urinary Gc-globulin (u-Gc-globulin) was reported as a promising novel biomarker of major
contrast material induced nephropathy-associated events (u-Gc-globulin/u-Cr in patientsdeveloping major adverse renal events (MARE)
vs. those without MARE were 125.68 ± 211.62
vs. 14.99 ± 38.10 ng/ml/mmol/l; p<0.001) (45).
Shoukry et al. have determined increased uGc-globulin levels by ELISA in diabetic patientsas an early diagnostic marker of diabetic nephropathy. Urinary Gc-globulin/u-Cr levels were

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more than 10 times higher in macroalbuminuric
patients compared to controls (1516.3 ± 228.6
ng/mg vs. 123.4 ± 28.2 ng/mg; p<0.001) (46).
Investigating the association between sepsisinduced acute kidney injury and Gc-globulin in
urine still remains an interesting challenge.
OROSOMUCOID
Orosomucoid (ORM) or α-1-acid glycoprotein is apositive acute phase protein. ORM is a 41-43kDa
heavily glycosylated protein (Figure 5) with several transport and immunomodulatory function(47). ORM has been described as part of the nonspecific defense system against excessive inflammatory response (48). ORM has anti-neutrophil
and anti-complement activity, it can inhibit apoptosis, macrophage activation, lymphocyte proliferation, superoxide generation, and platelet
aggregation as well (49). Its protective role was
Figure 5demonstrated also in several rodent models ofshock, inflammation and sepsis (50-52).
The normal orosomucoid concentration in human serum ranges between 0.5-1.2 g/L and it
can rise during acute and chronic inflammatory
diseases (53). In spite of the well-known fact that
serum orosomucoid (se-ORM) is a non-specificinflammatory marker, recently it has been described as a potential diagnostic and prognostic
biomarker of sepsis. Significantly higher levels
were found in sepsis than in SIRS and admission
se-ORM levels showed a good prognostic accuracy for sepsis mortality if combined with SOFA
score (AUC ROC: 0.878) (54).
ORM is also present in urine, but with much
lower concentrations than in serum, normally
ORM accounts for about 1-5 % of total proteinsin urine (<3 mg/L) (55, 56). Previous studies described slightly elevated u-ORM levels in

Crystal structure of human orosomucoid (alpha1-acid glycoprotein)

ORM contains a typical lipocalin fold with an eight-stranded beta-barrel. This structure is responsible for diverse
ligand-binding. Furthermore, ORM structure contains five N-linked glycosylation sites (47).

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diseases associated with chronic inflammatory activation, like autoimmune diseases,
diabetes mellitus and cancer (57-60). U-ORM
excretion can be elevated after acute inflammatory stimuli as well. Recently published datasuggest that u-ORM could be a promising
non-invasive marker for diagnosis of sepsis(61). About 100-times higher levels were
found in sepsis than in controls, and SIRS patients showed 10-fold higher u-ORM levels thancontrols. U-ORM was referred to urinary creatinine levels and a cut off value at 6.75 mg/mmolwith great sensitivity and specificity (94.7% and90.0%, respectively) has been described for diagnosis of sepsis. The diagnostic accuracy of uORM for sepsis (AUC ROC: 0.954) was similar to
PCT and higher than se-ORM. Furthermore, uORM levels correlated well with conventional inflammatory parameters. In this study, extremely
elevated u-ORM levels were found in septic patients with dialysis requirement (61). Anotherpaper demonstrated u-ORM above 40 mg/L as
an early predictor for acute kidney injury after cardiac surgery in children (AUC ROC 0.87).
Figure 6

U-ORM values were found to be strongly associated with severity of AKI (62).
In spite of the promising data, the exact mechanism of u-ORM elevation is not well explored.
Local renal processes due to systemic inflammation could play a crucial role, since extrahepatic
gene expression of ORM (leukocytes, endothelial cells, kidney, etc.) has been described (63).
Furthermore, glomerular and tubular dysfunction also may have a pivotal part.
U-ORM seems to be a more sensitive marker ofsepsis than se-ORM (Figure 6), providing clinically relevant information for real-time monitoring of inflammatory activation in a non-invasive manner.
CONCLUSION
The outcome of sepsis largely depends on early
diagnosis and the earliest possible beginning ofa consecutive adequate antibiotic therapy. For
definitive diagnosis, identification of pathogens
is still the gold standard however this approachquite often requires several hours or days leading to a delay in decision making.

Serum orosomucoid (A) and urinary orosomucoid (B) levels in sepsis

Urinary orosomucoid levels are referred to urinary creatinine and expressed in mg/mmol.
Based on (61).

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Therefore, measurement of fast responding
protein biomarkers of sepsis has gained a major
focus in the last decades. Unfortunately, mostof the protein biomarkers do not have proper
specificity even if they possess better sensitivity. For the assessment of overall tissue damage,
monitoring of the actin-scavenger system is apromising new entity. Urinary markers provide
a non-invasive tool for real-time monitoring of
septic processes. Orosomucoid determinationin urine might be a novel possibility for the early recognition of systemic inflammation. Since
sepsis is a heterogeneous clinical syndrome andnot a definitive disease a single marker alone
should never be satisfactory. Multi-marker approach and complex evaluation of the clinicalsigns and biomarkers should improve patientmanagement at the bedside.
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In this issue: Advances in the diagnosis of sepsis

Advances in the diagnosis of sepsis:
hydrogen sulfide as a prognostic marker
of septic shock severity
Miha Košir1, Matej Podbregar2,3

Emergency Department, University Medical Centre Ljubljana, Slovenia
Clinical Department for Anaesthesiology and Surgical Intensive Care,
University Medical Centre Ljubljana, Slovenia
3
Faculty of Medicine, University of Ljubljana, Slovenia
1

2

ARTICLE INFOABSTRACT

Corresponding author:
Prof. Matej Podbregar
Clinical Department for Anaesthesiology
and Surgical Intensive Care
University Medical Centre Ljubljana
Ljubljana, Slovenia
Phone: +38 64 021 5960
E-mail: podbregar@guest.arnes.si

Hydrogen sulfide (H2S) is a third known gasotransmitter. Most of the time it was knows as a poisonous gas.
In last 30 years, we are seeing change in its perception.
Scientists have discovered its major role in different organ systems. It is endogenously produced in various tissues and its production is influenced by many factors.
In normal, physiological conditions only 20% of H2S isin its free form. The role of H2S is very wide. It acts as asignaling molecule, has influence on vascular tone, inflammatory response, scavenges reactive oxygen species, can be cytoprotective and can even reduce theextent of myocardial ischemia. Different studies haveshown H2S has considerable influence in pathology ofsepsis and its outcome. High free plasma levels of H2S
are predictor of unfavorable outcome. Findings showthat moderate free plasma levels of H2S have protective effect. Paradoxical very low free plasma levels of
H2S, seen in patients with chronic heart failure, are also
predictor of severity of disease and poor outcome. We
presume that relationship between morbidity/mortality and concentration of H2S has a wide U-shape
curve dependence. New researches with discoveryof H2S agonists and antagonists could open new ways
in understanding different pathologies and ability to

Key words:
hydrogen sulfide, shock, sepsis

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Miha Košir, Matej Podbregar
Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

treat them. Recent advances in the identification of H2S agonists and antagonists may help in
forwarding our understanding of pathomechanisms and hence their treatment.


