Lab Communique March 2012| Volume 1

Pioneer

Technology

Advancement

Quality

Dia

gnos

is

Inno

vatio

n Invention

Cutting-edge

Bre

ak-t

hrou

gh

Research

Dev

elop

men

t

Trai

lbla

zer

As we come together to commemorate 60 years of

compassionate care, its time to let everyone know that

for over two decades the hospital's labs have been

delivering quality care with discipline and trust. This

memorable year was made all the more special, when

Hinduja Hospital emerged winners at the ICICI-

Lombard & CNBC TV18 Healthcare Awards in the multi-

specialty hospital (Metro) category.

As we enter the new era of technology and

advancement, Lab Communiqué brings to you some

trends that could set apart the credentials of this year

from the previous one. This issue features facets of

laboratory testing by skilled and exceptional new talent

in the Lab Medicine team (GenNext), who will be

nurtured to grow into specialists of the same stature as

their seniors. There are rules to be rewritten, new

challenges to address and new opportunities.

Happy reading!

Dr. Anita Bhaduri

ContentEditorial

Editor:

Dr. Anita Bhaduri, MD, Surgical Pathologist

For Editorial Enquiries write to us at:

info@hindujahospital.com

Printed and Published at:

Marketing Department

P. D. Hinduja National Hospital & M.R.C.,

Veer Savarkar Marg, Mahim,

Mumbai - 400016, (INDIA).

Tel.: +91-22-2445 1515/ 2444 2222/ 2444 9199

Fax: +91-22-24449151

Website: www.hindujahospital.com

Editorial

Dr. Anita Bhaduri

1 The Role of Frozen Section in the Evaluation of

Dr. Chitra Madiwale

9 Utility of Lymph Node Flow Cytometry in the

Diagnosis of Lymphomas

Dr. Tina Dadu

14 Why estimate Neurotransmitters?

Dr. Vipla Puri

21 Diagnosis of Paroxysmal Nocturnal

Hemoglobinuria: A Review

Dr. Kunal Sehgal

28 Transcon- 2011 Update

33 TB LPA Molecular Assays for MDR/XDR TB

Dr. Camilla Rodrigues & Mrs. Minal U. Paradkar

36 Biochemical Genetic Testing for Inborn Errors of

Metabolism

Dr. Alpa Dherai

41 Galactomannan Antigen Assay

Dr. Anjali Shetty

43 Conference Update

Ovarian Masses

The Role of Frozen Section in theEvaluation of Ovarian Masses

Dr. Chitra Madiwale,

Consultant - Histopathology

& Cytopathology

MD

Ovarian masses are not amenable to

pre-operative biopsy and their cytologic

sampling is limited to fluid examination,

if effusion is present. Additionally, their

work-up by tumor markers and imaging

studies may not be accurate. Since

surgical management depends on their

nature, the frozen section becomes a

valuable tool that provides a primary

diagnosis and a sense of direction to the

operating surgeon regards the extent of

surgery. It also determines whether there

is need for further work-up eg.

microbiologic examination, molecular

studies etc.

Indications for performing

frozen sections on ovarian

masses:

> Benign versus malignant mass.

> Primary versus metastatic tumor.

> Particular histologic type may be

necessary in some situations eg.

dysgerminoma requires accurate

intra-operative diagnosis as it requires

routine biopsies of the contralateral

ovary and lymph node sampling.

Similarly lymphomas & metastases

need to be recognized to avoid

unnecessary surgery.

Accuracy of frozen section

diagnosis of ovarian masses?

It would be logical to assume that a

perfect match between the frozen and

paraffin diagnosis would qualify as an

accurate diagnosis. While this is desirable

and happens quite often, it is not always

feasible for the surgical pathologist to hit

the nail on the head each time. The aim is

to achieve a fair agreement between the

two diagnoses with the frozen diagnosis

having no significant discrepancy that

would have influenced the intra-

operative management.

sufficiently sensitive and specific

(agreement rates ranging from 86.3% to

96%) with low false negative rates (2% to

6.8%) and more reassuringly, even lower

false positive rates (0 to 1%). It is

interesting to note that these figures are

comparable with the overall accuracy of

frozen section diagnosis in general

surgical pathology cases which varies

from 91.5% to 97.4%.

