February 2019ISSN 1650-3414

Communications and Publications Division (CPD) of the IFCC
Editor-in-chief: Prof. János Kappelmayer, MD, PhD
Faculty of Medicine, University of Debrecen, Hungary
e-mail: ejifcc@ifcc.org

The
Journal of theInternational
Federation ofClinicalChemistry andLaboratory
Medicine

Volume 30 Number 1

In this issue

Call for manuscript submissions on “Flow cytometry”
Katherina Psarra

4

Call for manuscript submissions
on “Improving the preanalytical phase in laboratory medicine”
Gabriel Lima-Oliveira5

The irreplaceable value of laboratory diagnostics:
four recent tests that have revolutionized clinical practice
Giuseppe Lippi

7

Factors affecting turnaround time in the clinical laboratoryof the Kathmandu University Hospital, Nepal
Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal

14

Hemolysis interference studies: the particular case of sodium ion
José Antonio Delgado, Daniel Morell-Garcia, Josep Miquel Bauça

25

Prevalence of anemia and associated factors in hospitalized children
attending the University of Gondar Hospital, Northwest Ethiopia
Bamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria,
Ayenew Addisu, Meaza Genetu

35

Waist circumference cutoff point determination for defining metabolic syndrome
in type 2 diabetes mellitus in Ethiopia
Shewit Hailemariam, Tadele Melak, Molla Abebe

48

In this issue

Critical issues and new trends on stat tests in clinical laboratory
Ariadna Arbiol-Roca, Dolors Dot-Bach 

59

Diamond Blackfan Anemia: genetics, pathogenesis, diagnosis and treatment
Getabalew Engidaye, Mulugeta Melku, Bamlaku Enawgaw

67

Gaucher disease: an underdiagnosed pathology
in the Eastern Moroccan population
Ouardia Bouayadi, Amina Lyagoubi, Adnane Aarab, Somiya Lamrabat,
Abdelilah Berhili, Mohammed Bensalah, Rachid Seddik

82

A rare case of non-secretory multiple myeloma:
a case report and literature review
Mohammed Bensalah, Somiya Lamrabat, Amina Lyagoubi,
Adnane Aarab, Ouardia Bouayadi, Rachid Seddik

88Increased bleeding risk in a patient with oral anticoagulant therapy
and concomitant herbal intake – a case report
Paul Gressenberger, Peter Rief, Philipp Jud, Katharina Gütl,
Viktoria Muster, Leyla Ghanim, Marianne Brodmann, Thomas Gary

95

Unusually low serum alkaline phosphatase activity in a patientwith acute on chronic liver failure and hemolysis
Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta

99

This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Call for manuscript submissions
for a thematic eJIFCC issue on “Flow cytometry”
Guest Editor for the “Flow cytometry” issue: Katherina Psarra

Multiparameter Flow Cytometry (FCM) is a recent
technology of very high performance developed
for a vast variety of diagnostic applications.
Is there really a limit of future cytometry? Will
the evolution and transformations arrive at alimit or will they go on and on? Will the effort
be the one that really matters? We hope to offer you some insight into this bright future with
an eJIFCC issue dedicated to cytometry.
We invite you to submit a paper on “Flowcytometry” to be published in this thematic
issue.
That way the message will be carried on to fellow
laboratorians about the importance, the magicand the charm of this evolving technology.
Submitted papers will be reviewed according to
the regular procedure of the eJIFCC.

Type of articles•

Original Papers•

Critical Reviews•

Case studies

Manuscripts to be submitted by e-mail to:•

the Editor-in-Chief: ejifcc@ifcc.org•

with a copy to the Guest Editor:
kpsarra@outlook.com

Guest Editor
Katherina Psarra
Immunology – Histocompatibility Department
Evangelismos Hospital
Athens, Greece

Important deadlines•

Deadline for submission of the tentative title(to the Guest Editor): April 1st, 2019•

Deadline for submission of the manuscript:
May 31st, 2019

Page 4eJIFCC2019Vol30No1pp004-004

This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Call for manuscript submissions
for a thematic eJIFCC issue on “Improving
the preanalytical phase in laboratory medicine”
Guest Editor for the “Pre-analytical phase” issue: Gabriel Lima-Oliveira

The pre-analytical phase encompasses all theprocedures before the start of laboratory testing. This phase of the testing process is responsible for a great deal, possibly the majority of
the laboratory errors.
Diagnosis, management, treatment of patientsand ultimately patient safety itself can be compromised by:•patient preparation;•

patient posture;•phlebotomy quality;•

kind/type of evacuated tube used to draw
blood samples;•

sample centrifugation;•

sample transportation;•

sample contamination;•

time to analyze;•

sample storage;•

and more…

professionals and to seek to guarantee patient
safety.
The electronic Journal of the IFCC (eJIFCC) is aplatinum open-access journal, i.e. there is nocharge to read, or to submit to this journal.
Our numerous high-quality articles, debates,
reviews, case studies and editorials are addressed to clinical laboratorians. We aim toassist the development of the field of clinicalchemistry and laboratory medicine worldwide.
Manuscripts are fully peer reviewed and immediately free to access and download fromwww.ifcc.org.
Submitted manuscripts shall be reviewed normally, according to the regular procedures ofthe eJIFCC.
As Guest Editor, I would like to invite researchers from a wide range of disciplines to contribute to papers on recent and innovative research
on Pre analytical phase.
Important deadlines

We aim to prepare a special thematic issue of theeJIFCC, entitled “Improving the preanalyticalphase in laboratory medicine”, to be publishedin November 2019, to inform the laboratory•

Deadline for submission of the tentative title(to the Guest Editor): May 1, 2019•

Deadline for submission of the manuscript:
July 31, 2019

Page 5eJIFCC2019Vol30No1pp005-006

Gabriel Lima-Oliveira
Call for manuscripts submission on “Improving the preanalytical phase in laboratory medicine”

Types of articles

Guest Editor•

Original Papers•

Critical Reviews•

Case studies

Gabriel Lima-Oliveira, Ph.D.
Researcher, University of Verona, Italy
Chair, COLABIOCLI WG-PRE-LATAM
Expert/Consultant, EFLM WG-PRE

Manuscripts to be submitted by e-mail to:•

the Editor-in-Chief: ejifcc@ifcc.org•

with copy to the Guest Editor:
dr.g.lima.oliveira@gmail.com

Page 6eJIFCC2019Vol30No1pp005-006

This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The irreplaceable valueof laboratory diagnostics: four recent tests
that have revolutionized clinical practice
Giuseppe Lippi

Section of Clinical Biochemistry, University of Verona, Italy

ARTICLE INFOABSTRACT

Corresponding author:
Giuseppe Lippi
Section of Clinical Biochemistry
University Hospital of Verona
Piazzale LA Scuro
37134 Verona
Italy
E-mail: giuseppe.lippi@univr.it

There is a common perception that laboratory medicine may be occasionally perceived as neglected discipline by clinicians, and that laboratory tests may beconsidered ordinary commodities. Although there
is still debate on the real contribution of diagnostic
testing in care pathways, many clinical diagnoses
cannot be made without laboratory data. In supportof evidence-based added value of laboratory diagnostics, this article aims to discuss the over-reaching
contribution of some recent tests to the clinical decision making, and the unquestionable role they haveplayed in revolutionizing clinical practice. These paradigmatic tests include highly-sensitive cardiac troponin immunoassays for diagnosing non-ST elevation
myocardial infarction, hemoglobin A1c for diagnosis
and therapeutic management of diabetes, procalcitonin for diagnosing severe bacterial infections andimproving antibiotic stewardship, along with natriuretic peptides for early diagnosing and managing
heart failure. It is advisable that altogether theseparadigms will help reaffirming the vital role of laboratory medicine in modern healthcare.

Key words:
laboratory testing, diagnosis,
clinical practice

Page 7eJIFCC2019Vol30No1pp007-013

Giuseppe Lippi
The irreplaceable value of laboratory diagnostics

INTRODUCTION
Laboratory medicine is conventionally defined
as a science devoted to generate clinically useful information by analyzing the concentration,
composition and/or structure of analytes in biological fluids [1]. Throughout the relatively long
history of this discipline as we currently know
it, and which probably commenced around the19th century [2], laboratory diagnostics is now
providing an almost invaluable contribution to
the clinical decision making. Although there
is still an open debate on the real influenceof diagnostic testing in care pathways, as mirrored by fierce controversies on the reiterated
assumption that clinical laboratory intervenes
in 70% of clinical decisions [3], it is now incontestable that many clinical diagnoses cannot
be made without laboratory data. In supportof the evidence-based added value of laboratory diagnostics, this article aims to discuss theover-reaching contribution of some recent tests
to the clinical decision making, and the unquestionable role they have played in profoundly
revolutionizing clinical practice.
CARDIAC TROPONINS
Cardiac troponins are essential componentsof the muscle contractile apparatus, including myocardial tissue. In myocardial cells, two
unique and exclusive isoforms of cardiac troponin I (cTnT) and cardiac troponin T (cTnT) arepresent, so that their immunochemical measurement allows to accurately establishing whether
or not the heart tissue has been injured, evenin the absence of open signs and symptomsof heart damage [4]. Unlike former methods,
the recent development of fourth-generation
highly-sensitive immunoassays has enabledmeasuring physiological concentrations of both
cTnI and cTnT, and to more accurately redefine
the diagnostic thresholds for identifying myocardial injury, thus including acute myocardial

infarction. According to recent guidelines andrecommendations, when the clinical presentation is suggestive for myocardial ischemia, adynamic elevation of cardiac troponins in theabsence of any other objective finding (e.g.,
normal electrocardiogram) is regarded as diagnostic of non-ST elevation myocardial infarction(NSTEMI) [5,6]. The first breakthrough occurred
after introducing high-sensitivity immunoassays
in routine clinical practice has been a substantial decrease in the number of diagnoses of unstable angina, in favor of an increment of thoseof NSTEMI, thus leading the way to hypothesizethat a requiem should be prepared for unstableangina [7]. On the other hand, the improved accuracy of these last generation, high-sensitivity
cardiac troponin immunoassays has contributed to amplify the rate of patients diagnosed
with NSTEMI (i.e., by 20-30%) [5,6], who would
have been earlier discharged with inaccurate
diagnosis and without appropriate medical or
pharmacological treatment. Is there any doubt
left that high-sensitivity cardiac troponin immunoassays have revolutionized the diagnostics of
myocardial infarction and improved the managed care of this condition? Certainly not.
HEMOGLOBIN A1c
Hemoglobin A1c, also known as glycated hemoglobin, results from the nonenzymatic binding ofhexose to the N-terminal amino acid of the hemoglobin molecule A1, which is contained into
the erythrocytes. Its concentration is hence directly proportional to the average blood glucose
level over the preceding 8-12 weeks [8]. Owing
to this important biological information, hemoglobin A1c has been for long used for monitoring glucose control in diabetic patients. A major
breakthrough has however occurred, when theAmerican Diabetes Association (AHA) has published updated recommendations for classification and diagnosis of diabetes [9], according
to which diabetes can now be diagnosed also

Page 8eJIFCC2019Vol30No1pp007-013

Giuseppe Lippi
The irreplaceable value of laboratory diagnostics

in the presence of a hemoglobin A1c value >48
mmol/mol (i.e., >6.5%) measured with an assay certified by the National Glycohemoglobin
Standardization Program (NGSP) and standardized to the Diabetes Control and Complications
Trial (DCCT) assay. The routine assessment ofhemoglobin A1c has hence enabled overcoming
many of the well-known drawbacks of plasma
glucose measurement (either fasting, randomor during an oral glucose tolerance test), which
essentially include the relative instability of
glucose concentration in uncentrifuged blood
samples, the high intra-individual variation ofblood glucose, as well as biological (i.e., acutestress, drugs) and analytical interference [10].
Moreover, the measurement of hemoglobin
A1c will now enable garnering a dual clinical information, since it not only allows to diagnosing diabetes, but will contextually provide important clinical information on medium-term
glycaemic control. Recent evidence supports
the conclusion that the measurement of fasting
plasma glucose may underestimate the real burden of diabetes compared to hemoglobin A1c
assessment, leaving this condition undiagnosed(and hence untreated) in up to one-third of prediabetic or diabetic patients [11,12]. Due to theclinical, social and economic burden caused bya delayed diagnosis of diabetes, it seems reasonable to conclude that routine assessment ofhemoglobin A1c has the potential to generate ahighly favorable impact on both diagnosis andmanagement of diabetes.
PROCALCITONIN
The greatest drawback in sepsis diagnostics isthat the current scoring systems based on integration of clinical and laboratory data, namely
the host systemic inflammatory response syndrome criteria (SIRS), the Sequential [Sepsisrelated] Organ Failure Assessment (SOFA) andthe quick SOFA (qSOFA) scores, have limited
diagnostic efficiency, because they have beenmostly validated for predicting prognosis anddeath [13]. Therefore, their use for identifying
sepsis would not permit an early diagnosis, andcould even leave some patients underdiagnosed/
untreated.
Procalcitonin is the 141 amino acids precursorof calcitonin, the leading hormone involved incalcium homeostasis [14]. In physiological conditions, procalcitonin is produced by thyroid C
cells and then converted in the mature form
calcitonin into the circulation. In patients withsevere infections, the synthesis of procalcitoninoccurs also in many extra-thyroid tissues (i.e.,liver, kidneys, lungs, pancreas), thus boosting
an increase of its circulating concentration overthe physiological reference range (i.e., <0.05
ng/mL) [15]. This peculiar biological behavior is
now exploited for diagnosing severe infections,especially sepsis.
The number of studies and meta-analyses whichhave analyzed the diagnostic performance of
procalcitonin for both diagnosing and managing
sepsis has exponentially increased over the past
decade. According to a recent meta-analysis
published by Tan et al [16], procalcitonin displays 85% diagnostic accuracy (with 0.80 sensitivity and 0.77 specificity) for diagnosing sepsis,
which appears sensibly higher than that of C
reactive protein (e.g., 73%, with 0.80 sensitivity and 0.61 specificity). Even more importantly,
in another recent meta-analysis published byMeier et al [17], procalcitonin-guided antibiotic
management was found to be effective to significantly shorten the duration of antibiotic therapy (mean variation, -2.86 days), thus representing a valuable step forward toward reducing
the worldwide burden of antibiotic resistance[18]. It is also worthwhile mentioning here thatprocalcitonin-guided antibiotic management
seems also associated with substantial economic savings, as recently highlighted by Schuetz etal [19]. It is hence reasonable to conclude thatthe use of this simple and rapid test holds great

Page 9eJIFCC2019Vol30No1pp007-013

Giuseppe Lippi
The irreplaceable value of laboratory diagnostics

promise to consistently improve clinical outcomes (i.e., earlier diagnosis), reduce the riskof antibiotic resistance (i.e., tailored therapy,
shortened administration), but may also generate favorable revenues on healthcare budgets.
NATRIURETIC PEPTIDESNatriuretic peptides are a family of protein
hormones exerting a vast array of metabolic
functions, including natriuresis, diuresis, vasodilation and improved insulin sensitivity [20].
Among the four members of this family, b-typenatriuretic peptide (BNP) and the N-terminal
fragment of pro-BNP (NT-proBNP) are producedin the left ventricular myocardium in response
to myocyte distension due to pressure overload
or volume expansion [21]. This important biological property has catalyzed the measurementof both BNP and NT-proBNP in the diagnosticsof heart failure. In the 2016 guidelines of theEuropean Society of Cardiology (ESC) [22], themeasurement of BNP or NT-proBNP has been
included among the essential diagnostic tests
in heart failure, alone or in combination withechocardiography. The reason underlying thisassumption is that increased values of these
peptides will help accelerating the diagnosis,
identifying patients needing additional cardiac
testing or accurately and safely ruling out heart
failure in those with non-diagnostic values.
Notably, in the meta-analysis of Roberts et al[23], both BNP and NT-proBNP displayed excellent performance for diagnosing acute heart
failure (0.95-0.99 sensitivity and 0.94-0.98 negative predictive value, respectively). In anotherrecent meta-analysis published by Pufulete
et al [24], BNP-guided therapy was found to beeffective in reducing by nearly 20% the numberof further readmissions for heart failure. Even
more importantly, in heart failure patients
aged <75 years, BNP-guided therapy was also
associated with 24% higher median survival
and 13% quality-adjusted life-years gain [25].

Finally, stronger evidence was also found that
BNP-guided care may be a cost-effective option
to clinically-guided care in patients with heart
failure and impaired ejection fraction [26]. It
is hence undeniable that BNP-driven care has
the great potential to improve the diagnosis ofheart failure, lower the risk of developing left
ventricular systolic dysfunction and ameliorate
the quality of life of heart failure patients.
CONCLUSIONS
There is a common perception that laboratory
medicine may be occasionally perceived as aneglected discipline by clinicians [27]. The validity of this assumption is reflected by the many
publications in which a deep knowledge of thereal significance of laboratory tests is lacking, sothat diagnostic testing is finally considered an
ordinary commodity.
For example, in a recent article published in
JAMA Internal Medicine, Morgan et al concluded
that high-sensitivity troponin testing often yields
a high number of false-positive results in patients with suspected myocardial infarction [28].
In another recent article published in the British
Medical Journal, O’Sullivan et al hypothesizedthat many of the vitamin D tests ordered in the
UK are unnecessary screening, and this conclusion was supported by the evidence that test
prescriptions have increased by over 50% between 2000/1 and 2015/16 in that country [29].
These assumptions symbolize a limited appreciation of the actual significance and implication oflaboratory tests. Regarding cardiac troponins, anincreased value is indeed an essential criterion
for diagnosing myocardial injury, although a concentration above the diagnostic threshold does
not disclose the type of underlying cardiac damage. Therefore, cardiac troponin testing can beordered for many important reasons other thanfor diagnosing myocardial infarction, such as inpatients with myocarditis, cardiac contusion,

Page 10eJIFCC2019Vol30No1pp007-013

Giuseppe Lippi
The irreplaceable value of laboratory diagnostics

Table 1

Paradigmatic examples of recent laboratory tests
which have revolutionized clinical practice
Test

DiseaseHighly-sensitive
cardiac troponin immunoassays

Diagnosis of non-ST elevation myocardial
infarction (NSTEMI)

Hemoglobin A1c

Diagnosis and therapeutic managementof diabetes

Procalcitonin

Diagnosis of severe infections (sepsis)and antibiotic stewardship

Natriuretic peptides

Early diagnosis and therapeutic managementof heart failure

cardiotoxicity. It is disappointing to infer thatthe clinical use of cardiac troponins remains still
uncertain nearly 20 years after the publicationof the first universal definition of myocardial infarction [6].
As regards vitamin D, the increased number ofrequests shall be interpreted according to thetemporal trend of vitamin D deficiency. A recent
study, analyzing the trends in diagnosing vitamin
D deficiency in the UK, has concluded that thiscondition has increased by over 15-fold between
2008-2014, as consequence of many environmental factors [30]. Since vitamin D deficiency
not only is a major contributor of skeletal health(by lowering the risk of osteoporosis and fractures), but also seems to play an essential rolein decreasing the risk of many human pathologies (e.g., cancer, cardiovascular disease, autoimmune and infectious diseases, and so forth)[31], an increasing number of prescriptions is
not certainly unexpected or unreasonable, andcannot be straightforwardly associated withinappropriateness.
This paradigmatic examples underscore the factthat clinical reasoning is unavoidable for accurate interpretation of laboratory test results, andthat a deep knowledge of the real significance ofeach laboratory test is essential for preventing a
deplorable underestimation of the added valueof in vitro diagnostic testing.
In conclusion, although it is predictable that theextent to which laboratory testing informs theclinical decision making will remain controversial [3], it cannot be denied that the contribution of laboratory medicine in modern healthcare remains pivotal, since it helps predicting
susceptibility to disease, making accurate diagnoses, prognosticating and monitoring diseases[32], and will become even more important in
the future for the ongoing diffusion of disruptive technologies (i.e., genomics, proteomics,
theranostics) and personalized (precision) medicine [33].
The four paradigmatic cases described in thisarticle (Table 1) represent just some arbitrary examples of how irreplaceable is the value of laboratory diagnostics, and in which way some diagnostic tests have recently revolutionized clinicalpractice. Indeed, many other examples could be
brought here, even more straightforward thanthose discussed in this article. It is advisable that
altogether these paradigms will help reaffirming
the vital role of laboratory medicine in modern
healthcare [34].

Page 11eJIFCC2019Vol30No1pp007-013

Giuseppe Lippi
The irreplaceable value of laboratory diagnostics

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6. Thygesen K, Alpert JS, Jaffe AS, (2018) Fourth Universal
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16. Tan M, Lu Y, Jiang H, et al (2018) The diagnostic accuracy of procalcitonin and C-reactive protein for sepsis: A
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27. Plebani M (2018) Quality and future of clinical laboratories: the Vico’s whole cyclical theory of the recurring
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31. Wacker M, Holick MF (2013) Vitamin D - effects on skeletal and extraskeletal health and the need for supplementation. Nutrients 5:111-148.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Factors affecting turnaround timein the clinical laboratory of the Kathmandu
University Hospital, Nepal
Rajendra Dev Bhatt1, Chandani Shrestha2, Prabodh Risal3

Department of Clinical Biochemistry Laboratory, Dhulikhel Hospital, Nepal
Kathmandu University School of Science, Nepal
3
Department of Biochemistry, School of Medical Sciences, Kathmandu University, Nepal
1
2

ARTICLE INFOABSTRACT

Corresponding author:
Rajendra Dev Bhatt
Department of Clinical Biochemistry
Dhulikhel Hospital
Kathmandu University Hospital
Dhulikhel
Nepal
E-mail: bhattdev.rajendra@gmail.com

BackgroundKey words:
turnaround time (TAT), quality service,
total testing process (TTP)
Competing interests:
Authors declared that they had
no conflicts of interest.

The turnaround time (TAT) as defined by most of
the laboratories is the time interval between thespecimens received in the laboratory to the time ofreports dispatched with verification. Nearly 80% of
hospital-attached clinical laboratories receive complaints about delayed TAT. Reporting in time is a crucial indicator of quality services along with accurate,
precise and reliable reports, thus each clinical laboratory should identify affecting factors to eliminate
them for the enhancement of quality services.
Methodology
Dhulikhel Hospital-Kathmandu University Hospitalis a tertiary care hospital, where this observationaldescriptive study was conducted in 2017. Requested
tests received on database in the Department ofClinical Biochemistry Laboratory along with test requisition form (TRF) were carefully screened for any
possible error. When analysis of individual patient’s
tests was completed, results of individual parameters were entered in the database manually. TAT wasPage 14eJIFCC2019Vol30No1pp014-024

Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

calculated as a time period between specimens
received to analysis completed. Once test analysis has completed it was immediately followedby verification.
Results
A total of 36,108 patients’ reports generated
from the Department of Clinical Biochemistry
Laboratory during study period were analyzed.
Nearly 36% of reports exceeded the predefined
TAT in case of stat tests, while around 7% of reports were out of predefined TAT in case of routine tests. Among prolonged TAT, around 75% ofreports were delayed due to various extra analytical reasons and approximately 48% of total
delayed reports were found only due to error
by cash unit.
Conclusion
The major reasons of delayed laboratory reports
were due to time consumed to fix the pre-analytical errors created by other departments
rather than laboratory itself. Cash unit alone
has the highest degree of error in total testingprocess and it is the most significant factor for
prolonged TAT. However reasons for prolonged
TAT may vary with hospital to hospital depending upon different factors.


INTRODUCTION
Most of the medical decisions are made on thebasis of laboratory findings 1. So, clinical laboratory findings must be accurate and well-timed.
Waiting for laboratory reports for long timeis often disappointing for patients and clinicians too. Hence, it is better for each laboratory to have its own turnaround time (TAT) 2-5.
Accuracy, reproducibility and punctuality havetheir own grounds in the field of clinical laboratory science 6,7. However, in general clinicallaboratories focus on accuracy and reliabilityof the test reports and pay less attention to theprompt release of laboratory reports 8. Early
diagnosis and appropriate treatment of thepatients is an outcome of the calculated accuracy and well-timed execution of the work 9, 10.
Analysis of the test report in time can also be
an important factor for the patients to cut out
their expenses by shortening the time of their
hospital stay 9, 10.
The total/therapeutic TAT is the time “from veinto brain”, it is the interval between the test request and the therapeutic decision, while thelaboratory TAT starts when the sample arrives
at the laboratory and ends when the report isreleased after the validation of the results 11, 12.
However, many laboratories confine their definition of TAT to intra-laboratory due to limitations in control of extra laboratory factors 13.
TAT varies from laboratory to laboratory and also
depends on the varying explanation of the laboratories and the clinicians 11, 14. Furthermore, TAT
can also be categorized on the basis of test types
and patient’s priority (outpatients, inpatients,
emergency) 11. Total Testing Process (TTP) in clinical laboratory, as described by Lundberg, hasnine steps namely, ordering, collection, identification, transportation, preparation, analysis,
reporting, interpretation and action 15.
A study done by the College of American
Pathologists, CAP Q-Probes, in 1998, foundthat, 41% of the laboratories defined emergency TAT as the interval between sample arrivaland result reporting, 27% defined it as the timefrom test ordering to reporting of the results
and 18% defined it as the interval between
sample collection and result reporting 16.
TAT is a measure of punctuality and is often regarded as one of the determinants of laboratory
efficiency 9. Humans, being impatient by nature,need everything to be done rapidly including
the clinical laboratory reports. Thus, they prefer

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospitalthe hospitals that guarantee fast service and do
not make them wait for long hours for their test
reports and proper diagnosis, treatment andmanagement of their problems. These facts
thus prove TAT crucial for both, medical as wellas commercial point of view 6.

their Test Requisition Form (TRF) available at
Department of Clinical Biochemistry Laboratoryof Dhulikhel Hospital in six months’ time fromMarch to August 2017 were analyzed.

Total laboratory testing process is divided into
three phases, namely; pre-analytical, analyticaland post-analytical, and TAT depends on thesethree phases 17. The pre-analytical phase refers to the time period between requisition oftest to the sample being reached to the handsof professionals and prepared for analysis.
The analytical phase is the period of measurement; this is the interval between the beginning of the measurement (actual testing) andthe confirmation of the test results. The postanalytical period indicates the time from result
verification or printing to the time when thephysician actually observes the results 18, 19, 20.
Among these three phases, pre-analytical andpost-analytical phases contribute to nearly 96%of the TAT 9 and factors may vary depending
upon the infrastructures of the institution, degree of automation, and experience and attribution of the employee 21, 22. Dhulikhel HospitalKathmandu University Hospital (DH-KUH) has
predefined TAT of one hour for tests requested
from emergency department and intensive careunit (ICU) while two and half hour for tests requested from outpatient departments and indoor depart­ments.

Inclusion criteria

Selection criteria
All the criteria matched specimens and TRF received at the Department of Clinical Biochemistry
Laboratory of Dhulikhel Hospital-Kathmandu
University Hospital were analyzed.
Exclusion criteria
a. Specimens for fasting and postprandial
blood glucose measurement. (As there isno system to record time of postprandial
sample reception)b. Sudden addition or cancellation of the tests
via telephone by clinician.
c. Test for Sex Hormone Binding Globulin,
Vitamin B12, Folic acid and Anti Cyclic
Citrullinated Peptide. (These tests are performed twice a week only)
d. Test for hemoglobin or serum protein
Electrophoresis. (Performed weekly only)
Data collection and TAT calculation

METHODOLOGY

The time of sample arrival and time of analysis
completed were recorded in Excel tables and thedifferences were calculated. Simultaneously, if
any obstacle was found in TTP, it was mentioned
in TRF and was noted in the spread sheet.

