Assiut University Hospital Article 2

WELCOME MESSAGE FROM THE HEAD OF CLINICAL
PATHOLOGY DEPARTMENT – ASSIUT UNIVERSITY
Prof. Azza M Ezz Eldin
Head of Clinical Pathology Department
I would like to extend a warm welcome to the reader of the second issue
of Clinical Pathology Department newsletter.
On this occasion I would like to give you an idea about our development
plans for Assiut University-Clinical Pathology Department. In 2013, a
plan for lab development was set by the Clinical Pathology
Department. This plan has two main goals. The first goal is to prepare
qualified lab personnel while the second one is to improve environmental
situations in order to improve the service approved by the department to
ultimately achieve patient satisfaction. To achieve these goals, the
department launched many projects on its way to get lab accreditation. The first stage of the projects
targeted the development of the following units: blood bank, autoimmune lab in the immunology unit,
hemolytic anemia lab in hematology unit and hormone lab in women health hospital. Microbiology lab
also included in this stage. In 2015, two more labs were included in the hematology unit (homeostasis
and flow cytometry laboratories).
The second stage was directed to reconstruction of the infrastructure and preparing a central lab
service. We are very excited to announce that this stage is intended to be finished by the end of
2017. We hope this will be a forward step into a better future for the clinical pathology department and
for the hospital as well.

ANTIBIOTIC RESISTANCE
Prof. Hebat Allah
Rashed
Director of
Microbiology
Laboratory
One cannot deny the
importance of
antibiotics in saving
many lives, but people
forget to use them with caution. The misuse and
overuse of antibiotics ultimately develops
bacterial resistance to antibiotics.
Although scientists and physicians have warned
the public repeatedly, antibiotic prescriptions in
several countries remain extremely high.
Since the making of penicillin during WWII,
chemists and scientist have been greatly
enthusiastic about creating and discovering new
lines of antibiotics. However, killing the bacteria
alone is not enough. For an antibiotic to reach the
market successfully, it needs to be manufactured
in a biologically compatible and safe manner.
That being said, despite discovering many new
elements, only few of these elements can be used
realistically.
It is essential that doctors as well as patients
understand the need for using a specific drug for
a specific target. Surely, awareness needs to be
raised about the issue that using a non-specific
spectrum of antibiotics leads to the development
of resistance.
Moreover, the misuse and abuse of antibiotics is
widespread and not at all confined to the
developing countries. The western countries also
allow antibiotic prescriptions for common colds.
Furthermore, the abuse of antibiotics in
agriculture to fatten the cattle is now causing the
spread of resistant strains. This leaves the current
antibiotics in a state of uselessness.
There is also the problem of people moving all
over the world in a matter of days or even hours.
That being known, the resistance which develops
somewhere can quickly develop somewhere else
across the globe.
There are many ways that we can control this
issue before it gets out of hand. Other than
raising awareness, governments should work
together to establish international guidelines for
antibiotic prescriptions and administration.
ght aid in utilizing phages for the treatment of
bacterial infections.
In some countries, such as India, governments
are urging stricter guidelines for antibiotic
administration and dispensing. It is a prolonged
process that will need time, but the sooner the
governments take action, the better.
However, sadly, one of the major issues causing
antibiotic resistance is the misuse of antibiotics.
In many countries, antibiotics are sold over the
counter, which leads to people treating
themselves from a common cold or flu by using
antibiotics. The majority of people do not
understand that the properties of antibiotics are
useless in many cases and the excessive use is
only adding to the resistance in their body. Also,
in developing countries, people who are poor
may not complete the full dose of an antibiotic,
which will also lead to resistance.