Published audits indicate that gyne-

cologic frozen section diagnosis is

1

The frozen section

becomes a valuable tool

that provides a primary

diagnosis and a sense of

direction to the operating

surgeon regards the

extent of surgery.

Pathologic problems in ovarian

frozen section diagnosis:

While in the hot seat at frozen section the

pathologist has the following concerns:

Situa of false

negative diagnosis): Am I under-

diagnosing a malignancy? Am I unable to

recognize features that may portend a

more aggressive behavior?

Situation- 2: (Creates risk of false positive

diagnosis): Am I over-diagnosing this

lesion as malignant as or more aggressive

than what it actually is?

Situation-3: (Creates a state of

confusion): I am unable to recognize

what this lesion is.

Problems with borderline

ovarian tumors:

Correct recognition of borderline tumors

is critical in order to avoid under-

treatment or over-treatment. In the

elderly, hysterectomy with bilateral

salpingo-oophorectomy with surgical

staging is necessary. However, their

occurrence in the reproductive age group

tion-1: (Creates risk

is more common and calls for fertility-

conserving measures, for example

unilateral salpingo-oophorectomy or

even cystectomy (especially in case of

bilateral tumors) with removal of the

affected portion of the ovary. These

procedures can be coupled with surgical

staging. While there are succinct & well -

defined histologic criteria for the

diagnosis of these tumors, their

recognition at frozen section is relatively

difficult and accounts for maximum

discrepancies between frozen and

permanent diagnoses amongst all

ovarian tumors. Tempfner et al analysed

96 cases of borderline tumors and

reported an agreement between frozen

and paraffin in 71.9% cases only.

> Borderline serous tumors (BST) &

borderline mucinous tumors (BST) are

among the most common borderline

tumors & the following points help

us to understand the diagnostic

challenges:

! Mucinous tumors form the largest

ovarian masses and are notoriously

heterogeneous in their histology with

benign, borderline and malignant

areas being present in the same tumor.

Remember, that logistically it is nearly

impossible to sample large masses

extensively at the time of frozen

section. Thus, based on the few frozen

sections studied, the impression

created could be of a benign or

borderline sub-type while more

extensive sampling on paraffins may

2

Correct recognition of

borderline tumors is

critical in order to avoid

under-treatment or

over-treatment.

Figure 1: Ovarian Borderline tumor

disclose that the benign tumor is

actually borderline or that the

borderline tumor actually has frankly

malignant areas.

! BSTs are relatively more consistent in

their histology as compared to BMTs.

Yet 20% of BSTs may harbour invasive,

low grade serous carcinoma foci that

could escape sampling at frozen.

! Peritoneal implants of invasive & non-

invasive types, non-destructive type of

stromal micro-invasion and even

lymph node metastases are all

described in borderline tumors and

can add to the diagnostic confusion.

However, except for destructive

stromal invasion, none of the others

have an adverse impact on the

outcome. At this juncture, it is

interesting to note that both invasive

and non-invasive implants may be

present in the same case. This

underlines the need for multiple

omental & peritoneal sampling by the

surgeon.

! Grossly BST & BMT may look like cysts

with fairly innocuous appearance or

they may produce rather striking

appearing masses. BMT may show

intricately honeycomb or solid areas in

the cyst wall. BST can show polypoid

and papillary fronds with presence of

grape-like structures secondary to

stromal edema. This appearance is not

only restricted to the cyst lining

but may also be apparent on its

outer aspect. In this situation, the

pathologist may be convinced of a

frozen diagnosis of BST, but may find it

difficult to convince the surgeon of the

same, owing to the fairly alarming

intra-operative appearance.

Problems with metastatic

tumors & other tumor types:

Ovarian metastases may be the initial

manifestation of an unknown primary.

Recognition of metastatic nature of the

tumor obviates the need for aggressive

surgery. Problems arise because

metastatic tumors can closely resemble a

variety of primary ovarian tumors, both

on gross and histologic examination. In

particular these are serious mimics of

mucinous ovarian tumors.

