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Lab Communique 1
Nucleic Acid Testing (NAT) Page 3
Volume 1
Food Intolerance and Food Sensitivity
Page 11
Inborn Errors of Metabolism
Page 15
Help prevent thalassemia - Jago re...
Page 23
Vol 12
From the Editor’s Desk
Important Lab Numbers
Contents 2 Editor’s Desk
3 Nucleic Acid Testing (NAT) Dr. Anand Deshpande
11 Food Intolerance and Food Sensitivity Dr. Vipla Puri
15 Inborn Errors of Metabolism Dr. Tester F. Ashavaid
21 Funded Research / Projects in the Department of Laboratory Medicine
23 Help prevent thalassemia - Jago re... Dr. Sharmila Ghosh
Department Of Laboratory Medicine ..............
Location- Jamuna Clinic (Hinduja Clinic)
Stat Lab – Reception..............................2444 7327
Stat Lab – Biochemistry .........................2444 7328
Blood Bank ............................................2444 7308
Donor Room .........................................2444 7306
Ground Floor
O.P.D. Blood Collection .........................2444 7079
Location - Lalita Girdhar Building (S1 Bldg)
Biochemistry Lab ................................ 2444 7935/
.....................................................2444 7931
Hematology Lab ....................................2444 7947
RIA Lab ................................................2444 7948
Microbiology/Serology Lab ................. 2444 7793/
.....................................................2444 7794
Histopathology /
Cytopathology Lab................................2444 7797
NAT Lab .............................................. 2444 7610/
.....................................................2444 7611
Home Collection Service ................3981 8181/
................................................... 6766 8181
Hinduja Poison Center .................... 2446 4600
Lab Medicine Fax Number ............... 2444 2318
Assistant Manager - Diagnostics Services
Dr. Preeti Goraksha ...............................2444 7942
Quality Co-ordinator .........................2444 7943
Chetna Patil ..........................................2444 7943
Ext 7143
It is my pleasure to
present this very
first edition of our
Lab Communique.
It was a long-felt
need to present the
current, new and
research activities of
the Hinduja Hospital
Laboratory Medicine department, which
has the unique distinction of being the 1st
hospital laboratory in Asia to be accredited
to the College of American Pathologists
since 2006.
This initiative will serve the need of
increasing awareness about a quality-
conscious hospital-based Laboratory`s
core testing practices,areas of research
interest and available facilities for
Postgraduates,General practitioners and
Specialists.
In this issue, some of the Laboratory
Consultants have put together the need
for awareness of newer technologies in the
laboratory setup with an emphasis on the
approach to some common disorders. We
hope this will serve to keep you abreast
with modern-day Laboratory practices.
Dr Anita S. Bhaduri, M.D.
Surgical Pathologist
Vol 12
Produced at the marketing cell of Hinduja Hospital. For feedback and comments write to [email protected]
Lab Communique 3
Nucleic Acid Testing (NAT) - Adding a layer of safety in blood transfusion
Dr. A. Deshpande, Consultant Transfusion Medicine & Hematology, MD – Pathology E-Mail - [email protected] Contact No.: 24451515 Ext 8307/8308
Blood transfusion provides an
essential and life-saving support
in modern healthcare. When used
correctly it can save lives. However,
it can cause acute or delayed
complications and also carries the
risk of Transfusion Transmitted
Infections (TTIs) such as HIV, HBV,
HCV and other diseases.
The five tests mandatory by Food
& Drugs Administration (FDA) for
donated blood units are HBsAg,
HIV Ab, HCV Ab, VDRL and
Malarial parasites. Current testing
methods carried out in India are
based on the principle of enzyme
immunoassay (EIA). EIA- based
blood screening tests detect
virus-induced antibodies or viral
antigens, not the virus itself. The
main problem is that the body takes
some time to produce detectable
amount of antibodies that can be
detected by these methods. This
period from the time someone is
infected to the time that enough
antibodies are produced for the
laboratory to detect, is called the
‘seroconversion window’ or
‘window period’. In some cases
such as HCV it could be as high as
60 days or more. In addition, the
EIA screening has a very high rate
of false positives that unfortunately
results in perfectly healthy donors
being labelled as “positive”. Hence,
in these techniques, test specificity
is sacrificed somewhat to gain
more sensitivity.
Over two billion people worldwide
are infected with HBV, which is
the leading cause of liver disease.
Of these, more than 350 million
are chronically infected, with a
higher risk for liver cancer and
liver cirrhosis. Currently available
screening technologies are
designed to detect core antibodies
or surface antigens. However,
these infection indicators do not
appear until upto eight weeks after
an infection. Thus HBV presents a
Figure 1: Window period reduction by NAT
Dr. Anand Deshpande M.D.
Vol 14
higher residual risk of transmission
by transfusion than HCV or HIV and
the HBV infection window period
is the real issue in the transfusion
setting. Countries like India with
a high prevalence of HBV have
the highest risk of transfusion
transmitted HBV and probably
would be the most to gain from
HBV NAT. In addition, worldwide
170 million people are infected
with HCV and 40 million with HIV.
These are a serious global concern.
Blood is processed into blood
components to enable more than
one patient to benefit from a single
donation. Thus a single unit of
blood collected from a donor in the
window period of infection may be
transfused in upto four recipients
or may be added to pools of more
than 1,000 units to manufacture
blood -derived products.
The greatest threat to the safety
of blood supply is donation by
‘seronegative’ donors during
‘window period’ of initial infection
and detectable seroconversion.
Window period samples have low
viral load. Detection of very low
viral load samples requires highly
sensitive assays.
Hence the Nucleic Acid
Amplification Testing (NAT)….!
With NAT the window period is
shortened considerably, as it is a
highly sensitive and specific test
that detects very low levels of viral
RNA or DNA that may be present in
donated blood. With the use of NAT
systems, the ‘window period’ for
detection of HIV is reduced from
20.3 days to 5.6 days, for HCV it
is reduced from 58.3 days to 4.9
days and for HBV from 53.3 days
Fig 2: Pre-Amplification Area - NAT Laboratory, PDHNH
Countries like
India with a high
prevalence of HBV
have the highest
risk of transfusion
transmitted HBV
and probably would
be the most to gain
from HBV NAT.
Lab Communique 5
to 35.4 days.