Study design

ProcedureThis is a cross-sectional, descriptive and observational study based on the data obtained
from the Department of Clinical Biochemistry
Laboratory of Dhulikhel Hospital-Kathmandu
University Hospital. Obtained data were closely
analyzed to observe current TAT and factors affecting prolonged TAT. All the samples along withIn the specimen collection unit, collection time
was mentioned in TRF and collected samples
and TRFs were transported to the Departmentof Clinical Biochemistry Laboratory by trained
staff. The TRFs and samples were then received
by technician in the Department of Clinical
Biochemistry Laboratory and verified according

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospitalto pre-structured standard operating procedure(SOP). The sample receiving time and parameters were immediately entered in Midas Version
3.2. If any possible error was noticed during
sample receiving process, that was mentioned
over TRF of respective patients. The sample receiving time entered in Midas is automaticallyprinted on the report as sample received time.
After completion of the individual patient’s
Figure 1

tests, the observed values for all the requested
parameters were manually entered in reporting
database software Midas. If any problem happened during analysis e.g. test was repeated orsudden machine breakdown, they were mentioned on the TRF without any delay by concerned staffs. The time when observed value isentered in Midas, appears as test analysis completed time in the patient’s report.

Flow chart of the Total Testing Process (TTP)

Patient’s Registration

Figure 1 presents the flow chart of the total testing process in Dhulikhel Hospital-Kathmandu University Hospital.

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

Any factors that were encountered during thewhole process, from receiving the samples toreleasing the reports, might be responsible forprolonged TAT. They were mentioned on the TRF
by concerned staff and were closely monitoredby the author and research assistant. Every
day all the criteria matching reports generated
from the Department of Clinical Biochemistry
Laboratory were analyzed for current TAT, together with the reasons of prolonged TAT mentioned on the TRFs. TTP was performed, as shownin Figure 1.
Ethical considerations
The research proposal was submitted to theInstitutional Review Committee (IRC) of the
Kathmandu University School of Medical
Sciences, and data collection was started aftergetting ethical clearance.
RESULTSThis study was conducted in the year 2017 by
analyzing a total of 36108 samples along with
their TRF. Out of those, only 24644 samples
were fit for the actual study. 11464 (31.74%)
samples with TRF were not suitable because
those requested tests had less than 20 minutes
TAT which is practically not possible. This had
happened because technical staff of clinical laboratory did not follow the standard operatingprocedure of TTP. Especially, in the late evening
and night, technical staffs had done analysis oftests without receiving sample electronically in
Midas, instead the samples were electronicallyentered only after completion of analysis or in
between of analysis. So, the observed TAT wasvery short which is not possible in real situation(Table 1).
When 24644 patients’ reports were analyzed for
TAT and affecting factors, 2434 (9.8%) of themhad prolonged TAT in comparison to predefined
TAT. But only 2010 patients out of 24644 were

found to have reasons documented on TRF forstretched TAT. When specific reasons for prolonged TAT were analyzed in 2010 patient’s
reports, 973 (48.4%) patients’ reports were delayed due to problem created in cash unit either
incomplete payment or payment not according
to test requested in TRF.
Nearly half of the total affected TAT was observed due to problem in cash unit and unfortunately patients from all the departments
had to pay before test proceeds. Departmentof Clinical Biochemistry Laboratory reporting
database is designed in such way that reportcannot be generated unless a proper payment
is made therefore the cash unit is the most important factor for prolonged TAT in Dhulikhel
Hospital.
Repetition of tests is another leading factor of
prolonged TAT in the Department of Clinical
Biochemistry Laboratory of DH-KUH which is494 (24.5%) out of 2010 patients. 313 (15.5%)
patients did not get their reports on predefinedtime due to sample related factors. Visually detected hemolyzed specimens were absolutely
rejected which resulted in delayed reports of
210 patients. It contributed delay of 10.44%
patients’ report. Furthermore, lingering oflaboratory reports were due to poor inventory, failure of analyzers either due to irregular
maintenance or lack of properly functioning
analyzers were noticeable factors responsiblefor delay in 230 (11.4%) patients’ reports.
Table 2 clearly illustrates the number of samples
from different departments in a particular timeframe and their mean TAT. The frequency of long
TAT is quite different among samples from ER/
ICU and OPD/Indoors. Frequency of abnormal
TAT for samples received from ER/ICU is fivetimes higher than samples received from OPD
and other indoor departments.
Bar graph in Figure 2 reflects that repetition oftest analysis for reconfirmation and consultation

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

Table 1

Specific reasons and their frequencies for prolonged TATSpecific reason

NumberFrequency(%)

Payment Missing(Incomplete payment)

542

26.96

Wrong payment(Not according to tests prescribed)

297

14.77

Wrong registration(Paid in another patients account)

127

6.31

Excess payment(Mostly double payment)

07

0.34

Critical value reconfirmationand consultation

494

24.57

Hemolyzed

210

10.44

Insufficient

40

1.99

Not Received

17

0.84

Unlabelled

15

0.74

Unsuitable Specimen/test

10

0.49

Marked Lipemic

08

0.39

Mislabelled

07

0.34

Clotted06

0.29

Out of Stock/Not Provided/Supplied

101

5.02

Expired20

0.99

Random Breakdown

66

3.28

Preventive Maintenance Schedule

23

1.14

Reporting
Software Breakdown

Reporting System (Midas) Down

20

0.99

Total

-

2010

-

Factors

Payment for Tests
in Cash Unit

Tests Repetition

Specimen related

Reagents related

Machine Breakdown

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

Figure 2

Factors affecting TAT in ER and ICU patients (%)

In the case of the laboratory reports of ER and ICU patients, the leading cause of TAT prolongation is the repetition of test
analysis and consultation of the results.

Table 2

Number of samples from different departments and their average TAT
Departments

ER and ICU

OPD and Indoors

Total Number of Samples

2403

22241

Prolonged TAT Samples

881 (36.66%)

1553 (6.98%)

Average TAT of Total Samples

1 hour 3 minutes

1 hour 21 minutes

Average TAT of prolonged TAT samples

1 hour 45 minutes

4 hours 30 minutesof observed value of samples received from
ER/ICU is the most prominent factor covering
around 45% for prolonged TAT. Factors related
to billing and registrations are the second mostimportant factors for delaying laboratory reports which is followed by specimen and laboratory information system related factors.

As shown in Figure 3, reports of tests requested
from OPD and other wards had prolonged TAT
due to different factors. Out of all five major
factors for delayed reports, billing in cash unit
is the highest, covering around 57%. Specimens
related factors were the second highest, affecting nearly 15% of the delayed TAT.

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

When affecting factors for prolonged TAT wereanalyzed for individual departments, they varied according to types of services they provide.
Such as the nature of service provided in emergency room (ER) and intensive care unit (ICU),
where prompt action is needed and of course
the laboratory reports must be delivered in timeand must have less TAT than other departments

As shown in bar graph, 48% of reports requested
from ER or ICU were delayed due to time wasted
in reconfirmation of obtained critical values oftest. Mostly reconfirmation was done by repeating the tests or informing laboratory consultant
before releasing such reports. Payment related

Factors affecting TAT in case of OPD and indoor patients

Re
pe
titi
on

of
Te
st
an
aly
sis
an
d

co
ns
ul
ta
tio
n

Figure 3

like general wards and outpatients departments(OPD).

In case of the laboratory reports of OPD and indoor patients, the TAT prolongation is mainly billing and registration
related.

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospitalissues were found to be the second most significant factor (32%) for long TAT for tests ordered from ER and ICU followed by sample related issues like hemolysis, low volume, wrong
sample etc. playing role in delaying reports of
17% patients.
In addition to the above-mentioned factors,
transportation of the sample is also one of thefactors affecting total TAT. Transportation time
can be varied, which can be from specimen collection center to the laboratory or from otherwards. It is not actually affecting laboratory
TAT but it may affect the total TAT. Hence, it
may lead to patients’ prolonged stay in hospitaland dissatisfaction, ultimately affecting hospital service. In general, average transportation
time of collected specimens from the collection
unit to the Department of Clinical Biochemistry
Laboratory was found 24 minutes.
Analyzing factors responsible for prolonged
TAT in ER and ICU, this study shows critical value reconfirmation and consultation time playing a major role for delay in predetermined
TAT covering 48% of the total whereas billing
and registration related issues were the next
significant reason (32%) for the same. Sample
related issues contributed around 17% to thetotal delay in TAT.
DISCUSSION
How punctually a hospital provides service to thepatient is an important determinant of quality ofthat hospital. In this study samples along with
their TRFs were analyzed to generate relevant
information about the TAT. Statistical analysis
was applied for samples taken from ER and ICU
departments and OPD and indoor departments.
The average TAT for the samples received fromall departments was 1 hour and 19 minutes.
Specific analysis for individual departments
showed average TAT for ER and ICU departments
and OPD and indoor departments as 1 hour and3 minutes and 1 hour and 21 minutes respectively. The study carried out by F. Bilwani et al.
showed that only 2.03% of the stat samples had
longer TAT than acceptable range 6 while ourstudy suggested that for ER and ICU departments a total of 36.66% of all samples investigated showed prolonged TAT.
This variation in TAT was mainly due to the delayed entry of the time at which the report was
generation. The laboratory technicians generally inform the ER and ICU department immediately via phone call and enter the data later
when they are relatively free.
Tests requested from OPD and indoor departments showed nearly four times less prolonged
TAT in comparison to emergency and ICU, andthe ratio of samples showing TAT prolongationstood at 6.98%.
A study performed by K. P. Chauhan et al. suggested that percentage of specimens exceeding
TAT in 2011 was 6.4% which decreased to 4.6%
by year 2012 14. The slightly higher prolonged
TAT in our case was due to the registration andbilling issues, analyzer errors, inventory of reagent related issues and sample related issues
along with reconfirmation and consultation
time. In this study, among all factors involved
for excessive TAT, preanalytical factors were responsible for nearly 75% of the delay whereas
around 24% of the delay was due to analyticalfactors.
Similarly, a study done by KN. Desai et al. suggests that 74.2% of the samples were delayed
due to preanalytical phase 7.
In contrast to this result, a study performed by
F. Bilwani et al. emphasizes that most of thedelays were due to analyzer error constituting
40% of the total specimens 6.
In our study analyzer error was responsible for
only 6.83% of the TAT prolongation observed exclusively for OPD and indoor departments.

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Rajendra Dev Bhatt, Chandani Shrestha, Prabodh Risal
Factors affecting turnaround time in the clinical laboratory of the Kathmandu University Hospital

CONCLUSION
Achievement of quality service is not simply possible in hospital attached laboratories withoutfinding the factors for prolonged TAT and immediate improvement of that area by hospitalmanagement. However, factors affecting TAT inclinical laboratories may vary from institute to
institute depending upon institutional infrastructure, their own setup, policy, system and attributes of employees working in different departments of the hospital. In case of the Departmentof Clinical Biochemistry Laboratory of Dhulikhel
Hospital-Kathmandu University Hospital, the major reasons for delay in laboratory reports were
due to the time burnt out to fix the preanalyticalerrors created by other departments and cash
unit alone was the major factor with highest degree of error in total testing process.


Acknowledgements
We would like to express our sincere thanks to
Mr. Prabin Gyawali, who had the courage to pioneer predefined TAT in the Department of Clinical
Biochemistry Laboratory of Dhulikhel Hospitaland most probably for first time in Nepal as well.
We are also thankful to all staff of Dhulikhel
Hospital, especially of the Department of Clinical
Biochemistry Laboratory and IT Department.

4. Froom P, Barak M. Auto-validation of complete bloodcounts in an outpatient’s regional laboratory. Clin Chem
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5. Manor PG. Turnaround times in the laboratory: a review of the literature. Clin Lab Sci . 1999;12(2):85-9.
6. Bilwani F, Siddiqui I, Vaqar S. Determination of delay
in turn around time (TAT) of stat tests and its causes: an
AKUH experience. J Pak Med Assoc. 2003;53(2):65-7.
7. Desai KN, Shah M, Patel K, , Ranapurwala M, Chaudhari
S, Shah M. Determination of Turn Around Time (TAT) in
NABL (National Accredited Board of Laboratory) accredited hematology and clinical pathological laboratory. Int J
Adv Res (Indore). 2013;1(6):192-96.
8. Kappelmayer J, Toth J. Clinical laboratories - production factories or specialized diagnostic centers. eJIFCC.
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9. Angeletti S, De Cesaris M, Hart JG, Urbano M, Vitali
MA, Fragliasso F, et al. Laboratory automation and intralaboratory turnaround time: experience at the university hospital campus bio-medico of Rome. J Lab Autom.
2015;20(6):652-8.
10. Holland LL, Smith LL, Blick KE. Reducing laboratory
turnaround time outliers can reduce emergency department patient length of stay: an 11-hospital study. Am J
Clin Pathol. 2005;124(5):672-4.
11. Hawkins RC. Laboratory turnaround time. Clin biochem Rev. 2007;28(4):179-94.
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1):S111-27.
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2. Antal-Szalmas P, Ivady G, Molnar A, Hevessy Z, Kissne Sziraki V, V Olah A, et al. [“Turnaround time”: a new parameterfor the characterization of the overall efficacy of laboratory
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18. Steindel SJ, Jones BA. Routine outpatient laboratory
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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hemolysis interference studies:
the particular case of sodium ion

José Antonio Delgado1, Daniel Morell-Garcia1,2, Josep Miquel Bauça1,2

1
2

Department of Laboratory Medicine, Hospital Universitari Son Espases, Palma, Balearic Islands, Spain
Institució d’Investigació Sanitària de les Illes Balears (IdISBa), Spain

ARTICLE INFOABSTRACT

Corresponding author:
Josep Miquel Bauça
Hospital Universitari Son Espases
Ctra. de Valldemossa, 79
07010 Palma, Mallorca
Spain
Phone: +34 871205876
E-mail: pepmiquel@gmail.com

BackgroundKey words:
hyponatremia, hemolysate,interference, interferogram
Competing interests:
All authors declare no conflicts of interest.

Despite many studies assessing hemolysis interference in almost every clinically relevant magnitude,
sodium has poorly been assessed. Our aim was toevaluate hemolysis interference on plasma sodium,using different strategies of hemolysis preparation, atdifferent baseline sodium ion concentrations and bias
specifications.
Methods
Two different strategies were used for the preparation of hemolysis from lithium heparin blood samples. Repeatibility was calculated at two levels foreach strategy and interferograms were outlined forboth approaches at sodium concentrations between
130-145 mmol/L. Results were interpreted according
to different specifications: reference change value,
RiLiBÄK, Westgard’s database, RCPA-QAP and CLIA.
Results
The coefficients of variation of the hemolyzed samples
using the first strategy were lower than for the second strategy (0.23-0.78% vs 0.57-48.6%, for 0.2 g/dL
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José Antonio Delgado, Daniel Morell-Garcia, Josep Miquel Bauça
Hemolysis interference studies: the particular case of sodium ion

free Hb and 0.28-0.44% vs 0.40-135.1%, for
0.9 g/dL free Hb). Statistically significant differences were seen when comparing the slopes ofthe pairs of interferograms at each sodium concentration obtained by both strategies (p<0.001
for 130 mmol/L; p=0.068 for 135 mmol/L;
p=0.002 for 140 mmol/L and p=0.001 for 145
mmol/L). Hemolysis cut-off values were generally independent of the sodium concentration.

spectral (especially in spectroscopic methods,
due to an overlapping of absorption spectra) andchemical (due to a release of components fromred blood cells which alter the in vivo concentration of the analyte) (6). Other hemolysis-derived
interfering mechanisms may be due to othercauses (e.g. magnesium in the measurement of
total calcium concentration, or adenylate kinase
in the measurement of creatine kinase activity).

Conclusions

A great number of studies have assessed the effect of hemolysis on almost every clinically relevant analyte. Towards a minimization of variability and a higher reproducibility, guidelines
have been published both for the performanceof such studies and for the in vitro simulationof hemolysis and the handling and processingof such blood specimens (7–10). The quantification of the degree of hemolysis is also fundamental for the proper management of samples
and test results (11,12).

Reproducibility of hemolysate preparation is procedure-dependent. A greater standardization isneeded for the preparation of a true hemolysate
to better quantify the degree of interference ofclinically relevant analytes, especially those withhigher complexity such as sodium. We found aconcentration-independent cut-off value for thehemolysis index that allows the establishmentof a single and robust value in every laboratory,
according to their quality specifications.


INTRODUCTION
In the preanalytical phase, the in vitro lysis of
red blood cells (hemolysis), which implies a release of hemoglobin and other intraerythrocyte
elements, is the main cause of interference andrejection in the biochemical analytical methods
worldwide (1–3). In vitro hemolysis depends
mainly on blood sample drawing techniquesand subsequent treatment (agitation, transportation, storage), whereas in vivo hemolysis may have at least 50 causes (4,5), including
Gram-positive bacteria, parasites, toxins or autoimmune disorders. Genetic disorders such as
sickle-cell disease or glucose-6-phosphate dehydrogenase deficiency may also lead to hemolytic
crises with high free hemoglobin levels in blood.
There are two central mechanisms of interference by hemolysis in clinically relevant tests:

In spite of their almost universal applicability forbiomarkers in laboratory medicine, there are a
few exceptions still needing a thorough examination, and the paradigm of such exceptions
is sodium ion. As previously reported, in vitro
hemolysis is known to negatively interfere withsodium due to a diluting effect (13,14), as theintracellular concentration of sodium is significantly lower than the concentration in serum or
plasma. The degree of hemolysis in a sample isfrequently assessed by measuring the free hemoglobin in serum or plasma.
The preparation of a true hemolysate is crucial
for the performance of studies assessing hemolysis interference. This term refers to the absence of unhemolyzed red blood cells or otherintact cells after the preparation of hemolysate.
One of the procedures most commonly was
first described by Meites (15), and includes awater-dilution step before freezing and thawing
an anticoagulated blood sample. Other usefulPage 26

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Hemolysis interference studies: the particular case of sodium ion

strategies in literature include microwave radiation, ultrasounds or mechanic lysis (16).
Nevertheless, the preparation of valid hemolysate for the study of sodium in serum or plasma should not have a water-addition step, as
it would decrease the concentration of the ion
in the solution, hence altering the (direct) relationship with the hemolysis index. According toliterature, intraerythrocyte sodium concentration is 10-15 mmol/L (17,18), whereas sodium
concentration in distilled water is negligible. As
a result, the water-dilution step in the preparation of a hemolysate would alter the relationshipbetween hemoglobin (hemolysis index) and sodium, as well as with any other intraerythrocyte
biomolecule. If including this step, the greater
water volume added for the preparation of thehemolysate, the greater interference observed(negatively). The equilibration with distilled water would impede the detection of the strictly
negative effect of hemolysis.
Our aim was to comprehensively assess themagnitude of hemolysis interference of plasma
sodium, using different strategies of hemolysate preparation, different baseline sodium ion
concentrations and different bias specificationsin their interpretation.
METHODS
Two different study procedures were suggested
in this study to parallelly assess and quantify
the magnitude of interference of hemolysis on
plasma sodium (Figure 1).
A total of 40 volunteers were recruited for the
performance of this study: 20 for the first approach, and 20 for the second.
The first approach consisted in blood extraction
into two simultaneous 3.5-mL lithium heparintubes without gel from each volunteer (ref.
368884, BD Vacutainer). One of them was directly centrifuged (10min 1500g; Sample A)

while the other was previously frozen-thawed
3 times to induce hemolysis and subsequently
centrifuged (Sample B). Both plasma samples
A+B were mixed in different proportions, starting from [1000 µL A + 0 µL B] to [1000 µL A + 200µL B] (greater proportions of B yielded excessive
hemolysis, not quantifiable by the analyzer).
Sodium ion was measured by indirect potentiometry, whereas hemolysis index was analyzed
by dichromatic spectrophotometry at different
wavelengths and calculated using an algorithm(Architect c16000 platform, Abbott Diagnostics,
USA). Sodium in sample A (pure) was taken as
reference. The experiment was performed atfour concentrations of sodium: approximately
130, 135, 140 and 145 mmol/L. None of thesamples was seen to by hyperlipidemic.
The second approach consisted in the simulation of hemolysis by removing the supernatantand the buffy coat of a plasma heparin tube,
freezing-thawing it 3 times and further centrifuging it (10min 1500g). No addition of distilledwater or washing step was carried out. Pools oflithium heparin plasma were prepared at different sodium concentrations (approximately 130,
135, 140 and 145 mmol/L) using plasma from
5 different participants for each, and aliquoted
into 1-mL tubes. Increasing volumes of the hemolyzed supernatant (5, 10, 20, 25, 30, 35 and40 µL) were added to each aliquot, which were
further centrifuged to remove possible intact
red blood cells.
Repeatibility of both strategies was assessedby measuring 10 times the hemolysis index at
5-minute intervals in 10 samples, at two different hemolysis indices (approx. 0.2 and 0.9 g/dL)
for each strategy in different days, and calculating the coefficients of variation.
Interferograms were outlined for both approaches and different initial sodium concentrations,according to guidelines (7).

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Hemolysis interference studies: the particular case of sodium ion

Figure 1

Strategies for the evaluation of hemolysis interference

SAMPLE A

10min 1500g
centrif.
Mix supernatants indifferent proportions

Strategy 1(20 volunteers)
3x

10min 1500g

freezethaw

centrif.

SAMPLE B

centrif.
freezethaw

removesupernatant

H

Strategy 2

Add different volumes ofhemolysate (H) to plasma(20 volunteers)

10min 1500g
centrif. and keepsupernatant only

The critical hemolysis index causing interference
was evaluated in every interferogram as thedifference from the baseline value, and established according to five different performance
specifications:•the reference change value in our laboratory(RCV), which integrates the within-subject
biological variation (0,6%) and our analytical coefficient of variation (0,67%);•

the RiLiBÄK specification of 3%;•

the desirable quality specification of 0.73%
for total error on the Westgard database(available at: https://www.westgard.com/
bio­data­base1.htm);•

the allowable limit of performance of 2%
by the Royal College of Pathologistsof Australasia (RCPA-QAP)(available at: https://www.westgard.com/
rcpa-bio­chemistry.htm); and•the CLIA specification of ±4 mmol/L(available at: https://www.westgard.com/
clia.htm).

The Shapiro-Wilk’s test was used to assess normality, and outliers were removed using theReed/Dixon´s test. The Fisher´s F-test was carried
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José Antonio Delgado, Daniel Morell-Garcia, Josep Miquel Bauça
Hemolysis interference studies: the particular case of sodium ionout to compare variances and the Student’s t-test
was used in order to compare the slopes and intercepts of both hemolysis-preparing strategies
at each sodium concentration. Statistical significance was set at 5%. The software SPSS v.20 wasused for all statistical analyses.

Sodium concentration was seen to decrease withhemolysis in every concentration assessed, independent of the hemolysis strategy (Figure 2).
When comparing the slopes of the pairs of interferograms at each sodium concentration obtained by both strategies, statistically significant
differences were detected (Table 2).

RESULTSAs outlined in Table 3, when the approach basedon hemolysis generation in whole blood, centrifugation and mixing in different proportions
with a paired normal plasma was used (strategy 1), the mean hemolysis index exceeding the
RCV specification for a sodium concentrationof 130 mmol/L was 0.95 g/dL hemoglobin (CI
95%: 0.88-1.02), while this critical value is reduced when a more strict limit is used, such as
the one suggested on the Westgard database

The coefficients of variation for the repeatedmeasures were 0.26-0.78% and 0.28-0.44% forhemolysis levels obtained by the first strategy(parallel extraction and mixing in different proportions), and 0.57-48.6% and 0.40-135.1% for
the samples obtained using the second strategy(hemolysate generation and subsequent addition to a normal plasma) (Table 1).
Table 1

Repeatability (coefficients of variation) of hemolysis index
obtained by different strategies
Plasma [Na+](mmol/L)Low Hemolysis Assay(0.2 g/dL Hb)

High Hemolysis Assay(0.9 g/dL Hb)

Strategy 1

144.1

0.26-0.78%

0.28-0.44%

Strategy 2

144.3

0.57-48.6%

0.40-135.1%

Table 2Comparison of slopes and intercepts of interferograms
obtained at a specified sodium concentration(y: deviation from baseline as %; x: free hemoglobin in g/dL)

Plasma [Na+](mmol/L)

Regression equation
strategy 1

Regression equation
strategy 2

p-value(slope)130y = –0.003x+0.088

y = –0.004x–0.136<0.001*135

y = –0.002x–0.198

y = –0.003x–0.076

0.068

140

y = –0.002x–0.272

y = –0.003x+0.013

0.002*

145

y = –0.002x–0.190

y = –0.004x–0.046

0.001** Significant at p<0.05

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José Antonio Delgado, Daniel Morell-Garcia, Josep Miquel Bauça
Hemolysis interference studies: the particular case of sodium ion

Figure 2

Interferograms for different sodium concentrations
using both hemolysis preparing strategies

STRATEGY 1

STRATEGY 2

130 mmol/L

130 mmol/L
0

0
0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

1,4

% Variation% Variation

0,2

0,4

0,6

-2
-3

1,2

1,4

-2
-3

-5

-5

-6

Free Hb (g/dL)

Free Hb (g/dL)

135 mmol/L

135 mmol/L

0

0
0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

-1

% Variation-1% Variation

1,0

-4

-4

-2
-3
-4

-2
-3
-4

-5

-5

Free Hb (g/dL)

Free Hb (g/dL)

140 mmol/L

140 mmol/L

0

0
0

0,2

0,4

0,6

0,8

1

1,2

1,4

-1

% Variation-1% Variation

0,8

-1

-1

-2
-3

0

0,2

0,4

0,6

0,8

1

1,2

1,4

-2
-3
-4
-5

-4

-6

-5

-7

Free Hb (g/dL)

Free Hb (g/dL)

145 mmol/L

145 mmol/L

0

0
0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

% Variation% Variation

-2
-3

0,4

0,6

-2
-3
-4

-4
-5

0,2

-1

-1

-5

Free Hb (g/dL)-6

Free Hb (g/dL)

Percentage deviation from sodium baseline value according to increasing hemolysis in the samples.

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0,8

1,0

1,2

José Antonio Delgado, Daniel Morell-Garcia, Josep Miquel Bauça
Hemolysis interference studies: the particular case of sodium ion

Table 3Hemolysis interference cut-off on plasma sodium
for different bias specifications
Free hemoglobin (g/dL)[Na+]
130mmol/L[Na+]
135mmol/L[Na+]
140mmol/L[Na+]
145mmol/L

CI for samples usedin Strategy 1(130.7-133.0)(136.2-137.7)(140.3-142.7)(145.6-148.5)

CI for samples usedin Strategy 2(131.7-133.1)(135.7-137.7)(139.3-142.5)(145.8-147.0)

Strategy 1

0.95(0.88-1.02)

0.96(0.88-1.04)

0.99(0.90-1.08)

1.04(0.95-1.13)

Strategy 2

0.61(0.56-0.67)

0.86(0.80-0.92)

0.77(0.70-0.84)

0.70(0.62-0.78)

Strategy 1

1.13(1.05-1.22)

1.17(1.07-1.26)

0.93(0.84-1.01)

0.84(0.74-0.94)

Strategy 20.74(0.68-0.80)

1.04(0.97-1.12)

0.93(0.84-1.01)

0.84(0.74-0.94)

Strategy 1

0.30(0.38-0.49)

0.22(0.30-0.45)

0.20(0.27-0.46)

0.24(0.33-0.48)

Strategy 2

0.15(0.18-0.32)

0.23(0.31-0.42)

0.23(0.26-0.42)

0.21(0.23-0.40)

Strategy 1

0.88(0.81-0.94)

0.84(0.77-0.90)

0.81(0.74-0.88)

0.86(0.78-0.93)

Strategy 20.56(0.51-0.61)

0.76(0.70-0.81)

0.65(0.59-0.71)

0.59(0.52-0.66)

Strategy 1

1.17(1.08-1.26)

1.17(1.08-1.26)

1.17(1.06-1.27)

1.17(1.06-1.28)

Strategy 2

1.17(1.08-1.26)

1.17(1.08-1.26)

1.17(1.06-1.27)

1.17(1.06-1.28)

RCV(2.5%)RiLiBÄK
(3%)

Westgard(0.73%)

RCPA-QAP(2%)

CLIA(±4mmol/L)

Confidence intervals are shown in parentheses. Abbreviations: RCV: reference change values; RCPA-QAP: Royal College of
Pathologists of Australasia Quality Assurance Program.