All categories of primary ovarian tumors

are endowed with not only a rich variety

of patterns but what complicates matters

is that they often share these patterns

among themselves as well. Now, we

know that the basic art of histopathology

is all about pattern recognition. This

overlapping plethora of patterns in

combination with the fact that frozen

sections are much thicker than paraffin

sections and also have freezing artifacts

3

Pathologist may be

convinced of a frozen

diagnosis of BST, but

may find it difficult to

convince the surgeon of

the same, owing to the

fairly alarming intra-

operative appearance.

Figure 2: Frozen Section of metastasis of breast carcinoma to ovary

that obscure finer details, can cause

diagnostic confusion. Thus, the surgical

pathologist needs to aware of the various

histologic nuances that may be subtle

and are imbibed only after being exposed

to numerous cases, in order to embark on

the correct diagnostic path.

Guidelines to improve the

accuracy of frozen section

diagnosis:

> If feasible, the entire ovarian

massshould be sent for frozen section.

> More sections may be sampled to

Tempfer et al emphasize

that majority of misread borderline

tumors have diameter of over 5cm.

Wang et al recommend at least one

section for every 10cm

enhance sensitivity for focal border-

line changes.

tumor dia-

meter in mucinous neoplasms. It is

preferable to defer diagnosis in cases

of very large mucinous neoplasms.

Grading of immature teratomas is best

decided on paraffin sections.

> Look at the nuclear grade during

frozen evaluation of suspected

borderline tumors. High grade nuclear

atypia and abnormal mitoses are

never a feature of borderline tumors

and their presence should prompt

more histologic sampling to search for

high grade invasive carcinoma

elsewhere in the tumor specimen.

If there is no invasive component,

then their presence is indicative of

high grade ovarian intra-epithelial

carcinoma.

> Awareness of the implications of your

diagnosis is important as outlined in

the following table.

4

Tumor type Intra-operative management

Epithelial Ovarian Ca + Borderline tumors after childbearing

TAH + BSO + tumor debulking + full surgical staging

Borderline tumors in young Fertility conserving surgery; cystectomy in some + surgical staging

Mucinous tumors Always assess status of appendix. Other principles as above

Malignant Germ cell tumors Surgical management principle similar to epithelial Ca. Fertility preservation important in dysgerminoma

Sex-cord stromal tumors Reproductive age & Stage I disease - Unilateral salpingo-oophorectomyAfter completion of child-bearing - TAH+BSO+standard surgical staging

Mucinous tumors form

the largest ovarian

masses and are

notoriously

heterogeneous in their

histology.

Obiakor et al recommend that in order to

avoid pitfalls it is preferable that even in

cases diagnosed as benign epithelial

tumors at frozen section, the omentum

and peritoneum should be carefully

examined as a small proportion may have

small foci of borderline change or focally

invasive disease.

> Think of metastasis when:

! There are bilateral, solid ovarian

tumors.

! Bilateral mucinous tumors that are

<10cm size cm in size.

! Multinodular growth pattern with

involvement of superficial cortex.

! Haphazardly arranged glands amidst

desmoplastic stroma.

! Sections from the ovarian hilum show

vascular emboli.

It is also a good habit to examine the

appendix in all ovarian mucinous tumors

to exclude possibility of a primary in that

organ.

> Remember that borderline ovarian

tumors are independent entities and

do not represent precursor lesions of

high grade invasive carcinomas.

Borderline tumors show K-Ras

mutations and are genetically

different from high surface epithelial

ca rc i n o m a s w h i c h s h o w p 5 3

mutations. Thus, some BST may

transform into a low grade serous

carcinoma (which also shows K-Ras

mutation). However, BST are never the

precursor for high grade serous

carcinomas. Post-operative therapy is

also tailored based on this fund-

amental difference.

>

solid areas, sampling of dissimilar

areas etc is essential.

> Good communication, common

sense, an ability to put evidence

together and a trained eye are

important assets for the pathologist.