NAT is being effectively utilized in
Australia, Indonesia, HongKong,
Korea, Malaysia, New Zealand,
Singapore, Thailand, Japan,
Europe, Middle East, Africa, France,
Germany, Israel, Italy, Spain,
Switzerland, UK, America, Brazil,
Carribean, Canada and a host of
other nations. A look at the NAT
experience of various countries
shows that every country has
benefitted from this technology.
What is Nucleic acid amplification
testing (NAT) and how does it work?
Nucleic acid Amplification Testing
(NAT) is a highly sensitive method
of testing blood that is used to
detect Hepatitis C, HIV and HBV
virus. NAT is a direct test which
detects the viral DNA or RNA.
It reduces the window period
by detecting low levels of viral
genomic materials that are present
soon after infection but before the
body starts producing antibodies
in response to a virus.
Genomic screening for infectious
agents using NAT is performed
with several in-vitro nucleic
acid amplification techniques,
e.g. Transcription –mediated
amplification (TMA), Polymerase
chain reaction (PCR) ligase chain
reaction and nucleic acid sequence
–based amplification. All these
techniques detect the presence
of infectious microorganism in
donor blood by amplifying the
nucleic acid sequences specific
to the microorganism,.giving it
a much higher level of sensitivity
and specificity than routine EIA
test. Thus the power of NAT
lies in its ability to detect viral
genomic nucleic acids rather than
the presence of antibodies. NAT is
used in addition to the antibody
test since in some individuals
theoretically the amount of virus
may have fallen below detectable
limits and antibodies could still be
detectable.
The NAT used in our institution
utilizes target amplification nucleic
acid probe technology for the
detection of HIV-1 RNA, HCV RNA,
and HBV DNA. The assay contains
reagents which may be used for
simultaneous detection of three
viruses or the individual viruses
HIV-1, HCV, and HBV (Triplex
assay).
The TMA assay involves three main
steps (i) target capture based sample
preparation, (ii) transcription-
mediated amplification, and (iii)
hybridization protection assay,
all performed in a single tube.
All three assays incorporate an
internal control to validate each
reaction.
During sample preparation, viral
RNA and DNA are isolated from
Nucleic acid
Amplification
Testing (NAT) is
a highly sensitive
method of testing
blood that is used
to detect Hepatitis
C, HIV and HBV
virus.
Vol 16
the amplicons. During the detection
steps the chemiluminescent signal
produced by the hybridized probe
is measured in a luminometer and
is reported as Relative Light Units
(RLU).
The assay differentiates between
the internal control and combined
HIV-1/HCV/HBV signals but
does not discriminate between
individual HIV-1, HCV and HBV
signals. Samples found reactive
in the Ultrio test are later retested
for HIV-1, HCV and HBV using
discriminatory assays.
Impact of NAT: A pilot project
in India at Apollo Indraprastha
Hospital, showed that out of
12,224 study samples 133 (1.09%)
were reactive by Ultrio Assay. 84
specimens via the use of target
capture. Oligonucleotides that are
homologous to highly conserved
regions of HIV-1, HCV and HBV
if present in the test specimen
are hybridized to the HIV-1 RNA,
HCV RNA or HBV DNA target. The
hybridized target is then captured
onto magnetic microparticles
that are then separated from the
specimen in a magnetic field.
Target amplification occurs via
TMA, which is a transcription-
based nucleic acid amplification
method that utilizes two enzymes.
Detection is achieved by
Hybridization Protection Assay
(HPA) using single-stranded nucleic
acid probes with chemiluminescent
labels that are complementary to
Fig 3: Post Amplification Area – NAT Laboratory, HH
The TMA assay
involves three main
steps (i) target
capture based
sample preparation,
(ii) transcription-
mediated
amplification, and
(iii) hybridization
protection assay,
all performed in a
single tube.
Lab Communique 7
samples were seroreactive but
NAT non reactive. There were 8
NAT yield cases – 1 HIV, 1 HIV-HCV
coinfection and 6HBV. Observed
NAT yield for all three cases was 1
in 1528 (0.065%). Similar studies
in other countries have also
demonstrated high yields. The
potential for NAT yield in India is
staggering given the prevalence
of these viruses in the population
5.7 million with HIV, 12 million
with HCV, and 40 million with
HBV (10 per cent of the world’s
HBV infected population) when
compared to other countries that
have already implemented the
technology. Data from this study
suggested that the NAT yield for
all three viruses in India could be
29 times higher than that observed
in Japan, and even higher for HIV-1
alone. In addition, India has a high
percentage of replacement blood
donors who are associated with
higher infection rates compared to
volunteer donors.
Our goal: To reach zero risk blood supply
We have come a long way in that
the donors were screened for
only syphilis and hepatitis B virus
before 1985. Now a series of
specific and non specific measures
are being introduced into the
screening of blood donations in
order to reduce the residual risk
of bloodborne viruses. The latest
measure has been viral nucleic
acid detection.
Given the high seropositivity
rate of HIV, HBV & HCV in India
and keeping in mind the high
percentage of first time donors
and replacement donors, it is likely
that adding NAT to the current
screening test, will have significant
reduction in TTI, making our blood
safer.
Blood safety has been an integral
part of the quality system at our
transfusion service. Our mission
is to provide safe and high quality
blood and blood components to
all patients requiring transfusion.
Implementation of NAT screening
of all blood donations in
Hinduja Hospital since March 2009
is another step towards ensuring
safe & effective transfusions.
Blood safety has
been an integral part
of the quality system
at our transfusion
service. Our mission
is to provide safe
and high quality
blood and blood
components to all
patients requiring
transfusion.
Vol 18
Achievements
DR. ANAND DESHPANDE
MD, Consultant, Transfusion Medicine & Hematology
• PostgraduateinPathologyfromNagpurUniversity.
• SeniorresidencyinHaematologyatPGI,Chandigarh.
• HasbeentrainedinperipheralbloodstemcelltransplantatUniversityof
Minnesota, Minneapolis USA.
• Recipient of prestigious BGRC oration of Mumbai Hematology Group
(MHG) in 2007.
• Scientific Advisory committee member (SAC) National Institute of
Immunohematology, ICMR, Mumbai.
• HassetupNucleicAcidAmplificationTesting(NAT)laboratoryatHinduja
Hospital for screening donors’ blood units. This is the first laboratory in
entire Maharashtra.
• HasbeeninstrumentalinsettingupHLAlaboratoryatHindujaHospital,
which is now carrying out tissue typing using molecular technique.