INTRODUCTION
Hydrogen sulfide (H2S) is a long-known substance.
In normal conditions, it is a gas with a very
characteristic odor of rotten eggs (1,2). In the
16th century it was the cause to eye inflammation and bacterial infection in sewer workers.
The toxic effect of H2S is in its ability to bind tocytochrome C oxidase. As such, inhibiting the mitochondrial respiratory chain and consequently
inhibiting cell energy production. A change inperspection came in 1989, when H2S was detected in mammalian brain. At the end of the century it was discovered that hydrogen sulfide has
ability to modulate vascular tone, neuronal function and also has cryoprotective abilities during
ischemia (3). With these discoveries, the hydrogen sulfide, beside nitric oxide (NO) and carbon
monoxide (CO), became a member of so called
gasotransmitters (4). In the last decade, numerous studies have investigated the role of H2S atdifferent diseases, ranging from chronic heart
failure to different types of shock. In one of thisstudies, Goslar and colleagues demonstrated
that total plasma sulfide is a marker of shock severity in non-surgical, critically ill adult patients
admitted into the medical ICU (5). Other animaland human studies confirm their findings.
ENDOGENOUS PRODUCTION
AND METABOLISM OF H 2S
To date we know four different pathways of H2S
production (Figure 1). Two of them are pyridoxal
5’-phosphate (vitamin B6) dependent enzymatic
pathway with cystathionine β-synthase (CBS)and cystathionine γ-lyse (CSE). The substrate
in both cases is L-cysteine (6). CBS is the main

H2S synthase in the nervous system, but is alsopresent in kidney, liver, brain, ileum, uterus, placenta and pancreatic islets. The production of
H2S is suppressed by NO and CO and enhanced
via CSB by S-adenosyl methionine. CSE is widely
present in different peripheral organs (kidney,
liver, thoracic part of aorta, ileum, portal vein,uterus, pancreatic islets, placenta), but it was
not detected in the brain. The activity of CSE isregulated by concentration of Ca2+ (7).
The third pathway consists of two enzymes, cysteine aminotransferase (CAT – aspartate aminotransferase) and 3-mercaptopyruvate sulfurtransferase (3-MST). This way of producing H2S
is present mainly in mitochondria. The base ingredient is L-cysteine. The activity of both enzymes is presumed to be suppressed under oxidative stress conditions, like in mitochondria.
Other way to regulate the production of H2S via
CAT/3-MST is with Ca2+ ions. In the absence ofCa2+ the production of H2S is maximal and lowered in concentration depended manner (6,7).
The fourth way of H2S synthesis is synthesis fromD-cysteine via D-aminoacid oxidase (DAO) and
3-MST. This is happening in kidney and cerebellum. D-cysteine is not endogenous produced molecule and can be only provided from food – exogenous pathway. Because of this, it is thought, that
D-cysteine has a therapeutic potential to increase
H2S production in cerebellum and kidney (6,7).
As already mentioned H2S can stop (inhibit)
mitochondrial respiratory chain by inhibiting
cytochrome c oxidase. This happens when H2S
concentration is high. Similar happens with NO,
CO and cyanide. On the other side, it is very interesting that it can also be enzymatically metabolized in mitochondria and as that can be asource for generating ATP. This can only happen
in very low concentrations (less than 10 μM) of
H2S. In that conditions, it also increases mitochondrial O2 consumption and ATP production.
High concentrations have opposite effect (6).

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Miha Košir, Matej Podbregar
Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

Figure 1

Pathways of endogenous production of H2S (simplified scheme)

Legend
CSE: cystathionine γ-lyse; CSB: cystathionine β-synthase; CAT: cysteine aminotransferase;
3-MST: 3-mercaptopyruvate sulfurtransferase; DAO: D-aminoacid oxidase

PHYSIOLOGICAL ROLE OF H 2S
Hydrogen sulfide is known to have a large numberof pharmacological effects in various cell types
and tissues. Its effect mostly depends on plasma
level of H2S. Higher plasma levels have pros andcons, which will be described in following lines.

Also, low levels, below than normal present inhealthy individual, can indirectly predict increased
morbidity and mortality. This was shown in study
by Kovačić and colleagues where they have demonstrated that total plasma sulfide in patients withchronic heart failure (CHF) vary with NYHA stages.

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Miha Košir, Matej Podbregar
Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

Lowest sulfide levels were present in NYHA classIV (2.67 [2.22-4.31] μM) compared to NYHA classII (5.84 [4.33-8.00] μM), along with negative correlation with pro-BNP and pulmonary arterysystolic pressure (8). High levels are often associated with cytotoxic mechanisms, which are associated with generation of free radicals, depletionof glutation, release of intracellular iron and proapoptotic actions (8,9). Based on different data
we can assume, that morbidity/mortality, among
other factors, depends also on plasma concentration of hydrogen sulfide. Correlation can be illustrated with wide U shape curve (Figure 2).
Because of the topic of the article we will focus
on the cytoprotective and vascular effects (dysfunction). This is crucial to understanding theH2S effect during septic shock.
Figure 2

Cytoprotective effectEvidence shows that H2S has wide cytoprotective effect in various tissues and organs. Low
levels of H2S are presumed to exert antiapoptotic and antinecrotic mechanisms (9). In nervous
system, it increases the levels of glutathione (amajor intracellular antioxidant) and, as such,protects cells from oxidative stress. Similar effect is seen in kidney and heart (7). During
heart ischemia the energy levels drop. Because
there is lack of oxygen the anaerobic respiration
starts and lactic acid starts to accumulate. The
consequence is reduction of intracellular pH(10). Na/K-ATPase cannot function and there isaccumulation of sodium in the cell. This leads
to reversal of Na/Ca antiporter and intracellularand mitochondrial calcium accumulation. At theGraph presenting allegedly correlation between total plasma sulfide
and morbidity/mortality

CHF: chronic heart failure

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Miha Košir, Matej Podbregar
Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

reperfusion, oxygen delivery restores, but the
respiratory chain complexes are in reduced
state and a large quantity of reactive oxygen
species is observed. All these events lead topromotion of cell death by necrosis (11). Withstudies where endogenous production of H2S
was enhanced or exogenous H2S donors wereadministered, scientists observed that H2S was
successful in attenuating myocardial infarction
following ischemic-reperfusion injury, promoted angiogenic responses and inhibited fibrosis
during heart failure. H2S also acts as a ROS scavenger (7). A study by Elrod JW et al. from 2007
demonstrated that exogenous administrationof H2S or overexpression of CSE, which enhance
H2S production, reduced myocardial infarct sizeand preserved left ventricular function after
ischemic-reperfusion injury. H2S can reduce mitochondrial respiration to induce a state known
as a “suspended-animation” in which cellularrespiration and oxygen demand are reduced.
Consequently, the oxidative stress is reduced
and mitochondrial function is preserved (3).
Vasodilatory effectHydrogen sulfide has also vasodilatory effect,
which is concentration-dependent. H2S induces
the relaxation of vascular smooth muscle via activation of KATP channels. The consequence is hyperpolarization of the cell and the effect is relaxation of the cell (4). Although the effect of H2S is
not only dependent on KATP channels. Its function
can also be modified by local oxygen concentration (5). High expression of KATP channels leads tovascular dysfunction (hypotension) during sepsis.
One other pathway has also been demonstrated
to induce vasodilatation with H2S. H2S can also
increase intracellular concentration of cyclic adenosine monophosphate (cAMP). This causes
activation of protein kinase A and relaxation ofsmooth muscle (12).

Evidence suggest that there is a synergistic action of NO and H2S in the modulation of vasculartone (13). H2S can activate nitric oxide synthaselocated on endothelia (eNOS) and augment
NO bioavailability (3). NO donor was shown toincrease the expression of CSE and by that increase the conversion of L-cysteine to H2S (14).
HYDROGEN SULFIDEAND ITS ROLE IN SEPTIC SHOCK
By definition, since 2016, sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Septic shockshould be defined as a subset of sepsis in which
particularly profound circulatory, cellular, andmetabolic abnormalities are associated with agreater risk of mortality than with sepsis alone(15). During this time, many endogenous factors contribute to development of symptoms
we usually see in septic patient. There is substantial production of cytokines, eicosanoids,
reactive oxygen species and nitrogen species.
All this overproduction leads to severe systemic
inflammatory response with hypotension andvital organs hypoperfusion and the end result
can be multiorgan failure. Secondary to that
there is also loss of vascular responses to endogenous and exogenous catecholamines.
In 2011, Goslar and colleagues reported thatduring the state of shock plasma sulfide values
are very elevated. Based on our results it canbe concluded that total plasma sulfide is equivalent to lactate as a predictor of ICU survival.
As described above we confirmed the findingsthat H2S is involved in regulation of vasculartone and the association between plasma H2S
concentration and extent of tissue damage.
Higher total plasma sulfide was inversely correlated with blood pressure and cardiac function.
Patients with higher total plasma sulfide during
ICU admission had higher mortality comparedto patients with lower levels. According to ourPage 138