IMMUNOPHENOTYPING: A QUITE TRICKY PROCEDURE
Prof. Maged Salah
MAHMOUD
Professor of Clinical
Pathology
Immunophenotyping is a
powerful technique used
for the diagnosis of
diseases and of particular
importance in the diagnosis
of acute leukemias and
lymphomas. It can be
done by either immunocytochemistry or flow
cytometry. The latter is being more popular and
becoming increasingly used in many laboratories.
Despite the great power of the technique, in certain
situations it can be greatly confusing or even
misleading given the following facts:
Firstly, the probe used for detection, which in this
case is the monoclonal antibodies. These are usually
obtained from different commercially available
sources, which are sold as “monoclonal” but in many
instances when these monoclonal antibodies are used
in a Western blot, a clear cross reactivity is observed,
meaning a cross reactivity with more than a single
molecule or epitope. This is quite understandable by a
simple look to the procedure by which monoclonal
bodies are raised. The first step is immunization of a
laboratory animal by a small synthetic peptide, usually
without checking its homology with other proteins in
the proteome of the organism against which the
antibody will be raised. As stated before this would
result in cross reactivity with other proteins/epitopes
in the sample that will add a true signal “not noise”
and this would not be recognized even by experienced
operator.
Secondly, the fluorescent label used to tag this
“monoclonal” antibody. With the vast array of
fluorophores available, comparison of their excitation
and emission wave lengths shows a considerable
overlap. It would be impossible to avoid the “bleed
through” of fluorescence on the fluorescence channels
of any given machine available in the market, even
with the use of highly sophisticated dichroic
filter/mirrors. This issue becomes of more importance
and a more likelihood in case of using multicolor
analysis protocols.
Thirdly, the mere fact that most of the examined
molecules, with very few exceptions, are normal
molecules “CDs” present on normal cells, and not
actual tumor markers. These molecules are not mere
concrete pillars on the cell surface, but they are the
products of genes susceptible to up- and downregulation
in response to the various stimuli delivered
to the cells from their microenvironment, and sample
storage and processing.
Fourthly, the nature of the analyzed sample, the
viability of the cells, and the degree of processing
needed before data acquisition. As a matter of fact,
apoptotic and necrotic cells lose the expression of
several molecules once they start their cell death
program. This is of utmost importance in interpreting
data collected from stored samples, refrigerated
samples, intracellular molecules that need fixation and
permeabilization, and much more in samples collected
by any means of homogenization from solid tissues or
paraffin-embedded sections. What adds to the problem
of this type of samples is that necrotic cell debris
causes noise, thus reducing the signal/noise ratio and
the quality of results.
Next, is the interpretation of results in view of a vague
language of the report like “positive” or “negative”
without reference to the meaning if positive means for
example 20 or hundred percent, and if negative means
zero or a certain figure below a cut-off value. What
makes a further confusion is the reporting the
fluorescence intensities without mentioning their exact
source i.e. from the whole analyzed cells in sample or
from a certain gated population, even how much this
gated population represents from the whole sample.
Moreover, the appearance of several scoring systems
that use arbitrary scores without reference to these
“positive” and “negative” scores are based on how
many positive cell percentages, and more important is
that assumed phenotype applies to how many cases
for a certain disease. Also, the emergence of the so
called biphenotypic leukemia as a morphological type
that in best conditions denotes ambiguity of diagnosis
and eventually management plans.
The impressive information afforded by
immunophenotyping imposed itself as a an important
diagnostic and typing requirement for acute leukemia,
but eventually, the huge literature of flow-cytometric
immunophenotyping data presented to the scientific
community, with some degree of inconsistency and
lack of consensus agreement, has driven the WHO to
gradually stalemate it from the guidelines for the
classification of acute leukemia.

Thromboelastography in Clinical
Coagulation Management and
Transfusion Practice
In conclusion, understanding the above mentioned
comments do not mean at all that we should abandon
immunophentyping, but rather points to the great
importance of a careful intellectual interpretation of
the results with thoughtful consideration of the results
in the context of integrated clinical and laboratory
results of the case in question.
Human myeloma cell line showing induction of CD45
expression by IL-6 stimulation
(Mahmoud et al, Blood 1998, 92 (10): 3887-3897
Human myeloma cell line showing variable levels of
expression of a CD19 transgene
(Mahmoud et al, Blood 1999, 94 (10): 3551-3558).
Prof. Nabila M.Thabet
Professor of Clinical Pathology
In the recent years , thromboelastography has become
a popular monitoring device for hemostasis and
transfusion management in major surgery , trauma
and hemophilia. Thromboelastography is performed
in whole blood and assesses the viscoelastic property
of clot formation under low shear condition.
Thromboelastography can be performed with a variety
of activator and inhibitors at different concentrations
representing the most important factors for different
intervals and clot formation variables .furthermore,
fibrinogen levels and platelet counts have a major
influence on thromboelastographic variables.
Aside from clot formation, Thromboelastography has
been used to estimate thrombin generation, platelet
function, and clot dissolution by fibrinolysis, all of
them with some limitations.
Thromboelastography has been successfully used for
patient coagulation
assessment,
hemostatic therapy
and transfusion in
trauma,
perioperative care,
and assessing
bleeding in
hemophilic patients.
Thromboelastograp
hy based algorithms
reduce both
transfusion
requirements and
blood loss in
cardiac surgery,
liver
transplantation, and
massive trauma.
Fig (1) represents Thromboelastometry (TEM): (A)
normal trace and appearance; in hemophilia (B) before
and (C) after factor VIII infusion; hypercoaguable
status, and (F) thrombocytopenia.
Parameter definitions: clotting time (CT) ;Clot
formation time (CFT); alpha angle(°); clot formation
rate (CFR ) ; maximum clot firmness (MCF)- lysis
index (Ly60)- that takes place after maximum
firmness (% remaining clot firmness ).

STEM CELLS FOR BLOOD TRANSFUSION
Prof. Maha Atwa
Director of Central
Blood Transfusion
Services
Researchers are working
on a collaborative
research project that aims
to generate a limitless and
infection-free supply of
red blood cells in the lab
from human embryonic
stem cells for use in clinical blood transfusion.
Blood
Each adult has about five liters of blood making up
approximately eight percent of a person’s body
weight. Blood consists of a fluid known as plasma and
several different types of cells; red blood cells, white
blood cells and platelets. Red blood cells carry oxygen
from the lungs to the other tissues; white blood cells
fight infection; and platelets are vital for clotting that
prevents us bleeding to death. Oxygen is essential for
all the organs in your body particularly your brain and
muscles. So if you lose a lot of blood or don’t make
enough because of a disease. Oxygen levels can fall
so low that you could die.
How we get blood
• Transfusion is the transfer of blood from one
person to another
• It is used to replace blood lost due to serious
injuries, illness, operations and child birth
• Supply depends on donors and is often limited or
insufficient
• Worldwide 150,000 woman die each year due to
blood loss from giving birth
• There are no good alternatives to carry oxygen
around the body
• Donor blood may, rarely, transmit diseases to a
patient
• Donor blood must be matched to the patient’s
blood
Blood transfusion
Blood transfusions are given to people who have lost
blood following a serious accident or surgery and to
people with life-threatening diseases where the
normal process of blood cell production has gone
wrong. However, there is a shortage of blood donors
and despite safety measures patients in need of blood
transfusions are at risk of getting infectious diseases
and having an immune reaction to blood that isn’t
their own, these problems might be solved using
human embryonic stem cells that are free from
infection, can be grown continuously in the lab and
can be turned into many different cell types, including
red blood cells.
Stem cell lab making future medicines
Stem cells
Human embryonic stem cells and induced pluripotent
stem cells can make any cell type in your body.
Scientists are excited about the possibility that many
diseases and injuries may be treated by cell
replacement therapies. For example insulin producing
cells to treat diabetes; replacing damaged spinal cord
cells after paralysis, or building new bone and
connective tissues to repair joints or bones that have
been badly broken. Stem cells can also be used to
make cells that can be used to find and test drugs
which will result in better, safer drugs and a reduction
in animal testing.
How we might use stem cells in the future
Study development of disease
Scientists study stem cells to understand what controls
normal and abnormal development from a fertilised
egg to a human being. This will help them understand
diseases caused by abnormal cell division, such as
cancer and birth defects.