• Has set up many Therapeutic apheresis procedures at Hinduja
Hospital including Therapeutic Plasma Exchange (TPE) & Therapeutic
Erythrocytapheresis (TEA). This is one of the first centres to carry out
TEA in India.
• HasalsoindroducedunexpectedRedBloodCellantibodyscreeningof
the patients. This is one of the first centres in Mumbai to introduce the
technique.
• Has received many awards including Best Paper award in National
conferences & Dr. H. M. Bhatia award, Dr. L. D. Sanghvi award (twice),
Dr. Hiranandani & Dr. A. J. Desai award (twice) of Mumbai Hematology
Group.
• Hasdelivereda largenumberof scientific lectures invariousCMEs&
Conferences.
• HasorganizedCMEin2006&2008–“Transfusion&Beyond”atHinduja
hospital which we are planning every alternate year.
• Ongoingprojects:-*Autoimmunehepatitis.
• OccultHepatitisB&HepatitisGVirusinfections.
E-mail: [email protected]. Contact No.: 24451515 Ext 8307/8308
Lab Communique 9
In India, current use of flow cytometry in a clinical setting has largely centered
on CD4 counts, HLA-B27 analysis, and leukemia immunophenotyping. There
has been a growing interest in extending the use of this tool to study PNH,
flow cytometry cross-match, platelet function disorders, RBC disorders,
multiple myeloma, solid tumors, body fluid analysis etc. However, limited
familiarity with standardized protocols and nuances of analysis is a gap that
has impeded routine usage of these assays for patient care.
It is to bridge this gap a two-day Symposium on “Advanced Clinical
Applications of Flow Cytometry” was held on February 25th & 26th, 2009
at Hinduja Hospital, Mumbai. The Symposium was organized by the
department of Hematology and was attended by 124 participants. The
teaching program was focused primarily on the needs of existing flow
cytometry users. It provided an opportunity to new enthusiasts of the
technology and physicians to get sensitized to the wide applications of flow
cytometry.
A number of eminent international and national speakers gave talks in their
area of expertise.
• Thekeynote lecturewasdeliveredbyDr.AttilaTarnok,Editor-in-chief,
“Cytometry Part A” University of Leipzig, Germany who spoke on How to
present flow cytometry data and Pediatric cell immunity.
• Dr.EdnaD’souzafromNIIH,MumbaispokeonFlowsortingforantenatal
diagnosis
• Dr.AmarDasgupta,COO,ReligareSuper,MumbaiandDr.RenuSaxena,
Head, Dept of Hematology from AIIMS, New Delhi discussed use of flow
cytometry in bleeding and platelet function disorders.
• Dr.Manisha Madkaikar and Dr.Prabhakar Kedar from NIIH, Mumbai
presented their work on PNH and RBC membrane disorder respectively.
• Transplant flowcytometry,an importantemergingapplicationof flow
cytometry was covered by Dr.N.K.Mehra, from AIIMS, New Delhi who
spoke on flow cytometry crossmatch and Dr.Paresh Jain, Scientific
advisor, BD Bioscience, who spoke on Absolute CD34 stem cell counting.
• Presentations in Oncology were made by Dr.Sujata Iyer, from BD
Biosciences, USA who spoke on BCR-ABL fusion protein detection,
Symposium on Advanced Clinical Applications in Flow cytometry
Dr. Shanaz Khodaiji
Dr. Shanaz Khodaiji - Consultant Hemotology, M.D. (Path & Micro), D.C.P. E-Mail - [email protected]. Contact No. 24451515 Extn – 7144
Hinduja Lab Newsletter 9
Vol 110
Dr.Avtar Krishnan from University of Miami, Florid, USA, who spoke on
Flow Immunocytochemistry of body fluids and Dr.Suchitra Swaminathan
from NIIH, Mumbai, spoke on Signal transduction studies in AML.
• Some miscellaneous topics were discussed by Dr.Manisha Madkaikar
who spoke on Flow cytometry in diagnosis of primary immunodeficiency
disorders, Dr.Sandeep Shah, from Ahmedabad who spoke on
Accreditation of a flow cytometry lab and Dr.Tina Dadu, from B..L.Kapoor
Hospital, New Delhi who presented her experience on Flow cytometry of
lymphnodes specimen.
This symposium was a first of its kind as it covered newer clinical applications
in flow cytometry and thus expanded the horizon for flow cytometry users
in India. The delegates were extremely happy with the scientific content as
well as the hospitality displayed by our hospital which went a long way in
making it a grand success.
Vol 110
Lab Communique 11
Food Intolerance and Food Sensitivity
Dr. Vipla Puri, Consultant Radioimmunoassay, Ph.D - Consultant RIA Laboratory Email: [email protected] Contact No. 24451515 Extn - 7148/7149
Bringing innovation at Hinduja
We are the first hospital based lab
to start food intolerance test called
‘Genarray’ based on Microarray
Technology. The technology
originally invented for studying
DNA and gene expression is
now being exploited to practical
diagnostic tests for early detection
of food intolerances by measuring
food specific IgG levels in blood.
With the imminent availability of
the sensitive technology in our
hospital we can now diagnose food
intolerance to 200 specific foods
in patients presenting with various
symptoms from general lethary,
weight gain, dermatitis, arthritis to
irritable bowel syndrome.
What is food intolerance
Food allergies and food intolerances
overlap considerably.Intolerances
are negative reactions to food
that do not involve the immune
system, such as lactose intolerance
or sensitivity. Food intolerance
is an exaggerated or abnormal
physical reaction to a food or food
additive caused by some chemical
or enzyme deficiency in the body.
Causes of food intolerance
Several factors may cause a person
to have an adverse reaction to food.
Sometimes a person’s body will
react to chemicals found naturally
in foods. For example: Some people
get headache after eating a certain
type of cheese and other foods
containing tyramine. Psychological
factors play a significant role in
food intolerance.
Carbohydrate intolerance:
Sucrose, fructose, milk, sugar,
lactose, or glucose are problems
for many people digesting
these compounds although
carbohydrates furnish most of the
energy needed in a healthy diet.
Celiac disease: This disorder is
caused by an intolerance to gluten,
the protein found in wheat and
other grains. In celiac disease, the
cells lining the small intestine are
damaged and prevent the normal
absorption of food constituents,
particularly fats. Celiac disease
involves an immune response but
does not involve IgE, an antibody
responsible for allergic reaction.