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Miha Košir, Matej Podbregar
Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

findings an increase in total plasma sulfide of
1μM resulted in a 5,8% higher probability of
ICU mortality (5). In 2008, Zhang found that endogenous production of H2S is time-dependent
during sepsis. Over production was only present in early stages and peaked 4-8 hours after
CLP (coecal ligation and puncture) (16).
Correlation between plasma H2S levels and sepsis was also shown in a different study by Zhang
and coworkers, where they showed significant
increase in plasma H2S levels in mouse models
of LPS (bacterial endotoxin lipopolysaccharide)
induced sepsis along with increased CSE gene
expression and activity of the enzyme (17).
This increase in gene expression, enzymatic
activity and consequently H2S levels was reduced with administration of dexamethasone.
Dexamethasone also reduced activity of inducible nitric oxide synthase (iNOS) (18).
The importance of H2S in pathogenesis of sepsis
is also reported in studies where propargylglycine (PAG), a CSE inhibitor, was administered.
There was a significant decrease in plasma H2Slevels with better survival rates. Furthermore,high H2S levels during sepsis may be responsible
for hemodynamic collapse in septic shock (4).
Increased endogenous production of H2S starts
in the acute phase of shock. During septic shock,there is also increase of NO levels because of
upregulation of iNOS. This higher NO levels can
induce CSE expression, which leads to higher
H2S levels – a positive feedback loop (4). Further
studies are needed to elucidate the role of H2Ssynthesis, where it may mediate inflammationor an anti-inflammatory mechanism.
Contrary to the data above there are some reports which are suggesting that higher (probably moderately high?) H2S levels act as cytoprotective and may prevent multiorgan failure (4).
It is known that H2S acts as a ROS scavenger invitro. The reduction of oxidative stress can be

result of inactivation of ROS producing enzymes
or by direct “rummaging” of ROS (4, 19).
H2S can also act as an endogenous modulatorof inflammation. As already described duringsepsis there is an increase in H2S production.
Inhibition of H2S production decreased systemic inflammatory response and reduced multiorgan failure in sepsis. Suggesting that H2S has
proinflammatory properties (16). Leukocytes
and their interaction with endothelium havea main role in pathogenesis of sepsis. Studies
utilizing H2S donors (sodium hydrogen sulfide
and sodium sulfide) showed reduced infiltration and adherence of leukocytes to the endothelium through the activation of KATP channels.
H2S donors also reduced edema formation.
Suppression of endogenous H2S production
have the opposite effect. Suppression can beachieved with NSAID, which reduce expressionof CSE (14).
MEASUREMENT OF ENDOGENOUS H 2S
AND ITS DIFFICULTIESThere are several methods to measure sulfide
concentrations in live biological systems – head
space gas analysis, derivatization methods(penta­fluorobenzyl bromide or N,N-dimethylp-phenylenediamine sulfate), spectrophotometry, direct measurement in solution with asilver sulfide or polarographic sensor and mostrecently developed colorimetric system using asilver-embedded Nafion/polyvinylpyrrolidone(PVP) membrane (20, 21). Different methods
give very variable results and there is no consensus which method is the most accurate andbest represent the true value of sulfide in living
system. Direct and precise measurement in living cells and fluids still remains a challenge.
Sulfide is naturally subjected to oxidation, it
has lipophilic property, is slightly soluble in water and acts as a weak acid. Beside free plasma
H2S there are also other forms of sulfide in the

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Advances in the diagnosis of sepsis: hydrogen sulfide as a prognostic marker of septic shock severity

body – mostly sulfane sulfur (elemental sulfur,
thiosulfate, persulfide, thiosulfonate, polysulfides, polythionates) and acid-labile sulfur. The
latter is mostly present in iron-sulfur clusters inproteins. The level of H2S is also influenced by
pH and temperature – at physiological pH and
37°C, around 20% of sulfide is present as H2S,
and if the temperature drops to 25°C 40% of
sulfide is present as H2S. Main problem today is
how to measure the gaseous form of H2S (5, 20,
21). Measurement of plasma H2S is still a challenge and new methods have to be developed.
Most widely used method is indirect measurement of H2S in plasma. In this case plasma is
mixed with distilled water, trichloroacetic acid,zinc acetate, N,N-dimethyl-p-phenylenediamine
sulfate in 7.2 M HCl and FeCl3 in 1.2 M HCl. This
results in a blue color reaction – methylene blue
formation. The supernatant is then spectrophotometrically measured. The calibration curveof NaHS (3.125-100 μM) or Na2S (0.699-139.86μM) are used to calculate the sulfide concentration (5, 16, 21).
A recently developed method enables selective detection of H2S in living cells using a silver
embedded Nafion/polyvinylpyrrolidone (PVP)
membrane and a colorimetric detection method. Silver and H2S form Ag2S which is brown incolor. Then absorbance at 310 nm is measured(22).
With gas chromatography, we are able to detect
sulfide at physiological levels, but it can falsely
rise sulfide levels by liberating loosely-boundsulfide because of irreversible sulfide binding or
shifts in phase transition equilibria (21).
Hartman and Dcona have described a methodfor direct measuring of H2S. It is based on conversion of profluorescent 8-azidopyrene-1,3,6trisulfonic acid (N3-PTS) to 8-aminopyrene1,3,6-trisulfonic acid (APTS) by H2S. Then the
fluorescence at 435 nm is measured (23).

CONCLUSION
The knowledge about role of hydrogen sulfide
in sepsis is still fragmental. High endogenous(over)production of H2S during sepsis has multiple implications. It contributes to smoothmuscle cell dysfunction, which leads to hypotension. This causes circulatory failure, myocardial dysfunction, organ injury and at the end
multi organ failure. Contrary to all negative effect, higher H2S production also has some beneficial effects. It improves endothelial function,
prevents adhesion of leukocytes and thrombocytes, stimulates the host defense system.
H2S acts as reactive oxygen species scavenger
and reduces oxidative stress, preserves mitochondrial function consequently leading to lessapoptotic cells. H2S has U-shaped effects curve;
in moderate elevation of H2S production during(early stages of) sepsis can be beneficial in termof host defense and other protective effect; on
the other hand, the extensive overproduction
has harmful effects.
All physiological stimulations which influence
H2S production and maintain a certain level ofsubstance in the tissue are not known. Next
step can be discovering and synthesis of H2S agonists and antagonists, which will influence thepathogenesis of several diseases where the roleof H2S apparently has been implicated.
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3. Polhemus DJ, Calvert JW, Butler J, Lefler DJ. The cardioprotective actions of hydrogen sulfide in acute myocardial infarction and heart failure. Scientifica (Cairo).
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4. Coletta C, Szabo C: Potential role of hydrogen sulfide in
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5. Goslar T, Marš T, Podbregar M. Total plasma sulfide asa marker of shock severity in nonsurgical adult patients. Shock. 2011; 36(4): 350-5. doi: 10.1097/SHK.
0b013e31822bcfd0.
6. Bełtowski J. Hydrogen sulfide in pharmacology andmedicine - An update. Pharmacol Rep. 2015; 67(3): 64758. doi: 10.1016/j.pharep.2015.01.005.
7. Kimura H. Signaling Molecules: Hydrogen Sulfide andPolysulfide. Antioxidants & Redox signaling. 2015; 22(5):362-76. doi: 10.1089/ars.2014.5869
8. Kovačić D, Glavnik N, Marinšek M. Total plasma sulfide
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9. Jha S, Calvert JW, Duranski MR, Ramachandran A, Lefer
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13. Coletta C, Papapetropoulos A, Erdelyi K, Olah G, Módis K, Panopoulos P, et al. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium dependent vasorelaxation. Proc
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14. Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S,
Cirino G, Wallace JL. Hydrogen sulfide is an endogenous
modulator of leukocyte-mediated inflammation. FASEB J.
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Consensus Definitions for Sepsis and Septic Shock(Sepsis-3). JAMA. 2016; 315(8): 801-10. doi:10.1001/
jama.2016.0287.
16. Zhang H, Moochhala SM, Bhatia M. Endogenous hydrogen sulfide regulates inflammatory response by activating the ERK pathway in polymicrobial sepsis. J Immunol. 2008; 181(6): 4320-31.
17. Zhang H, Zhi L, Moore PK, Bhatia M. Role of hydrogen sulfide in cecal ligation and puncture-induced sepsis
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290(6): 1193-201. doi: 10.1152/ajplung.00489.2005
18. Li L, Whiteman M, Moore PK. Dexamethasone inhibits lipopolysaccharide-induced hydrogen sulfide biosynthesis in intact cells and in an animal model of endotoxic shock. J Cell Mol Med. 2009; 13(8B):2684-92. doi:
10.1111/j.1582-4934.2008.00610.x.
19. Suzuki K, Olah G, Modis K, Coletta C, Kulp G, GeröD, et al. Hydrogen sulfide replacement therapy protects
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20. Wintner EA, Deckwerth TL, Langston W, Bengtsson A,
Leviten D, Hill P, et al. A monobromobimane-based assay to measure the pharmacokinetic profile of reactive
sulfide species in blood. Br J Pharmacol. 2010; 160(4):941–57. doi: 10.1111/j.1476-5381.2010.00704.x.
21. Shen X, Kolluru GK, Yuan S, Kevil CG. Measurement of
H2S in vivo and in vitro by the monobromobimane method. Methods Enzymol. 2015; 554:31-45. doi: 10.1016/
bs.mie.2014.11.039.
22. Ahn YJ, Lee YJ, Lee J, Lee D, Park HK, Lee GJ. Colorimetric detection of endogenous hydrogen sulfide production
in living cells utilizing silver-embedded polymer membrane. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2017; 177: 118-24. doi: 10.1016/j.
saa.2017.01.040
23. Hartman MCT, Dcona M. A new, highly water-soluble,
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A validation study of after reconstitution stabilityof diabetes: level 1 and diabetes level 2 controls
Shyamali Pal