with reviews and audits. A key element was the
identification of non-conformances and a
Corrective Action System to prevent
reoccurrences. Specific quality improvement
methodologies were not prescribed.
Like ISO 9001QMS .ISO 14001 EMS, ISO
15189 etc…….
Test new medicines
Stem cells can become specialized cells. These
specialized cells could replace the scarce source of
dead or donor tissue currently used to test new
medicines. This would also reduce the need for animal
testing.
Replace damaged cells and treat disease
Stem cells are already used in the treatment of
extensive burns, and to restore the blood system of
some patients with leukemia and other blood
disorders. Scientists hope stem cells can be used to
replace cells lost in many other devastating diseases
for which there are currently no sustainable cures, e.g.
motor neuron disease, Parkinson’s and liver failure.
QUALITY PHILOSOPHY AND
MANAGEMENT STRATEGIES
1- W. Edwards Deming.
Knowing as a (Quality establisher) he established
14 concepts for quality. And he invented the
Deming Cycle which known as a PDCA Cycle.
2- Joseph M. Juran (Juran Trilogy).
(Co-author of Quality Control Handbook 1951)
he focused in three components: Planning,
Control, and Improvement (PCI).
3- Total quality management (1980).
How to solve a problem using SPC Statistical
Process Control (by collect and analyze data)
Check sheets , Pareto diagrams , Histograms ,
Run charts , Flow charts , Cause and effect
diagrams , Force field analysis and Scatter
diagrams
4- Quality system and standards (1987).
To be certified, businesses needed to document
their quality system and insure adherence to it
5- The Malcolm Baldrige National Quality Award
(1987).
Awards are given in five categories
Manufacturing, service, Small business, Health
Care, and Education.
6- Six Sigma
It’s a Statistical Methods developed by Motorola
Company in Japanese.
The Six Sigma concept has developed into a
methodology that focuses on process
improvement and variation reduction (VR),
through the use of a measurement-based strategy.
This strategy is realized through the application
of a Six Sigma improvement project. This Six
Sigma methodology is often accomplished
through the use of sub-methodologies: Define
Measure, Analysis, Improve, and Control
(DMAIC).
There are many levels of these methods Orange
belt, Green belt, Black belt and Master black Belt
(champion)
7- Just in time, Lean Manufacturing, Poka-Yoke,
and others.
Poka Yoke is a Japanese term literally defined as
'mistake proofing'. Developed by (Toyota)
Company. Poka-Yoke is often used in the context
of designing devices, typically simple and
inexpensive, that prevent defects from being
made or, if they are made, from moving to the
next process.
Poka-Yoke is fooling proofing, which is the basis
of the Zero Quality Control (ZQC) approach,
which is a technique for avoiding and eliminating
mistakes. Generally this technique is used in
manufacturing process but has much wider uses,
such as; offices - order and invoice processing,
hospitals - drug dispensing, aircraft maintenance
- particularly with processes having the potential
of inducing catastrophic in-service failures...
reduce the billing error . Reduce the cost of waste

MEASUREMENT UNCERTAINTY IN CLINICAL LABORATORIES
Eng. Mahmoud A. Eltayeb
Chair of Arab
Accreditation Cooperation
ARAC
What is measurement
uncertainty?
In ordinary use the word
'uncertainty' does not inspire
confidence. However, when
used in technical sense as in
'measurement uncertainty' it carries a specific
meaning.
It is a parameter, associated with the result of
measurement that defines the range of values that
could reasonably be attributed to the measured
quantity.
Uncertainty is defined as the range of values usually
centered on the measured value that contains the true
value with stated probability.
When uncertainty is defined as above, it indicates
confidence interval which is the range of values that
corresponds to the stated uncertainty, and also
indicates a confidence level which is the probability
associated with the confidence interval.
Knowledge of the uncertainty shows if the result is
accepted within limits defined in specification or
regulation.
Measurement uncertainty and medical laboratory
accreditation
The standard ISO 15189: 2012 [Medical laboratories
— Requirements for quality and competence]
specifies the requirements for evaluating and
reporting uncertainty of measurement. The problems
presented by these requirements vary in nature
depending on the technical field.
Accreditation bodies are responsible for ensuring the
accredited laboratories meet the requirements of ISO
15189.
Accreditation bodies are harmonizing their
implementation of the requirements for expressing
uncertainty of measurement through the regional
organizations and the International Laboratory
Accreditation Co-operation (ILAC).
Sources of measurement uncertainty
• Environmental conditions.
• Measuring system
• Operator bias
• Values assigned to the measurement standard and
the reference materials.
• Measurement set up, methods and procedures.
• Variations in repeated measurements.
In medical laboratories, measurement uncertainty is
concerned only with those sources that arise from
within the measuring system i.e. from sample
preparation to production of measurement result.
Hence, pre- and post-measurement uncertainties are
not included in the estimation of measurement
uncertainty.
Why is measurement uncertainty important?
The uncertainty is a quantitative indication of the
quality of the result. It gives an answer to the
question, how does the result represent the value of
the quantity being measured? It allows users of the
result to assess its reliability.
Who evaluates measurement uncertainty?
Uncertainty evaluation is best done by personnel who
are thoroughly familiar with the measurement process
within the laboratory and understand the limitations of
the measuring equipment and the influences of
external factors. Personal should keep records
showing the assumptions that were made and sources
of information for estimation of component
uncertainty values.
Basic knowledge of mathematics and statistics is
required to determine measurement uncertainty.