Common symptoms are bloating
and gas.
Toxicological reactions and
food poisoning: Foods may
contain toxins that are naturally
part of the food or that were added
by mistake during manufacturing,
shipping or handling. For instance,
Dr. Vipla Puri Ph.D.
Vol 112
certain fish like tuna or mackerel
contain histadine which is
converted to histamine with
improper storage and if consumed
can cause adverse reactions.
Food poisoning, on the other hand,
results from contamination of
food with bacteria or other micro
organisms.
Symptoms may include vomiting,
diarrhea, dehydration and
unconsciousness.
Pharmacological effects: Some
foods produce symptoms that
resemble reaction to drugs. For
example, caffeine found in coffee,
tea and other products may cause
a rapid heartbeat, sleeplessness
and other effects.
Psychological reactions:
Psychological factors play a role in
food intolerance, causing people to
react to a particular food because of
smell, presentation and memories.
Symptoms of food intolerance
Food intolerance can cause a
surprisingly wide variety of
symptoms. However, certain
features concerning the timing
and occurrence of symptoms are
helpful when trying to identify
possible causes. Symptoms are
usually multiple, caused or made
worse by food intolerance.
Respiratory: Asthma, rhinitis,
glue ear.
Gastro – intestinal: Infantile
colic or colitis,Crohn’s disease,
recurrent abdominal pain, diarrhea,
constipation, irritable bowel
syndrome.
Skin: Eczema, Urticaria.
Nervous System: Headache,
migraine, hyperactivity.
Heart: Palpitations.
Musculo skeletal: Unexplained
joint pain, some kinds of arthritis,
unexplained muscle pain.
Psychiatric: Somatisation
disorder, fatigue, hypersomnia.
In practice, when food intolerance
is involved, such conditions rarely
exist alone, for example, a typical
sufferer may have migraine,
unexplained fatigue, bloating,
abdominal pain along with muscle
and joint pain.
Food intolerance – The common
culprits
The foods that tend to cause
intolerance reactions in sensitive
people include:
• Dairyproducts, includingmilk,
cheese and yogurt.
• Chocolate.
• Egg–particularlyeggwhite.
• FlavourenhancerssuchasMSG
(Monosodium glutamate)
• Foodadditives.
• Strawberries, citrus fruits,
tomatoes.
• Wine–particularlyredwine.
Foods may
contain toxins
that are naturally
part of the food or
that were added
by mistake during
manufacturing,
shipping or
handling. For
instance, certain fish
like tuna or mackerel
contain histadine
which is converted
to histamine with
improper storage
and if consumed
can cause adverse
reactions.
Lab Communique 13
What is the treatmentOnce the offending food is
identified by high levels of IgG
using this sensitive technology,
food intolerance can be managed by
cutting the food out of one’s diet For
example : Babies or children with
a lactose intolerance can be given
alternative soya milk, almond milk,
Adults may
somestimes be able
to tolerate small
amount of offending
foods, but should
experiment to work
out alternative
suitable diet to avoid
symptoms
and nutrient
deficiencies.DR. VIPLA PuRI
Ph.D - Consultant RIA Laboratory
• AwardedWHO fellowship towork as a visiting scientist atKarolinskaHospital, Stockholm, Sweden and at the Department of Biochemistry, University of Paris, Bicetre.
• InvitedbyUNDP /UNFPA /WHO/World Bank Special ProgrammeofResearch, Development and Research Training in Human Reproduction, Geneva as Advisor and Member of Task Force on Laboratory Methods.
• Visiting Research Assistant Professor, Department of Obstetrics andGynaecology, Washington University School of Medicine, St Louis, MO, USA.
• Invitedtoreviewmulticentretrialresultson“ScreeningforPre-eclampsia;evaluation of predictive abilities of angiogenic factors for pre-eclampsia. WHO – Geneva
• RecipientofICMR–ResearchGranttoworkonFoamCellFormationinLeprosy.
• RecognisedResearchGuideUniversityofMumbai.
• ReceivedbestpaperawardformyworkonHyperhomocystenemiaanindependent risk factor for Osteoporosis in men.
• DevelopedandStandardized3newassaysforthediagnosisofMovementDisorders, Pure Red Cell Aplasia and Multiple Sclerosis. Relevant documents for filing ‘PATENT’ have been submitted.
• Established newer technologies of Microarray to diagnose FoodIntolerance and Chemiluminescence to diagnose various allergies.
rice milk or hypo allergenic milk
formula instead of cow’s milk.
Adults may sometimes be able to
tolerate small amount of offending
foods, but should experiment to
work out alternative suitable diet
to avoid symptoms and nutrient
deficiencies.
Email: [email protected]. Contact No. 24451515 Extn - 7148/7149
Achievements
Vol 114
Quiz
Dr. Anjali Shetty MRCP, FRCPath, Consultant MicrobiologyEmail: [email protected]. Contact No. : 24451515 Ext 7156
Q1. The diagnosis of Clostridium difficile infection is made by
a. A positive stool culture for Clostridium difficile
b. Presence of antibodies in blood to Clostridium difficile
c. Detection of Clostridium difficile toxin in faeces
d. All of the above
Q2. The following helps to reduce antimicrobial resistance
a. Once started one must complete at least a 5 day course of antibiotics
b. Using lower doses of antibiotics for prolonged periods
c. Using a combination of antibiotics to treat all infections
d. None of the above
Q3. The following statement is true
a. Blood cultures should be collected when the pt spikes a fever
b. Blood cultures should be collected after the patient spikes a fever
c. Blood cultures should be collected before the pt spikes a fever
d. Timing of blood culture collection need not coincide with fever
spikes
Q4. The urine sample sent to diagnose Schistoma hematobium
should be
a. Early morning sample
b. Mid-stream sample
c. Terminal urine sample
d. Any of the above
Q5. A patient presents with fever of 1 day, mild rash, severe back
pain. The platelets are 90,000, WBC is 2,200, L-75, N-20, M-4,
and E-1. The preferred test for Dengue is?
a. Dengue IgM
b. NS1 antigen
c. Dengue IgG
d. Total IgM
Dr. Anjali Shetty MRCP, FRCPath
Answers
Q1 c Q2 d Q3 d
Q4 c Q5 b
14 Vol 1
Lab Communique 15
Inborn Errors of Metabolism
Dr. Tester F. Ashavaid, Consultant Biochemistry, Head, Department of Laboratory Medicine, Joint Director - Research., Ph.D, FACB, CSi, PGDBA (Fin). Email: [email protected] Contact No. 24451515 Extn - 7135 / 7136
Inborn Errors of Metabolism (IEM)
are a heterogeneous group of
disorders caused due to single
gene defect which may manifest
immediately after birth, or within
few days or weeks after birth.