R B Diagnostic Private Limited, Lake Town, Kolkata, India

INFO

ABSTRACT

Corresponding author:
Shyamali Pal
R B Diagnostic Private Limited
P- 857, Block- B, Lake Town
Kolkata 700089
India
E-mail: shy23_pal@yahoo.co.in

Objective

Key words:
HbA1c, six sigma, decision charts,
total allowable error, HPLC
Limitation of the study:
Involvement of more participant laboratories
could produce better scope of assessmentof after reconstitution stability. But
laboratories using same commercial lot was
not available except one laboratory, thedata from which has been supplemented.
Ethical issues:
The laboratories involved in the study used
controls exclusively for validation study. For
regular use to validate patient results the
manufacturer’s instruction has been followed.

The after reconstitution stability of L1 & L2 is 7 days
when stored at 2°-8°c and tightly capped. The total
content of the vial is 500 µL and per test requirementis 5 µL. Hence, in 7 days laboratories would consume
only 35 µL wasting 365 µL which is 73% of the expensive control samples and such wastage should be ideally prevented. The study of after reconstitution stability proved the stability of the control samples up to
90 days resulting in proper utilization of L1 & L2.
Materials and methods
The L1 & L2 controls were reconstituted using 500µL deionized water. The vials were allowed to standfor 5-10 minutes, swirled 8-10 times to maintain thehomogeneity. Aliquots of 10 µL were prepared. One
aliquot of one control level being used per day, bothlevels on alternate day. 5 µL control is mixed with 1.5
mL diluent and HbA1c was tested in D10 system by
HPLC method. Therefore, 500µL may be utilized up to
45 days approximately provided the extended use ofcontrol run is not affecting quality of test results. The
minimum number of samples tested by the laboratory from a single vial is 45 and maximum 46.
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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Results
Three lots were tested of which one lot has
been tested in two laboratories. Mean, SD,
CV%, TAE, %Bias, z-score and sigma calculations
were done. The medical method decision charts
were created for all lots based upon normalized
operational specifications which showed excellent precision in both control levels. Number ofrejections in the study was nil.
Conclusion
The extended use of controls is validated.
Abbreviations (in alphabetical order)
L1: Lyphochek Diabetic control level 1
L2: Lyphochek Diabetic control level 2
HbA1c: Glycosylated Hemoglobin
HPLC: High Performance Liquid
Chromatography
TAE: Total Allowable Error

and on 2009, Clinical and Laboratory Standards
Institute (CLSI) published the TAE of all clinical chemistry analytes (3). It has been recommended to use the CLSI table for TEA or to use
95% confidence interval of the limit of possible
analytic error whichever is appropriate/less (4).
If the result of reconstituted sample remains
within the range provided by manufacturer apparently it may be said that the after reconstitution period may be extended. To validate theextended stability after reconstitution, Sigma
should to be calculated and approved finallyfrom medical method decision chart (5, 6). The
medical method decision chart is based on sigma calculations and operating point ≥ 5 sigma is
the marker of excellence of performance.
MATERIALS AND METHODSINTRODUCTION
The IUPAC technical committee recommended
validation study in a single laboratory provided
the study is appropriate to the fitness of purpose and the validated document is continually
verified (1). The recommendation suggested
to estimate measurement trueness, recovery
and linearity. In the present study, the validation of stability of reconstituted analyte was inquestion. So, the number of rejections, CV% as
an indicator of measurement trueness, %bias
and z-score are the parameters to be estimated(2). As the values of the analyte have been provided in the manufacturers insert so TAE may
be obtained from the same. In 1974, Westgard,
Carey and Wold introduced the concept of TAE
to provide a quantitative approach for judging
the acceptability of method performance (2)Lyphochek Bilevel Diabetes Controls (L1 &L2)
were reconstituted using 0.5 ml deionized water. Calibrated auto pipette was used for reconstitution. The stopper was replaced and thecontrol samples were allowed to stand for 5-10
minutes. The vials were gently swirled 8-10
times before preservation in aliquots to maintain homogeneity. 10 µL reconstituted controls
were kept in each aliquot. The aliquots were
preserved at 2°C-8°C (7). One aliquot was takenout every day and 5 µL of reconstituted sample
was mixed in 1.5 mL diluent. The aliquot was
brought to room temperature and swirled before mixing with diluent. The L1 & L2 aliquots
were used on alternate day. Hence approximately 45 aliquots of one level control would
be consumed in 90 days. Three vials of Lot numbers 33870, 33890 and 33920 were tested for
90 days generating 135 numbers L1 & L2 datai.e., 135 number results generated per level per
lot. Lot 33920 have been tested in two different laboratories (incidentally common lot was
supplied) hence 135 number data of both levels
could have been accumulated from two laboratories. Both level control samples of same lot

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

were reconstituted and aliquoted on the same
day and time. HbA1c was measured in BIORAD
D10 system by Ion Exchange HPLC method. As
the objective was to establish after reconstitution stability of control samples hence method
and mode needed to be user specific and widely accepted.

and manufacturer’s target mean is expressed in% (8). When TAE is more than 20% as per CLSI
guideline 20% has been considered as optimum
TAE (4). From TAE, %Bias and CV% Sigma is calculated (TAE-%Bias/CV%). Method decision
charts were plotted on the basis of calculations
from Westgard Website.

STATISTICAL CALCULATIONSRESULTS AND DISCUSSION

Statistical calculations were as per the guidelineof Westgard et al (4). Mean, SD, CV%, %Bias of
every lot have been calculated. Bias is the difference of laboratory mean and Peer groupmean. The manufacturer’s mean of Lot 33870
were 5.4% (L1), 9.9 % (L2). The peer mean were
5.38% and 9.79%. The manufacturer’s mean
for lot 33890 were 5.3% and 9.8%. Peer mean
were 5.48 & 9.79. The manufacturer’s meanfor 33920 were 5.4 & 9.8 and peer mean were
5.42% and 9.84% respectively. Peer means
were used for statistical calculations as per
CLSI guideline and Westgard’s rule. TAE is thepercentage of total allowable deviation range.
The ratio of manufacturer’s allowable deviation

The after reconstitution stability was tested inthree consecutive lots 33870, 33890 & 33920.
The bias was calculated considering peer mean
as target mean. As per Multi QC rule ± 3SD
would be considered as acceptable allowableerror (TAE) keeping the option of one day 2-3SD
result in the warning range when the laboratory
uses its own laboratory mean [8]. In lot 33870
calculated TAE (from peer mean and SD) of L1
was 20.9 and in 33920 both level values were28.5 and 20. But as per CLSI guideline optimum
TAE should be 20% [Table 1]. So, instead 20.9 &28.5 the TAE was considered to be 20. No result
was found to have exceeded ± 2SD. Hence number of rejection/warning range result in three

Table 1

Control

HbA1c Peer results of lots 33870, 33890, 33920

Lot
numberLevel

Target (%)

Peer SD

Range(±3SDX2)