Personal should ensure that the measurement process
is under statistical control before determining
uncertainty.
Basic steps for evaluation of measurement
uncertainty
Measurement uncertainty can be estimated by two
different approaches
a) The bottom-up approach
This approach depends on the principles of ISO Guide
to the Expression of uncertainty in measurement
(GUM) and based on a comprehensive categorization
of the measurement in which each potential source of
uncertainty is identified and quantified. The estimates
of uncertainty, expressed as standard deviations
(standard uncertainties), are assigned to individual
components of the procedure which are
mathematically combined to provide the “combined
standard uncertainty” of the result then the expanded
uncertainty is obtained as follows:
• Identify all uncertainties in the measurement
process.
• Classify type of uncertainty:
Type A uncertainty which is evaluated by statistical
analysis of series of observations. It is the standard
deviation of the mean.
Type B uncertainty which is evaluated by means
other than statistical analysis of series of
observations.
internal quality control and Proficiency testing data, to
estimate the standard uncertainty associated with the
result.
• The combined standard uncertainty is calculated
from the impression component and the bias
component when bias component is significant
relative to the impression component.
• The impression component data should be
collected from testing QC material for a
minimum period of six months to ensure that
variations due to different operators, reagents and
calibrator lots, recalibrations, routine instrument
maintenance are captured. For new methods, a
minimum of 30 replicate determinations of
appropriate QC material is required to calculate
an interim standard deviation but this is
dependent on the frequency of analysis.
• The expanded uncertainty is obtained by
multiplying the combined standard uncertainty
by a coverage factor k in similar to bottom- up
approach
• The measurement results and the expanded
uncertainty are reported.
This approach is preferred for routine medical
laboratory tests and considered more practical than the
bottom-up approach. However, it is the laboratory’s
decision to use the method most appropriate for their
circumstances and supported by the available data.
• Quantify individual uncertainty by various
methods.
• Obtain the Combined standard uncertainty by
Root Sum Square (RSS method).
• Obtain the expanded uncertainty by multiplying
combined standard uncertainty by appropriate
coverage factor K.
o For K=1 the confidence level is 68%
o For K=2 the confidence level is 95%
o For K=3 the confidence level is 99%
Coverage factor 2 is often used for confidence
level 95 % .
• Document in an uncertainty budget.
• Report the measurement results and the
expanded uncertainty .
b) The top – down approach
The top-down approach uses available laboratory test
performance information, such as method validation,

INSULIN-LIKE GROWTH FACTOR 1
Prof. Hanan Omar
Professor of Clinical
Pathology
Insulin-like growth factor
1 (IGF-1), also called
somatomedin C, is a
protein (consists of 70
amino acids in a single
chain with three intramolecular
disulfide bridges) that in humans is
encoded by the IGF1 gene with a molecular weight of
7,649 daltons. It has also been referred to as a
"sulfation factor" and its effects were termed "non
suppressible insulin-like activity" (NSILA) in the
1970s.
IGF-1 is a hormone similar in molecular structure to
insulin. It plays an important role in childhood growth
and continues to have anabolic effects in adults. A
synthetic analog of IGF-1, mecasermin, is used for the
treatment of growth failure.
Clinical significance
Dwarfism
Rare diseases characterized by inability to make or
respond to IGF-1 produce a distinctive type of growth
failure. One such disorder, termed Laron dwarfism
does not respond at all to growth hormone treatment
due to a lack of GH receptors. The FDA has grouped
these diseases into a disorder called severe primary
IGF deficiency. Patients with severe primary IGFD
typically present with normal to high GH levels,
height below 3 standard deviations (SD), and IGF-1
levels below 3 SD. Severe primary IGFD includes
patients with mutations in the GH receptor, postreceptor
mutations or IGF mutations, as previously
described. As a result, these patients cannot be
expected to respond to GH treatment.
People with Laron syndrome have strikingly low rates
of cancer and diabetes.
Acromegaly
It is a syndrome that results when the anterior
pituitary gland produces excess growth hormone
(GH). A number of disorders may increase the
pituitary's GH output, although most commonly it
involves a tumor called pituitary adenoma, derived
from a distinct type of cell (somatotrophs). It leads to
anatomical changes and metabolic dysfunction caused
by elevated GH and insulin-like growth factor 1 (IGF-
1) levels.
Diagnostic test
IGF-1 levels can be measured in the blood in 10-
1000 ng/ml amounts. As levels do not fluctuate
greatly throughout the day for an individual person, It
is used by physicians as a screening test for growth
hormone deficiency and excess in acromegaly and
gigantism.
Interpretation of IGF-1 levels is complicated by the
wide normal ranges, and marked variations by age,
sex, and pubertal stage. Clinically significant
conditions and changes may be masked by the wide
normal ranges. Sequential management over time is
often useful for the management of several types of
pituitary disease, under nutrition, and growth
problems.
As a therapeutic agent
Patients with severe primary insulin-like growth
factor-1 deficiency (IGFD) may be treated with either
IGF-1 alone or in combination with IGFBP-3.
Mecasermin is a synthetic analog of IGF-1 which is
approved for the treatment of growth failure. IGF-1
has been manufactured recombinantly on a large scale
using both yeast and E. coli.
Research
Aging
Signaling through the insulin/IGF-1-like receptor
pathway is a significant contributor to biological
aging in many organisms. Cynthia Kenyon showed
that mutations in the daf-2 gene double the lifespan of
the roundworm, C. elegans. Daf-2 encodes the worm's
unified insulin/IGF-1-like receptor. Despite the
impact of IGF1-like on C. elegans longevity, direct
application to mammalian aging is not as clear as
mammals lack dauer developmental stages. It is also
inconsistent with evidence in humans
There are mixed reports that IGF-1 signaling
modulates the aging process in humans and about
whether the direction of its effect is positive or
negative
Neuropathy
Therapeutic administration of neurotrophic proteins
(IGF-1) is associated with potential reversal of
degeneration of spinal cord motor neuron axons in
certain peripheral neuropathies.
Cancer
The IGF signaling pathway is implicated in some
cancers. People with Laron syndrome have a lessened
risk of developing cancer. Dietary interventions and
modifications such as vegan diets shown to down