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Hemolysis interference studies: the particular case of sodium ion

for desirable quality specifications for total error(0.30 mg/dL hemoglobin). When the approachbased on the preparation of hemolysate andsmall-volume addition into normal samples wasfollowed (strategy 2), lower hemolysis cut-off
values were obtained at most concentrations.
At a sodium concentration of 130 mmol/L, ahemolysis of 0.61 g/dL Hb was shown to interfere when the RCV specification was used, whilesmall amounts as 0.15 g/dL Hb were seen to interfere when the Westgard’s specification was
followed.
There is no fixed tendency in the hemolysis cutoff at different sodium concentrations. Hemolysis
cut-off values are generally independent of thebaseline sodium concentration.
DISCUSSION
There is currently an objective and evident improvement in patient safety thanks to the automatization in the measurement of hemolysis
index in the clinical laboratories, which replaces
behind the visual inspection for the decision
upon their adequacy (19). The optimal hemolysis index cut-off value for each clinically relevant
test is method- and instrument-dependent, andalways subject to the previously defined quality
specifications in every specific laboratory (5). In
hemolysis interference studies, the procedure
for the preparation of hemolysate is of utmost
importance (20).
Hemolysis-preparing strategies
In our study, two different strategies were used toassess hemolysis interference at different plasma
sodium concentrations. The first strategy, basedon a parallel blood drawing, freeze-thawing one
whole-blood sample, centrifuging and mixing indifferent proportions with non-hemolyzed plasma, showed a better repeatability than the second strategy, based on hemolysate generationand subsequent addition to a normal plasma.

This better repeatability yields a greater reproducibility and robustness of the first strategy.
Hemolysis index cut-off establishment
Many studies may be found in literature assessing hemolysis interference for chemistry analytes, although sodium ion is only assessed invery few of them.
Steen and colleagues (21) assayed two different
sodium concentrations (127 and 140mmol/L),
using an hemolysate prepared by osmotic disruption with distilled water, and could not detect any significant difference in the values,
thus interpreting an absence of interference. As
stated, the inclusion of a water-dilution step inthe preparation of the hemolysate could mostprobably add a bias in the results.
Another approach by Lippi et al (22) followed afreeze-thaw procedure for hemolysis preparation and, with a critical difference set at ±0.3%,
found that even small amounts of hemolysis(0.016 g/dL of hemoglobin) could interfere in
the measurement of sodium (seen at [Na+] =140.1±1.5mmol/L).
Another study by Saldaña and collaborators(23) introduced a washing step with NaCl in
the preparation of the hemolysate. After correcting for the dilution, an hemoglobin concentration of 0.21 g/dL was shown to induceinterference.
A third study evaluated one single sodium concentration (146 mmol/L) and found a deviationof 2% from baseline when hemolysis was 0.66
g/dL. Greater concentrations of free hemoglobin were not assessed (24).
In our study, given the interval of sodium concentrations, we found a concentration-independent
cut-off value for the hemolysis index, at different
bias specifications. This allows the establishmentof a single cut-off value for hemolysis in every laboratory, according to their quality specifications.

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Hemolysis interference studies: the particular case of sodium ion

Apparent hyponatremia
There are varied and important clinical implications derived from a falsely reduced plasma
sodium result due to hemolyzed samples,
which is the main cause of preanalytical rejection of samples (14,25). In addition, there is noconsensus whether sample transportation to
the laboratory may influence the degree of invitro hemolysis, whether by pneumatic tube or
not (26–28).
Hyponatremia, defined as a sodium concentration <135 mmol/L, is reported to have an incidence between 4-19% of outpatients, being
also associated with a mortality increase (29).
After the diagnosis of a in vivo hyponatremia,
a comprehensive differential diagnostic study
is essential to determine the exact etiology, including volemia, sodium clearance and serum
osmolality, among other biochemical tests (30).
The treatment of hyponatremia needs to be directed to correct the etiologic cause and restore
blood sodium levels with a rate 6-12 mmol/L in
the first 24 hours, as a greater rate would trigger an osmotic demyelination (31). Therefore,robust and reliable sodium results by the laboratory are crucial not only for the diagnosis ofelectrolyte imbalance disorders, but also for thedecision of treatment strategies and follow-up.
The wide range seen in previous bibliography
for the establishment of a hemolysis cut-off for
sodium interference highlights the challenging
aspect of this cation. Our study is the first tocomprehensively assess the impact of hemolysis on plasma sodium, including two strategies
for sample preparation, several sodium concentrations (130 to 145 mmol/L), and interpretingsuch results according to different deviation
specifications. The inclusion of different specifications in the establishment of a cut-off value
makes our approach of greater applicability forlaboratories worldwide. The main limitations of
our study relate to the use of a single analyticalplatform, given that hemolysis index is measured by different spectroscopic methods andmathematical algorithms among clinical laboratories, as well as the low number of samples included, although in line with previous literature.
CONCLUSIONS
Our study brings to light the importance of theproper preparation of hemolyzed samples forthe interference quantification in such a particular case as sodium ion. The negative effect by
dilution seen in this case makes the protocols
including water-dilution steps unsuitable for sodium ion studies, and other more appropriate
strategies need to be followed. The establishment of a valid and concentration-independent
hemolysis cut-off value will lead to more reliable laboratory results for sodium ion results.
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Hemolysis interference studies: the particular case of sodium ion

8. CLSI C56-A Hemolysis, Icterus, and Lipemia/Turbidity
Indices as Indicators of Interference in Clinical Laboratory
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20. Gómez Rioja R, Alsina Kirschner MJ, Álvarez Funes
V, Barba Meseguer N, Cortés Rius M. Hemólisis en las
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9. CLSI GP44-A4 Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests; Approved Guideline - Fourth Edition.

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11. Luksic A, Nikolac Gabaj N, Miler M, Dukic L, Bakliza A,
Simundic A. Visual assessment of hemolysis affects patient safety. Clin Chem Lab Med. 2018;56(4):574–81.
12. Lippi G, Cadamuro J, von Meyer A, Simundic AM.
Practical recommendations for managing hemolyzed
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13. Dimeski G. Interference Testing. Clin Biochem Rev.
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1986;24:127–39.
25. Llopis MA, Trujillo G, Llovet MI, Tarrés E, Ibarz M,
Biosca C, et al. Quality indicators and specifications for
key analytical-extranalytical processes in the clinical laboratory. Five years’ experience using the Six Sigma concept. Clin Chem Lab Med. 2011;49(3):463–70.
26. Petit M, Mine L, Pascreau T, Brouzes C, Majoux S, Borgel D. Preanalytical influence of pneumatic tube delivery
system on results of routine biochemistry and haematology analysis. Ann Biol Clin. 2017;75(6):703–12.
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28. Sodi R, Darn SM, Stott A. Pneumatic tube system induced haemolysis: assessing sample type susceptibility tohaemolysis. Ann Clin Biochem. 2004;41:237–41.
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2013;62(1):139–49.
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Schrier RW, Sterns RH, et al. Diagnosis, evaluation, andtreatment of hyponatremia: expert panel recommendations. Am J Med. 2013;126(10 suppl 1):S1–42.
31. Braun MM, Barstow CH, Pyzocha NJ. Diagnosis andmanagement of sodium disorders: hyponatremia andhypernatremia. Am Fam Physician. 2015;91(5):299–307.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Prevalence of anemia and associated factors
among hospitalized children attending the
University of Gondar Hospital, Northwest Ethiopia
Bamlaku Enawgaw1, Yaregal Workineh2, Sisay Tadesse2, Eyuel Mekuria2,
Ayenew Addisu3, Meaza Genetu4
Department of Hematology and Immunohematology, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia
2
Department of Medical Laboratory Sciences, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia
3
Department of Medical Parasitology, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia
4
Department of Immunology and Molecular Biology, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia
1

ARTICLE INFOABSTRACT

Corresponding author:
Meaza Genetu
P.O. Box 196
Department of Immunology
and Molecular Biology
School of Biomedical and Laboratory Sciences
College of Medicine and Health Sciences
University of Gondar
Ethiopia
E-mail: m.genetu@yahoo.com

BackgroundKey words:
anemia, hospitalized children,
associated factors, Gondar,
Northwest Ethiopia

Anemia in children continues to be a major publichealth challenge in most developing countries, particularly in Africa.
In the early stages of life, it leads to severe negative
consequences on the cognitive functions as well as
growth and development of the children, which may
persist even after treatment.
Objective
The main aim of this study was to assess the prevalence and associated factors of anemia among hospitalized children attending at university of Gondar
comprehensive and specialized referral hospital,
Northwest Ethiopia.
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Bamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria, Ayenew Addisu, Meaza Genetu
Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

MethodBACKGROUND

A cross sectional study was conducted on 384
hospitalized children, between February andJune, 2018. Data of socio demographic characteristics and clinical conditions of the study individuals were collected using questionnaire after
taking appropriate written informed consent
and assent. Then 3 mL of blood was collected
for complete blood count analysis and also stool
examination was done for intestinal parasites.
Data were coded, cleared and entered into SPSS
version 20 for analysis.

Anemia is one of the major public health concerns that cause significant morbidity and mortality in children worldwide (1). It is one of themost prevalent public health problems in theworld, affecting both affluent and poor countries with major consequences of human healthas well as social and economic developments(1,2). Even its prevalence has shown decrement
across the regions; still it remains a serious public health problem around the globe (3).

Bivariate and multivariate logistic regression
models were used to identify associated factorsof anemia. P-value ≤ 0.05 was considered as statistically significant.
ResultThe overall magnitude of anemia among hospitalized children was 58.6%; of them 56.4%
were males. Of anemic children, 28% had mild,
51.1% moderate and 20.9% severe anemia. The
magnitude of anemia among children aged 6-59
months, 5-11 years and 12-14 years were 54.1%,
58.9% and 67.5%, respectively.
In this study, anemia was positively associatedwith parasitic infection (AOR= 2.541; 95% CI:
1.363, 4.737), not eating meat and animal products (AOR = 1.615; 95% CI: 1.014, 2.574).
Conclusion
Anemia among hospitalized children in thisstudy was found to be a severe public healthproblem. It was strongly associated with intestinal parasitic infection and not eating meat andanimal products.
Focused polices and strategies should be designed to reduce anemia among hospitalized
children in Ethiopia.
Although anemia occurs at all stages of humanlife cycle, its prevalence is higher especiallyin younger preschool aged children due to increased demand of iron for fast growth (4).
It adversely affects the cognitive and physicaldevelopment of children which in turn results
in a significant impairment of work capacity andschool educational performance (3, 5-7).
The magnitude of anemia in children substantially varies across the world’s regions, whereby
the global prevalence is estimated to be 42.6%,
and its magnitude in Africa, South East Asia,
America and European regions is 62.3%, 53.8%,
23.3% and 22.9%, respectively. Globally, on average, around 9.6 million children are severely
anemic (1).
Anemia with prevalence of ≥ 40%, 20-39.9%,
5-19.9% and <5% in the community is categorized as severe, moderate, mild and no publichealth problem, respectively (8).
The 2008 WHO report also has revealed that
more than half (56.3%) of the world’s preschool
aged children reside in developing countries
where anemia is a severe public health problem
including sub-Saharan Africa with a prevalenceof 40% and above (7, 9, 10).
Similarly, the 2015 WHO report, from the global
anemia prevalence in 2011 showed that thehighest figure (42.6%) was in children compared to other age groups around the globe

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Bamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria, Ayenew Addisu, Meaza Genetu
Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

and its prevalence in Africa and Ethiopia was
62.3% and 50%, respectively (1).
Anemia is associated with socioeconomic, biological, environmental and nutritional factors.
Nutritional deficiencies (iron deficiency, vitamin
B12 and folate deficiency), lack of awarenessamong the mothers about the problem together with their low educational status, unhealthy
food habits and parasitic infestations were considered the main factors associated with anemia among children (1, 3, 4, 7, 11-13).
The prevalence of anemia among hospitalized
children in different parts of the world has beenstudied; 33.2% in Lebanon (14), 61.6% in Turkey(15), 56.3% in Uganda (16), 83.2% in Southern
Tanzania (17), and 77.2 % in Mwanza, Tanzania(13). However, in our country Ethiopia, thereis lack of information about the prevalence
and risk factors of anemia among hospitalized
children.
Most studies in Ethiopia focus on the prevalence of anemia under age five and for school
aged children (18-20). Therefore, this study
was aimed to determine the prevalence andassociated factors of anemia among hospitalized children attending university of Gondar
referral and comprehensive hospital, Gondar,
Northwest Ethiopia. Identifying risk factors anddetermining the magnitude of anemia haveparamount importance to reform the regionaland national public policy for ensuring sustainable improvement.
METHODS
Study setting and population
A cross sectional study was done among 384
hospitalized children aged 6 months to 14 years
at university of Gondar comprehensive and specialized referral hospital, between February andJune, 2018. Gondar is located at a distance of 737
km from the country’s capital, Addis Ababa–in

the North Gondar zone of the Amhara regional
state, in North-Western Ethiopia. The city‘s latitude and longitude coordinates are 12°03′61″
N and 37°02′81″ E, respectively; and it is locatedat elevation of 2,133 m above sea level. Children
with chronic kidney disease, active bleeding,
HIV/AIDS, those who have undergone surgery
in the previous one month and who had chronic
illness were excluded from the study.
Data collection proceduresUpon obtaining a signed consent form and additional assent from children aged 7 years andabove, the children who fulfilled the inclusion
criteria were enrolled into the study. A pre-tested and standardized questionnaire was used
to collect social demographics and social economic history. The caretaker/ family’s level ofincome was assessed by asking the caretaker
how much they earned per month including
donation or handout from other persons or organizations. Other clinical data of hospitalized
children were also collected by nurses working
in pediatric ward.
Laboratory analysis
Hematology analysis
About 3mL of venous blood was collected fromeach subject for the analysis of hematologicalparameters; hemoglobin (Hgb), hematocrit (%),
mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red blood cell count (RBC) and red
cell distribution width (RDW) were determinedusing Cell-Dyn1800 automated blood analyzer.
Stool examinations
For intestinal parasite examination, stool samples were collected and both wet mount andconcentration technique were employed by anexperienced laboratory technologist.

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Anemia definition and severity classification
Since the study area altitude is 2133 meter above
sea level, results of Hgb values were adjusted
by subtracting 0.8g/dl to its respective sea level
as it is recommended by WHO. Then Hgb cutoff
value 11g/dl for children 6-59 months, 11.5g/dl
for children 5-11 years and 12g/dl for children
12-15 years were considered to define anemia.
Regarding to its severity of anemia, Hgb value of
10.0-10.9 g/dl, 7.0-9.9 g/dl and less than 7 g/dl
were considered as mild, moderate and severe
anemia, respectively for children aged 6-59
months and Hgb value of 11.0-11.4 g/dl for children 5-11 years and 11.0-11.9 g/dl for children
aged 12-14 years was considered as mild typeof anemia while Hgb value of 8.0-10.9 g/dl andbelow 8.0 g/dl were considered as moderate
and mild type of anemia for both age groups;
children aged 5-11 years and 12-14 years (8).
Statistical analysis methods
Data were cleaned, edited, checked for completeness and entered into SPSS version 20 statistical software for analysis. Descriptive statistics were used to summarize the characteristicsof the study population. To determine factors
Table 1

associated with anemia binary logistic regression analysis was done; and odds ratio with its
95% confidence level was used to determine thestrength of association between the predictors
and dependent variables. A p-value of less than
0.05 were considered as statistically significant.
RESULTS
Socio-demographic and clinical characteristics
of study participants
In this study, a total of 384 hospitalized children
aged between 6 months and 15 years were
included, of which 212 (55.2%) are males ingender and 55.7% were from rural setting. The
median age of the children was 5 years with interquartile range (IQR) of 2-10 years. Nearly half(45%) of the hospitalized children were in theage group between 6 months to 5 years. From
the total hospitalized children, 18% were infected with intestinal parasites, 20% were hospitalized for five or more days and 21.6% (83/384)
were malnourished. From malnourished hospitalized children, 45.8% (38/83) were severely
malnourished. Regarding educational status,
58.1% of mothers and 51.6% fathers of hospitalized children were unable to read and write.

Socio-demographic and clinical characteristics
of hospitalized children attending University of Gondar
comprehensive and specialized referral hospital, Northwest Ethiopia
Characteristics

Sex

Age in years

Frequency

Percentage

Male

212

55.2

Female172

48.8

0.5- 5

172

45.0

5-11

129

33.1

12-14

83

21.9

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Residence

Nutritional status

Intestinal
parasite infection

Lengthof hospitalization

Maternal
educational status

Paternal
educational status

Monthly householdincome in ETB

Rural

214

55.7

Urban

170

44.3

Severe malnutrition

38

9.9

Moderate malnutrition

45

11.7

Normal

301

78.4

Yes

69

18.0

No

315

82.0

Up to 2 days

112

29.2

3-5 days

195

50.8

Above 5 days

77

20.0

Unable read and write

223

58.1

Primary education

44

11.5

Secondary education

47

12.2

College/ University

70

18.2

Unable read and write

198

51.6

Primary education

42

10.9

Secondary education

57

14.8

College/ University

87

22.7

<1000

66

17.2

1001-2000

181

47.1

Above 2000

137

35.1

ETB: Ethiopian Birr; 28ETB = 1 $USD.

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

For nearly two thirds (64.3%) of the hospitalized
children, their family/care giver monthly household income was below 2,000 Ethiopian Birr (70US dollars) (Table 1).
Prevalence of anemia
The mean (± SD) value of altitude adjusted Hgb
level among hospitalized children was 10.5 ± 2.6
g/dL, with values ranging from 2.3 g/dL to 19.3 g/
dL. The overall prevalence of anemia in this study
was 58.6% (225/384), with 65.9% (141/214) and
49.4% (84/170) magnitude among rural and urban hospitalized children, respectively.
Of the 225 anemic cases, 127 (56.4%) were males.
Of the total anemic hospitalized children, 28%(63/225), 51.1% (115/225) and 20.9% (47/225)
had mild, moderate, and severe anaemia,
Figure 1

respectively. Ninety-three (41.3 %) anemic cases
were in the age group between 6 months to 5
years, and the rest 76 (33.8%) and 56 (24.9%) arein the age group 5 to 12 years and 12 to 14 years,
respectively.
The most common type of anaemia was the
microcytic hypochromic anemia observed in
62.2% (140/225) of children, followed by 36.9%(83/225) with normocytic normochromic anaemia. Two children (0.9%) had macrocytic normochromic anaemia. There was no significant
difference in the prevalence of anemia between
females and males which was 57.0% and 59.9%,
respectively. The prevalence of anemia washigher among older children (65.7%) in 12-15
years, and it had shown increased as the child’s
age increased (Figure 1).

Distribution of anemia among different age groupsof hospitalized children

80.0%
67.5%

70.0%

PERCENTAGE

60.0%
50.0%

Anemic
Non-anemic

58.9%

54.1%
45.90%

41.10%

40.0%

32.50%

30.0%
20.0%
10.0%
0.0%

6-59 months

5-11 years

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12-15 years

AGEBamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria, Ayenew Addisu, Meaza Genetu
Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Factors associated with anemia
In order to determine child related factors thatwere associated with anemia, bivariate logistic
regression analysis followed by multivariate logistic analysis was done.
As indicated in Table 2, in the bivariate logistic
regression analysis, children related factors suchbeing 12-15 years old, infection with intestinal
parasites, not eating meat and animal products,
edema and fever were significantly associated
Table 2

with anemia. Then those variables having p-value less than 0.25 were subjected to multivariate
logistic regression.
In multivariate logistic regression analysis, being
infected with parasitic infection (AOR = 2.541;
95% CI: 1.363, 4.737) and not eating meat andanimal products (AOR = 1.615; 95% CI: 1.014,
2.574) were statistically associated with anemia. In this study, vomiting (AOR = 0.446; 95%
CI: 0.208, 0.96) and diarrhea for more than

Association of child related factors
with anemia among hospitalized children

Characteristics

Sex

Age in years

Nutritional
statusIntestinal
parasites

Hospitalization
lengthAnemic

Non-anemic

COR (95% CI)

AOR (95% CI)

Male

127 (59.9)

85 (40.1)

1-

Female98 (57)74 (43)0.886(0.589,1.333)

-

0.5-5

93 (54.1)

79 (45.9)

1

1

5-11

76 (58.9)

53 (41.1)

1.218(0.768,1.932)

1.02(0.603,1.726)

12-15

56 (67.5)

27 (32.5)

1.762(1.018,3.049)

1.53(0.829,2.822)

Malnourished

49 (59)34 (41)1.024(0.625,1.677)-

Normal176 (58.5)

125 (41.5)

1

-

Yes

50 (72.5)

19 (27.5)

2.105(1.187,3.734)

2.541(1.363,4.737)*

No

175 (55.6)

140 (44.4)

1

1

Up to 2 days

69 (61.6)

43 (38.4)

1

1

3-5 days

105 (53.8)

90 (46.2)

0.727(0.453,1.167)

0.685(0.414,1.132)

Above 5 days

51 (66.2)

26 (33.8)

1.222(0.666,2.242)

1.29(0.681,2.442)

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Do youeat meat

Edema

Fever

Vomiting

Diarrhea>1 weekYes

101 (51.8)

94 (48.2)

1

1

No

124 (65.6)

65 (34.4)

1.775(1.177,2.678)

1.615(1.014,2.574)*

Yes

71 (70.3%)

30 (29.7)

1.982(1.219,3.225)

1.56(0.898,2.71)

No

154 (54.4)

129 (45.6)

1

1

Yes

27 (45.8)

32 (54.2)

0.541(0.31,0.946)

0.606(0.324,1.136)

No

198 (60.9)

127 (39.2)

1

1

Yes

18 (46.2)

21 (53.8)

0.571(0.294,1.112)

0.446(0.208,0.96)*

No

207 (60)

138 (40)

1

1

Yes

14 (43.8)

18 (56.3)

0.52(0.25,1.079)

0.402(0.178,0.907)*No

214 (58.8)

150 (41.2)

1

1* Indicates statistically significant association;
COR: Crude Odds Ratio; AOR: Adjusted Odds Ratio; CI: Confidence interval

Table 3

Association of family/caregiver related factors
with anemia in among hospitalized children

Characteristics

Residence

Maternal
educational
statusAnemic

Non-anemic

COR (95% CI)

AOR (95% CI)

Urban

84 (49.4)

86 (50.6)

1

1

Rural

141 (65.9)

73 (34.1)

1.977(1.309,2.988)

1.638(0.844,3.177)

Unable read &write

144 (64.6)

79 (35.4)

1.823(1.059,3.137)

1.657(0.299,9.182)

Primary
education

21 (47.7)

23 (52.3)

0.913(0.429,1.942)

0.908(0.191,4.305)

Secondaryeducation

25 (53.2)

22 (46.8)

1.136(0.542,2.382)

1.113(0.278,4.455)

College /University

35 (50)35 (50)

1

1

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Paternal
educational
statusMotheroccupation

Fatheroccupation

Monthly income
in ETB

Unable read& write125 (63.1)

73 (36.9)

1.673(1.005,2.786)

0.318(0.064– 1.588)

Primary
education

25 (59.5)

17 (40.5)

1.437(0.682,3.03)

0.455(0.103,2.003)

Secondaryeducation

31 (54.4)

26 (45.6)

1.165(0.597,2.276)

0.558(0.156,1.996)

College /University

44 (50.6)

43 (49.4)

1

1

Government
employee

32 (51.6)

30 (48.4)

0.66(0.38 -0 1.145)

1.112(0.241,5.128)

Private12 (41.4)

17 (58.6)

0.437(0.201,0.949)

0.467(0.166,1.315)

House wife

181 (61.8)

112 (38.2)

1

1

Government
employee

40 (48.2)

43 (51.8)

11

Farmer134 (63.5)

77 (36.5)

1.871(1.119,3.127)

1.553(0.305,7.892)

Private51 (56.7)

39 (43.3)

1.406(0.772,2.56)

1.955(0.463,8.255)<1000

43 (65.2)

23 (534.8)

1.842(1.004,3.381)

1.528(0.569,4.102)

1001-2000

113 (62.4)

68 (37.6)

1.638(1.044,2.569)

1.283(0.563,2.926)

Above 2000

69 (50.4)

68 (49.6)

1

1

ETB = Ethiopian Birr, 28.40 ETB = 1 $USD;
COR: Crude Odds Ratio; AOR: Adjusted Odds Ratio; CI: Confidence interval.

one week (AOR = 0.402; 95% CI: 0.178, 0.907)
were also showed a statistically significant association with anemia. This is due to the factthat during vomiting and diarrhea, there will
be a decrease in body fluid and mask the lowlevel of Hgb value.
Family/care giver-related factor association withanemia was determined as indicated in Table 3.

In bivariate logistic analysis, being rural in residence, having no formal maternal education
and a monthly income below 2,000.00 Ethiopian
Birr (28 Ethiopian Birr = 1 $USD) were statistically associated with anemia among hospitalized children. However, there is no family/caregiver related factors associated with anemia in
multivariate logistic regression analysis.

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

DISCUSSION
The findings from this study indicate that theprevalence of anemia among hospitalized children aged 6 months to 15 is high. The prevalence
was found to be 58.6 %. The overall prevalence
is higher than reported from other studies inIndia (21), Lebanon (14), Nepal (22) and Nigeria(23), where the prevalence were between 32
and 50%. The variations in percentage in different regions might be due to heterogeneity ofthe studied population, dietary habits, different
nutritional status and incidence of worm infestation in a defined geographical spots (21).
In our study, prevalence of anemia was elevated as age increase and it was quite high amongolder children aged 12-14 years (65.7%). Thisfinding is contrary to that describe by others
where the prevalence of anemia was highest
among children under age of five (14). The highest prevalence in age group between 12 and 15
may be attributed to low intake of iron rich foodor inappropriate dietary choices in children,
poor iron absorption due to iron absorption inhibiting factors such as tannine in tea which reduces hemoglobin synthesis or inadequate ironabsorption enhancers (vitamin C and hydrochloric acid) (24). Based on our finding, anemia
prevalence in children under five which is 54.1%
is lower than the result found in Ghana, Brazil,
Bangalore, and Tanzania (13, 17, 25-27).
Different studies had different result on the association between anemia and gender. This study
found insignificant difference in anemia prevalence between male and female, 57.0% and59.9% respectively. While others have demonstrated that the prevalence of anemia can vary
between male and female children (28). They argued that the high prevalence of anemia in boys
may be due to the faster growth of preschoolboys than girls that has high iron demand whichcannot be met by diet alone.