TAE (%)= Range x 100/Peer mean

Peer

InsertPeer

InsertPeer

InsertPeer

Insert

L1

33870

5.38

5.4

0.184

0.2

1.104

1.2

20.0

22.22

L2

33870

9.79

9.9

0.257

0.4

1.542

2.4

15.6

24.24

L1

33890

5.48

5.3

0.166

0.2

0.996

1.2

18.2

22.64

L2

33890

9.87

9.8

0.245

0.4

1.47

2.4

14.9

24.5

L1

33920

5.42

5.4

0.184

0.2

1.104

1.2

20.0

22.22

L2

33920

9.84

9.8

0.328

0.4

1.968

2.4

20.0

24.5

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Table 2

Statistical evaluation of HbA1c values of Lot 33870
Deviation
from
%
CV%
peer
Bias
mean

N

Mean
HbA1c
(%)

SD

182

5.47

0.127 2.32

0.09

Tests
per
vial

1.67

45+46

AllowAllowable Critical
Sigma z-score able
impre- error*
bias*
cision*

8.0

0.49

8.3

11.6

-0.641

6.3

-1.13

19

13

-1.911+46+45182

9.52

0.191 2.01

-0.27

2.96

45+46
+46+45*Allowable Bias- Normal Operational Specifications along Y-axis. Calculated as: %Bias*100/TAE (%)*Allowable Imprecision- Normal Operational Specifications along X-axis. Calculated as: CV%*100/TAE (%)
N- Number of tests performed
Critical error – Calculations obtained from “Quality Control Grid Calculator”. Critical error< ±2.0 shows excellent performance

Table 3

Statistical evaluation of Lot 33890
Deviation
from
%
CV%
peer
Bias
mean

N

Mean
HbA1c
(%)

SD

180

5.56

0.127 2.285

0.08

Tests
per
vial

1.46

45+45

AllowAllowable Critical
Sigma z-score able
impre- error*
bias*
cision*

7.3

0.48

8

12.5

-0.834

5.9

-0.82

13.6

14.7

-1.337+45+45
183

9.67

0.262

2.2

-0.20

2.02

46+46
+45+46lots was zero. The peer mean & SD were considered for TAE calculation as they are narrower
than manufacturer’s range (Insert value), basedon statistical calculation of worldwide results
and system and method specific.
Two lot results showed intra laboratory precision findings. After reconstitution stability of Lot
33870 & 33890 were tested using two L1 & two
L2 vials for 90 & 92 days. One aliquot tested onalternated day so 45 aliquots consumed within
90 days and 46 consumed by 92 days. The sigma
values were 8.0, 6.3 & 7.3, 5.9 [Tables 2 & 3].

Z-Score calculation is recommended for accuracy check of internal quality control [9] and inboth lots both level z-score are within ± 2 [Tables
2 & 3].
The inter performance testing of one lot has been
done in two laboratories with lot no. 33920. Bothlaboratory tested 3 vials of L1 & L2 for 90 days
obtaining total 270 results [Tables 4 & 5]. The
sigma values were above 6 and z-scores within
limit. The sigma and z-score of all three lots satisfies the criteria of standard performance.

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Table 4

N

Mean
HbA1c
(%)

135

5.49

Statistical evaluation of Lot 33920 (Laboratory 1)

SD

Deviation
from
%
CV%
peer
Bias
mean

Tests
per
vial

0.17

2.5

45+45

0.07

1.3

AllowAllowable Critical
Sigma z-score able
impre- error*
bias*
cision*

7.5

0.27

6.5

12.5

-0.569

9.2

0.39

6.6

10.2

-0.336

+45
135

9.97

0.203 2.04

0.13

1.32

45+45
+45

Table 5

N

Mean
HbA1c
(%)

135

5.55

Statistical evaluation of Lot 33920 (Laboratory 2)

SD

Deviation
from
%
CV%
peer
Bias
mean

Tests
per
vial

0.23

2.2

45+45

0.13

2.4

AllowAllowable Critical
Sigma z-score able
impre- error*
bias*
cision*

8.0

0.5

12

11

-0.923

8.2

1.03

17.2

10.1

-1.37

+45
135

10.18

0.206 2.02

0.34

3.45

45+45
+45

The use of normalized operation specification
chart (NOPSPECs) is being done from the early
1990s. The chart describes the operational limits for imprecision and inaccuracy with respect
to a desired level of quality assurance for theparticular analyte [10]. The chart is the medical
decision chart to select a QC procedure (11, 12).
The idea of the medical decision chart is to express the values on the X and Y axis as % of TAE.
The X-axis is plotted as imprecisionmeasure% TAE
i.e., percentage of allowable imprecision and Y
axis Bias measure% TAE or percentage of allowablebias of the method to be validated (Charts 1-8).
The charts may be downloaded from Westgards
website. Sigma would be automatically calculated from the input of TAE (%), CV% & % Bias.

The medical decision chart is based on CLSI
guideline by Westgard et al where maximum
allowable bias is 20% and CV is 10% resulting
2 sigma. Considering allowable Bias measure%TAE
20 the colour codes in the charts showed theless the imprecision (CV %) the higher is thesigma value (2-6, colour codes red –green). The
method is validated if sigma is ≥4, good if sigma
is ≥5, excellent when sigma is ≥ 6. The sigma operating points below 6 are beyond the level of
excellence as above 6 sigma no grade has beenprescribed yet. Calculations were described below Table 2 and followed for all three lots. The
operational specification chart for both controls in 3 groups were evaluated (Tables 1-5,
Charts 1-8). Analytical accuracy for all the assay

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

performance are within optimum excellence
decision range (Charts 1-8). Hence, the diabetic
controls may be used for 3 months instead of 7days after reconstitution.
As per the QC calculator rule critical error up to
2 indicates excellent precision. In the present
study, the critical errors of all the lots are < ± 2.
The stability of the reconstituted control havesatisfactorily passed both operating points andcritical error criteria. So, the utilization of diabetic controls up to 90 days is acceptable if reconstituted control material is kept in aliquot at 2°-8°C
and one aliquot is being used once (13, 14).
CONCLUSION
1. The use of diabetic controls of BIORAD
for extended days is validated and verified. The controls may be used for approximately 90 days provided they arepreserved properly.
2. The study is having an important practical application value. The laboratory has
saved 5-fold expenditure i.e. 1 control in
7 days, hence 6 control vials in 45 days
approximately. Such cost saving would
be beneficial for the patient care services as the laboratory would be able toperform HbA1c at a patient beneficial
rate. HbA1c is a prognostic marker ofdiabetes so the aim of the laboratories
should be to offer the test at a moderate cost.
3. Any waste should be discouraged. The
study showed prevention of waste of
expensive controls.

REFERENCES
1. Thompson M, Stephen L R E, Wood R. Harmonized
guidelines for single lab validation of methods of analysis (IUPAC technical report). Pure and Applied Chemistry;
Vol.74:p.835-55,2009.
2. Westgard J O, Carey R N, Wold S. Criteria for judging
precision and accuracy in method development and evaluation. Clin. Chem; Vol.20: p.825-33, 1974.
3. Clinical and Laboratory Standards Institute (CLSI). Estimation of total analysis error for clinical laboratory methods. CLSI EP 21-A-2003.
4. Westgard J O, Westgard S A. Total analytic error discussed in Clinical Laboratory News. Clinical Laboratory
News, AACC; Sept.p.8-10, 2013.
5. Dong – Hyun J, Kang Ki-Ju. A method for optimal material selection aided with decision making theory. Science
Direct; Vol.21.p.199-206, 2000.
6. Westgard J 0. A method evaluation decision chart(Medx Chart) for judging method performance. ClinicalLaboratory Science; Vol.8, p.277-83, 1995.
7. Kit Inserts, BIORAD, Lot numbers 33870, 33890, 33920.
8. Westgard J O, Klee G G. Quality management. Chapter16 in Fundamentals of Clinical Chemistry, 4th edition.
Burtis C Ed, WB Saunders Company, Philadelphia, 1996,
p.211-23.
9. Asian B, Mudjet A, Cevlik T, Bolayrli M, Emerk K. Uncertainty in proficiency testing schemes: KBUDEK experience. Clin. Biochem, vol.42, issue 5, p.321, 2009.
10. Westgard J O. Charts of Operational Process Specifications (OPSpecs charts) for assuring precision, recovery
and Quality Control needed to satisfy proficiency testing
performance criteria. Clin. Chem. 38/7, p.1226-33, 1992.
11. Westgard J O, Stein B. An automatic process for selecting statistical QC procedures to assure clinical or analytical quality requirements. Clin .Chem.43, 0. 400-3, 1997.
12. Westgard J O. Six sigma quality design and control:
Desirable precision and requisite QC for laboratory measurement processes. Madison, Wis: Westgard QC: 2001.
13. Westgard J O. Managing quality vs. measuring uncertainty in the medical laboratory. Clin Chem Lab Med.48,
p.31-40, 2010.
14. Westgard J O. Critical error grid calculator. Westgard
website: tools.westgard.com/qccalculator.html