egulate IGF-1 activity, has been associated with
lower risk of cancer. However, despite considerable
research, perturbations specific to cancer are
incompletely delineated and clinical drug trials have
been unsuccessful.
When is it ordered?
IGF-1 testing may be ordered, along with a GH
stimulation test, when:
• A child has symptoms of GH deficiency, such as
a slowed growth rate and short stature
• Adults have symptoms that a health practitioner
suspects may be due to a GH deficiency, such as
decreased bone density, fatigue, adverse changes
to lipid levels, and reduced exercise tolerance.
• However, testing for IGF-1 deficiencisis not
routine in adults who have these symptoms; GH
and IGF-1 deficiency are only very rare causes of
these disorders.
An IGF-1 also may be ordered when a health
practitioner suspects that someone has an underactive
pituitary gland and at intervals to monitor those on
GH therapy.
Less commonly, IGF-1 testing may be ordered, along
with a GH suppression test, when a child has
symptoms of gigantism or when an adult shows signs
of acromegaly.
When a GH-producing pituitary tumor is found, GH
and IGF-1 are ordered after the tumor is surgically
removed to determine whether the entire tumor has
been extracted. IGF-1 also is ordered at regular
intervals when someone is undergoing the drug and/or
radiation therapy that frequently follow tumor
surgery.
IGF-1 levels may be ordered at regular intervals for
many years to monitor a person's GH production and
to watch for pituitary tumor recurrence.
What does the test result mean?
A normal level of IGF-1 must be considered in
context. Some people can have a GH deficiency and
still have a normal IGF-1 level.
If a decrease in IGF-1 is suspected to be due to a more
general decrease in pituitary function
(hypopituitarism), then several other endocrine glands
and their pituitary regulating hormones will need to be
evaluated to decide on appropriate treatment. Reduced
pituitary function may be due to inherited defects or
can develop as a result of pituitary damage following
conditions such as trauma, infections, and
inflammation.
Decreased levels of IGF-1 also may be seen with
nutritional deficiencies (including anorexia nervosa),
chronic kidney or liver disease, inactive/ineffective
forms of GH, and with high doses of estrogen.
IncreasedIGF-1:
Elevated levels of IGF-1 usually indicate an
increased production of GH. Since GH levels vary
throughout the day, IGF-1 levels are a reflection of
average GH production, not of the actual amount of
GH in the blood at the time that the sample for the
IGF-1 measurement was taken. This is accurate up to
the point at which the liver's capacity to produce IGF-
1 is reached. With severely increased GH production,
the IGF-1 level will stabilize at an elevated maximum
level.
Increased levels of GH and IGF-1 are normal during
puberty and pregnancy but otherwise are most
frequently due to pituitary tumors (usually benign).
If IGF-1 is still elevated after the surgical removal of
a pituitary tumor, then the surgery may not have been
fully effective. Decreasing IGF-1 levels during
subsequent drug and/or radiation therapies indicate
that the treatment is lowering GH production. If levels
of IGF-1 become "normalized," then the person is no
longer producing excess amounts of GH. When
someone is undergoing long-term monitoring, an
increase in IGF-1 levels may indicate a recurrence of
the pituitary tumor.
DecreasedIGF-1:
If the IGF-1 level is decreased, then it is likely that
there is a GH deficiency or insensitivity to GH. If this
is in a child, the GH deficiency may have already
caused short stature and delayed development and
may be treated with GH supplementation. Adults will
have an age-related decrease in production, but lower
than expected levels may reflect a GH deficiency or
insensitivity.