In recent decades hundreds of
new inborn errors of metabolism
have been discovered. Some of
the major classes of inborn errors
of metabolism are disorders
of carbohydrate metabolism
(e.g. Glycogen storage disease),
amino acid metabolism (e.g.
Phenylketonuria, Maple Syrup Urine
Disease, Glutaric acidemia type –
1) organic acid metabolism (e.g.
Alkaptonuria), fatty acid oxidation
& mitochondrial metabolism
(e.g. Glutaric Acidemia type –
2), peroxisomal function (e.g.
Zellweger Syndrome), Lysosomal
Storage Disorders (e.g. Gaucher’s
Disease, Niemann Pick Disease) ,
purine or pyrimidine metabolism
(e.g. Lesch-Nyhan Syndrome),
steroid metabolism (e.g. Congenital
adrenal hyperplasia) mitochondrial
function (e.g. Kearns-Sayre
Syndrome).
All of us need to be aware that
apparently every healthy newborn
has a significant risk of being
maimed or killed by an inborn
error of metabolism which can
be eliminated by early diagnosis.
However routine laboratory
investigations and qualitative
screening tests are insufficient to
confirm the diagnosis.
Disorders of amino acid metabolism
result from defects either in the
synthesis of or breakdown of amino
acids, or in the body’s ability to
get the amino acids into the cells.
We at Hinduja Hospital, perform
quantitation of amino acids from
plasma, urine & cerebrospinal
fluid (CSF) by High Performance
Liquid Chromatography (HPLC).The
aminoacidogram obtained is then
correlated with the case history of
the patient in order to rule out any
metabolic disorder. An HPLC system
offers accurate & reproducible
results, along with high resolution,
precision and sensitivity. Keeping
into consideration our patient’s
convenience, we offer various
HPLC packages for amino acid
analysis. These include:
1. Plasma Amino Acid Analysis,
urinary Amino Acid Analysis,
CSF Amino Acid Analysis:
Analysis of amino acids in
various physiological samples
such as plasma, urine and CSF
is a valuable diagnostic tool &
is central to the investigation of
Dr. Ashavaid, T.F. Ph.D, FACB, CSi, PGDBA (Fin).
Vol 116
possible neurometabolic disorder.
Quantitative amino acid analysis
by HPLC provides confirmation of
the identity and concentration of
amino acids by providing accurate
information on the levels of
amino acids that may be present
in subnormal concentrations.The
presence of characteristic pattern
of elevated amino acids is very
useful in diagnosis of these rare
disorders.
2. Tyrosinemia package: The
amino acids reported in this package
are - Plasma tyrosine, Urinary
tyrosine & Urine Nitrosonaphthol
test (Qualitative).Tyrosinemia is
a genetic disorder characterized
by elevated blood levels of amino
acid tyrosine. There are three
types of Tyrosinemia Tyrosinemia
– I, Tyrosinemia – II, Tyrosinemia
¬– III. The body needs adequate
supplies of tyrosine to make
many important brain chemicals
that helps regulate appetite,
pain sensitivity, body’s response
to stress, normal functioning
of thyroid, pituitary & adrenal
glands, Low levels of tyrosine
may lead to hypothyroidism, low
blood pressure, chronic fatigue &
sluggish metabolism.
3. Phenylketonuria (PKu)
package: The amino acids
reported in this package are –
Plasma Phenylalanine, Plasma
Tyrosine & FeCl3 test (Qualitative).
Phenylketonuria is a rare genetic
disorder caused by the buildup
of excess phenylalanine in the
blood. The metabolic pathway in
Phenylketonuria is the conversion
of phenylalanine into another
amino acid, tyrosine and this
pathway is affected due to the
deficiency of enzyme phenylalanine
hydrolase. The body needs both
phenylalanine as well as tyrosine
to make three neurotransmitters
– epinephrine, dopamine &
norepinephrine. A shortage of
either of the two amino acids could
leave the patient vulnerable to host
of mental disorders, anxiety or
chronic fatigue.
4. Sulphur Amino Acids
package: The amino acids
reported in this package
are - Plasma Cystine, Plasma
Methionine, Serum Homocysteine
& Urine Homocysteine.The
amino acids Cystine, Methionine
and Homocysteine are sulphur
containing amino acids and
the metabolism of all these are
closely related. Both methionine
and cystine are required for the
production of the body’s most
abundant natural antioxidant,
glutathione, which helps neutralize
toxins in the liver.
Recent studies have suggested
that people who have elevated
homocysteine levels have a much
greater risk of heart attack or
Quantitative
amino acid analysis
by HPLC provides
confirmation of
the identity and
concentration of
amino acids by
providing accurate
information on
the levels of amino
acids that may be
present in
subnormal
concentrations.
Lab Communique 17
stoke than those with average
levels. Vitamin B6, B12 and folate
are necessary to metabolize
homocysteine and the patients
who are deficient in these vitamins
may have increased levels of
homocysteine.
5. Non Ketotic Hyperglycinaemia
(NKH) package: The amino acids
reported in this package are -
Plasma Glycine, CSF Glycine and
ratio of CSF Glycine to Plasma
Glycine.It is an autosomal recessive
inborn error of glycine metabolism.
Being a glucogenic amino acid, it
helps in supplying the body with
glucose needed for energy, thereby
regulating blood sugar levels.
6. Maple Syrup urine Disease
(MSuD) package: The amino
acids reported in this package are
- Plasma Leucine, Plasma Isoleucine
and Plasma Valine. It is caused due
to deficiency of branched chain
alpha keto acid dehydrogenase
complex (BCKDH). This disease is
characterized in an infant by the
presence of sweet smelling urine,
with an odor similar to that of maple
syrup. Keeping MSUD under control
requires careful monitoring of
blood chemistry and involves both
a special diet & frequent testing.