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 1

Method decision chart of L1, 33870

Test or Analyte

HbA1c,33870,L1

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
20.0
Offset0.0
Method Performance
Bias (% diff)
1.7
Imprecision (% CV)
2.3
Sigma Metric
8.0

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.33
4.00
5.00
6.67
10.00

Medical Decision Chart

25.0

6 Sigma
5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )20.0

3 Sigma
2 Sigma
Operating Point

15.0

10.0

5.0

0.0

0.0

2.0

4.0

6.0

8.0

10.0

Allowable Imprecision (s, % )Page 148

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12.0

Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 2

Method decision chart of L2, 33870

Test or Analyte

HbA1c,33870, L2

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement

Dr.Shyamali Pal

Sigma Limits

S

Allowable Total Error

15.6

6

2.60

Offset

0.0

5

3.12

4

3.90

Method Performance
Bias (% diff)

3.0

3

5.20

Imprecision (% CV)

2.0

2

7.80

Sigma Metric

6.3

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 3

Method decision chart of L1, 33890

Test or Analyte

HbA1c,33890,L1

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
18.2
Offset0.0
Method Performance
Bias (% diff)
1.5
Imprecision (% CV)
2.3
Sigma Metric
7.3

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.03
3.64
4.55
6.07
9.10

Medical Decision Chart

20.0

6 Sigma

18.0

5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )16.0

3 Sigma

14.0

2 Sigma
Operating Point

12.0
10.0
8.0
6.0
4.0
2.0
0.0

0.0

2.0

4.0

6.0

8.0

Allowable Imprecision (s, % )Page 150

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10.0

Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 4

Method decision chart of L2, 33890

Test or Analyte

HbA1c,33890,L2

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
14.9
Offset0.0
Method Performance
Bias (% diff)
2.0
Imprecision (% CV)
2.2
Sigma Metric
5.9

s
2.48
2.98
3.73
4.97
7.45

Medical Decision Chart

16.0

Allowable Inaccuracy (bias, % )Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

6 Sigma

14.0

5 Sigma

12.0

3 Sigma

4 Sigma
2 Sigma

10.0

Operating Point

8.0
6.0
4.0
2.0
0.0

0.0

2.0

4.0

6.0

Allowable Imprecision (s, % )Page 151

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 5

Method decision chart of L1, 33920 (Laboratory 1)

Test or Analyte

HbA1c,33920,L1, Laboratory1

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
20.0
Offset0.0
Method Performance
Bias (% diff)
1.3
Imprecision (% CV)
2.5
Sigma Metric
7.5

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.33
4.00
5.00
6.67
10.00

Medical Decision Chart

25.0

6 Sigma
5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )20.0

3 Sigma
2 Sigma
Operating Point

15.0

10.0

5.0

0.0

0.0

2.0

4.0

6.0

8.0

10.0

Allowable Imprecision (s, % )Page 152

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 6

Method decision chart of L2, 33920 (Laboratory 1)

Test or Analyte

HbA1c,33920,L2,Laboratory 1

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
20.0
Offset0.0
Method Performance
Bias (% diff)
1.3
Imprecision (% CV)
2.0
Sigma Metric
9.2

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.33
4.00
5.00
6.67
10.00

Medical Decision Chart

25.0

6 Sigma
5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )20.0

3 Sigma
2 Sigma
Operating Point

15.0

10.0

5.0

0.0

0.0

2.0

4.0

6.0

8.0

10.0

Allowable Imprecision (s, % )Page 153

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 7

Method decision chart of L1, 33920 (Laboratory 2)

Test or Analyte

HbA1c,33920,L1,Laboratory 2

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
20.0
Offset0.0
Method Performance
Bias (% diff)
2.4
Imprecision (% CV)
2.2
Sigma Metric
8.0

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.33
4.00
5.00
6.67
10.00

Medical Decision Chart

25.0

6 Sigma
5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )20.0

3 Sigma
2 Sigma
Operating Point

15.0

10.0

5.0

0.0

0.0

2.0

4.0

6.0

8.0

10.0

Allowable Imprecision (s, % )Page 154

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Shyamali Pal
A validation study of after reconstitution stability of diabetes

Chart 8

Method decision chart of L2, 33920 (Laboratory 2)

Test or Analyte

HbA1c,33920,L2,Laboratory 2

AnalystMethodology

Ion Exchange HPLC

Date

Quality Requirement
Allowable Total Error
20.0
Offset0.0
Method Performance
Bias (% diff)
3.5
Imprecision (% CV)
2.0
Sigma Metric
8.2

Sigma Limits
6
5
4
3
2

Dr.Shyamali Pal

s
3.33
4.00
5.00
6.67
10.00

Medical Decision Chart

25.0

6 Sigma
5 Sigma
4 SigmaAllowable Inaccuracy (bias, % )20.0

3 Sigma
2 Sigma
Operating Point

15.0

10.0

5.0

0.0

0.0

2.0

4.0

6.0

8.0

10.0

Allowable Imprecision (s, % )Page 155

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12.0

Case report: A toddler with anasarca
caused by congenital nephrotic syndrome

Tumelo M. Satekge1, Olivia Kiabilua1, Gertruida van Biljon2, Komala Pillay3,
Tahir S. Pillay1,4
Department of Chemical Pathology1, Faculty of Health Sciences, University of Pretoria
and NHLS Tshwane Academic Division, Pretoria, South Africa
2
Department of Paediatrics, University of Pretoria and Steve Biko Academic Hospital, South Africa
3
Division of Anatomical Pathology, University of Cape Town and Red Cross Children’s Hospital and NHLS,South Africa
4
Division of Chemical Pathology, University of Cape Town, South Africa
1

INFO

ABSTRACT

Corresponding author:
Prof. Tahir S. Pillay
Department of Chemical Pathology
Faculty of Health Sciences
University of Pretoria
Arcadia, Pretoria, 0007
South Africa
Phone: 012-319-2114
E-mail: tspillay@gmail.com

Congenital nephrotic syndrome is a rare inherited
disorder arising from defects in the proteins of thecells in the glomerular basement membrane and develops either in utero or at birth. The clinical presentation is the result of massive protein loss in the urine
with associated compensatory mechanisms.

Key words:
congenital nephrotic syndrome,
proteinuria, generalised edema
Disclosures:
No conflicts of interest declared.
TS, OK and TSP conceived the idea andwrote and edited the manuscript. GvB was
the paediatrician managing the patient andcontributed to the manuscript. KP was theanatomical pathologist who analysed thebiopsies and contributed to the manuscript.

Here we present a clinical case of a female toddler
with a history of anasarca (severe generalised edema) from birth and who presents with the classicalbiochemical laboratory findings of nephrotic syndrome, together with the more pronounced featuresthat arise from protein loss including abnormal thyroid function testing and a marked hypercholesterolaemia. Renal biopsy indicated congenital nephrotic
syndrome of the Finnish type. This clinical-diagnostic
case report represents an example of the broad spectrum of pathophysiological findings of a severe congenital nephrotic syndrome.