BIOMARKERS IN MULTIPLE SCLEROSIS: AN UP-TO-DATE
OVERVIEW
Clinical Chimestry Unit
Clinical Pathology Departement
Multiple sclerosis (MS) is a condition where the CNS
of a person presents a special kind of distributed glial
scars (sclerosis) which are a remaining of a previous
inflammatory demyelination. Multiple sclerosis is the
most common reason of neurological disability among
young adults.
In multiple sclerosis, localized destruction of myelin
occurs in the CNS. Alterations of the immune system
have been implicated in this process, by the
demonstration of myelin-reactive T lymphocytes and
the oligoclonal bands (OCB) in CSF reflecting local
synthesis of immunoglobulins (figure 1). Patients with
multiple sclerosis have a restricted immune response
with predominately IgG1 subclass. Genetic linkage
studies have indicated a variety of associations, but
the strongest is with HLA-DR2 (human leukocyte
antigen) genes. During the last decades, the effort of
establishing satisfactory biomarkers for multiple
sclerosis has been proven to be very difficult, due to
the clinical and pathophysiological complexities of
the disease. According to their pathophysiological
implication in MS pathogenesis, up-to-date
biomarkers are divided into three subgroups, geneticimmunogenetic,
laboratorial, and imaging. According
to the clinical application they further subdivided into
markers aid in diagnosis, biomarkers of
demyelination—neuroinflammation relapse,
biomarkers of prognosis-disability progression and
biomarkers of therapeutical response
Biomarkers aid in diagnosis includes:
• HLA-DRB1*1501
Is the mainly responsible allele for attributing genetic
risk in MS population. Positive correlation of HLA-
DRB1*1501with OCB in the CSF of MS patients was
found.
• OCB IgG in CSF
Positive OCB IgG in the CSF of patients with
clinically isolated syndrome (CIS) was found to
duplicate the risk of progression to clinically definite
multiple sclerosis (CDMS). Their diagnostic
sensitivity is high but they lack in specificity.
• Kappa Free Light Chains (KFLC) in CSF
KFLC high CSF levels have been reported in MS and
considered as highly predictive for CIS conversion to
CDMS.
• Measles-Rubella-Zoster Endothecal Reaction
(MRZ Reaction)
MRZ IgG reaction in CSF displays, compared to OCB
IgG, a higher specificity for MS diagnosis and higher
prognostic value of progression from CIS to CDMS.
• Vitamin D:
Vitamin D suppresses Th1 immune response in
multiple levels and enables the production of many
neurotrophic factors. 25-Hydroxyvitamin D levels in
untreated MS patients correlate inversely with
radiologic disease activity.
• Antibody against potassium channel protein
KIR4.1
It was reported that a subset of MS patients
have a seropositive anti- KIR4.1.
Biomarkers of demyelination neuroinflammation
relapse:
• Myelin Basic Protein (MBP):
MBP and its fragments are found in large quantities in
the CSF of MS patients during relapse. A significant
correlation of decrease in CSF-MBP, contrastenhancement
in MRI, suggest an association with
myelin breakdown in MS.
• αB-Crystalline
Immunohistochemical analysis of demyelinating
lesions revealed increased expression of this protein.
αβ-Crystalline is a heat-shock protein which forms
aggregates during stress. It is considered as primary
target molecule for T-cells in MS. Its mechanism of
action encompasses activation of IL-17, IL-10, IL-13,
TNF, and Chemokines CCL5 and CCL1.
• Neuronal Cell Adhesion Molecule (N-CAM)
Constant CSF elevation of N-CAM has been reported
immediately after MS relapse, showing negative
correlation with clinical improvement.
• Brain-Derived Neurotrophic Factor (BDNF)
Lower CSF-BNDF levels in secondary progressive
MS (SPMS) patients comparatively to relapsing
remitting MS (RRMS) patients have been reported.
Low BDNF levels are considered to contribute in
demyelination and axonal damage progress.
• Fetuin-A