Morphological classification of anemia showed
microcytic hypochromic anemia was the most
predominant type occurring in 62.2% of children. This finding is similar to the study donein Tanzania, and it is assumed that iron depletion is the main factor responsible for the highpercentage of microcytic-hypochromic anemia(13). On the other hand, normocytic normochromic anemia was the second type of anemia
in this study. This may be due to acute blood
loss, drug therapy and chronic diseases (anemia
of inflammation) (29, 30).
Child related factors like being infected withparasitic infection and not eating meat and animal products were statistically associated withanemia. The lower immune response in children
compared to adults, poor hygiene and environmental conditions favor the susceptibility ofchildren to parasitic infections (31). In our finding, children with parasitic infestation are twoand half times more likely to have anemia thanchildren who are free from parasitic infection,(AOR= 2.541; 95% CI: 1.363, 4.737). This finding
is similar to study done in Bangladesh (32). The
mechanism behind this relation can be due tothe fact that the parasite directly induces irondeficiency through blood loss by mechanical rupture of host capillaries and arterioles followedby the release of a battery of pharmacologically
active polypeptides including anticoagulants,
antiplatelet agents, and antioxidants (31).
Not eating meat and animal products were also
found to be related to a risk of acquiring anemia in our study and this is similar to the study
done in Uganda (12). Iron can be found in two
forms in foods heme and non-heme. Heme ironis only found in animal products, whereas nonheme iron is only found in plants (33). Iron deficiency and/or low bioavailability account forhalf of the anemia in developing countries (34)
accessed 28 November, 2018.

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Bamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria, Ayenew Addisu, Meaza Genetu
Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

In this study univariate analysis showed anemiathat was significantly associated with maternal
related variables like being rural in residence(COR= 1.977 (1.309, 2.988)), have no formal
education (COR= 1.823 (1.059, 3.137)) and low
monthly income (COR= 1.842 (1.004, 3.381)),but in multivariate analysis this factor was not
significant statistically. Since mothers are mostly care givers for the child, maternal education
has always been linked to many child health outcomes. It may also affect health decision making and thus influence the probability of a childmeeting certain nutrition-related requirements.

significantly associate with anemia. Deworming
and interventions like iron supplementation andnutritional education activities are important todecrease the prevalence of anemia.

In addition, anemia among children was also
associated with household income. Children
living in household with lower monthly income(<2000 Ethiopian Birr) were more likely to haveanemia compared to those with higher income.
Similar finding was from study conducted in
Brazil and northern Ethiopia (9). The reason
might be due to children from poor households
are less likely to get iron-rich foods.

Hgb: Hemoglobin

This study has some limitations in that we lackdata on the prevalence of blood parasite like
plasmodium falciparum, one of the risk factors
considered to be the principal cause of severe
anemia in malaria-endemic areas in Africa. The
clinical diagnoses of the children were also notincluded in this study therefore clinical causesof anemia could not be identified.

WHO: World Health Organization

CONCLUSIONS
Prevalence of anemia in hospital admitted children aged between 6 months and 15 years ishigh and it was found to be a severe public healthproblems. Being more common in children may
predispose this vulnerable population to future
infections, hematological and developmental
disorder. Microcytic, hypochromic and normocytic normochromic pattern were the common
morphological types of anemia. Parasitic infection and not eating meat and animal products

Abbreviations
AOR: Adjusted odds ratio
COR: Crude odds ratio
EDTA: Ethylene Die-amine Tetra-acetic Acid
FA: Fanconi Anemia
HCT: Hematocrit
IDA: Iron Deficiency Anemia
MCH: Mean Cell Hemoglobin
MCHC: Mean Cell Hemoglobin Concentration
MCV: Mean Cell Volume
RBC: Red Blood Cell
RDW: Red Cell Distribution widthEthical approval and consent to participate
Ethical clearance and approval was granted
from University of Gondar, Collage of Medicine
and Health Sciences, School of Biomedical andLaboratory Sciences Ethical Review Committee.
Permission letter was also obtained from thehospital administrators. The purpose of theresearch was explained to the study subjects
and written informed consent and assent was
obtained from each hospitalized children andtheir care givers and then those who were willing to participate were included in the study.
Participation was fully voluntarily, refusal at
any time during data collection was permitted.
Confidentiality was kept. When haemoglobin
level of the children was below 11 g/dl, the result was communicated with the physicians.

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Bamlaku Enawgaw, Yaregal Workineh, Sisay Tadesse, Eyuel Mekuria, Ayenew Addisu, Meaza Genetu
Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

Availability of data and materials
All data generated or analyzed during this study
are included in this published article.
Competing interests
The authors have declared that they have no existing competing interests.
Funding
No specific funding was received for this work.
Authors’ contributions•

BE & AA: conceived and designed
the experiments•

YW, ST, EM, MM: performed the experiments•

BE & MG: analyzed and interpreted results•

All authors contributed to the writing andediting of the manuscript and approved thefinal version submitted.

Acknowledgments
The authors would like to extend their gratitude to the University of Gondar comprehensive
and specialized referral hospital pediatrics ward
staffs for their support during this study. We also
thank the data collectors, children and their parents/caregivers who participated in this study.

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Prevalence of anemia and associated factors in hospitalized children from Gondar, Northwest Ethiopia

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Waist circumference cutoff point determination
for defining metabolic syndrome
in type 2 diabetes mellitus in Ethiopia
Shewit Hailemariam1, Tadele Melak2, Molla Abebe2
1
2

Ayder Comprehensive Specialized Hospital, Mekelle University, Tigray Region, Northern Ethiopia
Department of Clinical Chemistry, School of Laboratory Medicine, College of Medicine and Health Sciences,
University of Gondar, Ethiopia

ARTICLE INFOABSTRACT

Corresponding author:
Tadele Melak
P.O. Box 196
College of Medicine and Health Sciences
Department of Clinical Chemistry
University of Gondar
Amhara Regional State
EthiopiaPhone: +251921576005
E-mail: 0923tadie@gmail.com

BackgroundKey words:
cutoff point, diabetes mellitus,
metabolic syndrome,
waist circumference

Metabolic syndrome (MetS) is a complex disorder char­ac­terized by a cluster of interrelated cardiovascularrisk factors. So far, cutoff point variability of waist circumference was documented to define MetS.
Objective
To determine the classification power and cutoff pointof waist circumference to define MetS among patientswith type 2 diabetes.
Methods
An institution-based cross sectional study was conducted from March to April 2017 at Ayder Comprehensive
Specialized Hospital among patients with type 2 diabetes. Using systematic sampling technique, 520 participants were enrolled into the study. Data were collected by checklist, anthropometric measurements
and biochemical analyses. Data were entered to
Epi-info 3.5.1 and transferred to SPSS 20 for analysis.
Participants having more than one abnormal MetS
components were categorized as patients and thePage 48

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Waist circumference cutoff point estimation for metabolic syndrome in Ethiopia

others were considered as control. The classification power of waist circumference to distinguish patients from controls was determined
by ROC curve analysis. Waist circumference cutoff points were determined by taking the pointthat had a maximum youden index.
Results
Among the 520 participants, 308 (59.2%) werefemales. The mean age of the participants was
56 ± 10.8 years for males and 55 ± 11.4 years
for females. The classification power of waist
circumference was 0.67 (0.58-0.75) for maleand 0.63 (0.52-0.73) for females. The optimal
waist circumference cutoff point to distinguishpatients from controls were 95.5 cm (sensitivity 39.8%, specificity 86.3%, p< 0.001) for
males, and 87.5 cm (sensitivity 73.1%, specificity 54.5%, p< 0.017) for females.
Conclusion
The positive predictive value of waist circumference was 93% for females and 90% for males inNorthern Ethiopia using 87.5 and 95.5 cm points
cut-off for females and males, respectively.


INTRODUCTION
Metabolic syndrome (MetS) is a complex disorder characterized by a cluster of interrelated
factors that increase the risk of cardiovascular disease (CVD). In type 2 diabetic patients(T2DM) and those with other non-communicable diseases (1), MetS increases the complication rate (2).
Various international organizations are using
five parameters including elevation of waist circumference (WC), blood pressure (BP), triglycerides (TG), fasting blood glucose (FBG), andreduced HDL-C level to define MetS (3).

Elevated WC is characterized by the accumulation of fat in the visceral part of the abdomen
and plays a central role in the pathogenesis of
MetS (4).
To precisely assess the visceral fat volume, abdominal computed tomography (CT) has been
considered as the most accurate and reproducible technique (5). However, CT scans are costly
and time consuming (6). Because of this limitation, a variety of alternative methods are applied to assess abdominal obesity.
Waist to hip ratio and WC are correlated withabdominal imaging. Among them, WC is considered to be simple and inexpensive measurewith excellent correlation with the CT results(7). In addition, it is used to define MetS basedon various international criteria and different
scholars indicted that WC appears to be the best
central obesity indicator than BMI and waist to
hip ratio (8).
Many organizations and expert groups recommend WC as one of the criteria to define MetS.
International Diabetes Federation (IDF), particularly, needs elevation of WC as an obligatory criterion with a different cutoff point in accordancewith ethnicity and gender. Recent publications
report that WC cutoff point is quite variable
among different ethnic groups and between
genders. However, based on ethnicity and gender, until now, the IDF established WC cutoff
point for the general populations for two regions only to define MetS, for the European andAsian populations.
In this regard, the African region has no cutoff
point for WC to define MetS yet (9). A numberof studies support the necessity of locality based
WC cutoff points. Consequently, this study
aimed to determine WC cut points among T2DM
patients in Ayder Comprehensive Specialized
Hospital (ACSH), Northern Ethiopia.

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Waist circumference cutoff point estimation for metabolic syndrome in Ethiopia

MATERIALS AND METHODS
Samples and design
An institution based cross-sectional study was
conducted among T2DM patients from March
to April 2017 in ACSH. Five hundred twenty participants were selected based on systematic random sampling techniques.
Data collection
Data were collected on the basis of self-administered standardized questionnaires andthe measurement of anthropometric, bloodpressure and biochemical parameters. A selfadministered standardized questionnaire wascustomized on the basis of the WHO STEP wise
approach to non-communicable disease risk factor surveillance (10).
Measurements
Blood pressure was measured after the participants had rested for at least 5 minutes. The
measurements were taken using left arm at theheart level, using BP monitor MB 300-D digital
instrument. In each case, the mean of two results was used for analysis (11).
BMI was calculated as weight (kg) divided byheight squared (m2). WC was taken by positioning the non-elastic measuring tape midway between the lower rib margin and the iliac crest.
It was measured to the nearest 0.5 cm (12).
Fasting (8 to 12 hour) 5 mL venous blood was
also collected in clot activated test tubes. Serumwas separated within 30 minutes (2000 rpm, 5minutes). FBG, TG and HDL-C were determinedusing a Pentra C400 clinical chemistry analyzer.
Data analysis and interpretation
The data were entered into EpiData 3.1 and thentransferred to SPSS version 20. Data cleaning wasdone before analysis, descriptive and summary
statistics were also performed. Participants who
had one or more abnormal MetS components(excluding WC and FBS), were coded as caseand the others as control (9, 13). The classification potential (AUC) of WC to distinguish cases
from control was determined using ROC curvefor both sexes separately. Sensitivity and specificity of WC to distinguish cases from controls
was calculated at several WC points. For each
WC point Youden index (YI) was calculated. The
WC points, having the maximum YI was taken as
the optimum cutoff point of WC to classify casefrom controls. In all conditions, P-value of < 0.05
was considered as statistically significant.
RESULTS
Among the 520 participants, 308 (59.2%) werefemales. The mean (± SD) ages of the participants
were 56 ± 10.8 and 55 ± 11.4 years for males andfemales, respectively. A majority of the participants, 457/520 (87.9%), were from the urban
area (Table 1).
The mean duration of diabetes co-morbidity after diagnoses was 4.7 ± 2.9 years for males and
4.6 ± 2.5 years for females. All of the study participants are taking anti-diabetic drugs. Seventy
two (35%) male and 144 (46.8%) female participants were overweight and obese (Table 2).
Four hundred thirty six patients were investigated for MetS (275 females and 161 males).
Reduced HDL-C was the most typical abnormalcomponent of MetS in both genders, it was present in 221 (42.5%) patients. High blood pressure
was detected in 157 patients (30.2%) (Table 3).
The classification power of WC to define MetS
was characterised by AUC. The value of AUC
was 0.67 (95% CI, 0.58-0.75) for males and 0.63
(95% CI, 0.52-0.73) for females, respectively(Figure 1).
The classification power of WC to define the reduced HDL-C was lower as compared to the other MetS components in both sexes (0.60 in males
and 0.53 in females) (Table 4).

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Waist circumference cutoff point estimation for metabolic syndrome in Ethiopia

Among men, WC at a cut-off value of 95.5 cms
yielded the highest YI (0.261) with a corresponding sensitivity of 39.8% and specificity of 86.3%.
Table 1

For women, the WC at a cut-off value of 87.5 cms
yielded the highest YI (0.276) with a sensitivityof 73.1% and specificity of 54.5 % (Table 5).

Socio-demographic characteristics of patientswith diabetes mellitus at Ayder Comprehensive Specialized Hospital,
Northern Ethiopia, 2017 (N=520)
Male
n (%)

Female
n (%)

Total
N (%)

30-39

11 (5.2)

18 (5.8)

29 (5.6)

40-49

45 (21.2)

81 (26.3)

26 (24.2)

50-59

8 (36.8)

95 (30.8)

173 (33.3)

60-69

55 (26.0)

76 (24.7)

131 (25.2)≥ 70

23 (10.8)

38 (12.4)

61 (11.7)

Governmental

80 (37.7)

68 (22.1)

148 (28.5)

Non-governmental

19 (9.0)

13 (4.2)

32 (6.1)

Self employee

68 (32.0)

56 (18.2)

124 (23.8)

House wife

0 (0.0)128 (41.5)

128 (24.6)

Farmer22 (10.4)

11 (3.6)

33 (6.35)

No

23 (10.9)32 (10.4)

55 (10.6)

Single17 (8.0)

9 (6.2)36 (6.9)

Married181 (85.4)

212 (68.8)

393 (75.6)

Divorced

8 (3.8)19 (6.2)

27 (5.2)

Widowed6 (2.8)58 (18.8)64 (12.3)

Tigrie208 (98.1)

305 (99.0)

513 (98.6)

Other

4 (1.9)3 (1.0)7 (1.4)

Variables

Age ( years)

Occupation

Marital status

Ethnicity

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Educational level

Residence

Religion

Table 2

No formal schooling

29 (13.7)

79 (25.7)

108 (20.7)

Less than Primary school

14 (6.6)

43 (13.9)

57 (10.8)

Primary school completed

47 (22.2)

60 (19.5)

107 (20.6)

Secondary school completed

38 (17.9)

53 (17.2)

91 (19.0)

College/University completed

84 (39.6)

73 (23.7)

157 (28.9)

Rural

29 (13.7)

34 (11.1)

63 (12.2)

Urban

183 (86.3)

274 (88.9)457 (87.8)

Orthodox

191 (90.1)

288 (93.5)479 (92.1)

Muslim18 (8.5)

18 (5.8)

36 (6.9)

Other

3 (1.4)2 (0.7)5 (1.0)Frequency of clinical, behavioral and anthropometric measurementsof T2DM patients at Ayder Comprehensive Specialized Hospital,
Northern Ethiopia, 2017 (N=520)
Male
n (%)

Female
n(%)

Total
N (%)

Anti-DM Only

123 (58.0)

170 (55.2)

293 (56.3)

Anti-DM +Anti-HT

34 (16.0)

27 (8.8)

61 (11.7)

Anti-DM+Anti-Dyslipidemia

25 (11.8)

58 (18.8)

83 (16.0)

Anti-DM + HT +Anti-Dyslipidemia

30 (14.2)

53 (17.2)

83 (16.0)

Less than one year

20 (9.4)

25 (8.1)

45 (8.7)

1-5 years

101 (47.6)

154 (50.0)

255 (49.0)

6-10 years

39 (18.4)

75 (24.4)

114 (21.9)

More than10 years

52 (24.5)

54 (17.5)

106 (20.4)

Variables

Medication status

Duration of diabetescomorbidity (Yrs)

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Waist circumference cutoff point estimation for metabolic syndrome in Ethiopia

Yes

46 (21.7)

14 (4.5)60 (11.5)

No

166 (78.3)

294 (95.5)

460 (88.5)

Yes

5 (2.4)0 (0.0)5 (1.0)No

207 (97.6)

308 (100)

515 (99.0)

Under weight

11 (5.0)

10 (3.2)

21 (4.0)

Normal weight

129 (60.0)

154 (50.0)

283 (54.5)

Over weight65 (31.7)

116 (37.7)

181 (34.8)

Obese

7 (3.3)28 (9.1)

35 (6.7)

Alcohol consumption

Cigarette Smoking

BMI

DM: diabetic mellitus; HT: hypertension.

Table 3Frequency of individual components of MetS
among T2DM patients in Ayder Comprehensive Specialized Hospital,
Northern Ethiopia, 2017 (n=520)
Male

FemaleTotal (N%)

Variablesn (%)

95 % CIn (%)

95 % CIHigh TG

79 (37.3)

30.7-44.0

134 (56.5)

37.1-49.0

213 (40.9)high BP75 (35.4)

28.9-42.3

82 (26.6)

21.2-31.3

157 (30.2)

low HDL-C

116 (54.7)

47.6-61.7

105 (34.1)

20.6-47.6

221 (42.5)

Clusters of abnormal components

At least 1 component

161 (75.9)

70.2-81.6

275 (89.3)

85.4-92.4

436 (83.8)

At least 2 components

78 (36.8)

30.7-43.5

131 (42.5)

36.7-47.7

209 (40.2)

At least 3 components

57 (26.9)

20.6-32.9

99 (32.1)

27.3-37.2

156 (30.0)

All components

26 (12.3)

7.9-16.7

45 (14.6)

11.0-19.2

71 (13.6)

BP: Blood Pressure; HDL-C: High density lipoprotein cholesterol; TG: Triglyceride.

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Figure 1

ROC curves of WC cutoff value to discriminate cases
from controls among T2DM patients for both sexes
in Ayder Comprehensive Specialized Hospital, Northern Ethiopia

Table 4Characteristics of AUC value of the ROC curves WC to define casesand MetS components among T2DM patients in Ayder Comprehensive
Specialized Hospital, Northern Ethiopia, 2017 (N=520)

Sex

Male

FemaleMetS
components

AUC(95% CI)

Sensitivity

Specificity

P value

Hypertension

0.67 (.60 -.75)

0.51

0.76

≤0.001Hypertriglyceridemia

0.67 (.60 -.74)

0.49

0.76

≤0.001

Low HDL-C

0.60 (.52 -.67)

0.39

0.73

0.013

Cases

0.67 (.58 -.75)

0.40

0.86

≤ 0.001

Hypertension

0.63 (.56 -.70)

0.79

0.33

0.001

Hypertriglyceridemia

0.57 (.52 -.65)

0.75

0.34

0.010

Low HDL-c0.53 (.44 -.61)

0.72

0.59

0.017

Cases

0.63 (.52-.73)

0.73

0.45

0.01

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Table 5

Performance of different WC cutoff-points for discriminating patientsfrom controls among T2DM patients at Ayder Comprehensive Specialized
Hospital, Northern Ethiopia, 2017 (N=520)
WC cutoffpoints (cm)Sensitivity(95%CI)

Specificity(95%CI)

PPV(95%CI)

NPV(95%CI)

Youden
index

90

63.4

60.8

83.6

34.4

0.242

92

54.4

70.6

85.3

32.7

0.250

Male

94

44.1

80.4

87.7

31.3

0.245

(n=212)

95.5

39.8

86.3

90.1

31.2

0.261

98

27.9

88.2

88.2

27.9

0.161

100

21.7

92.2

89.7

27.2

0.139

80

88.4

24.2

87.3

20.0

0.126

82

84.4

30.3

91.0

18.9

0.147

Female

85

81.4

36.4

91.4

19.0

0.178

(308)

87.5

73.1

54.5

93.0

19.6

0.276

88

70.9

54.5

92.8

18.4

0.254

90

61.8

57.6

92.4

15.3

0.194

Sex

CI: confidence interval; PPV: positive predictive value; NPV: negative predictive value.

DISCUSSION AND CONCLUSIONS
Central obesity is the major component of metabolic risk factors. WC is considered to be simple
and inexpensive measure with excellent correlation with the results of CT. As a good indicator of
visceral fat, WC is widely used to predict the outcome of MetS. WC cutoff values are age, genderand ethnicity specific for MetS.

significant power for both sexes (P-value <0.001
and <0.017 for males and females, respectively), but it had a poor classification power tocategorize patients from controls in both sexes.
The optimal WC cutoff value was 87.5 cm (sensitivity 73.1% and specificity 54.5%) for females
and 95.5 cm (sensitivity 39.8% and specificity
86.3 %) for males for identifying patients fromcontrols.

Therefore, in the current study, WC had classified patients from controls with statisticallyEven though the classification power of WC wasstatistically significant, it had poor classification

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power for males (AUC=0.67; 95% CI: 0.58-0.75),
and for females (AUC =0.63; 95% CI 0.52-0.73)
to discriminate cases from controls (14). Similar
to our study, the classification power of WC toclassify patient from control was poor in a study
conducted in Benin, where, the AUC was 0.67
for males and 0.68 for females (15). Moreover,in Egypt it was also 0.69 for males and 0.63 forfemales (16).
On the contrary, in several studies such as inAngola (0.85 in males and 0.79 in females) (17),and in Congolese adults (0.899 in males and0.844 in females) (18), WC had a good classification power to classify patient from control inboth sexes.
In our study, the optimal WC cutoff point thatbest predicts cases was 95.5 cm for the T2DM
males, which is relatively higher than the value
for females. The finding from the present study
is slightly higher than the IDF recommended value for males, which is 94 cm (9).
In contrast to our study, WC cutoff point in urban African teachers was 90 cm in men (19) andthe same to that in black South African T2DM
male patients were also 90 cm (20). These values were somewhat different to that found in
T2DM male patients in our study area.
The WC cutoff value for T2DM females determined in this study is 87.5 cm which is similar
to the study in University employees in Angola(87.5 cms) (21).
The result from this study was higher than therecommended cutoff points to define cases for
African females, which is ≥80 cms. Whereas our
finding is lower than the study conducted with
African females, which was 98 cm (19) and a instudy from Egyptian adults (96.25 cms) (16).
The present study suggested that the WC cutoff
point for females is lower than males, which isin agreement with the recommended value.

Some studies show that the WC cutoff point
for African populations is higher in rural South
African females (92 cms) than males (86 cms)
(22), in Cape Town 94 cm for females and 83.9
cm for males (23).
Similarly, in Benin, the cutoff is 94 cm for females
and 80 cm for males, which is the exact reverseof the recommended value (24). Furthermore, a
study in Congolese community also shows higher WC value for females (99 cms) compared tothe males (95 cms) (18).
In general, several studies in Africa that determined WC cutoff points showed a higher WC
value in females as compared to males. On thecontrary, our finding is higher in males than females, which is in agreement with the IDF.
On the other hand, a study from Tunisian adults
showed equal value of WC for both males
and females which was 85 cm (25). Although
the WC value in adult Egyptian females (96.2cms) wasn’t higher than males (100.5 cms), it
is higher than the IDF recommended value for
African females (16).
Further study in prospective cohort is better todetermine the optimal WC cutoff point among
T2DM patients to define MetS. In addition,
studies which can consider the confounders
such as lipid lowering medications, age of theparticipants and duration of diabetes are advisable in order to increase the classification power of WC.


List of abbreviations
AUC: Area Under the Curve
BMI: Body Mass Index
BP: Blood Pressure
CT: Computed Tomography
CV: Cardiovascular

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Shewit Hailemariam, Tadele Melak, Molla Abebe
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CVD: Cardiovascular Disease

Conflict of interests

FBG: Fasting Blood Glucose

The authors declared no conflict of interests.

HDL-C: High Density Lipoprotein cholesterol

Funding

HT: Hypertensive

No specific fund for this research work.

IDF: International Diabetes Federation

Acknowledgments

IR: Insulin Resistance

The authors would like to acknowledge the study
participants and the Ayder Comprehensive Spe­cialized Hospital.

LDL-C: Low Density Lipoprotein cholesterol
MetS: Metabolic syndrome
NCD: Non Communicable Disease

NCEP/ATPIII: National Cholesterol Education
Program Adult Treatment Panel III

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NT: Normotensive

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ROC: Receiver Operating Characteristics
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VAT: Visceral Adipose Tissue

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Ethical issue
Ethical clearance was obtained from the Insti­tu­
tional Review Board of the University of Gondar.
The purpose and importance of the study wereexplained to the participants. The data were collected after a full written consent was received.
Author’s contribution
SH conceived the idea, carried out the proposal
writing, participated in the data collection, data
analysis and drafted the manuscript. TM and MA
participated in data analysis and interpretationof the findings. TM participated on final writeup of the paper. All authors reviewed the manuscript and approved for publication.

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Axelsson J, Vasselai P, et al. Waist circumference and visceral fat in CKD: a cross-sectional study. Am J Kidney Dis.
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8. Ma W-Y, Yang C-Y, Shih S-R, Hsieh H-J, Hung CS, Chiu
F-C, et al. Measurement of Waist Circumference Midabdominal or iliac crest? Diabetes care. 2013;36(6):1660-6.
9. Alberti K, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI,
Donato KA, et al. Harmonizing the metabolic syndrome.
Circulation. 2009;120(16):1640-5.

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Shewit Hailemariam, Tadele Melak, Molla Abebe
Waist circumference cutoff point estimation for metabolic syndrome in Ethiopia

10. WHO. The WHO STEPwise approach to noncommunicable disease risk factor surveillance (STEPS): WHO STEPwise approach surveillance- Instrument 3.1.

visceral fat and metabolic syndrome in a Congolese community: further research is still to be undertaken. Pan Afr
Med J. 2013;14(1).

11. Ranasinghe P, Cooray DN, Jayawardena R, Katulanda P.
The influence of family history of Hypertension on diseaseprevalence and associated metabolic risk factors among
Sri Lankan adults. BMC Public Health. 2015;15(1):1-9.

19. Prinsloo J, Malan L, De Ridder J, Potgieter J, Steyn H.
Determining the waist circumference cut off which best
predicts the metabolic syndrome components in urban
Africans: the SABPA study. Exp Clin Endocrinol Diabetes.
2011;119(10):599-603.