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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

INTRODUCTION
Congenital nephrotic syndrome (CNS)
CNS manifests normally at birth to 3 months oflife and is associated with inherited defects in
one of the three components of the glomerular basement barrier namely fenestrated endothelium, glomerular basement membrane andpodocytes (1-3). Mutations in podocytes leadto congenital nephrotic syndrome of the Finnish
type (CNS-F) where podocyte foot processes
form filtration slits covered by the slit diaphragm(1-4). The responsible NPHS1 gene was the firstdescribed and encodes for nephrin, a type - 1
transmembrane protein of the immunoglobulin
superfamily and is a pivotal component of theslit diaphragm. Almost 50 mutations so far havebeen described in the NPHS1 gene and the autosomal recessive gene maps to 19q13.1 (4, 5).
CNS-F presents with massive non-selective proteinuria in utero allowing for prenatal screening (1). Other features include premature birth(35 – 38 weeks) with low birth weight and neonatal edema as they were present in this case.
Other mutations may occur in podocin (NPHS2)
and is associated with autosomal recessive,steroid-resistant focal segmental glomerulosclerosis (FSGS). Autosomal dominant FSGS canresult from TRCP6 and CD2AP mutations causing defects in the podocyte. Also, mutations
in Wilms´ tumor suppressor gene can occur in
the Denys-Drash syndrome (glomerulopathy
in combination with Wilms’ tumor and malepseudohermaphroditism). Although the common histological finding is mesangial sclerosis,
this diagnosis was excluded by the absence of
ambiguous genitalia and abdominal mass (2, 4).
CASE REPORTClinical features
A 15-month-old first-born female toddler presented with a history of generalised body swellingsince birth and neurodevelopmental delay. The

first presentation was at 2 weeks after full-term
normal vaginal delivery with low birthweight of
2.3 kg. The mother was HIV-positive during pregnancy and received anti-retroviral drugs. She reported unsatisfactory growth of the new-bornwith motor and speech delay. The child was fed
breast milk until 4 months and afterwards with
infant formula. On examination, she was below
the 3rd percentile for height and weight (weight
6.5 kg; length 64 cm and head circumference 44.5
cm). Despite having Harrison’s sulci, the child
was un-distressed with no dysmorphic features.
There were generalised edema and hypoplastic
teeth. Central nervous system examination revealed delayed social and motor milestones withno smile, reaching or sitting and decreased tone
and muscle power with normal reflexes. Basedon these clinical presentations, congenital nephrotic syndrome was suspected and specificlaboratory testing was performed and renal biopsy was done for immuno-histochemical workup to establish diagnosis.
Laboratory findings
The urine dipstick resulted with 3+ for proteinuria,
no signs of haematuria. Blood testing showed asignificant depressed C3 level of 0.638 g/L (reference
interval 0.9-1.8 g/L) and hypoalbuminaemia of
2.0 g/L (reference interval 27-43 g/L) indicating
nephrotic syndrome (NS). Whole blood countrevealed anaemia with haemoglobin of 8.0 g/L
and there was thrombocytosis with 656 x 10⁹/L.
Plasmatic coagulation tests were found within
normal ranges, as well as liver enzymes and renal
function testing and electrolytes. Total- and LDLcholesterol levels were markedly increased; TSH
and PTH were also significantly elevated together
with hypocalcaemia and hyperphosphatemia as
outlined in Table 1. Serologic testing for active
infections including hepatitis, toxoplasmosis,
anti-streptolysin-O titer, cytomegalo virus andsyphilis gave negative results, HIV-antibody ELISA
was negative and HIV-RNA was not detectable.

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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

Histological and immunochemical findings
A renal biopsy showed mesangial hypercellularity, focal microcystic dilatations of the tubules
and a mild interstitial inflammatory infiltrate(Figure 1A). 4+ foot process effacement and increased tubular lipid droplets were visualisedon electron microscopy. Immunofluorescence
displayed focal 1+ IgM mesangial positivity.

ig

Clinical course

1

After establishing diagnosis, optimal supportive
treatment including Enalapril p.o., intravenous
albumin, furosemide, low salt intake, high caloric- and protein diet were given along withcalcitriol and thyroxine. Poor compliance to thyroxine contributed to neurodevelopment delay.
Treatment was not effective and was hamperedby persistent proteinuria.
Unilateral nephrectomy was performed showing
massive lymphoplasmocytic inflammatory infiltrates, focal segmental glomerulosclerosis
with microcystic tubular dilatation, hypertensive
vascular changes and ascending pyelonephritis(Figure 1B). Unfortunately, renal function dramatically deteriorated with volume overload,
subsequent bacterial peritonitis and the patientdied from septic shock.

Figure 1A Renal biopsy histology showing mesangial hypercellularityand focal microcystic dilatations of tubules

A
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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

Figure 1B Left kidney histology post nephrectomy demonstrating interstitial
lymphoplasmocytic inflammatory infiltrates, microcystic dilatations oftubules, fibrosis and tubular atrophy

B
DISCUSSION
Generalised edema can result from renal disease, liver disease, allergic reactions, cardiac
disease, malnutrition or protein-losing enteropathy. In children, the most common cause isrenal disease. First, evaluation can commencewith simple tests such as urine dipstick andurine microscopy to identify proteinuria andcasts. In our case, edema is a hallmark of NS but
the pathogenesis is not fully understood (6, 7).
Selective loss of albumin leads to hypoalbuminemia and decreased capillary oncotic pressure
resulting in net fluid flux outwards and renal

losses not compensated with increased albumin
production in the liver. Sequestration of fluid in
the interstitium triggers renal sodium reabsorption and fluid retention to maintain intravascular volume and blood pressure balanced (the“underfill” hypothesis) (6, 7).
In NS associated with glomerulonephritis the extent of proteinuria is variable and a reduction in
GFR is common. Intravascular fluid volume is typically normal or expanded because of inappropriately stimulated sodium and fluid retention
together with decreased GFR results in an “overfill” state. Distinguishing between these two

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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

states is critical to the management of edema in
NS. Protein loss in the urine leads to further complications including hypogammaglobulinaemia.
In NS, there is increased risk of thromboembolism owing to increased pro-coagulants anddecreased anticoagulants (urinary loss of antithrombin III, plasminogen and protein S) (8). In
our patient, we found thrombocytosis which isone of the prothrombotic factors in NS; however the rest of the coagulation profile was unremarkable and there were no clinical signs ofa thromboembolic state. As part of nephrotic
syndrome workup to exclude secondary causes,
relevant infections particularly in the presenceof pre-natal HIV exposure were sought and testing was found negative for all pathogens tested.
The markedly elevated total and LDL cholesterol is probably due to over-compensated lipoprotein synthesis for reduced plasma oncoticpressure due to depleted albumin (9). This isassociated with an increased risk of cardiovascular events (9). The hypothyroid state in thenephrotic syndrome could also be a contributory factor to the hypercholesterolemia. The
elevation of Apo B containing lipoproteins is aprominent feature in nephrotic syndrome andin severe cases is accompanied by hypertriglyceridemia (9). Thyroid hormone alterations inCongenital Nephrotic syndrome of the Finnish
type (CNS-F) without renal impairment are well
documented (10). Thyroid binding globulin islost with proteinuria leading to low total T4 andT3. Correspondingly, free thyroid hormones are
expected to be normal or even elevated withnormal TSH. In severe CNS-F, as in this case, FT4
is lost in urine proportionally with massive proteinuria without commensurate compensationby the thyroid gland. This in turn stimulates the
hypothalamic-pituitary axis as demonstrated byhigh elevated TSH (10). Low FT3 in this case canbe explained by the catabolic nature of NS as a
non-thyroidal illness characterised by impaired
peripheral conversion of T4 to T3.

There was persistent hypocalcaemia with noother overt signs of rickets. She had poor neurodevelopment including motor milestones,
Harrison sulci and hypoplastic teeth. Vitamin
D-binding protein loss is a likely cause for
calcidiol depletion owing to its greater affinity for 25OHD 3. However, 1.25(OH)2D 3 and24,25(OH)2D3 can also be low in patients withnephrotic syndrome without renal impairment(11). This can be explained by depletion of the
precursor in the form of 25-OHD3 and to a lesser
extent to loss of binding protein in severe cases.
Tubular damage as a result of exceeded reabsorption of filtered protein can lead to destabilisation of 1α-hydroxylase resulting in low calcitriol level (11). It has been hypothesized that
corrected total calcium overestimates hypocalcemia; this was also evident in our case (Table1). Ionised calcium and vitamin D metabolites
were unfortunately not measured. Depleted total calcium was associated with an elevated PTH
on more than two occasions (Table 1) indicativeof secondary hyperparathyroidism leading tobone demineralization (11).
The histology was typical of CNS-F where immunofluorescence did not detect immune deposits. Microcystic changes of proximal and distal
tubules are common findings in CNS of Finnish
type, but it may also be present in other histological subtypes of NS. Mostly, CNS-F shows
slower progression even with marked proteinuria and renal function tests can be normal
early on. If signs of deterioration are present
despite optimal supportive treatment, bilateralnephrectomy, dialysis and subsequent transplantation should be the definitive approach toprevent further severe deterioration by reversing the biochemical abnormalities and hormonal disturbances with minimal risk of recurrence
after surgery (12). However, in our case a rapid
dramatic deterioration occurred with septic
shock leading to fatal outcome.