Saving Lives:………Case Studies
Case 1: A 3 year old male
child presented with history of
Tyrosinemia type – 2 disorder,
was suffering from watering of
eyes, epithelial haze, multiple, fine
pin point , dot shaped infiltrates
on corneas. On quantification by
HPLC tyrosine levels were found
to be elevated [162 nmoles/ml -
reference range: 24 – 115 nmoles/
ml].The child was then treated with
Tyrex – 2 powder (phenylalanine &
tyrosine free) along with other diet
as prescribed by the dietician. On
follow up the blood tyrosine levels
were found to be still elevated but
compared to previous reports,
they were low [128 nmoles/ml].
The patient was continued with the
same diet and on follow up, the
tyrosine levels are now found to
be normal.
Case 2: A 3 year old female
presented with known case of
Phenylketonuria, had history of
developmental delay. She was on
restricted diet and on quantitation
by HPLC the phenylalanine levels
were found to be normal. Later
on she had difficulty in walking
& talking. Eczema & skin rashes
developed all over her body, also
her hair color got changed from
black to golden .On quantitation
the phenylalanine levels were
found to be elevated [994 nmoles/
ml - Reference range: 26 – 91
nmoles/ml, due to improper diet.
She was then asked to follow the
diet strictly. On follow up, amino
acid quantitation showed decrease
Recent studies
have suggested
that people who
have elevated
homocysteine levels
have a much greater
risk of heart attack
or stoke than
those with average
levels.
Vol 118
in phenylanine levels.
Case 3: An 18 days old male child,
suffering from severe acidosis was
admitted to hospital on day 3 of
life. Blood gas analysis show severe
metabolic acidosis & his respiratory
rate was increased. Blood ammonia
levels were elevated, urinary
Methylmalonic Acid was positive.
Hence the clinician was suspecting
Methylmalonic Aciduria (MMA)
which is a disorder of amino acid
metabolism involving defect in
conversion of methylmalonyl CoA
to succinyl CoA and this conversion
takes place in presence of vitamin
B 12.On quantitation the levels of
isoleucine, valine, methionine &
threonine were low and the levels
of tyrosine, leucine & phenylalanine
were elevated because patients
with MMA have problems breaking
down & using certain amino and
fatty acids from food they eat. A
specific diet was prescribed. On
follow up after 4 months all the
amino acids were found to be
normal. Blood ammonia levels
were also normal.
Amino acid analysis aids
in the diagnosis of dietary
protein adequacy and amino
acid balance, forms of protein
intolerance, nutritional
deficiencies (vitamins, minerals)
renal & hepatic dysfunction,
psychiatric abnormalities,
reduced detoxification capacity,
susceptibility to occlusive arterial
disease and many inherent
disorders in amino acid metabolism.
The hidden impairments in amino
acid metabolism are problematic
& often go undiagnosed which
may or may not be expressed
as specific symptoms. They may
silently increase the susceptibility
to a degenerative disease or they
may be associated with, but not
causative for, a disease. Hence
quantitative amino acid analysis
by HPLC is necessary in timely
diagnosis of inborn errors of
metabolism and helps in thorough
nutritional and metabolic workup.
Thus, Amino acid analysis by HPLC
is adequate to suggest the diagnosis
of inborn errors of metabolism, due
to marked increase in the levels of
amino acids in plasma, urine or
CSF, thereby helping the clinicians
to promptly initiate appropriate
therapy for the patient. Hence there
is a need to create awareness in
general population on the ill effects
of inborn errors of metabolism
and the need to prevent them.
This would certainly benefit the
society as a whole in reducing and
preventing psycho-social burden
of the medical consequences due
to inborn errors of metabolism.
The concept that “metabolic
or genetic disorders are very
difficult to diagnose and if
diagnosed, it is impossible to
treat” no more stands.
The hidden
impairments
in amino acid
metabolism are
problematic & often
go undiagnosed
which may or may
not be expressed as
specific symptoms.
They may silently
increase the
susceptibility to a
degenerative disease
or they may be
associated with, but
not causative for, a
disease.
Lab Communique 19
DR. (MISS) TESTER FRAMROZE ASHAVAID
Ph.D, FACB, CSi, PGDBA (Fin).
• PrincipalInvestigatorforLTMTInstitutionawardfor2years“StudyofIndian genome to assess the risk factors in coronary heart disease ” , 2001.
• Invited to Judge poster session at the 15thWorld Congress ofInternational Society of Heart Research held at Winnipeg, Canada, from 6th – 11th July,2001.
• LadyTataMemorialTrust(LTMT) awarded Institutionalgrant for 18months. For research project entitled “ Development of Multilocus Assay for Candidate Markers in Indian Patient s with Cardiovascular disease risk, (Sanctioned 2003)
• DepartmentofBiotechnology(DBT),Govt.ofIndiaawardedInstitutionalgrant for 3 years research project entitled “The genetic basis of atherothrombotic coronary artery disease in the Indian population. (Sanctioned, 2003).
• AppliedBiosystemsawardedSNPGenotypingreagentsworth$3150forresearch work on “Single Nucleotide Polymorphism and Risk of Coronary Heart Disease in the Indian Population”. {2003)
• DaiichiPureChemicals(Japan)andAccurexBiomedicalPvt.LtdIndiahavesupported a project entitled “Measurement of lipids, lipoproteins, and apolipoproteins in healthy Indian population”. (2003)
• DBT Task force meeting held at Dept.of Biotechnology the researchproject entitled “ The genetic basis of coronary artery disease in Indian Population” (2005).
• AppointedontheInspectionCommitteeofUniversityofMumbaitoinspectthe institutions for M.Sc, Ph.D.
• Invited lecturer for MRCOG Part I (Biochemistry) conducted in UK.through teleconference in 2003.
• PostgraduateguidetoM.Sc.andPh.DstudentsinAppliedBiologyfromUniversity of Bombay.
Achievements
Email: [email protected] Contact No. 24451515 Extn - 7135 / 7136
Vol 120
From Left to Right: Dr. Anand Deshpande (Transfusion Medicine & Hematology), Dr. Chitra Madiwale (Histopathology & Cytopathology), Dr. Sharmila Ghosh (Hematology), Dr. Tester Ashavaid (Biochemistry), Dr. R. B. Deshpande (Histopathology & Cytopathology), Dr. Anita Bhaduri (Histopathology & Cytopathology), Dr. Camilla Rodrigues (Microbiology), Dr. Vipla Puri (Radioimmunoassay), Dr. Anjali Shetty (Microbiology) and Dr. Shanaz Khodaiji (Hematology).
