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 info@hindujahospital.com

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 - dr_adeshpande@hindujahospital.com 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: dr_adeshpande@yahoo.co.in. 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 - dr_skhodaiji@hindujahospital.com. 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: dr_vpuri@hindujahospital.com 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: dr_vpuri@hindujahospital.com. Contact No. 24451515 Extn - 7148/7149

Achievements

Vol 114

Quiz

Dr. Anjali Shetty MRCP, FRCPath, Consultant MicrobiologyEmail: dr_ashetty@hindujahospital.com. 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: dr_tashavaid@hindujahospital.com 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: dr_tashavaid@hindujahospital.com 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: dr_vpuri@hindujahospital.com. 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_ sghosh@hindujahospital.com 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_ sghosh@hindujahospital.com. 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).