12. European Health Risk Monitoring (EHRM). National
Public Health Institute Recommendation for indicators,
international collaboration, protocol and manual of operations for chronic disease risk factor surveys 2002.
13. Grundy SM, Brewer HB, Cleeman JI, Smith SC, Lenfant C. Definition of metabolic syndrome. Circulation.
2004;109(3):433-8.
14. Hajian-Tilaki K. Receiver operating characteristic(ROC) curve analysis for medical diagnostic test evaluation. Caspian J Intern Med. 2013;4(2):627.
15. Agueh VD, Sossa C, Ouendo DM-E, Paraizo NM, Azandjemè C, Kpozehouen A, et al. Determination of the optimal waist circumference cut-off points in Benin adults. O
J Epi. 2015;5(04):217.
16.Assaad-Khalil SH, Mikhail MM, Aati TA, Zaki A, Helmy
MA, Megallaa MH, et al. Optimal waist circumference
cutoff points for the determination of abdominal obesity and detection of cardiovascular risk factors among
adult Egyptian population. Indian J Endocrinol Metab.
2015;19(6):804.
17. Magalhães P, Capingana DP, Mill JG. Prevalence of themetabolic syndrome and determination of optimal cutoff values of waist circumference in university employees from Angola: cardiovascular topic. Cardiovasc J Afr.
2014;25(1):27-33.
18. Katchunga PB, Hermans M, Bamuleke BA, Katoto PC,
Kabinda JM. Relationship between waist circumference,

20. Kalk WJ, Joffe BI, Sumner AE. The waist circumference of risk in black South African men is lower than inmen of European ancestry. Metabol Syndr Relat Disord.
2011;9(6):491-5.
21. Magalhães P, Capingana DP, Mill JG. Prevalence of themetabolic syndrome and determination of optimal cutoff values of waist circumference in university employees
from Angola. Cardiovasc J Afr. 2014; 25(1):27-3.
22. Motala AA, Esterhuizen T, Pirie FJ, Omar MA. The prevalence of metabolic syndrome and determination of the
optimal waist circumference cutoff points in a rural South
African community. Diabetes care. 2011;34(4):1032-7.
23. Peer N, Steyn K, Levitt N. Differential obesity indices identify the metabolic syndrome in Black men andwomen in Cape Town: the CRIBSA study. J Pub Health.
2016;38(1):175-82.
24. Mabchour AE, Delisle H, Vilgrain C, Larco P, Sodjinou
R, Batal M. Specific cut-off points for waist circumference
and waist-to-height ratio as predictors of cardiometabolicrisk in Black subjects: a cross-sectional study in Benin andHaiti. Diabetes Metab Syndr Obes. 2015;8:513.
25. Bouguerra R, Alberti H, Smida H, Salem L, Rayana C,
El Atti J, et al. Waist circumference cutoff points for identification of abdominal obesity among the Tunisian adult
population. Diabetes Obes Metab. 2007;9(6):859-68.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Critical issues and new trends
on stat tests in clinical laboratory
Ariadna Arbiol-Roca, Dolors Dot-Bach

Laboratori Clínic Territorial Metropolitana Sud (LCTMS), University Hospital of Bellvitge,
L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain

ARTICLE INFOABSTRACT

Corresponding author:
Ariadna Arbiol-Roca
Laboratori Clínic Territorial
Metropolitana Sud (LCTMS)
University Hospital of Bellvitge
Feixa Llarga s/n, 08907
L’Hospitalet de Llobregat
BarcelonaSpain
E-mail: ariadna.arbiol@bellvitgehospital.cat

Meeting Report on the IX European Symposium
on Clinical Laboratory and In Vitro Diagnostics
Industry (Barcelona)

Key words:
stat tests, symposium, clinical laboratory

The IX European Symposium of the Clinical Laboratory
and In Vitro Diagnostics Industry, entitled “Stat Tests in
Clinical laboratory”, took place in Barcelona, Catalonia(Spain), between May 17–18, 2017.
The scientific program was structured in several roundtables that dealt with the following topics: emergency
laboratory models, accreditation of stat tests by ISO
15189, critical issues of stat tests and the new proposals of the in vitro diagnostics industry for emergency
laboratories. The aim of the Symposium was the discussion of the transformation that stat tests havegenerated on clinical laboratories in terms of organization, turnaround time, accreditation, and probableevolution of these laboratories coming years.

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Critical issues and new trends on stat tests in clinical laboratory

INTRODUCTION
The IX European Symposium of the ClinicalLaboratory and In Vitro Diagnostics Industry,
entitled “Stat Tests in Clinical laboratory”, washeld in a most welcoming yet modern environment at the old gothic Hospital de la Santa
Creu. The event was co-organized between
the Catalan Association of Clinical Laboratory
Sciences (ACCLC) and the Catalan Society ofBiology (SCB). The symposium was sponsoredby the International Union of Pure and Applied
Chemistry (IUPAC) and under the auspices of theEuropean Federation of Clinical Chemistry andLaboratory Medicine (EFLM) and International
Federation of Clinical Chemistry and Laboratory
Medicine (IFCC).
The topic of stat testing in clinical laboratories
was chosen as an opportunity to discuss the current state of emergency laboratories and also
analyse the probable evolution of these laboratories in the coming years. Stat (from the Latin
statim, immediately, but also considered as anacronym for “short turnaround time”) identifies
laboratory tests that should be made available
within a defined, as short as possible time, according to clinical necessity (1,2).
There are several reasons that make stat testing
an interesting topic for a Symposium. Mainly,
organizational and economic aspects: collecting blood outside scheduled activity, the needof quick sample transportation to the laboratory in some cases with pneumatic tube systems, activation of specific paths for managing
specimens with priority over routine samples,in many of laboratories entails exclusive staff,
specific instrumentation and the need to maintaining back-up instrumentation (3).
EMERGENCY LABORATORY MODELS
Currently, three laboratory models are adopted
for the management and performance of stat
tests. Each of these solutions is related to thesize and type of hospital or institution in which
they are employed (2).
Generally, in small- and medium-size laboratories, ordinary and stat tests are integrated, andtheir analysis is performed in the same placeusing the same instrumentation. The situationin large laboratories is rather more heterogeneous, with the majority of organisations continuing to separate stat from ordinary tests,
using different instrumentation, personnel andlocations. An intermediate option also exists, inwhich stat test analyses are semi-integrated in
an automated core chain with routine samples,
all of which are processed at the same time.
However, each approach requires specific workflow processes, leading to different timelinessto produce a validated result. Generally speaking, the stat testing process should be structured to fit the context of care in which the testing services are required.
There was a strong discussion regarding how toprioritise these stat analyses in order to provide
an adequate turnaround time (TAT), such as
for samples in a specific emergency chain andbranches of the chain for the highest-priority
samples. It was concluded that large laboratories tend to incorporate stat tests in central automation areas (core), prioritising and adapting
circuits to obtain response times appropriate to
the needs of each situation.
For decades, stat tests have been performed
in dedicated laboratories, either at stand-alone
satellite locations or aggregated with central
laboratories. However, considering that today all
first-line tests can be managed rapidly via totallaboratory automation (TLA) (24-hour laboratories), the traditional model appears outmoded
for several reasons.
Firstly, a dedicated emergency laboratory represents a source of duplication of analytical platforms that perform the same assay in both emergency and ordinary situations, as well as the

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duplication of operating staff and, sometimes,
the duplication of orders for the same test.

ACCREDITATION OF STAT TESTSBY ISO 15189

Secondly, from an analytical perspective, parallel
processing of tests across more than one laboratory location within a healthcare setting requiresthat the analyser alignment be checked continuously to assure comparability of patient results.
Although ensuring that the difference between
results produced for the same test in stat andcentral laboratories does not exceed a clinically
acceptable difference is often a challenge, it ismandatory for patient monitoring during hospitalisation. All this complexity represents a significant drain on laboratory resources and makes the
dedicated emergency laboratory/section model
outdated and more expensive than TLA, especially when the latter can manage modern testingprocesses effectively and with a short TAT (2).

The second roundtable discussion addressed issues regarding the accreditation of stat tests
by the International Standard ISO 15189. All
experts work at emergency laboratories, accredited by UNE-EN ISO 15189, and which
perform a high percentage of accredited stat
tests. Accreditation is a procedure by which
an authoritative body gives formal recognition
that an organisation is competent to carry out
specific tasks according to certain standards.
The National Accreditation and Certification
Authority (ENAC) is the agency appointed by
the Spanish government to operate as the only
national accreditation body. Accreditation ofstat test laboratories according to ISO 15189 is
becoming more and more a matter of course in
Catalonia. However, there are currently only accredited stat tests laboratories in the provinceof Barcelona, with those in Girona, Lleida andTarragona certified by ISO 9001:2008 or ISO
9001:2015. The essential difference between
certification and accreditation is that the latter,as well as management requirements, also refers to technical competence (5).

Finally, the chairman enquired as to the role ofpoint-of-care testing (POCT) in emergency laboratories. Bedside testing through implementation of point-of-care devices in the emergency department, intensive care unit or any otherward that more often would require urgent test
results for patient management.
The experts explained that POCT at satellite locations may represent a new laboratory model.
Stat tests may sometimes be performed nearan intensive care department as part of criticalanalyses used in the evaluation of vital functions. POCT is typically evaluated positively byclinicians because it allows a reduction in TAT
and a reduced length of stay in the emergency
department (4).
All the experts agreed that POCT should dependon laboratory staff because it is a stat test activity. The involvement of laboratory staff in the
management of POCT should therefore be total,from the choice of measurement system, staff
training, and quality control assurance to the integration of the results into the clinical historyof the patient.

To accredit or not to accredit, that is the question. All speakers agreed that the process ofaccreditation would improve the quality of lab
services due to the better documentation ofprocesses and the increased responsibilities orinterest of management. The first step prior toaccreditation is building an enthusiastic teamthat is educated regarding the development ofquality management systems.
There was an interesting debate as to whether
the accreditation of stat tests is more difficult
than the accreditation of routine analyses. The
former is certainly more laborious because
emergency laboratories contain many membersof staff and accreditation requires an increasein staff competence. Frustration of evaluated

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Ariadna Arbiol-Roca, Dolors Dot-Bach
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staff must be avoided, and they must be shownthat accreditation is a continuous process of improvement. Laboratory leaders must ensure that
staff technical competence is highly praised, as
this can provide a great boost to team spirit, as
well as instil a sense of achievement and pride in
their accreditation.
It was agreed that quality standards for stat
tests should be just as rigorous as those for routine tests. Moreover, internal and external quality controls must be available for all parameter
devices and must fulfil the same quality requirements specified for non-stat test analysis (5).
In addition, interchangeability studies must be
carried out when emergency laboratories are
separated from routine laboratories using different instrumentation. ISO 15189 requires that
results obtained using different devices are interchangeable in order to assess non-different
clinically relevant discrepancies (5).
It was deemed necessary to be proactive in risk
management and to make good use of brands
listed by ENAC in the final laboratory reports,
although the experts outlined the potential difficulties of their use following the regulations.
During the roundtable, it was also emphasisedthat laboratories should evaluate the impact ofwork processes and potential failures on examination results, as they can affect patient safety.
Laboratories should therefore modify these processes to reduce or eliminate the identified risks,
as well as document decisions and actions taken.
It was noted that the presence of the ENAC markon reports provides an assurance that the laboratory will be able to rely on this endorsement;
only laboratories that are actually accredited canmake use of the ENAC mark to indicate accredited status (5). However, the experts explained
there can be difficulties in using the ENAC markin final reports, with improper use potentiallyleading ENAC to bring legal action against thelaboratory.

Finally, the speakers encouraged the attendees
to consider and initiate actions that will leadthem to accreditation, arguing that they themselves had perceived internal improvements intheir processes and that they were very satisfied to have achieved this. Accreditation is aprocess that involves the whole laboratory andrequires both good support from management
and the positive assessment of clients (clinicians and patients).
Hence, higher quality laboratory testing associated with accreditation is expected to improve
patient care by aiding the timeliness and accuracy of medical decision making.
Accreditation programs can help drive improvements in the management of individual laboratories and laboratory networks and may also
have positive spill over effects on performance inother sectors of the health care system. In summary, laboratory accreditation can be achieved
through leadership, vision and hard work.
CRITICAL ISSUES OF STAT TESTSThe third debate was attended by a clinician
who shared their vision of the laboratory froman outside perspective. Critical issues discussed
included the non-conformities that laboratories detect in samples and the impact that they
may have on result interpretation. All speakers
agreed that a strict stance should be taken when
dealing with stat test non-conformities such as
haemolytic, poorly spotted or insufficient samples. Greater awareness should be promoted
among clinicians, as should the continued training of nursing staff involved in sample extraction. It became clear during the debate that in
special situations, such as with newborns, itwould be necessary to consider a differentiatedtreatment.
Currently, serum index measurement (haemolysis, icterus and lipaemia) is automated anddetermination of the interfering substances isPage 62

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Critical issues and new trends on stat tests in clinical laboratory

objective (6). This approach provides benefits
compared with subjective visual interpretation,such as increasing the traceability, efficiency andeffectiveness of the work process.
In any case, internal and external serum index
quality assurances are available for the vast majority of laboratories.
In summary, non-conformities must be dealt withand minimised systematically, as they can influence the reliability of test results, affect patient
safety, delay result acquisition and, ultimately,
are a drain on resources.
There was an interesting debate regarding thedefinition of turnaround time. Laboratories mostcommonly define turnaround time as the time
taken from specimen receipt in the laboratory
to the moment that test results are reported.
However, clinicians may also define turnaround
Table 1time as the period from test ordering to reporting (7).
Speakers and assistants agreed that laboratory
staff must be involved from the moment ofsample arrival in the laboratory with the aimof optimising hospital circuits. Laboratory staff
can help to achieve optimum turnaround timein a variety of ways, for example by introducing
a pneumatic tube system. Many studies have
proven the efficiency of this mechanism in reducing delays arising from human couriers (8).
The causes of poor satisfaction in laboratory
users include stat test turnaround times; thisparameter was considered by the majority as
the most important indicator of laboratory
functioning. Hospital computerisation, including the recording of time from test request,
sample collection, report generation and report
receipt by the clinician would help in generating

Turnaround times in stat laboratories

Parameters

TAT (min)

TAT goals (min)S. Angeletti

A. Dolci

AH. Lou

TL. Ellison

Wu, Nichols, Hawkins(Ref. 9)(Ref. 10)

(Ref. 11)

(Ref. 12)

(Refs. 13-15)

Blood gas

-

-

-

-

<15

Blood count cell

21

11-20

22

-

<20

P – Potassium

41

28-49

40

-

<30

P – Troponin

45

29-34

-

52

<60

Prothrombin time

42

36-44

34

-

<45

41

36-58

39

-

<45

P – glucose

40

-

-

55

<45

Urinalysis

-

-

-

-

<30

Literature

P – Creatinine or
P – Urea

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Ariadna Arbiol-Roca, Dolors Dot-Bach
Critical issues and new trends on stat tests in clinical laboratory

turnaround time data. Analysis of any outliersin turnaround times in a lab would then provide
an insight into potential causes of delays andthe areas that need improvement.
Table 1 shows summarizing data from previous
publications on turnaround times for stat test
results. Differences among laboratories havebeen shown. TAT goals for stat samples were
established by different recommendations fromclinical practice guidelines.
There are situations that these TAT have to be
improved, for example: sepsis code, stroke codeand myocardial infarction code. Emergency departments, primary care physicians, divisions ofcardiology, hospital administrations, and laboratory staff should work collectively to develop
an accelerated protocol for the use of biochemical markers in the evaluation of these patients.
Another topic discussed was the catalogue of astat test laboratory. It was argued that several
aspects should be considered before a stat test
is included in a catalogue, with the following
underlined: cost, knowledge of the property,
technological availability, human resources, andcharacteristics of the test in terms of positive
and negative predictive value, sensitivity andspecificity, and above all clinical utility.
The catalogue should be adapted to the laboratory needs of each hospital and it is essential toreach agreement with clinicians. The control ofdemand must be based on scientific evidence
and not guided by economic criteria alone.
In many laboratories, it is obvious that sometests are ordered as stat not for clinical needs
but rather for other organisational reasons,such as quick retesting due to previously unsuitable samples, requests for previously forgotten
tests, or a desire to receive routine results more
rapidly (16).
Sometimes, users may simply be unaware of
the difference between stat and ordinary tests.

However, logistical issues can never be considered a good reason for ordering stat tests, as
inappropriately ordered tests may degrade theability of the laboratory to deliver clinically urgent information in a timely fashion. Clinicians
not receiving results in the expected timeframe
typically order more urgent tests, further reducing laboratory effectiveness and increasing
costs (17).
Finally, a discussion took place regarding criticalresults, i.e. those results that must be immediately sent, either by phone or electronically, tothe patient care provider by laboratory staff. The
question arose as to who should define a result
as being of critical value; responses included
the requirement for consensus with clinicians,
published recommendations and laboratories’
professional experience.
An expert outlined a project involving theCatalan Institute of Health (ICS) and other centres. As part of the project, a descriptive crosssectional study was undertaken by laboratory
professionals and clinicians from hospitals andprimary care centres, with biological parameters reviewed whose values - are likely to beconsidered as possibly critical - and therefore
must be reported. In addition, the critical limits
considered and actions carried out after obtaining a critical result were also reviewed. From
this approach an agreement between the participating laboratories was reached that subsequently formed the basis for the construction of
an initial model, based on consensus with clinical practitioners.
To achieve consensus with clinicians, a Delphi
model was applied in real time according to the“Health Consensus” methodology.
The model used data from a questionnaire thatwas sent to clinicians to value and identify, according to their criteria, when a result should beconsidered critical. The document prepared will
be available soon.

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PROPOSALS OF THE IN VITRODIAGNOSTICS INDUSTRYFOR EMERGENCY LABORATORIESThe final debate was performed with the collaboration of the in vitro diagnostics industry,
who outlined potential technological solutions
for emergency laboratories.
Initially, each representative of the in vitro diagnostics industry explained their technological
proposal for stat test laboratories. All speakers
agreed that each laboratory and hospital is different and that flexible solutions should be soughtfor each model.
Currently, three laboratory models are available: ordinary and stat tests fully integrated into
automated chains; stat test laboratories independent from ordinary test laboratories; and anintermediate option in which stat test analyses
are semi-integrated in an automated core chain
with routine samples, all of which are processedat the same time.
Many laboratories are currently considering
the installation of total laboratory automation
systems for routine clinical chemistry and laboratory haematology testing. The advantages of
total laboratory automation include cost reduction and improved turnaround time. Such automated pipelines integrate pre-analytics (checkin, sorting, centrifugation and aliquoting) andpost-analytics (storage and disposition), offering the possibility of bulk input, volume detection, aliquoting and storage. All speakers statedthat their companies are attempting to find solutions with which to prioritise the processingof stat test samples within automated chains.
The moderator and attendees asked the industry representatives to promote the development of computer-based tools that would allow
the calculation of turnaround time and allow
users to determine exactly how long each process lasts, including centrifugation, aliquoting,processing and report global delivery time. They
also advised that the industry focus their efforts
on achieving improvements in pre-analyticalprocesses. Computer enhancements should
also be made available for communicating critical results either through mobile telephony or
messaging via tablets and other devices. All
speakers agreed that they encounter many
problems regarding the protection of data when
installing such systems in hospitals. Finally, they
were asked to work to offer technological andother solutions so that stat test laboratories canobtain accreditation, according to UNE-EN ISO
15189, for the analyses carried out.
Finally, the in vitro diagnostics industry representatives acknowledged that it is necessary to
work together to solve both current needs andthose that may arise in the future.
CONCLUSIONS
We are moving towards a new model of healthcare system, in which every patient has a critical pathology and treatment is “emergent” forall. In this scenario, laboratories must consider
every test a stat test, so that the separation between routine and stat processes is abolished.
Automation, and particularly total laboratory
automation, represents a formidable tool withwhich to both meet increasingly demanding
critical needs and, even more importantly, improve patient outcomes.
In conclusion, in laboratory medicine, althoughtechnology can be used to improve clinical effectiveness and patient outcomes, it must be
managed by qualified laboratory professionals.
REFERENCES
1. Lippi G, Caputo M, Banfi G, Buttarello M, Ceriotti F,
Daves M, et al. Recommendations for the detection andmanagement of critical values in clinical laboratories. Biochim Clin. 2008;32:209–16.
2. Soffiati G, Giavarina D. Stat laboratory testing: integration or autonomy? Clin Chem Lab Med. 2010;48(7):927-30.

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3. Lippi G, Mattiuzzi C, Plebani M. Stat testing utilizationin clinical laboratories. National survey of Italian Societyof Clinical Biochemistry and Molecular Biology (SIBioC).
Clin Chem Lab Med. 2014;52(5):e79-84.

11. Lou AH, Elnenaei MO, Sadek I, Thompson S, Crocker
BD, Nassar B. Evaluation of the impact of a total automation system in a large core laboratory on turnaroundtime. Clin Biochem. 2016;49(16-17):1254-1258

4. Plebani M, Lippi G. Point of care testing: evolving scenarios and innovative perspectives. Clin Chem Lab Med.
2014;52(3):309-11.

12. Ellison TL, Alharbi M, Alkaf M, Elimam S, Alfaries M,
Nounou RA, Nasr R, Owaidah T. Implementation of totallaboratory automation at a tertiary care hospital in Saudi
Arabia: effect on turnaround time and cost efficiency.
Ann Saudi Med. 2018;38(5):352-357.

5. International Organization for Standardization (ISO).
Medical laboratories – particular requirements for quality and competence. ISO 15189:2012.
6. Simundic AM, Nikolac N, Ivankovic V, Ferenec-Ruzic
D, Magdic B, Kvaternik M, et al. Comparison of visual vs.
automated detection of lipemic, icteric and hemolyzed
specimens: can we rely on a human eye? Clin Chem Lab
Med. 2009;47(11):1361-5.
7. Salinas M, Lopez-Garrigos M, Gutierrez M, Lugo J, Llorca
F, Uris J. Stat laboratory timeliness management according
to clinician needs. Clin Chem Lab Med. 2011;49(2):331-3.
8. Guss DA, Chan TC, Killeen JP. The impact of a pneumatic tube and computerized physician order management on laboratory turnaround time. Ann Emerg Med.
2008;51(2):181-5.
9. Angeletti S, De Cesaris M, Hart JG, Urbano M, Vitali
MA, Fragliasso F, Dicuonzo G. Laboratory Automation
and Intra-Laboratory Turnaround Time: Experience at the
University Hospital Campus Bio-Medico of Rome. J Lab
Autom.2015;20(6):652-8.
10. Dolci A, Giavarina D, Pasqualetti S, Szoke D, Panteghini
M. Total laboratory automation: Do stat tests still matter?
Clin Biochem. 2017;50:605-611.

13. Wu AH, Apple FS, Gibler WB, Jesse RL, Warshaw MM,
Valdes R. National Academy of Clinical Biochemistry Standards of Laboratory Practice: recommendations for theuse of cardiac markers in coronary artery diseases. Clin
Chem. 1999;45(7):1104-21.
14. Nichols JH, Christenson RH, Clarke W, Gronowski
A, Hammett-Stabler CA, Jacobs E, et al. Executive summary. The National Academy of Clinical Biochemistry
Laboratory Medicine Practice Guideline: evidencebased practice for point-of-care testing. Clin Chim Acta.
2007;379(1-2):14-28.
15. Hawkins RC. Laboratory turnaround time. Clin Biochem Rev. 2007;28(4):179-94.
16. Scherrenburg J, van de Wijngaart DJ, Janssens PM.
Reducing the number of clinical stat phlebotomy orders:feasible or not? Clin Chem Lab Med. 2012;50(12):2141-8.
17. Lippi G, Simundic AM, Plebani M. Phlebotomy, stat
testing and laboratory organization: an intriguing relationship. Clin Chem Lab Med. 2012;50(12):2065-8.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Diamond Blackfan Anemia: genetics,
pathogenesis, diagnosis and treatment

Getabalew Engidaye1, 2, Mulugeta Melku2, Bamlaku Enawgaw2
1
2

Amhara Regional State Debre Berhan Health Science College, Debre Berhan, Ethiopia
Department of Hematology & Immunohematology, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia

ARTICLE INFOABSTRACT

Corresponding author:
Bamlaku Enawgaw
Department of Hematology &Immunohematology
School of Biomedical and Laboratory Sciences
College of Medicine and Health Sciences
University of Gondar
Ethiopia
E-mail: bamlak21@gmail.com

Diamond Blackfan Anaemia (DBA) is a sporadic inherited anemia with broad spectrum of anomalies that arepresented soon after delivery. It is inherited mainly inautosomal dominant inheritance manner and caused
by mutations and deletions in either large or small
ribosomal protein genes that results in an imbalance
between the biosynthesis of rRNA and ribosomal proteins, eventually the activation and stabilization of p53.
Diagnosing DBA is usually problematic due to a partial
phenotype and its wide inconsistency in its clinical expression; however, molecular studies have identified aheterozygous mutated gene in up to 50% of the DBA cases and corticosteroid drugs are the backbone treatment
options of DBA. Anomalies in bone marrow function in
DBA cases are broadly associated with both congenitaland acquired bone marrow failure syndromes in human. In this review different literatures were searchedin Medline (eg. PubMed, PMC, Hinari, Google scholar), OMIM, EMBASE by using search engines (Google,
Yahoo, Baidu Ask.com) and searching was performedby using search key words (DBA, ribosomopathies, Bone
Marrow Failure Syndromes, pure red cell aplasia). Only
human studies were included. This review is summarizing the current understandings of DBA.

Key words:
Diamond Blackfan Anemia,
ribosomopathies,
pure red cell aplasia,
ribosomal proteins,bone marrow failure syndromes

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Diamond Blackfan Anemia: genetics, pathogenesis, diagnosis and treatment

INTRODUCTION
Diamond Blackfan Anemia (DBA) is a sporadic
heterogeneous genetic disorder characterized by
red blood cell aplasia in association with skeletal
anomalies and short stature that classically appear soon after birth (1-4). Although the prominent feature of DBA is anemia (5), clinically it isa broader disorder and is manifested by growth
retardation and congenital malformations of thehead, heart, neck, upper limbs, and urinary system, which are present in approximately 30% to
50% of the DBA patients (6-9).
As shown by many studies, the incidence of DBA
was estimated to be 1-4 cases per 500,000 live
births in a year and it seems to be constant overtime. No seasonal disparity has been observed
as a function of the date of conception. The tendency of the disorder across the ethnicity of thepeople and in both genders, is almost comparable (10,11).
Chronic macrocytic-normochromic anemia, lowreticulocyte count, and decreased or totally absent erythroid precursors characterized by failure of erythropoiesis with normal productionof leukocytes and platelets in the bone marrow,are the main hematological features of DBA(2,3,12). Furthermore, a majority of the patients
have laboratory findings of increased mean
corpuscular volume (MCV), elevated erythrocyte adenosine deaminase activity (eADA) andpersistently elevated fetal hemoglobin (Hgb F).
However, these laboratory findings may not be
observed in some DBA cases; and even in thesame families, signs and symptoms may vary
among affected family siblings (13,14).
After a succession of studies, by now DBA has
been shown to be associated with both ribosomal and non-ribosomal mutations in genes
located on more than 11 chromosomes which
are responsible for encoding the ribosomal proteins (RP) (12,15).