Laboratory Medicine Consultants
Lab Communique 21
Funded Research / Projects in the Department of Laboratory Medicine (July 2007 to Date)
1. LDL and HDL subfractions, their genetic basis and the role of 5-Lipoxygenase
activator protein (FLAP) promoter polymorphism in Coronary Heart disease.
Dr. Tester F. Ashavaid
2. Early detection of Invasive fungal infections in Immunocompromised patients
Dr. Camilla Rodrigues
3. Molecular diagnosis of Cystic Fibrosis in Indian patients
Dr. Tester F. Ashavaid
4. Role of Lipoprotein (a) in Atherosclerosis in Indian population
Dr. Tester F. Ashavaid
5. Hepatitis G Virus and occult hepatitis B infection in healthy blood donor population
Dr. Anand Deshpande
6. A comparison of private and public healthcare’s effect on tuberculosis epidemic in
Mumbai, India.
Dr. Camilla Rodrigues
7. Role of Matrix metalloproteinase gene variants in Indian patients with coronary artery
Dr. Tester F. Ashavaid
8. To improve the diagnosis of Autoimmune Hepatitis.
Dr. Anand Despande
9. Hospital Acquired Pneumonia (HAP). Including Ventilator associated Pneumonia (VAP)
in Adults:-Etiology clinical outcome and antibiotic susceptibility pattern.
Dr. Camilla Rodrigues
10. Optimization of multiplex PCR for diagnosis of TB from clinical samples and
evaluating the utility of MIRU VNTR in genotypic and phylogenetic studies’
Dr. Camilla Rodrigues
11. Monitoring of minimal residual disease in ALL’.
Dr. Sharmila Ghosh
12. TB specific CD4 and CD8 T cell responses in the presence of persistent antigen - Does
prolonged in vivo exposure of TB specific T cell to MTB antigen lead to a level of
dysfunction in TB specific T cells?
Dr. Camilla Rodrigues
13. Direct susceptibility tesing of M.Tuberculosis with BACTEC MGIT 960 system
Dr. Camilla Rodrigues
14. Invitro susceptibility testing to anti tubercular experimental compounds.
Dr. Camilla Rodrigues
Lab Communique 21
Vol 122 2009 Vol 1 No. 122
Fibroblast Growth Factor 23 (FGF 23) in Bone Kidney Axis
FGF 23 has emerged as an important
moderator in the physiology of
phosphate homeostasis. Renal
phosphate wasting disorders
leading to hypophosphatemia are
among the causes of defective
mineralization of bone and growth
plate development.
Given the dramatic increase in
skeletal size during growth, the
need to preserve skeletal mass
during adulthood and the large
capacity of bone to store calcium
and phosphate, a complex systems
biology has evolved that permits
cross-talk between bone and
other organs to adjust phosphate
balance and bone mineralization in
response to changing physiological
requirements.
FGF 23 is a recently identified
‘Phosphatonin’ that is implicated
in systemic balance of phosphate
maintained by interaction of
intestines, bone and kidneys.
In clinical settings, excessive
activity of FGF 23 results
in hypophosphatemia low
1,25-Dihydroxy Vitamin D3 levels
and osteomalacia. Circulating FGF-
23 is a physiological regulator of
phosphate balance and as such
also a potential uremic toxin.
More recently, FGF 23 has been
implicated as an excellent indicator
of the complex derangements of
Calcium Phosphate metabolism
induced by chronic kidney disease
and probably also a valuable
surrogate parameter of the
deranged mineral metabolism.
Sensitive measurement of human
FGF 23 in circulation is now
considered as an important
diagnostic tool for the evaluation of
patients with a variety of different
hypophosphatemic disorders
including oncogenic osteomalacia,
X-linked hypophosphatemic rickets,
bone and mineral homeostasis,
and early and advanced renal
failure. FGF 23 has also been
implicated as a predictor of the
risk of mortality in the first year of
hemodialysis and in patients with
renal transplant.
We, at Hinduja Hospital are the
first to start this test since it is
now established that FGF 23 is
also an important marker for the
therapeutic approach in Chronic
Kidney Disease patients.
Dr. Vipla Puri, Ph.D
Dr. Vipla Puri, Consultant Radioimmunoassay, Ph.D - Consultant RIA Laboratory Email: [email protected]. Contact No. 24451515 Extn - 7148/7149
22 Vol 1
Lab Communique 23
Help prevent thalassemia – Jago re …..
Dr. Sharmila Ghosh, Consultant Hematology, MD – Pathology. E-Mail: dr_ [email protected] Contact No. 24451515 Extn - 8013/8014
Thalassemia refers to a spectrum
of diseases characterized by
the reduction or absence in the
synthesis of the globin chains
of hemoglobin. The disease was
first noted in the Mediterranean
population, and this geographical
association explains its naming by
Whipple and Bradford in 1932 as
“Thalassa” which in Greek means
the sea and “Haema” is Greek for
blood.
Worldwide, approximately 15
million people are estimated to
suffer from thalassemic disorders.
Reportedly, there are about 240
million carriers of b- thalassemia
worldwide, i.e. 1.5% of world
population, and in India alone,
the number is approximately 30
million with 50% in S.E.Asia. The
burden of hemoglobinopathies in
India is high with nearly 12,000
infants being born every year with
a severe disorder. These numbers
imply that every hour 1 child is
born who will suffer with this
genetic disorder. The carrier rate
for b thalassemia varies from 1-17
% in India with an average of 3.2 %.
This means that on an average 1 in
every 25 Indians is a carrier of
thalassemia. The distribution of
the thalassemia gene is not uniform
in India and the prevalence is very
high among certain communities
such as Sindhis and Punjabis
from Northern India, Bhanushalis,
Kutchis, Lohanas from Gujarat,
Mahars, Neobuddhists, Kolis and
Agris from Maharashtra, & Gowdas
and Lingayats from Karnataka etc.
and certain tribes in the northern,
western and eastern parts, with
lower incidence in the southern
tribes. There is a genetic, ethnic
and regional diversity of the
hemoglobin variants as well as of
the genetic mutations in India.
Thalassemia exists in 3 forms:
• Thalassemia trait or the
asymptomatic carrier stage–
The carrier does not exhibit
any symptoms and leads an
absolutely normal life
• Thalassemia intermedia–
Genotypically the patient is
similar to a thalassemia major
but differs phenotypically in
that they do not require regular
transfusions
• Thalassemia major– In
thalassemia major, the
production of -globin chains is
severely impaired, because both
b-globin genes are mutated.