A calcium regulating surface glycoprotein, protein , s
coding messenger RNA is overexpressed in MS
patients CNS, resulting in its high concentrations in
active demyelinating lesions.
• Cytokines and Adhesion Molecules
Inflammatory activity in active demyelinating lesion
lead to the liberation of many different cytokines that
can be used as biomarkers of disease activity they
include serum levels of IFN-γ-TNF-α, IL-6, IL-10,
sICAM-1and sVCAM-1.
Biomarkers of Axonal loss- neurodegeneration
• Soluble Molecule Nogo-A
Nogo-A is a CNS myelin component that inhibits
axonal repair. Its presence in CSF of MS patients
constitutes a bad prognostic marker of axonal repair.
Nogo-A is adequately specific for MS, as it could not
be isolated in other autoimmune or infectious
neurological disorders.
• N-AcetyloAspartate (NAA)
NAA is an aminoacid, highly expressed in neurons.
CSF-NAA reduction correlates adequately with
disability progression. NAA could be helpful in
differential diagnosis between MS and NMO.
• Tau Protein
Tau is a cytoskeleton protein whose basic
responsibility is microtubular stabilization. High CSF
levels in MS patients have been reported.
Simultaneous elevation in Tau and NF-H values in
CSF, in patients with CIS, has a 70% predictive value
of conversion to CDMS, which is superior to the
predictive value of MRI.
GFAP is a structural protein of the astrocytes whose
CSF levels increase in association with gliosisastrocytosis.
High CSF values have been found in
SPMS patients, but rarely in RRMS patients, and
seem to correlate well with disability progression.
• OCB IgG, KFLC, MRZ reaction and NAA,
NF-H reflect adequately disability
progression in MS.
Biomarkers of therapeutical response:
• HLA-DRB1* 10401, 0408, 1601
polymorphisms have higher risk for
developing neutralizing antibodies against
IFN-β.
• Vitamin D increased serum levels associated
with IFN-β responders.
• Fetiun A and NF-L levels are reduced in CSF
and considered biomarkers for good
responders to Natalizumab treatment.
• BDNF increased CSF levels observed in
Glatiramer Acetate responders, correlating
well with clinical improvement.
Biomarkers of differentiation from NMO:
CSF NAA, GFAP, and CSF: serum albumin ratio
(AR) can also help when there is a possibility for
NMO.
Biomarkers of prognosis-disability progression:
• TOB-1
TOB-1 gene has a role against T-cell multiplication,
keeping autoreactive cells in a dormant state. Its
decreased expression leads towards a more intense
immune response. TOB-1 polymorphisms represent
an independent factor influencing the progression
from clinically isolated syndrome (CIS) to clinically
definite multiple sclerosis (CDMS).
• Neurofilaments (NFs):
Neurofilaments are major axonal cytoskeleton
proteins consisting of three subunits (light chain/NF-
L, intermediate chain/NF-M, and heavy chain/NF-H).
NF-H chains seem to correlate better with disease
progression, with significant elevation recorded only
in progressive disease forms.
• Glial Fibrillary Acidic Protein (GFAP)
Figure 1: The top sample is the normal cerebrospinal
fluid (CSF) and the second lane is the CSF
concentrated 80-fold from a patient with multiple
sclerosis and the corresponding serum diluted 1:3 is
immediately below. The γ- region of the CSF from
this patient has several densely staining oligoclonal
bands which are not in the corresponding serum.

BIOLOGICAL RISK ASSESSMENT
Prof. Sohair Kamel Sayed
Professor of Clinical
Pathology
Identifying potential
hazards in the laboratory is
the first step in performing
a risk assessment. No one
standard approach or
correct method exists for
conducting a risk
assessment; However, several strategies are available,
such as using a risk prioritization matrix, conducting a
job hazard analysis; or listing potential scenarios of
problems during a procedure, task, or activity. The
process involves the following five steps:
1. Identify the hazards associated with an infectious
agent or material.
2. Identify the activities that might cause exposure
to the agent or material.
3. Consider the competencies and experience of
laboratory personnel.
4. Evaluate and prioritize risks (evaluate the
likelihood that an exposure would cause a
laboratory-acquired infection [LAI] and the
severity of consequences if such an infection
occurs).
5. Develop, implement, and evaluate controls to
minimize the risk for exposure.
Standardization of the risk assessment process can
greatly improve the clarity and quality of this process.
Step 1. Identify the hazards associated with an
infectious agent or material.
The potential for infection, as determined by the most
common routes of transmission (i.e., ingestion by
contamination from surfaces/fomites to hands and
mouth; percutaneous inoculation from cuts, needle
sticks, nonintact skin, or bites; direct contact with
mucous membranes; and inhalation of aerosols).
The frequency and concentration of organisms
routinely isolated, as determined by specimen type, •
patient data (of individual or the hospital population),
epidemiologic data, and geographic origin of the
specimen;
Intrinsic factors (if agent is known) as: Pathogenicity,
virulence, mode of transmission, infectious dose,
Form (stage) of the agent and resistance to antibiotics.
Indicators of possible high-risk pathogens that may
require continuation of work in a biological safety
cabinet (BSC), such as
-Slowly growing gram-negative rods or gramnegative
coccobacilli;
-Slow growth in blood culture bottles (i.e., positive at
≥48 hours), with small gram-negative rods or gram
negative coccobacilli;
Step 2. Identify activities that might cause
exposure to the agent or material.
1. The facility (e.g., biosafety level (BSL) -2, BSL-3,
open floor plan [more risk] versus separate areas or
rooms for specific activities [less risk], sufficient
space versus crowded space, workflow, equipment
present);
2. The equipment (e.g., in the case of uncertified
biosafety cabinets (BSCs), cracked centrifuge
tubes, improperly maintained autoclaves,
overfilled sharps containers, Bunsen burners);
3. Potential for generating aerosols and droplets.
Aerosols can be generated from most routine
laboratory procedures but often are undetectable.
The following procedures have been associated
with generation of infectious aerosols e.g.
manipulating needles, syringes and sharps,
Manipulating inoculation needles, loops, and
pipettes and manipulating specimens and cultures
4. Use of sharps;
5. Improperly used or maintained equipment;
Examples of possible hazards are decreased
dexterity or reaction time for workers wearing
gloves, reduced ability to breathe when wearing
N95 respirators, or improperly fitting personal
protective equipment (PPE).
Step 3. Consider the competencies and experience
of laboratory personnel.
Age (younger or inexperienced employees might be at
higher risk); Genetic predisposition and nutritional
deficiencies, immune/medical status (e.g., underlying
illness, receipt of immunosuppressive drugs, chronic
respiratory conditions, pregnancy, nonintact skin,
allergies, receipt of medication known to reduce
dexterity or reaction time);
Education, training, experience, competence; Stress,
fatigue, mental status, excessive workload;
Perception, attitude, adherence to safety precautions;
and The most common routes of exposure or entry
into the body (i.e., skin, mucous membranes, lungs,
and mouth).
Step 4. Evaluate and prioritize risks.
Risks are evaluated according to the likelihood of
occurrence and severity of consequences :