Molecular mechanisms underlying the causal
consequence between RP haploinsufficiency
and anemia have not yet been clearly elucidated. A generally documented pathogenetic
hypothesis implies that a defective ribosome
biosynthesis leads to apoptosis in erythroid
progenitors which in turn is leading to erythroid
failure. This mechanism has been named ‘‘ribosomal stress’’, and there are indications that it
may be signaled through p53. All genes identified to be mutated in DBA encode ribosomal
proteins which are involved in either the small(RPS) or large (RPL) subunits of these proteins
and the scarcity of these proteins can cause thedevelopment of the disease (16). The disordersof ribosome synthesis or associated genes thatlead to disrupted ribosomal biosynthesis (ribosomopathy) are more than one independentribosomal protein mutation i.e., all patients of
DBA may not show the same RP mutations (6).
Corticosteroids, transfusion therapy and stem
cell transplantation are the current options forthe treatment of DBA (17,18).
The main aim of this narrative review is to provide the reader with a comprehensive knowledge on DBA genetics, pathogenesis, diagnosis and treatment. Different literatures weresearched in Medline (e.g., PubMed, PMC,
Hinari, Google scholar), OMIM, EMBASE by using search engines (Google, Yahoo, Baidu Ask.
com) and searching was performed by using
search key words (Diamond Blackfan Anemia,
Ribosomopathies, Pure red cell aplasia, Ribo­somal proteins, Bone marrow failure syndromes).
Only human studies were included.
MOLECULAR PATHOPHYSIOLOGYOF DIAMOND BLACKFAN ANEMIA
Impaired ribosomal biogenesis in DBA
Recently, DBA is a well-recognized inheritedbone marrow failure syndrome, frequently
caused by alterations in ribosomal protein (RP)

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genes. Otherwise it rarely results from the mutation of the hematopoietic transcription factor
gene, GATA1. After the preliminary descriptionsof heterozygous RPS19 mutations in a subsetof DBA cases, a substantial progress has been
made over the past decades for improved explanation of the genetic causes of DBA (13). With agrowing emphasis on the RP genes, the search
for DBA-related genes, which was initially basedon classic genetics techniques including cloningof cytogenetic abnormalities and extended linkage analysis, has shifted to target resequencingof the known RP genes (19-21).
These advanced studies have recognized bothlarge and small subunits of RP gene anomalies
which currently includes but is not limited to
RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17,
RPS19, RPS24, and RPS26 as a mutated genein multiple families of DBA patients (8,22,23).
Changes in larger number of RPs have also been
identified in isolated patients or families, including RPL3, RPL7, RPL9, RPL14, RPL19, RPL23A,
RPL26, RPL35, RPL36, RPS8, RPS15, RPS27A,
RPL18 and RPL35 (24-26).
Mutations in RP genes have been confirmed tobe the direct cause of faulty erythropoiesis andconsequently anemia, and are found in morethan half of DBA cases (27). The RPS19 gene was
the formerly known mutated small ribosomal
protein and is still the most frequently mutated gene in DBA patients which accounts about
25% of total DBA patients (13). More than 50%
of RPS19 mutations are either deletions of one
RPS19 allele, or insertional, frame shift, splice
site, or nonsense mutations, which lead to an
untimely termination of RPS19 protein synthesis. This results in a deficiency of RPS19 protein
which is termed as ‘‘haploinsufficiency’’ in human cells (28,29). Again, greater than half of themissense mutations termed as ‘‘class I’’ whichcause the RPS19 protein to be folded improperly and rapidly aimed at premature degradationof ribosomal proteins consequently leading to
RPS19 haploinsufficiency (30).
RPS19 protein plays an important role in 18SrRNA maturation and 40S synthesis in humancells (29,31,32). Mutations associated with
RPS19 can disrupt the pre-rRNA processing of
the 18S rRNA and pre-40S subunits, leading to
reduced production of 40S ribosomal subunits.
Finally, the decreased expression of RPS19 isable to imitate many aspects of the DBA phenotype (21,35). As a result, this knockdown of
RPS19 by RNA interference can cause a severe
defect that alters the normal primary humanhematopoietic progenitor differentiation andproliferation of the erythroid progenitor (EP)cells (33). Furthermore, the defective erythropoiesis in DBA caused by RPS19 deficiency canbe rescued by ectopic overexpression of exogenous RPS19. All of these strongly support thenotion that RPS19 haploinsufficiency resulted
from RPS19 modifications could be the central
pathogenic mechanism in the underlying DBA
pathology suggesting the feasibility of RPS19
gene augmentation to treat DBA cases (31).
Thus, RPS19-lacking cells suffer from a relatively
shortage of 40S rRNA and has a reduced ability
for translation initiation. RPS19 deficiency canlead to increased apoptosis in hematopoietic
cell lines and bone marrow cells. Suppressionof RPS19 prevents cell proliferation and early
erythroid differentiation, but not late erythroid
maturation in RPS19-deficient DBA cell lines.
Haploinsufficiency of RPS19 has been shown ina subset of patients and appears to be sufficient
to cause DBA (20,22).
Advanced studies showed that the RPS19Dsk3
mouse recapped the human DBA phenotype insofar as a hypoproliferative, pro-apoptotic anemia
with growth hindrance. Given existing data, it is
now generally believed that DBA resulted from an
intrinsic cellular flaw in which erythroid progenitors and precursors are greatly sensitive to demisePage 69

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by apoptosis (36,37). The phenotypes observedin zebrafish and mouse DBA models DBA can be
partly or entirely rescued by alterations in p53, robustly proposed that p53 stabilization and activation plays a noteworthy role in the pro-apoptotic
phenotype of cells with RP haploinsufficiency (38).
DBA have led to the formulation of “ribosomal
stress” hypothesis in which reduced RP synthesis activates p53 that induces the downstream
events and leads to cell cycle termination or
apoptosis. Finally, this phenomenon results inthe DBA phenotype of anemia, deprived growth
and results in congenital abnormalities (16).
There are numerous potential mechanisms bywhich a faulty ribosome assembly or nucleolar stress might signal to p53 stimulation. One
interesting mechanism in the pathophysiology
of DBA is the interaction of ribosomal proteins
with Murine Double Minute (MDM2) which is a
powerful controller of p53 level and its activity.
MDM2 is a ring finger ubiquitin ligase that interacts with and enhances the degradation of p53.
In this interaction, the large proteins of the 60S
subunit namely RPL5, RPL11, and RPL23 havebeen presented to bind to MDM2 and reduces
the activities of MDM2 which in turn results in
p53 stabilization (39).
The heterozygous DBA mutations result in lossof function in a single copy of ribosomal protein
gene. Thus, the pathophysiology of DBA is now
attributed to ribosomal malfunction and thesemutations in ribosomal genes have been identified in approximately 50% of DBA patients (40).
The large number of ribosomal proteins mutated in DBA fail to cluster in any specific regionof the ribosome. Haploinsufficiency or reduced
expression of a ribosomal protein results in decreased levels of the cognate 40S or 60S subunit
and a defect in processing of the ribosomal RNA
precursors (33,41,42).
Although DBA is considered as a ribosomopathy,
it can be also caused by non-RP gene mutations.

GATA1 encodes a transcription factor which isessential for erythroid differentiation and therefore it is not surprising to state that this gene isinvolved in DBA pathogenesis (43). The mutationin the GATA1 gene is a Guanine-Cytosine (G→C)
transversion at location 48,649,736 on the X
chromosome. By applying whole-exome sequencing, aberration in the zinc-finger transcription factor gene “GATA1”, has been identified to
be one of the possible causative agents for thedevelopment of DBA. This G→C transversion isassociated with X-linked form of DBA, leading to
the substitution of leucine to valine at amino acid74 of GATA1. This aberration affects GATA1 splicing processes and leads to termination of the
full-length GATA1 protein level and synthesis of ashort isoform what we call GATA1 short (GATA1s)(11). While comparing the two isoforms, there is
no exon 2 in GATA1s consequently the absenceof a transactivation domain. Full-length GATA1
has a crucial role in humans for the differentiation of erythroid cells. During erythroid differentiation, full-length GATA1 enhances the synthesisof erythroid genes by silencing megakaryocytic
or other hematopoietic lineage-specific genes.
This up and down regulation of GATA1 is vital
during the megakaryocyte erythroid progenitor(MEP) commitment program to megakaryocyticand erythroid differentiation (44,45).
While the specific role of GATA1s in DBA pathophysiology remains unclear, studies suggest thatfull-length GATA1 protein level could be an important driver of DBA pathophysiology. Researchers
used short ribonucleic acids (shRNA) against
several RPs in normal CD34+ cells to induce RP
deficiency to confirm the role of GATA1 in DBA
patients.
Thereafter they observed a reduction of fulllength GATA1 protein level and erythroid maturation defects. On the other hand, increasing
the GATA1 protein level could partly rescue defects in DBA associated RP haploinsufficiency(46,47).

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Mechanisms of erythroid failure
due to ribosomal protein deficiency
DBA typically presents with erythrocyte aplasia
in the first one year of life and both quantitativeand qualitative defects of erythroid progenitors contribute to the abnormal erythropoiesis.
Majority of the genes responsible for the development of DBA are ribosomal proteins suggesting that insufficiency in ribosomal function
may be the underlying cause of red cell aplasia in DBA cases. Ribosomal haploinsufficiency
leads to disrupted ribosome biosynthesis anda consequent cytoplasmic buildup of numerous free RPs which results in the stabilization
and activation of p53 (38,48-50). In ribosomal
stress conditions, several cytoplasmic free RPs
such as RPL11, RPl5, RPL23, RPS7, and RPS27
will bind to Murine double minute (MDM2) andhinders its interaction with p53, leading to thestabilization of p53 (51-53). This pathway has
been shown to be upstream of apoptosis andcell-cycle arrest, eventually which leads to DBA
erythroid hypoplasia (54,55).
Homozygous inactivation of p53 in RPS19 mutant mice totally corrected the DBA erythroid
hypoplasia, further corroborating the MDM2/
p53- mediated DBA pathogenesis caused by
RPS19 mutations and cytoplasmic free RPL26
could influence the translation of p53 mRNA
(30,56). However, it is believable that permanent overexpression of RPS19, by gene transfer
may result in a persistent activation of MDM2
and/or inhibition of p53 (55).
Moreover, when global translation was blocked
by inhibition of the translation initiation factor Eukaryotic Transcription Initiation factor
4- Erythroid (eIF4E), the investigators observeda down regulation of full-length GATA1 while
the other proteins persisted unaffected. Theseobservations suggest that ribosome biosynthesis faults could impact translation of specificproteins, as GATA1. Thus, in addition to thecytoplasmic free RPs, full length GATA1 level reduction may participate in the stabilization of
p53 and, consequently, the DBA erythroid cell
phenotype (47).
Although the mechanism by which mutations in
the RP genes caused explicit defects in erythroid
cell maturation is not copiously understood,
several lines of evidences indicate that p53 activation induced by ribosomal malfunction may
be fundamental for the pathogenesis of DBA(56). Disruption of 40S biosynthesis provoke therelease of RPL11 and other RPs into the nucleoplasm results in the binding with MDM2. Thisphenomenon can compromise MDM2 activity;
thereafter the consequent accumulation of p53.
The exhibition of phenotypes such as growth retardation, macrocytic anemia with reticulocytopenia and increased apoptosis in bone marrowprogenitors by RPS19 mutant mice suggested
the presence of a direct link between DBA phenotypes and accumulation of p53 (51).
The pathophysiology for erythroid defect in DBAmight therefore be because of the failure of aparticular protein to achieve a threshold levelat a critical stage; for example, by disturbing the
stoichiometry of multi-protein erythroid-specific complexes or by a more selective influenceon the translation of a vital protein. A global reduction in translation could also be significant
for the pathophysiology of DBA (57).
In conclusion, defective erythropoiesis in DBA
is mostly the consequence of either ribosome
biogenesis defects due to mutations in ribosomal protein (RP) genes or as consequenceof mutation in the GATA1 gene, leading to a
reduction of full-length GATA1 and synthesis ofa short isoform, GATA1s. In normal conditions,p53 is degraded because of its interaction with
MDM2. In DBA, the ribosome biogenesis defectinduces a release of several free RPs in the cytoplasm, which can inhibit p53 degradation. p53
degradation is also inhibited by the reduction

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in full-length GATA1 synthesis (15). Treatment
includes gene therapy strategies such as gene
addition (to increase copy numbers of the wildtype form of the mutated proteins), gene silencing (leading to the degradation of the mutatedmRNA) and genome editing (such as zinc-finger
nucleases, transcription-activator like effector
nucleases, or clustered, regulator).
GENETICS AND INHERITANCEOF DIAMOND-BLACKFAN ANEMIA
InheritanceAccording to the report of recent studies, approximately 40 – 45% of DBA cases are hereditary which are inherited with autosomal dominant inheritance which mean that a single copyof altered gene in each cell is adequate to cause
the disorder (58) whereas the remaining 55 –60% of the DBA patients are sporadic, i.e., resulted from new aberrations in the gene which
occur in people who have no history of this disorder in their family (12).
Even though autosomal dominant inheritance isthe frequently observed pattern of inheritance,
autosomal recessive inheritance, which is defined as the presence of DBA siblings from unaffected consanguineous parents, with a lesser
frequency has been reported. DBA classicallypresents at 2 - 3 months of age; only 25% ofaffected offsprings are anemic at birth and hydrops is occasional (59,60).
Genetics
As shown in the pathophysiology of DBA, it
mainly arises from an abnormal ribosomal protein gene with the exception of the rare form
resulting from mutation of transcriptional factor GATA1 (6,13,22,24,25,28,61-63). The genes
encoding ribosomal proteins belonging to boththe large and small ribosomal subunits arefound to be mutated in DBA (7,11). This disorder is described by genetic heterogeneity,

disturbing different ribosomal gene loci. Morethan a decade of heterozygous mutations resulting in haploinsufficiency have been identified for the genes that encode ribosomal proteins (40) (Table 1).
Mutations in ribosomal genes account for 60 –70% of DBA patients. Among these ribosomal
gene mutations, about 20% of the cases involve
large deletions that require analysis of copy
number variation for detection. On the other
hand, overall it is estimated that around 35% of
the DBA cases remain yet genetically indeterminate. These ribosomal gene aberrations may
be inherited in an autosomal dominant pattern
or may arise spontaneously-linked mutations in
the transcription factor GATA1 (11).
GENOTYPE AND PHENOTYPE
CORRELATION
In general, the phenotypic spectrum of DBA embraces a wider domain of severity, even within
the same families, it ranges from the classicalsyndrome to individuals with a solitary increase
in eADA. No correlation has yet been foundbetween the identity of the DBA gene and hematological severity, including response to steroids. However, craniofacial abnormalities are
more linked to mutations affecting either RPL5
or RPL11 (22,29).
Clinical figures from European and American
DBA cases disclosed that the incidence ofmalformations in DBA patients having RPS19
mutations is 31% and this is not significantly
different from that of the entire DBA population (29,64,65). RPS19 mutations are foundin some first-degree families presenting onlywith isolated high eADA and/or macrocytosis.
However, large deletions at the 19q13 locus
and unbalanced translocation t (X; 19) are always linked to mental retardation, which points
to a contiguous gene syndrome. Conversely,
the patient with balanced translocation t(X;19)

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Table 1

Mutations of ribosomal protein genes in Diamond Blackfan Anemia

Sr.
No.

Study

Year

Gene

Chromosome

SubunitFrequency*

1

Mirabello et al

2017

RPL18
RPL35

19q13.33
9q33.3

60S
60S

2
2

2

Gazda et al

2012

RPS26

17p13

40S

6.5

3

Farrar et al

2011

RPS17

15q

40S

5†

4

Doherty et al

2010

5

Gazda et al

2008

RPS7
RPS10
RPL5
RPL11

2p25
6p
p22.1
p36.1-p35

40S
40S
60S
60S

1
2-5
7
6.5

6

Farrar et al

2008

RPL35A

3q29-qter

60S

2

7

Cmejla et al

2007

RPS17

15q

40S

5†

8

Gazda et al

2006

RPS24

10q22-q23

40S

2

9

Draptchinskaia et al

1999

RPS19

19q13.2

40S

25

10

-

-

Others-GATA1, RPS28-* Frequency may depend on the cohort size.
† Including gene deletions.

which interrupts RPS19, without loss of othergenes, has normal mental development (66).
Comparatively, mutations in RPL5 and RPL11
possibly will be accompanied by a more severe
phenotype than mutations in RPS19, particularly with respect to skeletal deformations (22).
Current studies advise that the patients withan RPL5 and RPL11 mutation have an increased
probability to present with craniofacial, thumband heart anomalies (9,22,61).
Physical anomalies are observed in up to 50% of
the cases with a wide range of severity. These areclassically craniofacial, including hypertelorism,
flat nasal bridge, and high arched or cleft palate.
Thumb abnormalities have also been seen in 20%of the DBA cases, including the usual triphalangeal thumb (67). In RPS5 alterations, craniofacial,congenital heart, and thumb defects are more
severe than those seen with pathogenic variants
in RPL11 and RPS19 (22,68). Curiously, patientswith PRL5 mutations incline to have cleft lip and/or plate or cleft soft palate, solitarily or in combination with other physical abnormalities.
Consistent with these reports, patients with
RPL5 mutations also had physical malformations and cleft palate, and in contrary, patients
without an RPL5 mutation presented with cleft
palate (69). Cleft lip and/or cleft palate wasalso reported in almost 50% of the affected babies with RPL5 pathogenic variants (22). Small

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gestational age was reported in individuals withan RPL5 and RPS19 pathogenic variant as well,which is higher in individuals with RPS5 pathogenic variant (68). Pathogenic variants in RPL11
are largely associated with thumb abnormalities(22); individuals with RPL11 pathogenic variants
have been also identified with cleft lip or palate(CL/P) (68).
Individuals with mutated variants of RPS10,
RPS19, RPS26 have not yet shown any genotypephenotype correlations (24). Till now, no genotype-phenotype correlations have been also
identified in persons with RPS29 pathogenic variants. DBA with mandibulofacial dystostosis wereidentified in mutants of RPS28 and TSR2 whereas
persons with the variants of RPL27, RPL31 and
RPL27 have no identified genotype-phenotype
correlations (70).
CLINICAL PRESENTATIONSAND DIAGNOSISClinical presentations
Hematologic features
DBA babies typically present with an erythrocyte aplasia in the first twelve months oflife, with the median age of exhibition of twomonths but occasionally its presentation may
be delayed up to adulthood. By definition, all
DBA patients are anemic (71), and it is present
at birth in only 15% of patients and fetal hydrops has occasionally been reported, suggesting that erythropoiesis is spared in utero perhaps this is due to transiently being rescued bymaternal or placental factors with a post-natal
switch from effective to ineffective erythropoiesis (17,60,72).
Red blood cells are usually macrocytic; reticulocyte counts are reduced or zero but theother hematological lineages are not involved
as a rule with the exception of slightly an abnormal lower leukocyte and increased platelet

counts reported at diagnosis. Bone marrowaspirates indicate isolated erythroblastopenia,(usually <5% of nucleated cells on bone marrow smears) in more than 90 percent of the patients. Another unusual bone marrow patternis erythroid hyperplasia with maturation detention; apparently normal numbers and maturation of erythroblasts have been exceptionallydescribed (17,73).
Bone marrow analysis also demonstrated normal cellularity and morphology except for theerythroid line in all patients (74). Erythroid entire aplasia and hypoplasia have been found in
DBA cases presented with erythroid maturationarrest with an elevated number of juvenile precursors and indicated dyserythropoietic morphology (71,74,75).
The colony assessment for BFU-E confirmed totally absent/reduced growth in 83% of patients.
Addition of stem cell factor (SCF) induced a noticeable increment of erythroid colonies in all
the tested subjects. The activity of eADA which
is a crucial enzyme in the purine salvage pathway, is usually high in DBA patients (71,74,76).
A moderately increased risk of developing hematological malignancies also exists and initialclinical manifestations such as pallor, shortnessof breath while suckling, failure to thrive andsystolic murmur are observed during infancy(16). The risk of developing solid tumors, myelodysplastic syndrome, or leukemia is elevatedin DBA patients (77).
Physical abnormalities
More than a third of the disordered personspresent with a variety of associated congenital
physical anomalies. Especially, thumb and upper limb malformations as well as craniofacial
anomalies including short stature are common.
A cute snub nose and wide spaced eyes, and other craniofacial anomalies are also seen. Other
defects frequently observed include urogenital

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anomalies, atrial or ventricular septal defects,
and prenatal or postnatal growth retardation.
A distinct facial appearance and triphalangeal
thumbs have been characteristically explainedin DBA (27,70,78,79).
According to some studies, the incidence andseverity of physical abnormalities have not been
gender-related, whereas other researchers revealed that a greater severity of abnormalities
was observed among males, compared to females (66).
Table 2

Diagnostic criteria
Diagnosing DBA is usually tough due to its partial
phenotypes and the wide inconsistency of clinical expressions (16,71). Having the variability,
the International Clinical Consensus Conference
stated diagnostic and supporting criteria for the
diagnosis of DBA (71) (Table 2). The diagnosis
of DBA is made when the requirements of major criteria outlined in Table 2, are fulfilled andthe Parvovirus infection and Fanconi’s anemia
are ruled out. However, some essentials are not

Diagnostic criteria for DBA*
Diagnostic criteria•

Normochromic, often macrocytic anemia developing in the first year of life•

Profound reticulocytopenia•

Normocellular bone marrow with selective deficiency of erythroid precursors•

Normal or slightly reduced leukocyte count•Normal or slightly increased platelet count
Major supporting criteria•

Gene mutation described in ‘‘classical’’ DBA•

Positive family history
Minor supporting criteria•

Elevated erythrocyte adenosine deaminase activity•

Congenital anomalies described in ‘‘classical’’ DBA•

Elevated HbF•

No evidence of another inherited bone marrow failure syndrome* Accepted by the DBA working group of the European Society for Paediatric Haematology and Immunology, ESPHI (17).

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included in these criteria such as the presenceof typical malformations, the response to steroids and the chronic course of the anemia canalso help to diagnose DBA (73).
Onset after the age of two years or the absenceof isolated bone marrow erythroblastopenia induce a big caution in the diagnosis of DBA. The
only identification of pathogenic mutants in oneof the DBA genes conclusively establishes a diagnosis of DBA (61,80). Furthermore, moleculardiagnosis enables the detection of carriers, andthe exclusion of hematopoietic stem cell transplantation from sibling donors living with themutations. However, determining the effects ofmissense mutations may be difficult, whereas
nonsense and frameshift mutations will probably be pathogenic in the majority of cases (73).
The bone marrow is classically normocellular,
with a scarcity of erythroid precursors. The erythroid burst-forming units (BFU-E) and erythroid
colony-forming units (CFU-E) in vitro are severely reduced, with relative sparing of the granulocyte-monocyte colony-forming units (CFU-GM)(81). The erythrocyte adenosine deaminase activity (eADA) levels are often elevated (74).
DBA may be misdiagnosed with transient erythroblastopenia of childhood (TEC). Therefore, toavoid misdiagnosing of DBA, TEC must be ruledout. TEC is a common disorder of children age
greater than one year. It is an acquired, shortlived failure of red cell production usually of amonth or so in duration. TEC, most likely a post
infectious, transient autoimmune IgG-mediateddisorder, typically occurs in children probably as
the result of infections acquired through contact(82). As in DBA, children with TEC often present
with profound anemia as a result of pure red cellaplasia. On the other hand, DBA is a dominantly
inherited disorder observed in children younger
than one year. It is characterized by presence ofcongenital anomalies; elevated MCV, fetal Hgb
and erythrocyte adenosine deaminase activity(75,76,83,84).

TREATMENT AND PROGNOSIS OF DBA
Heterogeneity of DBA is also shown in responseto treatment and in the follow-up of DBA cases.
In 2008, a group of veteran clinicians establisheda consensus for the diagnosis and treatment of
DBA. This document represents a gold standard
for therapeutic decisions. There are no obvious
phenotypic or genotypic differences between
remission and non-remission patients (71,83).
Although corticosteroid therapy is endeavoredwith varying regimens for almost all DBA cases,
treating the patients with RBC transfusion may
be used primarily in the first year of life to reduce corticosteroid associated side effects andadverse effects in neonates which may include
noteworthy growth disturbances. Even so there
is currently no way to select the patients whowill respond or not to the treatment, around
80% of the patients initially respond to steroids
but those DBA patients who initially responded
to this therapy suffered from many side effects.
Some DBA cases remained responsive to steroids, while efficacy vanished in others taking
the treatment (18,71,83). The mode of actionof corticosteroids in DBA is particularly obscure.
Apoptosis at the progenitor level seems to be
the cause of the anemia and corticosteroids
seem to have a non-specific anti-apoptotic effect in erythroid progenitors particularly at thecolony forming unit-erythroid (CFU-E) proerythroblast interface (84).
Patients who are not responsive to steroids or
are unable to tolerate the treatment may require
chronic RBC transfusion therapy. This therapy
is given to most patients every 35 weeks witha goal of sustaining the Hb level greater than 8
g/dL. Based on the child’s growth and function,
Hb values may need to be higher for some patients. Since the transfusion therapy of RBC ischronic, iron overload becomes challenging, so
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mandatory (71). Pediatric hematologists withexpertise in the treatment of DBA will frequently commence iron chelation with intravenousor oral agents after 15 units of red cell transfusions. Intravenous chelation is frequently needed for the establishment of nonmetallic portsthat are magnetic resonance imaging compatible (84,85).
Performing allogeneic Hematopoietic Stem Cell
Transplantation (HSCT) is another alternative tocure hematological aspects of DBA. This therapy
is performed when DBA patients become unresponsive to repeated transfusions or to prevent
iron overload and organ damage due to dependency of frequent transfusions. However, theadverse events due to HSCT may exceed fromthose adverse events due to iron overloading.
In the future, gene therapy is presumed promising for RPS19 deficient DBA patients. Other
treatments, have been used in DBA over thelast three decades. However, these drugs appear to be largely ineffective and there is currently no evidence that any of these has a majorrole in the management of DBA (71). The prognosis is generally good. However, complicationsof treatment and a higher incidence of cancer
may reduce life expectancy. Disease severity
depends on the quality and response to treatment. For patients undergoing regular transfusions, quality of life is clearly altered (85).

discovery of RPS19 as the foremost DBA geneled to an exciting scientific research in DBA andother ribosomal disorders. Even though there
is convincing evidence for the involvement of
p53 in the pathogenesis of DBA, no single proposed mechanism so far accounted for all facetsof DBA and yet none is mutually exclusive. Even
though a number of genes are responsible for
DBA, mutations in RPS19 are the major causesof impairment of ribosomal protein biosynthesis which lead to nucleolar disorganization andactivation of the p53 family. Many treatment
options have been tried over the past several
years with inconsistent success. Despite manytreatments having been tried for the management of DBA, chronic red blood cell transfusion,
corticosteroids and hematopoietic stem celltransplantation remained the cornerstones oftherapy.
Abbreviations
CL/P: Cleft of Lip or Palate
DBA: Diamond Blackfan Anemia
eADA: elevated erythrocyte
Adenosine Deaminase Activity
HDM: House Dust Mites
MDM: Murine Double Model
RP: Ribosomal Protein

CONCLUSIONSDBA is a clinically heterogeneous disorder accompanied by hypoplastic anemia and also
manifested by congenital malformations. Even
if Ribosomal protein haploinsufficiency havebeen coined as a primary causative agent of
DBA, non-ribosomal proteins such as GATA1
have been identified to have a vital role in thepathogenesis of DBA. The molecular mechanisms which are fundamental for the pathogenesis of DBA are still not entirely understood. The

RPL: Large Ribosomal Protein
RPS: Small Ribosomal Protein
TEC: Transient Erythroblastopenia of ChildhoodAvailability of data and materials
Data sharing not applicable to this article as nodatasets were generated or analysed during thecurrent study.

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Getabalew Engidaye, Mulugeta Melku, Bamlaku Enawgaw
Diamond Blackfan Anemia: genetics, pathogenesis, diagnosis and treatment

Competing interest
The authors declare that they have no competinginterests.
Authors’ contributions
MS & BE performed literature searching anddrafted the manuscript. MM involved in draftingof the manuscript along with MS & BE. All authors read and approved the final manuscript.