The severe imbalance of globin
chain synthesis results in
Dr. Sharmila Ghosh MD – Pathology
Vol 124
ineffective erythropoiesis and
severe microcytic, hypochromic
anemia. Clinical presentation of
thalassemia major occurs at 6
months of age. Affected infants
fail to thrive and become
progressively pale. Feeding
problems, diarrhea, irritability,
recurrent bouts of fever, and
progressive enlargement of the
abdomen due to splenomegaly
may occur. Patients are treated
by lifelong blood transfusion
every 15 to 30 days along with
iron chelation therapy. The
cost of treatment of a 4-year-
old thalassemic child is around
Rs.90,000-100,000 annually.
The only cure available today
is bone marrow transplantation
which is largely unaffordable to
the large majority of the Indian
children.
Prevention of thalassemia – the need of the hour.
There is an urgent need for the
prenatal diagnosis of thalassemia
to combat the burden of
hemoglobinopathies in India.
Thalassemia is an autosomal
recessive disorder which is
inherited from parents.
If one (1) parent has beta
thalassemia trait and the other
parent has normal hemoglobin A,
there is a 50 percent (1 in 2) chance
with each pregnancy of having a
child with beta thalassemia trait.
These are the possible outcomes
with each pregnancy.
• 50 percent (1 in 2) chance
of having a child with beta
thalassemia trait
• 50 percent (1 in 2) chance of
having a child without trait
The cost
of treatment
of a 4-year-old
thalassemic child is
around Rs.90,000-
100,000 annually.
The only cure
available today
is bone marrow
transplantation
which is largely
unaffordable to the
large majority of the
Indian children. What if both parents have Beta Thalassemia trait?
If both parents have beta
thalassemia trait there is a 25
percent (1 in 4) chance with each
pregnancy of having a child with
Beta Thalassemia disease. Beta
Thalassemia disease is a lifelong
illness that can result in serious
health problems. These are the
possible outcomes with each
pregnancy.
• 25 percent (1 in 4) chance
of having a child with beta
thalassemia disease
Lab Communique 25
• 50 percent (1 in 2) chance
of having a child with beta
thalassemia trait
• 25 percent (1 in 4) chance of
having a child without trait or
disease
Thalassemia produces a huge
psychological and financial drain
on patients and their families.
This dreaded disease can be
prevented by extensive screening
and counseling programmes. All
it takes is a simple blood test to
identify silent carriers and counsel
them so as to avoid marriages
between them.
Who then should be screened for thalassemia?
• Pre-marital youth (18-25 years
of age)
• Antenatal women in their first
trimester
• Parentsandextendedfamilyof
thalassemia major children
• Individuals belonging to the
high-risk communities
• Any individual with a raised
RBC count
In the screening for classical
beta thalassemia trait, the first
indicator is the blood film with
the classical phenotype being
the hypochromic microcytic
red blood cells and the red cell
indices showing a reduced mean
corpuscular hemoglobin (MCH)
and mean corpuscular volume
(MCV). The hallmark is the
presence of an elevated level of
Hb A2 which can be detected and
quantified by the high performance
liquid chromatography(HPLC),
the gold standard technology for
the screening of beta thalassemia
carriers today.
At Hinduja Hospital, we have
the facility for the screening
of hemoglobin variants by the
Cation-exchange HPLC (CE-HPLC)
system which is currently the
method of choice for screening
hemoglobinopathies and has
been established to be superior to
electrophoresis.
Highlights of this method:
• Fullyautomated
At Hinduja
Hospital, we have
the facility for
the screening of
hemoglobin variants
by the Cation-
exchange HPLC
(CE-HPLC) system
which is currently
the method of
choice for screening
hemoglobinopathies
and has been
established to
be superior to
electrophoresis.
Vol 126
• Allows the quantification of
HbA2, HbF, HbA along with that
of hemoglobin variants HbS,
HbD, HbE, HbC from a single
test.
• The time taken for analysis is
only 6 minutes.
• Strictattentionispaidtoquality
control which ensures the
accuracy of results.
• CE-HPLC can also be used
for prenatal screening
and prenatal diagnosis of
hemoglobinopathies.
It is a combined effort involving
obstetricians, pediatricians,
backed by excellent laboratory
infrastructure for screening
hemoglobinopathies by the HPLC
technology. Community awareness
programmes will not only help to
spread knowledge of the disease
but also remove the social stigma
associated with thalassemia.
CE-HPLC system in Hinduja Lab
HPLC generated chromatogram
Lab Communique 27
Achievements
DR SHARMILA GHOSH
MD. Consultant, Hematology
Awarded scholarship by the international jury for the International Society of Pediatric Oncology (SIOP) for participation and paper presentation at the regional and annual congress of SIOP, held at Shanghai and Geneva, 2006. Member of the organizing committee and faculty for SIOP 2007, held at Mumbai.
Publications: More than 20 publications in peer reviewed journals
ECFMG award for best post graduate speaker
Life Member
• IndianSocietyofHematologyandBloodTransfusion
• FlowCytometrySociety
• MumbaiHematologyGroup
E-Mail - dr_ [email protected]. Contact No. 24451515 Extn - 8013/8014
Vol 128
HematologyAPTT Inhibitor Screen
Blood lympho culture by cell cultureJak 2 mutation by PCR Qualitative
Blood BankCross matching & RBC ab screening
HCV genotyping
BiochemistryDPD Mutation analysis
TPMT mutation analysisDMD 22 mutation analysis
Biotinidase
Microbiology/SerologyCulture NTM atypical Mycobacterium
Influenza AntigenScrub Typhus IgM
RIA LabGlutamineHistamine
Plasma MetanephrinePlasma Nor Metanephrine
Fibroblast growth factor 23BAL Fluid CD4 C8 Counts
New Tests Introduced
For a price list and more information please contact the Assistant Manager Diagnostic Services, Dr. Preeti Goraksha at 24451515 Ext 7142 / Chetna Patil, Quality Co-ordinator at 24447943 Ext 7143
Printed and Published by Marketing Department Hinduja Hospital, Veer Savarakar Marg, Mahim, Mumbai - 400016 at Synergy Creations for free and private circulation.
Editor: Dr. Anita Bhaduri. Registered.(The publisher cannot be held responsible for errors or any consequences
arising from the use of the information contained in this newsletter).