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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

Table 1

Laboratory testing
ResultReference interval

Urine
Protein

4.9 g/L

Creatinine

0.6 mmol/L

Protein/creatinine ration

8.17<0.015 g/mmol

Urea and electrolytes
Sodium141

136-145 mmol/L

Potassium

5.2

3.4-4.7 mmol/L

Chloride112

98-107 mmol/L

Urea

27.0

1.1-5.0 mmol/L

Creatinine

124

15-31 umol/L

Bicarbonate

13

23-29

Anion gap

21

9-16

Liver function tests
Total protein

2648-70 g/L

Albumin<10

27-43 g/L

Total Bilirubin

6

6-21 mmol/L

Direct Bilirubin

1

0-6 mmol/L

Alanine transaminase (ALT)11

2-25 U/L

Aspartate transaminase (AST)28

0-49 U/L

GGT

129

15-132 U/L

ALP

191

48-406 U/L

Calcium

1.66

2.12-2.59 mmol/L

Corrected calcium

2.39

2.19-2.64 mmol/L(low albumin)

Magnesium

0.92

0.7-0.99mmol/L

Phosphate

2.13

1.10-1.95 mmol/L

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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

TSH

>100

0.95-6.52mIU/L

FT4

5.8

7.6-16.1pmol/L

FT3

2.9

4.5-10.5pmol/L

PTH

103.4

1.3-9.3pmol/L

LipidsTotal Cholesterol

13.36 mmol/L

Triglycerides

6.55 mmol/L

LDL-Cholesterol

9.19 mmol/L

HDL-Cholesterol

0.68 mmol/L

Complete blood count
WBC

20.56

6-18x10⁹L

Hb

8.0

10.7-13g/L

MCV

93.3

70-86fl

Haematocrit

0.255

0.32-0.420

Platelet656

180-440x10⁹L

Iron

4.9

9.0-21.5 umol/LTransferrin<0.70

1.49-3.82 g/LFerritin

34

36-84 ug/L

TAKE HOME MESSAGES /LEARNING POINTS••

Anasarca in neonates requires a systemic
approach to rapidly establish the correct
diagnosis. Bedside testing with urine dipstick is an essential screening tool to guide
further investigations; a 3+ proteinuria on
dipstick is highly suggestive of nephrotic
syndrome to be confirmed by appropriate
laboratory work-up.

necessitate aggressive supportive treatment in preparation for definitive treatment with surgery for restoration of growthand development.
•

Always exclude secondary causes of congenital nephrotic syndrome because theytend to occur more frequent than primarycauses showing better treatment response.

•

Various mutations in CNS have been described; CNS-F resulting from nephrin genemutations is not uncommon worldwide andaffects both sexes equally. Genetic counselling and antenatal screening for congenital nephrotic syndrome - Finnish type with

Nephrotic syndrome is characterised byproteinuria, edema, hyperlipidaemia, hypoproteinaemia and may be associatedwith coagulopathy. Early diagnosis will
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Tumelo M. Satekge, Olivia Kiabilua, Gertruida van Biljon, Komala Pillay, Tahir S. Pillay
A toddler with anasarca caused by congenital nephrotic syndrome

haplotype analysis is often required andshould therefore be considered early on.

slit area and is characteristically spliced in the human kidney. Am J Pathol 1999;155:1681-7.

•

In congenital nephrotic syndrome, thyroid
hormones and Vitamin D status must be
evaluated.

6. Siddall EC, Radhakrishnan J. The pathophysiology of
edema formation in the nephrotic syndrome. Kidney international 2012;82:635-42.

•

Renal biopsy is mandatory in CNS to further
differentiate and support diagnosis and tobetter estimate the severity of clinical progression regarding renal failure.

REFERENCES
1. Spahiu L, Merovci B, Jashari H, Kepuska AB, Rugova BE.
Congenital nephrotic syndrome - finish type. Med Arch
2016;70:232-4.
2. Rapola J. Congenital nephrotic syndrome. Pediatric nephrology 1987;1:441-6.
3. Wang JJ, Mao JH. The etiology of congenital nephrotic
syndrome: Current status and challenges. World J Pediatr
2016;12:149-58.
4. Tryggvason K. Unraveling the mechanisms of glomerular ultrafiltration: Nephrin, a key component of the slit
diaphragm. J Am Soc Nephrol 1999;10:2440-5.
5. Holthofer H, Ahola H, Solin ML, Wang S, Palmen T, Luimula P, et al. Nephrin localizes at the podocyte filtration

7. Bockenhauer D. Over- or underfill: Not all nephrotic
states are created equal. Pediatric nephrology
2013;28:1153-6.
8. Mahmoodi BK, ten Kate MK, Waanders F, Veeger NJ,
Brouwer JL, Vogt L, et al. High absolute risks and predictors of venous and arterial thromboembolic events inpatients with nephrotic syndrome: Results from a large
retrospective cohort study. Circulation 2008;117:224-30.
9. Wheeler DC, Bernard DB. Lipid abnormalities in thenephrotic syndrome: Causes, consequences, and treatment. Am J Kidney Dis 1994;23:331-46.
10. Ito S, Kano K, Ando T, Ichimura T. Thyroid function inchildren with nephrotic syndrome. Pediatric nephrology
1994;8:412-5.
11. Nielsen CA, Jensen JE, Cortes D. Vitamin d status isinsufficient in the majority of children at diagnosis of nephrotic syndrome. Dan Med J 2015;62.
12. Slaughenhoupt BL, Lohrasbi FF, Harrison HL, Van Savage JG. Urologic management of congenital nephrotic
syndrome of the finnish type. Urology 1998;51:492-4.

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Editor-in-chief
Gábor L. Kovács
Institute of Laboratory Medicine, Faculty of Medicine, University of Pécs, Hungary

Assistant Editor 				Case Editor			Harjit Pal Bhattoa				Department of Laboratory Medicine		
University of Debrecen, Hungary		Reinhard B. Raggam
Department of Internal Medicine
Division of Angiology, University of Graz, Austria

Editorial Board
Khosrow Adeli, The Hospital for Sick Children, University of Toronto, Canada
Borut Božič, University Medical Center, Lubljana, Slovenia
Edgard Delvin, CHU Sainte-Justine Research Center, Montréal, Québec, Canada
Nilda E. Fink, Universidad Nacional de La Plata, Argentina
Ronda Greaves, School of Health and Biomedical Sciences, RMIT University, Victoria, Australia
Mike Hallworth, Shrewsbury, United Kingdom
Andrea R. Horvath, Prince of Wales Hospital and School of Medical Sciences, University of New
South Wales, Sydney, Australia
Ellis Jacobs, Alere Inc., New York, USA
Allan S. Jaffe, Mayo Clinic, Rochester, USA
Bruce Jordan, Roche Diagnostics, Rotkreuz, Switzerland
János Kappelmayer, Faculty of Medicine, University of Debrecen, Hungary
Evelyn Koay, National University, Singapore
Gary Myers, Joint Committee for Traceability in Laboratory Medicine, USA
Maria D. Pasic, Laboratory Medicine and Pathobiology, University of Toronto, Canada
Maria del C. Pasquel Carrera, College of Chemists, Biochemists and Pharmacists, Pichincha, Ecuador
Oliver Racz, University of Kosice, Slovakia
Rosa Sierra Amor, Laboratorio Laquims, Veracruz, Mexico
Sanja Stankovic, Institute of Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia
Danyal Syed, Ryancenter, New York, USA
Grazyna Sypniewska, Collegium Medicum, NC University, Bydgoszcz, Poland
Jillian R. Tate, Queensland Royal Brisbane and Women’s Hospital, Herston, Australia
Peter Vervaart, LabMed Consulting, Australia
Stacy E. Walz, Arkansas State University, USA

				

			

				
		
		

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