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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gaucher disease: an underdiagnosed pathology
in the Eastern Moroccan population
Ouardia Bouayadi1,2, Amina Lyagoubi1,2, Adnane Aarab1,2,
Somiya Lamrabat1,2, Abdelilah Berhili1, Mohammed Bensalah1,2,Rachid Seddik1,2
1
2

Laboratory of Hematology, Mohammed VI University Hospital, Oujda, Morocco
Faculty of Medicine and Pharmacy, Mohammed the First University, Oujda, Morocco

ARTICLE INFOABSTRACT

Corresponding author:
Ouardia Bouayad
Central Laboratory
Mohammed VI University Hospital
Oujda
MoroccoE-mail: ouardiabouayadi@gmail.com

Gaucher disease (GD) is a lysosomal storage disease.
It corresponds to a congenital deficit in β-gluco­cere­brosidase. This pathology should be considered in thepresence of unexplained splenomegaly, with or without signs of haemorrhage, skeletal manifestations or
hepatomegaly.

Key words:
splenomegaly, thrombocytopenia,
Gaucher cells, β-glucocerebrosidase
Conflict of interest:
Authors declared no conflict of interest.

The diagnosis is based on the measurement of the
β-gluco­cerebrosidase activity but the preanalyticalprocess should be respected in order to avoid theunder-diagnosis of this disorder and the delay of its
management. We report two cases of Gaucher disease collected at Mohammed VI University Hospitaland Al Farabi regional hospital in Oujda. We have emphasized the need for a reference center for overloaddiseases.

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O. Bouayadi, A. Lyagoubi, A. Aarab, S. Lamrabat, A. Berhili, M. Bensalah, R. Seddik
Gaucher disease: an underdiagnosed pathology in the Eastern Moroccan population

INTRODUCTION
Gaucher disease is a lysosomal storage disease,
the transmission is autosomal recessive.
It follows a mutation in the GBA gene coding for alysosomal enzyme: β-glucocerebrosidase, whichhydrolyzes glucosylcerebroside (glycosyl ceramide) into ceramide (cerebroside) and glucose.
There are three subtypes of the disease. The
most common is type 1, known as the non-neuropathic form. Pancytopenia, hepatosplenomegaly and bone lesions occur as a result of glucocerebroside accumulation in the liver, lung, spleen
and bone marrow in these patients.
The two other phenotypes include a severe
neurological involvement in type 2 that affects
infants and is deadly; in type 3 the neurologicalinvolvement is less severe and is also associatedwith features of types 1 [1].
CASE 1
A 37-year-old male, was admitted to the internal medicine department of the regional hospital of Oujda for the management of anemic
syndrome with splenomegaly. The clinical examination found mucocutaneous paleness withvoluminous splenomegaly, the rest of the clinicalexamination is without particularity. Laboratory
tests revealed bicytopenia with normochromic
anemia (Hb at 6.3 g/dL) and thrombocytopenia at 140 G/L , leukocytes at 5 G/L . Liver andrenal functions were normal, the serologies ofhepatitis B, hepatitis C and HIV were negative.
The abdominal ultrasound showed a huge splenomegaly, a normal-size liver without signs of
portal hypertension. The myelogram showed amarrow, with cellular abundance a hyperplasiaof the erythroblastic line with the presence ofmany Gaucher cells. X-ray of the skull, thoracolumbar spine, and pelvis were normal. The
chest CT scan revealed the presence of alveolar syndrome with a small left pleural effusion.

The abdominal computed tomography showedsignificant heterogeneous splenomegaly withcompression of adjacent organs and a small fluid effusion in the Douglas cul-de-sac.
Subsequently, β-glucocerebrosidase [7.5 U/L(reference range: 6.5 - 10.5 U/L)], Chitotriosidase[2980 nmol/h/mL (normal value <120 nmol/h/mL)] and ferritin [833 μg/L (reference range:
males 20-200 μg/L)] measurement was done.
The diagnosis of Gaucher disease type 1 wasbased on clinico-biological data, the cytological aspect of the marrow and the results of thebiomarkers, without enzymatic evidence. The
patient was splenectomized; and the pathological examination of the spleen showed sub
capsular splenic infarction with presence ofGaucher cells.
CASE 2A 26-year-old male, with no significant medical history was admitted to the Department ofInternal Medicine of Mohammed VI University
Hospital for the management of splenomegalywith anemic and haemorrhagic syndrome. The
history of the disease dated back to 6 months
before admission by the progressive appearance of anemic syndrome constituted by cutaneous pallor, asthenia and fatigability, associated with a hemorrhagic syndrome demarked
by episodes of repeated epistaxis and diffusesbone pain. The symptomatology constituted
hepatic, colic and left hypochondrial pain.
Clinical examination found a cutaneomucous
pallor with splenomegaly exceeding the whiteline of the abdomen, without hepatomegaly orother associated signs. Blood test found a hemoglobin level at 12 g/dL (reticulocyte: 1.54%),
platelet count at 40 G/L and leucocytes at
3940/ μL. The serologies of hepatitis A, B and
C and leishmaniasis were negative. The myelogram showed the presence of many Gaucher
cells (Figure 1).

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O. Bouayadi, A. Lyagoubi, A. Aarab, S. Lamrabat, A. Berhili, M. Bensalah, R. Seddik
Gaucher disease: an underdiagnosed pathology in the Eastern Moroccan population

Figure 1

Gaucher cells on medullary smear

Gaucher cells

The histological aspect of the bone marrow biopsy(BMB) suggesting a storage disease (in particular
Gaucher disease). Abdominal CT showed portal
hypertension with huge heterogeneous splenomegaly including areas of necrosis. Standard radiography was normal and osteo-densitometry
displayed osteopenia.
The result of the β-glucocerebrosidase assay was
6 U/L (reference range: 6.5 - 10.5 U/L). But giventhat the bone marrow aspiration showed typical cytological image of Gaucher disease; a newmeasurement was requested which showed a
deficit at 1.6 μkat/kg (reference range: 4.2 to 8.1
μkat/kg). The levels of chitotriosidase, hexosami-­ adae and ferritin were 3380 nmol/h/mL, 1500nnmol/h/mL and 690 μg/L, respectively. The diagnosis of Gaucher disease type 1 was retained.
While waiting for enzymatic substitution treatment, the patient was treated by corticosteroids.
Beta-glucosidase activity was determined in thelymphocytes by the fluorimetric method using
a synthetic substrate 4-methylumbelliferyl-βglucopyranoside. Taurocholic acid was used as
an activator at pH 5.5. In parallel, the control activity of N-acetyl-β-D-glucosaminidase was performed using the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-β-D-glucopyranoside tovalidate the quality of the specimen.

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Gaucher disease: an underdiagnosed pathology in the Eastern Moroccan population

DISCUSSION
The diagnosis of metabolic disorders could be
difficult and time consuming. Pancytopenia, organomegaly (especially splenomegaly) and bone
symptomatology are the most typical signs oftype 1 Gaucher disease, the most common form[2]. Neurological signs are typical for types 2 and
3 of the disease [3].
For both patients, the diagnosis was made in
adulthood, at an average age of 32 years. These
data are consistent with previously published results [4]. However, Charrow and al [5] report, in
their study of the international register containing the records of 1698 patients, an average ageof diagnosis of 17.4 years.
It should be noted, however, that in this study,
50% of patients were diagnosed before the ageof ten, because of severe symptoms, requiring
special medical care and thus an earlier empiric
diagnosis was possible [6].
When the clinical presentation is insufficient andin the absence of signs of orientation in the family, Gaucher disease remains a diagnostic challenge. It is often not included in the differential
diagnosis of thrombocytopenia, and can present
a challenge even to an experienced hematologist [7].
However, the presence of the most common
initial symptoms, splenomegaly, cytopenia withcytological and biochemical exploration can leadto the diagnosis [8].
Demonstration of reduced enzymatic activityof β-glucocerebrosidase is required for definitive diagnosis of GD. However, our patients inwhom enzymatic assays was carried out displayed subnormal values of the GBA activity.
By analyzing the clinical and biological contradiction, we emphasize the importance of the preanalytical process, especially the one pertaining
to the maximum sample transportation time of
48 hours.

We reiterate the importance of standardizing diagnostic methods and setting up specialized laboratories. Given the critical role of the preanalytical phase, screening tests using dried blood spots
can be an optimal and alternative solution to the
enzymatic activity assay on blood specimens.
Under-diagnosis of Gaucher disease in developing countries is explained among other factors
by the lack of referral centers specialized in the
diagnosis and management of this pathology.
Apart from the pre-analytical and analytical problems, the case with normal β-glucocerebrosidase
activity can be explained either by the mutation of its activator “Saposin C” (PSAP), or it isa heterozygosity resulting in a borderline enzyme result (knowing that heterozygotes have
15 to 20% of the normal enzymatic activity of
β-Glucocerebrosidase) [9].
Chitotriosidase is a sensitive biological marker.
This is a good diagnostic guide and is especially
useful for monitoring the course of the disease[10]. It is also elevated during sarcoidosis, leishmaniasis and other storage diseases. Although,its rate is moderate compared to the values
found in the GD [11]. The assay of chitotriosidase activity was performed in our two patientsand in both cases showed high activity.
The evolution of Gaucher disease could be complicated by the appearance of haematologicaldiseases (malignant or not) [6,11], or solid cancers [6]. We also identified a patient aged 59
years, diagnosed with type 1 Gaucher disease,
who developed multiple myeloma with Ig G kappa
monoclonality after 23 years, unfortunately due
to lack of medical documents we did not report
his case (Figure 2).
The therapeutic management has been revolutionized by enzyme replacement therapy.
Because of its high cost, access to this therapy
remains limited in developing countries. The
clinical trials of gene therapy have given very
promising prospects.

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O. Bouayadi, A. Lyagoubi, A. Aarab, S. Lamrabat, A. Berhili, M. Bensalah, R. Seddik
Gaucher disease: an underdiagnosed pathology in the Eastern Moroccan population

Figure 2

Medullary smear showing Gaucher cells with plasma cells
in a patient with Gaucher disease complicated by multiple myeloma

Plasma cells

Gaucher cells

CONCLUSION
Gaucher disease is not exceptional in our country.
Type 1 is the most common. Given the frequency
igure BneousMedu
ary wemea
showinof consangumarriages,
insiston theimportanceof ahregionalp tie w
G uc registry
er diseand thec needmp
for the establishment of a reference center for
Gaucher disease and metabolic diseases in general, to allow early diagnosis and adequate care.
REFERENCES
1. Rahal F, N Brahimi, A Ladjouze-Rezig. Maladie de Gaucher. Batna J Med Sci 2015; 2:66-69

2. Zimran A. How I treat Gaucher disease. Blood. 2011;
118: 1463-1471.
3. Machaczka M, KämpeBjörkvall C, Wieremiejczyk J, etal. Impact of imiglucerase supply shortage on clinicaland laboratory
aucherce parameters
s with pin Norrbottnianpatients
sma cells in a with
Gaucher disease type 3. Arch Immunol Ther Exp (Warsz).
d b 63:m65-71.
lt
l
2015;
4. Mankin HJ, Trahan CA, Barnett NA, Laughead J, Bove
CM, Pastores GM. A questionnaire study for 128 patientswith Gaucher’s disease. Clin Genet. 2006; 69:209–17.
5. Charrow J, Andersson HC, Kaplan P, Kolodny EH, Mistry
P, Pastores G, et al. The Gaucher registry: demographics
and disease characteristics of 1698 patients with Gaucher’s disease. Arch Intern Med. 2000;160:2835–43.

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O. Bouayadi, A. Lyagoubi, A. Aarab, S. Lamrabat, A. Berhili, M. Bensalah, R. Seddik
Gaucher disease: an underdiagnosed pathology in the Eastern Moroccan population

6. Revest M, Perlat A, Decaux O, Lamyb T, Vérin M, Brissot P et all, Étude rétrospective sur dix ans des patients
atteints de maladie de Gaucher au CHU de Rennes. La Revue de médecine interne 30 (2009) 847–856.

9. Jebar K. La maladie de Gaucher: à propos d’un cas
et revue de la littérature, thèse de doctorat en médecine, faculté de médecine et de pharmacie-Oujda. 2016;p299.

7. Mistry PK, Sadan S, Yang R, et al. Consequences of diagnostic delaysin type 1 Gaucher disease: the need for
greater awareness among hematologists-oncologists andan opportunity for early diagnosis and intervention. Am J
Hematol. 2007; 82: 697-701.

10. Audemard A, Troussard X, Galateau-Salle F, Macro M. Maladie de Gaucher. Hematologie 2013; 19:
217-222.

8. Sokołowska B, Skomra D, Czartoryska B, et al. Gaucher
disease - of the possible causes of splenomegaly - casereport. Pol Arch Med Wewn. 2004; 112: 1107-1112.

11. Stirnemann J, Caubel I, Kettaneh A, Fain O, Belmatoug N. Aspects épidémiologiques, cliniques, biologiqueset thérapeutiques de la maladie de Gaucher. Presse Med
2003; 32:503-511.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

A rare case of non-secretory multiple myeloma:
a case report and literature review
Mohammed Bensalah1,2, Somiya Lamrabat1,2, Amina Lyagoubi1, 2,
Adnane Aarab1,2, Ouardia Bouayadi1,2, Rachid Seddik1,2
1
2

Hematology Laboratory, Mohammed VI University Hospital, Oujda, Morocco
Faculty of Medicine and Pharmacy, Mohammed the First University, Oujda, Morocco

ARTICLE INFOCASE REPORTCorresponding author:
Mohammed Bensalah
Boulvard Zaid Ben Soltan
Quartier Ben Khaldoune
Rue: N, Numéro: 29
Oujda
MoroccoE-mail: mohammed.bensalah99@gmail.com

Non-secretory myeloma (NSM) is a rare form of myeloma. It is defined as monoclonal plasmocytic proliferation of the bone marrow with the same clinical andradiological manifestations of myeloma. However,plasma cells are unable to secrete immunoglobulin(serum and urinary electrophoresis are negative andfree light chain measurement is unquantifiable).

Key words:
non-secretory myeloma,
free light chain, Kappa, Lambda

This variant of multiple myeloma (MM) usually posesa diagnostic challenge to the biologist and clinician.
We report a rare case of non-secretory myeloma
in a 76-year-old patient who was diagnosed at the
Mohammed V University Hospital Center in Oujda,
Morocco.

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

INTRODUCTION
Multiple myeloma is a hematological malignancy
characterized by the presence of clonal plasma
cells in the marrow that typically secrete an abnormal immunoglobulin causing a monoclonal
gammapathy. This serum protein is often characterized by an intact immunoglobulin (heavy
and light chain), or it may be characterized onlyby the light chain. In the urine, an intact immunoglobulin is also often present [1].
Myeloma is characterized by end-organ damage
as manifested by hematologic, renal, or bonecomplications [2].
Myeloma may be preceded by a premalignant
phase in which clonal plasma cells are present
but there is no evidence of end-organ damage: this is known as “monoclonal gammopathy of unknown significance” or “smoldering
myeloma” [3].
Non-secretory myeloma (NSM) is a rare clinical form of multiple myeloma with monoclonal
plasmocytic proliferation of the bone marrow
and the same clinical and radiological manifestations. However, in the case of non-secretory
myeloma, plasma cells are unable to secrete immunoglobulin (serum and urinary electrophoresis are negative and free light chain measurement is unquantifiable) [1].
CLINICAL-DIAGNOSTIC CASE
Mr. B.T., 76 years old, whose medical history
includes:•Chronic smoking for 25 years, weaned
35 years ago;•

Type 2 diabetes with oral antidiabetic drugs;•

Epilepsy treated with Phénobarbital,
0.75 mg/day.

The patient was admitted for mixed-type backpain, left intercostal neuralgia and left rib painthat was resistant to analgesics. Everything

evolves in a context of apyrexia and conservationof the general state.
The osteo-articular examination found pain in
the palpation of the lower back spine. The restof the clinical examination was without any
particularities.
The patient has benefited from a biological assessment which did not indicate a biologicalinflammatory syndrome (normal ertyhrocyte
sedimentation rate and CRP test) and the complete blood count with differential was withoutabnormalities. Serum protein electrophoresis
showed hypogammaglobulinemia at 3.7 g/L
and serum and urine immunofixations were
negative with a normal Kappa/Lambda ratio.
Renal and hepatic status was normal. (Table 1,
Figure 1)
Magnetic resonance imaging (MRI) of the thoracic spine showed suspicious-looking D9 vertebral
body compression with swollen pre-vertebral
soft tissue swelling and posterior wall retraction,
as well as a heterogeneous aspect of the cervicalvertebrae.
The myelogram revealed 85% medullary plasmo­
cytosis. (Figure 2)
Immunohistochemistry performed on osteomedullary biopsy showed medullary infiltration by myelomatous plasmocyte proliferation(CD138 positive) with a Kappa monotype.
Therapeutically, the patient was put on melphalan-prednisone-thalidomide (MPT)/Zometa protocol with a partial response (medullary plasmocytosis is of 18%).
DISCUSSION
Multiple myeloma is a hematological malignancy characterized by monoclonal plasmocytic proliferation invading the hematopoietic
bone marrow. Serum protein electrophoresis
shows either the presence of a narrow peakmigrating most often in the gamma globulin

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

zone for secreting myelomas, or hypogammaglobulinemia associated with Bence-Jones proteinuria for light chain myelomas. The study ofthe myelogram shows a plasmocytosis greaterthan 10%. This plasmocytic proliferation is accompanied by hematological, bone and renal
complications [4]. The contribution of Flow
Table 1

Cytometry (CMF) in the initial evaluation is limited. However, it plays a more important rolein the differential diagnosis of MM, where it
can be a useful ancillary tool in identifying unusual morphologic variants of myeloma, casesof prominent reactive plasmacytosis, or B-cell
non-Hodgkin lymphomas (NHLs) with extreme

Laboratory results
Parameter

Case

Reference range

Hemoglobin

14,6 g/dL

13-18 g/dL

Ertyhrocyte sedimentation rate

24

-

CRP

8,82 mg/L

0-5 mg/L

ALT

22 UI/L

0-55 UI/L

AST

25 UI/L

5-34 UI/L

Gamma-GT

25 UI/L

12-64 UI/L

LDH

383 UI/L

125-243 UI/L

Creatinine

8,44 mg/L

7,2-12,5 mg/L

Ca++

93 mg/L

88-100 mg/L

24-h proteinuria

48,98 mg/24h<500

Total protein

54 g/L

60-78 g/L

IgG

3,82 g/L

5,4-18,22 g/L

IgM<0,20 g/L

0,22-2,40 g/L

IgD<7 mg/L

7,7-132,10 mg/L

Free light chains Kappa-serum

6,57 mg/L

3,30-19,40 mg/L

Free light chains Lambda-serum

5,41 mg/L

5,71-26,30 mg/L

Kappa / Lambda Free light chains ratio

1,21

0,26-1,65

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

Figure 1

A: Serum protein electrophoresis showing hypogammaglobulinemia
B: negative serum immunofixation
C: negative urine immunofixation

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

Figure 2

Myelogram showing a medullary plasmocytosis

plasmacytic differentiation, among others [5].
CMF has also shown interest in accurately quantifying medullary plasmocyte infiltration andthe proportion of pathological plasma cells relative to total medullary plasma cells (ratio) [6].
MM is almost always preceded by an asymptomatic premalignant stage termed monoclonal gammopathy of undetermined significance(MGUS) [7-9]. MGUS is defined as a serum
monoclonal protein (non-IgM type) <30 g/L,
Clonal bone marrow plasma cells <10%, andabsence of end-organ damage such as hypercalcaemia, renal failure, anaemia, and bonelesions (CRAB) or amyloidosis that can be attributed to the plasma cell proliferative disorder [9]. About 80% of multiple myeloma originates from non-IgM immunoglobulin MGUS

(non-IgM MGUS), and 20% from light-chain immunoglobulin MGUS (LC-MGUS). In the eventof progression, IgM immunoglobulin MGUS
(IgM MGUS) usually evolves into Waldenstrom
macroglobulinaemia, but in rare instances IgM
MGUS can progress to multiple myeloma (IgM
myeloma) [9-14].
Non-secretory myeloma is a rare clinical formof multiple myeloma (2% to 4% of all cases) [1]with monoclonal plasmocytic proliferation of
the bone marrow and the same clinical and radiological manifestations. However, in the caseof non-secretory myeloma, plasma cells are unable to secrete immunoglobulin (serum and urinary electrophoresis are negative and free lightchain measurement is unquantifiable). (Table 2)

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

Table 2

Criteria for Non-Secretory Myeloma [1]Biopsy-proven clonal plasma cells<0,5 mg/dl serum protein by serum protein electrophoresis<200 mg/24hrs of light chain proteinuria by urine protein electrophoresis
Unquantifiable free light chain measurement
Evidence of CRAB criteria (hypercalcemia, renal dysfunction, anemia, bone disease)for symptomatic myeloma (which must be attributable to clonal plasma-cell infiltration)
This rare entity (NSM) must be distinguishedfrom oligosecretory myeloma, in which proteins are produced but at very low levels thatmake reliable measurement more challenging.
Oligosecretory multiple myeloma is often characterized by serum protein <10 mg/dL, urine
protein <200 mg/24 hrs, and free light chain values <100 mg/L (or 10 mg/dL) [15].
Two distinct types of NSM have been described.
The first group consists of patients who are“non-producers.” These are patients whose tumors may have defects in immunoglobulin synthesis. These tumors are not able to synthesize
or secrete a protein eventhough they might
have all the features of a plasma cell disorder[16]. In this category, we include the patients
who have no measurable protein in the bloodor urine, yet who still have a significant plasma
cell burden in the marrow and evidence of endorgan damage. In addition, even the dosage ofthe free light chain will not reveal measurable
disease as currently defined. The next categoryof non-secretory myeloma patients consists of
those whose tumors produce a protein but havedefects in secretion, possibly due to a mutationof the immunoglobulin gene thus explaining theabsence of secretion in a patient with non-secretory myeloma.
According to the recommendations establishedby the International Myeloma Workshop[3], theworkup for all newly diagnosed myeloma patients includes: routine chemistries including
LDH and beta-2-microglobulin, complete blood
cell count with differential, serum protein electrophoresis with immunofixation, quantitative
immunoglobulins (including IgD or IgE if suspected), 24-hr urine test with protein quantification and immunofixation, serum free lightchain assay, skeletal survey and positron emission tomography scan[1].
Response assessment in myeloma is typically
based on the absence of a detectable protein in
the blood or urine and a normal free light ratio.
Even with a response that meets these criteria,a more in-depth assessment using moleculartechniques, such as multi-parameter flow cytometry (MPF) [1,17,18], is able to evaluate theminimal residual disease (MRD) that likely willcontribute to relapse. Therefore, the IMWG included the evaluation of the residual disease of
MM by MPF in the therapeutic response criteria. However, MPF alone is probably not sufficient to assess total body myeloma burden. For
this reason, the pairing of imaging and moresensitive marrow assessment represents an optimal method by which to assess response totherapy and MRD, and will likely be applied to
all myeloma patients, not just non-secretory patients in whom the inability to use SPEP or UPEP
limits methods of response assessment [1].

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M. Bensalah, S. Lamrabat, A. Lyagoubi, A. Aarab, O. Bouayadi, R. Seddik
A rare case of non-secretory multiple myeloma: a case report and literature review

CONCLUSION
In conclusion, the absence of a monoclonal protein in serum and/or urine does not rule out thediagnosis of MM. Indeed, the diagnosis of nonsecreting myeloma should be made in patientswith clinico-biological and radiological characteristics of MM with a ratio of normal Kappa/
Lambda free light chains. As a result, this report
has now become a standard for the diagnosis of
non-secreting myeloma.
REFERENCES
1. Lonial S,Kaufman JL. Non-secretory myeloma: aclinician’s guide. Oncology (Williston Park). 2013
Sep;27(9):924-8, 930.
2. Lonial S, Miguel JF. Induction therapy for newly diagnosed multiple myeloma. J Natl Compr Canc Netw.
2013;11:19-28.
3. Dimopoulos M, Kyle R, Fermand JP, et al. Consensus
recommendations for standard investigative workup: report of the International Myeloma Workshop Consensus
Panel 3. Blood. 2011;117:4701-5.

9. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J,
Merlini G et al. International Myeloma Working Group
updated criteria for the diagnosis of multiple myeloma.
Lancet Oncol. 2014 Nov;15(12):e538-48.
10. Rajkumar SV, Dispenzieri A, Kyle RA. Monoclonal gammopathy of undetermined signifi cance, Waldenstrom
macroglobulinemia, AL amyloidosis, and related plasma
cell disorders: diagnosis and treatment. Mayo Clin Proc
2006; 81: 693–703.
11. Kyle RA, Durie BGM, Rajkumar SV, et al, and the International Myeloma Working Group. Monoclonal gammopathy of undetermined signifi cance (MGUS) andsmoldering (asymptomatic) multiple myeloma: IMWG
consensus perspectives risk factors for progression andguidelines for monitoring and management. Leukemia
2010; 24: 1121–27.
12. Landgren O. Monoclonal gammopathy of undetermined signifi cance and smoldering multiple myeloma:
biological insights and early treatment strategies. Hematology (Am Soc Hematol Educ Program) 2013; 2013:
478–87.
13. Schuster SR, Rajkumar SV, Dispenzieri A, et al. IgM
multiple myeloma: disease defi nition, prognosis, and differentiation from Waldenstrom’s macroglobulinemia. Am
J Hematol 2010; 85: 853–55.

4. Charlot-Lambrecht I, Salmon J.-H, Gagneux-Lemoussu L, Brochot P et J.-P. Eschard. Myélome multiple EMC
2011-01-01, Volume 6, Numéro 4, Pages 1-12.

14. Turesson I, Kovalchik SA, Pfeiff er RM, et al. Monoclonal gammopathy of undetermined signifi cance and risk oflymphoid and myeloid malignancies: 728 cases followed
up to 30 years in Sweden. Blood 2014; 123: 338–45.

5. Olteanu H. Role of Flow Cytometry in the Diagnosis
and Prognosis of Plasma Cell Myeloma. Surg Pathol Clin.
2016 Mar;9(1):101-16.

15. Durie BG, Harousseau JL, Miguel JS, et al. International uniform response criteria for multiple myeloma.
Leukemia. 2006;20:2220.

6. Gressier M, Chaquin M, Lhermitte L, Asnafi V,Macintyre
E, Brouzes C. Intérêt de la cytométrie en flux multiparamétrique 8 couleurs dans la prise en charge des gammapathies monoclonales. Ann Biol Clin 2013 ; 71 (3) : 313-23.

16. Decourt C, Galea HR, Sirac C, Cogne M. Immunologic
basis for the rare occurrence of true nonsecretory plasma
cell dyscrasias. J Leukoc Biol. 2004;76:528-36.

7. Landgren O, Kyle RA, Pfeiff er RM, et al. Monoclonal
gammopathy of undetermined signifi cance (MGUS)
consistently precedes multiple myeloma: a prospective
study. Blood 2009; 113: 5412–17.
8. Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A
monoclonal gammopathy precedes multiple myeloma in
most patients. Blood 2009; 113: 5418–22.

17. Paiva B, Martinez-Lopez J, Vidriales MB, et al. Comparison of immunofixation, serum free light chain, and immunophenotyping for response evaluation and prognostication in multiple myeloma. J Clin Oncol. 2011;29:1627-33.
18. Paiva B, Almeida J, Perez-Andres M, et al. Utility offlow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders.
Cytometry B Clin Cytom. 2010;78:239-52.