Likelihood of occurrence:
-Almost certain: expected to occur.
-Likely: could happen sometime.
-Moderate: could happen but not likely.
-Unlikely: could happen but rare.
-Rare: could happen, but probably never will.
• Severity of consequences:
Consequences may depend on duration and
frequency of exposure and on availability of
vaccine and appropriate treatment. Following are
examples of consequences for individual
workers:Colonization leading to a carrier state,
asymptomatic infection, toxicity,oncogenicity,
allergenicity, infection and disease
Step 5. Develop, implement, and evaluate controls
to minimize the risk for exposure.
• Engineering controls, if possible, first isolate and
contain the hazard at its source.
- Primary containment: BSC, sharps containers,
centrifuge safety cups, splash guards, safer sharps
(e.g., autoretracting needle/syringe combinations,
disposable scalpels), and pipette aids
- Secondary containment: building design features
(e.g., directional airflow or negative air pressure,
hand washing sinks, closed doors, double door
entry)
• Administrative and work practice controls
- Strict adherence to standard and special
microbiologica practices
- Adherence to signs and standard operating
procedures
- Frequently washing hands
- Wearing personal protective equipment (PPE) only
in the work area
- Minimizing aerosols
- Prohibiting eating, drinking, smoking, chewing gum
- Limiting use of needles and sharps, and banning
recapping of needles
- Minimizing splatter (e.g., by using lab "diapers" on
bench surfaces, covering tubes with gauze when
opening)
- Monitoring appropriate use of housekeeping,
decontamination, and disposal procedures
Implementing "clean" to "dirty" work flow
Following recommendations for medical
surveillance and occupational health,
immunizations, incident reporting, first aid,
postexposure prophylaxis
- Implementing emergency response procedures PPE
(as a last resort in providing a barrier to the hazard)
- Gloves for handling all potentially contaminated
materials, containers, equipment, or surfaces
- Face protection (face shields, splash goggles worn
with masks, masks with built-in eye shield) if BSCs
or splash guards are not available. Face protection,
however, does not adequately replace a BSC. At
BSL-2 and above, a BSC or similar containment
device is required for procedures with splash or
aerosol potential.
- Laboratory coats and gowns to prevent exposure of
street clothing, and gloves or bandages to protect
nonintact skin
- Additional respiratory protection if warranted by
risk assessment Job safety analysis
- One way to initiate a risk assessment is to conduct a
job safety analysis for procedures, tasks, or activities
performed at each workstation or specific laboratory
by listing the steps involved in a specific protocol
and the hazards associated with them and then
determining the necessary controls, on the basis of
organism suspected. Precautions beyond the
standard and special practices for BSL-2 may be
indicated in the following circumstances:
- Test requests for suspected Mycobacterium
tuberculosis or other mycobacteria, filamentous
fungi, bioterrorism agents, and viral hemorrhagic
fevers
- Suspected high-risk organism (e.g., Neisseria
meningitides
- Work with large volumes or highly concentrated
cultures
- Compromised immune status of staff
- Training of new or inexperienced staff
- Monitoring effectiveness of controls
Risk assessment is an ongoing process that requires
at least an annual review because of changes in new
and emerging pathogens and in technologies and
personnel.
- Review reports of
incidents,
exposures,
illnesses, and nearmisses.
- Identify causes and
problems; make
changes, provide
follow-up training.
- Conduct routine
laboratory
inspections.
- Repeat risk assessment routinely.

YOU CAN'T LIVE A POSITIVE LIFE WITH A NEGATIVE MIND
Prof. Sherif Helmy
Professor of Clinical
Pathology
Optimism has to be one
of the most valuable traits
that you can cultivate and
it will make you happier
and more popular. It is
most needed when you
are going through hard times and when you are
facing up to challenges. Of course it's more
difficult to remain optimistic when it feels like
everything is crashing down around you.
The Power of Positivity
Being positive results in happy changes in your
life. It means you will attract more people around
you and make them feel good about themselves
as they will enjoy your company. This makes
things better for you as you will have the support
of all your friends. At the same time if you are
able to remain optimistic then you will be happier
because you will genuinely believe that things are
going to get better. You will be more likely to
take the good types of risks and to attempt to get
yourself out of the situation rather than giving in.
Look for Positive Elements: Events make you
grow as a person. In other cases, your loss is
another person's gain. May be, it will open up
other opportunities. Just focus on a good outcome
from all the bad news.
Appreciate the Little Things: Whether it's
waking up healthy in the morning, going for a
picnic or talking to someone you love. We lose a
lot throughout our lives but there are some things
you can rely on.
Understand the Nature of Change: Don't waste
too much energy trying to fight the change, it is
inevitable. We go through different stages in our
lives, some full of chaos but they won't go on
forever. It's just a matter of time.
How to Remain Positive
Recognize the Value of Positivity: You need to
recognize just how valuable it is to be positive
and how unhelpful it is to be negative. This will
then give you the determination to drive out
negative thoughts.
Have a Plan: It will make you see a way out and
give you hope leading to optimism. Make sure
you are taking steps to improve matters.
Meanwhile have an alternative plan for each
outcome so that whatever happens you are ready
to deal with matters.
Face the Worst Case Scenario: Play through the
worst possible scenario in your mind. Most likely
it won't be as bad as you thought it would be.
You will still be able to cope. Suddenly, you
won’t be afraid anymore.