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Increased bleeding risk in a patientwith oral anticoagulant therapy
and concomitant herbal intake – a case report
Paul Gressenberger, Peter Rief, Philipp Jud, Katharina Gütl,
Viktoria Muster, Leyla Ghanim, Marianne Brodmann, Thomas GaryDivision of Angiology, Department of Internal Medicine, Medical University of Graz, Austria

INFO

CASE REPORTCorresponding author:
Paul Gressenberger
Division of Angiology
Medical University of Graz
Auenbruggerplatz 158036 Graz
Austria
Phone: +43 316 385 12911
Fax: +43 316 385 13788
E-mail: paul.gressenberger@medunigraz.at

We report the case of a 36-year old male, understable rivaroxaban therapy for 18 months, who wasadmitted to our emergency room with sudden onsetof hemoptysis.

Key words:
hemoptysis, ginger, rivaroxaban,
herbal intake

Anticoagulant therapy was given after recurrent
spontaneous deep vein thrombosis (DVT) and aheterozygous Factor-V-Leiden mutation was present.
There was no co-medication reported, however, thepatient reported a constant intake of three liters ofhome-brewn ginger tea per day in the last month.
The patient was hospitalized to further investigate
the reason of hemoptysis.

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P. Gressenberger, P. Rief, P. Jud, K. Gütl, V. Muster, L. Ghanim, M. Brodmann, T. Gary
Increased bleeding risk in a patient with oral anticoagulant therapy and concomitant herbal intake

INTRODUCTION
The use of Novel oral anticoagulants (NOACs) for
the treatment of patients with venous thrombotic events (VTE) is now standard of care anddemonstrated clinical efficacy and safety in numerous clinical studies (1,2).
Usually, these substances have a low risk for interaction with other medications compared tovitamin K antagonists (VKA), the standard medication given for the treatment of VTE events inthe decades before. However, little is known concerning interaction of NOACs with herbal medicinal products (HMP).
The use of HMP is frequent among the population (3). Ginger (Zingiber officinalis) is a very
common spice used worldwide because of its
aromatic taste (4). Zingerone (ZGR), a phenolicalkanone found in zingiber officinalis, has beenreported to have various pharmacological activities so that many people even use it as a medicine (4,5).
However, little is known about concomitant use
regarding potential interactions associated withnew oral anticoagulants like rivaroxaban or other factor Xa inhibitors (4,5,6). This may become
relevant since the prescription of NOACs significantly increased in the last years (6,7,8).
We report a case of a 36-year old male under rivaroxaban therapy who developed hemoptysis
after drinking about 2 to 3 liters of ginger tea
per day over a period of one month.
CLINICAL - DIAGNOSTIC CASE
A 36-year old male under continuous rivaroxaban therapy for 18 months was admitted toour emergency room with a sudden onset ofhemoptysis appearing for the first time. The
patient reported that two hours before he produced about 20 mL of sputum containing significant blood spots. He was free from respiratory tract infections the last month and did notreport any personal or family history consistent
with a bleeding diathesis. No other anticoagulant drugs, platelet aggregation inhibitors including NSAIDs, or any other pharmaceuticaldrugs were taken.
After reviewing a long and detailed medical history, it has been ascertained that the patient has
been consuming about 2 to 3 liters of homebrewn ginger tea on a daily basis for over a period of one month.
Anticoagulant therapy was prescribed to prevent recurrence of a VTE event, after he had
two spontaneous DVT events in the lower extremities, together with a heterozygous factor
V Leiden mutation status. The patient has been
under stable NOAC treatment for 18 months
without signs of bleeding. The patient was
hospitalized to further investigate a reason forhemoptysis.
Laboratory testing showed no abnormalities–
renal function was within reference range. The
HAS-BLED Score was 0 points, Chest - X-ray was
normal, a CT scan of the thorax was also unsuspicious, and ECG was found normal. The Ear
Nose Throat (ENT)-specialist excluded a possible bleeding source in the Naso-oro-pharyngeal
region.
Since increased consumption of ginger may also
affect platelet function, platelet function testing(PFT) was performed 3 days after hospitalization. A PFT is mainly used to detect hereditaryplatelet function disorders, also to test for efficacy of antiplatelet therapy such as acetylsalicylic acid (ASA) or clopidogrel.
For testing platelet function, the MultipleElectrode-Analyzer (MEA) using a multiplatesystem was used. The reference ranges supporting a sufficient antiplatelet effect of ASA are defined between 10 - 50 units using the ASPI test,
and 19 - 46 units for clopidogrel in the ADP test,
respectively.

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P. Gressenberger, P. Rief, P. Jud, K. Gütl, V. Muster, L. Ghanim, M. Brodmann, T. Gary
Increased bleeding risk in a patient with oral anticoagulant therapy and concomitant herbal intake

In our patient, values in the ASPI test were foundto be 69 units and in the ADP test 47 units. The
value for the ADP test can be interpreted as a relevant antiplatelet effect caused by ginger, whichis almost comparable to the effect of clopidogrelon the platelet function.
Because of the 2-fold thrombotic event and theheterozygous Factor-V-Leiden mutation, the patient was advised to continue rivaroxaban therapy as a long term medication. However, due tothe initial hemoptysis, the NOAC treatment ofrivaroxaban 20 mg was paused for 48h.
Furthermore, the patient was advised to significantly reduce ginger consumption because of its
obvious additional antiplatelet effect when usedin such high concentrations. The patient wasdischarged after 4 days in a good general condition, rivaroxaban was re-started, however in a
reduced maintenance dose.
DISCUSSION
The use of NOACs for several indications significantly increased in the last years. Compared
to VKA, NOACs have some beneficial effects. In
various clinical trials lower bleeding risk and lesshospitalization were reported. Furthermore noroutine laboratory monitoring is necessary (6).
However, little is known about concomitant useof herbal medication and potential interactions
with NOAC medication (4,5,6).
Our patient was admitted to our emergency
room with hemoptysis appearing for the firsttime under stable rivaroxaban therapy. The patient has been on rivaroxaban treatment 20 mg
for 18 months because of recurrent VTE events.
Bleeding complications are possible side effects
of anticoagulant treatment.
In the recent medical history no plausible causative reason for the hemoptysis was found. The
patient had no personal or familial history consistent with a bleeding diathesis. Neither had

he exposure to anticoagulant drugs other thanrivaroxaban, nor to platelet aggregation inhibitors including NSAIDs. The HAS-BLED score, reflecting the bleeding risk of the patient (9), was0 points under stable anticoagulation therapy
for 18 months - it seems unlikely that the anticoagulant treatment alone could have caused
the hemoptysis.
After obtaining an extensive medical history, it
has been ascertained that the patient increased
his intake of homebrew ginger tea up to 2-3 liters daily in the last month.
There are various effects of ginger reported on
the human organism, also affecting the coagulation system towards bleeding; an increased use ofginger in combination with the NOAC treatment
could therefore have perpetuated hemoptysis in
our patient (3,4,5,6). A platelet function testing
was performed on the third day after cessationof ginger intake. The result of the PFT showeda markedly decreased platelet aggregation pattern after ADP stimulation, revealing a possible
effect of ginger on platelet function which is almost comparable to the effect of treatment withclopidogrel.
Since recurrent spontaneous VTE events demonstrate an indication for long-term anticoagulation. Therefore, our patient received a subsequent dose reduction of Rivaroxaban. This wasconsidered to avoid further bleeding events as
it was recently reported to be meaningful in theso called EINSTEIN-CHOICE Study (10).
Furthermore, the patient was advised to lower
his ginger consumption.
To summarize, despite the delayed testing ofplatelet function after three days of withdrawalof ginger intake, we conclude that extensive ginger intake together with rivaroxaban therapy enhanced bleeding risk in our patient, this should
be considered in obtaining medical history in patients with unclear bleeding events under DAOK
therapy.

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P. Gressenberger, P. Rief, P. Jud, K. Gütl, V. Muster, L. Ghanim, M. Brodmann, T. Gary
Increased bleeding risk in a patient with oral anticoagulant therapy and concomitant herbal intake

TAKE HOME MESSAGES/LEARNING POINTS

cological Properties of Zingerone (4-(4-Hydroxy-3-methoxyphenyl)-2-butanone). Scientific World Journal. 2015;
2015: 816364•

The use of herbal medicinal products is common among the population. However, little
is known about its concomitant use and thepotential interactions associated with direct
oral anticoagulants.

5. Anti-factor Xa activities of zingerone with anti-platelet
aggregation activity.

Platelet function testing in such situation
seems meaningful and may help to support
the hypothesis drawn from medical history
in patients with unclear bleeding events.

7. Jobski K, Hoffmann F, Herget-Rosenthal S, Dörks M.
Oral Anticoagulants in German nursing home residents Drug use patterns and predictors for treatment choice. Br
J Clin Pharmacol. 2017 Nov; 17•

REFERENCES
1. EINSTEIN Investigators. Oral rivaroxaban for symptomatic venous thromboembolism. N Engl J Med. 2010 Dec
23; 363 (26): 2499-510
2. The Hokusai-VTE Investigators. Edoxaban versus Warfarin for the Treatment of Symptomatic Venous Thromboembolism. N Engl J Med. 2013 Oct 10; 369 (15): 1406-15
3. Agbabiaka TB, Wider B, Watson LK, Goodman C. Concurrent use of prescription drugs and herbal medicinal
products in older adults: a systematic review protocol.
Drugs Aging. 2017 Dec; 34 (12): 891-905
4. Ahmad B, Rehman MU, Amin I, Arif A, Rasool S, Bhat
SA, Afzal I, Hussain I, Bilal S, Mir Mu. A Review on Pharma-

6. Di Minno A, Frigerio B, Spadarella G, Ravani A, Sansaro
D, Amato M, Kitzmiller JP, Pepi M, Tremoli E, Baldassarre D.
Old and new oral anticoagulants: Food, herbal medicinesand drug interactions. Blood Rev. 2017 Jul; 31(4):193-203

8. Sommerauer C, Schlender L, Krause M, Weißbach S,
Rieckert A, Martinez YV, Reeves D, Renom-Guiteras A,
Kunnamo I, Sönnichsen A. Effectiveness and safety of vitamin K antagonists and new anticoagulants in the prevention of thromboembolism in atrial fibrillation in older
adults - a systematic review of reviews and the development of recommendations to reduce inappropriate prescribing. BMC Geriatr. 2017 Oct 16; 17(Suppl 1): 223
9. Kooiman J, van Hagen N, Iglesias Del Sol A, Planken EV,
Lip GY, van der Meer FJ1, Cannegieter SC, Klok FA, Huisman
MV. The HAS-BLED Score Identifies Patients with Acute
Venous Thromboembolism at High Risk of Major Bleeding
Complications during the First Six Months of Anticoagulant Treatment. PLoS One. 2015 Apr 23; 10 (4): e0122520
10. Weitz J et al. Rivaroxaban or Aspirin for Extended
Treatment of Venous Thromboembolism. N Engl J Med.
2017 Mar 30; 376 (13): 1211-1222

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This is a Platinum Open Access Journal distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Unusually low serum alkaline phosphataseactivity in a patient with acute on chronic liver
failure and hemolysis
Parul Arora1, Shekhar Singh Jadaun2, Prasenjit Das3, Shalimar2,
Sudip K. Datta1

Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India
Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
3
Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
1
2

INFO

CASE REPORTCorresponding author:
Sudip Kumar Datta
Department of Laboratory Medicine
All India Institute of Medical Sciences
New Delhi, 110029
India
Phone: 011-26594629
Cell: 9868397278; 9350179698
E-mail: dr.sudipdatta@gmail.com

A 28-year-old male with acute on chronic liver failure(ACLF) and hepatic encephalopathy had deranged liver function with curiously low level (0-15 IU/L) of serum alkaline phosphatase (ALP). Peripheral smear examination suggested hemolytic anemia. The findingof persistent low ALP, after ruling out pre-analyticalcauses, in ACLF has been reported in Wilson’s disease(WD) with/ without autoimmune hemolytic anemia(AIHA). Definitive evidences of WD were not seen in
our case. Positive DCT and histological features suggest a diagnosis of autoimmune hepatitis with secondary hemochromatosis and cholangitis. Low ALP
might not always be a determinant of bile duct pathology in patients of ACLF with AIHA.

Key words:
ACLF, autoimmune hepatitis,
hemolytic anemia, low ALP,
Wilson’s disease

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysis

INTRODUCTION
Acute on chronic liver failure (ACLF) is acute deterioration of liver function in patients with preexisting liver disease. The American Association
for the Study of Liver Diseases defined ACLF as:“acute deterioration of pre-existing chronic liverdisease usually related to a precipitating event
and associated with increased mortality at 3
months due to multi-system organ failure” (1).
Common causes of ACLF include active alcohol
consumption, reactivation of hepatitis B virus
infection, superinfection with hepatitis E virus,
autoimmune hepatitis flare, sepsis and superimposed drug or toxic injury.
In our case the patient presented with acuteliver failure and hemolytic anemia. However, he
was detected with chronic liver disease (CLD)
two years back. Most causes of ACLF were ruledout. However, an unusual finding of low serum
alkaline phosphatase (ALP) values (<5-15 IU/L)
was observed. Possible causes for the same inthe setting of ACLF were looked for.
CLINICAL – DIAGNOSTIC CASE
A 28-year-old man presented to the emergency
with jaundice for two weeks and progressively
increasing abdominal distension. Two days priorto admission he developed malena, decreased
urine output and drowsiness. His past records
revealed that he was diagnosed with CLD, however the etiology was unknown. He recovered
spontaneously during that episode and was asymptomatic till the present admission. His relatives denied any history of blood transfusion,
intravenous drug abuse, tattooing, promiscuity,
consumption of alcohol or smoking.
On assessment in casualty, the patient was
found to be icteric and hypotensive. His abdomen was distended; without any guarding or rigidity. Neurological examination suggested thepresence of grade III hepatic encephalopathy.
Kayser–Fleischer ring was not visualized on slit

lamp microscopy. Chest and cardiovascular examination was within normal limits.
On admission he was suspected to be a case of
ACLF, which was confirmed in the subsequent
two days. His blood investigations revealed:blood urea nitrogen (BUN): 59 mg/dL, serum
creatinine: 3 mg/dL, serum total bilirubin: 24.1
mg/dL, direct bilirubin: 17.8 mg/dL, aspartate
aminotransferase (AST) levels: 205 IU/L, alanine aminotransferase (ALT): 23 IU/L and ALP:
43 IU/L. Blood ammonia level was elevated to
135 µmol/L. Details of the serial liver function
are shown in Table 1. It was observed that serum ALP values decreased to very low (5-15
IU/L) to even undetectable levels over the next
few days.
Prothrombin time, INR and activated partial
thromboplastin time were prolonged (Table 1).
Hemoglobin and platelet counts were low- 6.4
g/dL and 50,000/mm3 respectively. Leucocytes
were normal in count and morphology. Peripheral
smear examination was suggestive of hemolytic
anemia. Direct Coomb’s test (DCT) was positiveand indirect (ICT) was negative.
Serum markers for viral hepatitis (HBsAg, Anti
HCV, Total anti-HBcAb, IgM HAV, IgM HEV andHEV RNA) were negative. Autoimmune markers
including anti-nuclear antibody, anti–smoothmuscle antibody, antibody for liver-kidney microsomal type-1 were negative. Total IgG levelwas 1590 mg/dL (RI: 840 -1700 mg/dL). Serumcopper was 53.5 ug/dL (RI: 70-140 ug/dL) andserum ceruloplasmin 17 mg/dL (RI: 20-60 mg/dL), both marginally on the lower side. 24 hours
urine copper estimation could not be done.
Urine routine microscopy showed the presenceof 12-15 RBC/HPF and 6-8 WBC/HPF. Bile pigments were present in urine. However, urine
culture was sterile. His esophagogastroscopy
showed grade-2 esophageal varices.
The patient was managed with broad-spectrum
antibiotics and other supportive measures.

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysis

Table 1

Laboratory parameters at admission and follow-up

Lab parameter

Reference
interval

2 monthsprior toadmissionOnadmission

Day
1

Day
3

Day
4

Day
6

Day
7

TLC (x103/μL)

4.0-10.0

7.1

-

11

4.9

4.5

6.3

8.1

N-Neutrophils (%)

-

65

82

69

75

84

86

L-Lymphocytes (%)

-

30

-

08

21

10

04

09

M-Monocytes (%)

-

01

-

08

06

11

07

04

E-Eosinophils (%)

-

03

-

01

03

03

04

01

B-Basophils (%)

-

01

-

01

01

01

01

00

RBC (x106/μL)

4.5-5.5

-

-

1.5

1.62

2

1.9

1.13

Hemoglobin (g/dL)

13-17

7.8

-

6

6.4

7.5

7.7

6.6

HCT (%)

40-50

22.4

-

17

17.9

21.2

22.5

15

RDW (%)

11.6-14.0

19.8

-

18.5

21.1

25.3

29.6

31.1

Platelet (x103/μL )150-410

118

-

60

50

80

40

20

BUN (mg/dL)

7-20

20

50

59

38

26

29

54

Creatinine (mg/dL)

0.6-1.2

1.1

2.4

3

1.5

0.8

1

2.9

Calcium (mg/dL)

9.2-11.0

7.9

-

8.5

9

8.3

8.7

9.4

Phosphate (mg/dL)

2.3-4.7

3.7

-

7.1

3.4

2.5

2.8

7.3

Uric acid (mg/dL)

4-8.5

3.3

-

4.5

1.9

1.3

1.5

4.1

Sodium (mEq/L)

136-142

120

126

132

137

149

-

Potassium (mEq/L)

3.8-5

5

7.6

4.3

4

3.5

-

Total Bilirubin (mg/dL)

0.1-1.2

4.8

24.35

24.1

26.6

29.5

37.7

38.6

Bilirubin conjugated(mg/dL)

<0.3

3.6

17.8

19

21.7

25.7

24.6

-

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysis

Total Protein (g/dL)

6-7.8

7

-

5.4

5.3

5.2

5.3

4.8

Albumin (g/dL)

3.2-4.5

2.1

-

2.1

2

2.5

2.7

2.5

Globulin (g/dL)

2.3-3.5

4.9

-

3.3

3.3

2.7

2.6

2.3

AST (IU/L)

8-40

87

-

68

205

174

88

291

ALT (IU/L)

4-45

12

-

17

23

17

10

79

ALP (IU/L)

80-270

179

-

51

43

Low

Low

Low

Arterial blood Ammonia(μmol/L)

10-35

-

-

-

121

135

-

128

Total IgG (mg/dL)

840 -1700

-

-

-

-

-

-

1590

However, his general condition worsened with
progressive jaundice and grade-IV encephalopathy. He later developed shock and died 7 days
after admission.
His postmortem liver biopsy showed features of
cirrhosis with marked activity [Figure 1A, B, F,& G]. Features of lympho-plasmacytic bile duct
injury [Figure 1C & E], interphase hepatitis, features of ascending cholangitis, ballooning of hepatocytes, significant canalicular and intra-cytoplasmic cholestasis [Figure 1D] were present.
Significant steatosis or deposition of copper
associated protein was not noted with orcein
stain. Perl’s prussian blue stain showed featuresof hemochromatosis, with grade-3 iron deposition in the hepatocyte cytoplasm, Kupffer cells
and bile duct epithelial cells [Figure 1H]. Basedon the overall features, histological possibilityof chronic cryptogenic hepatitis with cholangitis and secondary hemochromatosis weresuggested.
DISCUSSION
The index case had three characteristic features:first, hemolytic anemia on peripheral smear
with positive DCT; second, hemochromatosis in

histopathology; and third, very low to undetectable levels of serum ALP.
Hemolysis in the setting of liver failure can be
immune mediated or non–immune mediated.
Immune mediated hemolysis is seen in fulminant viral hepatitis, septicemia and autoimmune hemolytic anemia (AIHA). Non-immune
mediated mechanisms are implicated in microangiopathic hemolysis - disseminated intravascular coagulation in sepsis, disseminated malignancy, fulminant Wilson’s Disease (WD), viral
hepatitis, etc.
In the index case, although serum IgG levels
were normal and autoimmune profile was negative; clinical findings, positive DCT and histological features drive towards a possibility of an
autoimmune hepatitis with secondary hemochromatosis. Features of ascending cholangitis
and marked cholestasis in liver biopsy suggest
acute hepatic insult. The gradual rise of AST
was not accompanied with a parallel rise in ALT,
which points towards a non-hepatic pathology.
Due to the presence of hemolytic anemia, thepatient had grade-3 iron deposition in the hepatic parenchyma, including the bile duct epithelial cells. AIHA is generally a chronic disorder

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysis

which may progress to liver failure. Extrahepaticfeatures of autoimmunity and seroimmunologicchanges may be however absent in most cases.
The third interesting finding was a persistently
low serum ALP value despite presence of lymphocytic cholangiopathy, ascending cholangitis
as well as iron deposition in bile duct epithelial
cells. Methodological interferences in ALP assay due to anticoagulant contaminations, commonly with EDTA, were excluded on the basis ofother biochemical parameters like calcium andpotassium (2). A few cases have been reported
in association with WD where serum ALP wasundetectable (3,5). A recent report has identified two novel mutations in ATP7B gene which
encodes for a membrane-bound copper transporting ATPase in a 42 year woman with WD
and low ALP levels (4).
WD is manifested with impaired biliary copper
excretion resulting in positive copper balance inFigure 1liver. Defective copper incorporation in apoceruloplasmin leads to low blood ceruloplasmin levels which in this case was marginally low. Serumcopper was also observed to be marginally low.
Hepatic decompensation frequently occurs inthese patients.
Moreover, in severe liver failure, hemolytic anemia may develop as large amount of copper isreleased into the circulation due to hepatocellular necrosis. However, KF ring, a pathognomonic feature of WD was not visualized. In our
index case, the diagnosis of WD was not considered on histological examination, as there
was no macrovesicular steatosis, nuclear glycogenization or evidence of hepatic significant
deposition of copper associated protein.
Besides, hypophosphatasia, a rare, genetic disease, characterized by mutations in the tissue
non-specific alkaline phosphatase (TNSALP)
gene is reported to lead to diminished activity

Post-mortem liver biopsy photomicrographPhotomicrograph shows distorted lobular architecture with formation of complete nodules (arrows) (A x 40).
Interphase hepatitis (arrows) and lymphocytic bile duct injury are noted (arrows) (B x 100; C x 200).
Ballooned hepatocytes and canalicular cholestasis are noted (D x 200).
At places dense septal lympho-plasmacytic cell infiltrate are seen to cross the para-septal limiting plate and destroying
the hepatocytes (arrow) (E x 100).
Masson’s trichrome stain highlights the collagen band (arrows) (F x 40).
Reticulin stain also highlighting the septal fibrosis (J x 100).
Perl’s Prussian blue stain shows grade three iron deposit in the hepatocytes, focally in Kupffer cells and in the bile ductlining cells (arrows) (H x100).

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysisof the TNSALP enzyme in target tissues. Clinicalfeatures include low levels of ALP in serum andbone, osteomalacia and periodontal disease.
Although, this is commonly described in children, rarely it has also been described in adulthood, when it is most commonly characterized
by poor healing, recurrent metatarsal stressfractures, and bone pain. Our index case did nothave any of these manifestations and was safely
ruled out for the diagnosis of hypophosphatasia.
Potential interferences from any drugs in theassay of ALP were also ruled out. Drugs that arereported to interfere physiologically or analytically in ALP measurement include ibuprofen,
theophylline, cefoxitin, doxycycline, amphotericin B, tetracycline, antiepileptics, anticoagulants, lipid-lowering drugs, and inhibitors ofbone matrix formation and resorption. Our index patient was managed with broad spectrum
antibiotics- Piperacillin with Tazobactam. In addition, he also received Rifaximin, Lactulose andother supportive measures. Piperacillin with
Tazobactam is not known to cause lowering of
ALP levels, rather may sometimes cause transient increase in its levels.
In summary, peripheral blood and positive DCTof the patient indicate a possible presence ofAIHA. On histopathology, autoimmune hepatitis with secondary hemochromatosis was suggested. However, ALP is usually raised in casesof hemochromatosis. Hence, in the index case,whether a persistent low ALP level in the settingof hemochromatosis is caused by a sub-clinical
WD or AIHA remains debated.
Two cases with WD with superimposed autoimmune features have also been reported, however definitive evidences of WD and AIH wereobserved in those patients (6).
Therefore, it is important to highlight that, inpresence of the above two conditions; the serum alkaline phosphatase may not be a true determinant of any possible bile duct pathology.

LEARNING POINTS
1. Autoimmune hepatitis is an important cause
for ACLF and may present without the serological markers for the same.
2. Patients with AIH and WD have been reported and may present with overlapping
features of both the diseases.
3. Low ALP, although reported with WD may
not always be a pointer towards WD; especially in patients with overlapping features.
4. ALP might not always be a true determinantof bile duct pathology.
Abbreviations:
ACLF: Acute on Chronic Liver failure
CLD: Chronic Liver Disease
ALP: Alkaline phosphatase
BUN: Blood urea nitrogen
AST: Aspartate aminotransferase
ALT: Alanine aminotransferase
INR: International normalized ratio
DCT: Direct Coomb’s test
ICT: Indirect Coomb’s test
RI: Reference Interval
AIHA: Autoimmune hemolytic anemia
WD: Wilson’s Disease

REFERENCES
1. Olson JC, Kamath PS. Acute-on-chronic liver failure: concept, natural history, and prognosis. Curr Opin Crit Care.
2011 Apr;17(2):165–9.
2. Imafuku Y, Meguro S, Kanno K, Hiraki H, Nemoto U, Hata
R, et al. The effect of EDTA contaminated in sera on laboratory data. Clin Chim Acta. 2002 Nov;325(1–2):105–11.

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Parul Arora, Shekhar Singh Jadaun, Prasenjit Das, Shalimar, Sudip K. Datta
Low serum alkaline phosphatase activity in a patient with acute on chronic liver failure and hemolysis

3. Oosthuizen NM. Undetectable serum alkaline phosphatase activity in a patient with fulminant hepatic failureand hemolytic anemia. Clin Chem. 2011 Mar;57(3):382–5.

5. Shaver WA, Bhatt H, Combes B. Low serum alkaline
phosphatase activity in Wilson’s disease. Hepatol Baltim
Md. 1986 Oct;6(5):859–63.

4. Chen L, Li X, Zheng Z, Lu X, Lin M, Pan C, et al. A novel
ATP7B gene mutation in a liver failure patient with normal ceruloplasmin and low serum alkaline phosphatase.
Gene. 2014 Mar 15;538(1):204–6.

6. Milkiewicz P, Saksena S, Hubscher SG, Elias E. Wilson’s
disease with superimposed autoimmune features: reportof two cases and review. J Gastroenterol Hepatol. 2000
May;15(5):570–4.

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Editor-in-chief
János Kappelmayer
Department of Laboratory Medicine, University of Debrecen, 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, Abbott, Orlando, FL, USA
Allan S. Jaffe, Mayo Clinic, Rochester, USA
Bruce Jordan, Roche Diagnostics, Rotkreuz, Switzerland
Gábor L. Kovács, University of Pécs, Hungary
Evelyn Koay, National University, Singapore
Tamas Kőszegi, University of Pécs, Hungary
Janja Marc, University of Ljubljana, Slovenia
Gary Myers, Joint Committee for Traceability in Laboratory Medicine, USA
Tomris Ozben, Akdeniz University, Antalya, Turkey
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, PolandPeter Vervaart, LabMed Consulting, Australia
Stacy E. Walz, Arkansas State University, USA

				

			

				
		
		

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