When in doubt, remember the medical

dictum “Primum Non Nocere” ( First do

no harm). As is aptly stated by Lechago et

al: “A common situation that must be

pathologist

to help by trying to produce a diagnosis at

all costs.” If definitive diagnosis is not

possible then judicious deferral or a

generic diagnosis is the preferred route

An audit of gynecologic frozens

at Hinduja Hospital:

At Hinduja Hospital, we follow two vital

quality procedures in our practice of

frozen sections:

> Any discrepancy in the frozen and

paraffin diagnosis is explained and

reconciled in a comment at the end of

the final histopathology report.

> We conduct an annual audit of our

frozen cases so that we may learn from

our experiences.

At Hinduja Hospital a total of 3434 frozen

sections (by 3 experienced histopatho-

logists) were performed during the

period from January 2008 to May 2011,

of which 131 (3.8%) were gynecologic

frozens. Of these, 43 cases were for

uterine lesions and 88 were for ovarian

masses.

Correct sampling, for example-from

avoided is the understandable pro-

pensity of the conscientious

.

5

A common situation

that must be avoided is

the understandable

propensity of the

conscientious pathologist

to help by trying to

produce a diagnosis at

all costs.” If definitive

diagnosis is not possible

then judicious deferral

or a generic diagnosis is

the preferred route

6

Table 1: Analysis of frozen and paraffin diagnosis among published cases and a comparison with Hinduja Hospital's data

No of cases

Agreement between frozen & paraffins

False +ve at frozen False –ve at frozen Deferred diagnosis

Obiakor et al 311 93.8% Nil 3.5% 2.6%

Rose et al 383 92.7% 1% 6.3% Nil

Twaalhoven et al

176 86.3% 0.6% 6.8% 6.3%

Wang et al 299 96% Nil 2% 1%

HH cases 88 92 % 1.13% 5.6% Nil

Table 2: Comparison between frozen and paraffin diagno s among of ovarian massessi

Frozen Diagnosis Paraffin diagnosis Comments

Borderline serous Endometrioid carcinoma Underdiagnosis-grade and type of malignancy

Borderline mucinous tumor Mucinous carcinoma Underdiagnosis of grade of malignancy due to limited sampling.

Serous cystadenoma Borderline serous False negative due to limited sampling

Mucinous cystadenoma Borderline mucimous False negative due to sampling limitation

High grade serous carcinoma Metastases Misinterpretation of an overlapping pattern

Spindle cell tumor Endometriosis Falsepositive with non-neoplastic interpreted as neoplastic; the significant smooth muscle proliferation which often accompanies the endometriotic foci was interpreted as tumor

> Uterine lesions: Frozen section was

performed for assessment of uterine

lesions, either for primary diagnosis or

for assessment of depth of myoin-

vasion in endometrial carcinoma.

There was 95.3% concordance bet-

ween frozen and paraffin diagnosis.

> Ovarian masses: There was 92% con-

cordance between frozen and paraffin

diagnosis (81/88cases). An analysis of

the discordant diagnoses and a

comparison of our cases with those in

published literature are summarized

in Tables 1 & 2.

Take Home Message:

Frozen section diagnosis is not just about reporting histology on cryostat sections but represents an intra-operative

consultation in the true sense, with the pathologist having a duty to help the surgeon in the best possible manner.

Regardless of various advances in pathologic diagnosis, it will remain a time-tested diagnostic tool so long as surgeries are

performed. However, this tool must be judiciously utilized and is most effective when the pathologist and the surgeon are

aware of the advantages and limitations.

References:1. Obiakor F, Maiman M, Mittal K ey al: The accuracy of frozen section in the diagnosis of ovarian neoplasm. Gynecologic Oncology 1991; 43:61-63.

2. Twaalfhoven FCM, Peters AAW, Trimbos JB, Hermans J, Fleuren GJ: The accuracy of frozen section diagnosis of ovarian tumors. Gynecologic Oncology1991; 41:189-92.

3. Wang KG, Chen TC, Wang TY, Yang YC, Su TH: Accuracy of frozen section diagnosis in gynecology. Gynecologic Oncology 1998; 70:105-10.

4. Interinstitutional comparison of frozen section consultations. A college of American pathologists Q- probes study of 90,538 cases in 461 institutions. Arch Pathol Lab Med 1996; 120: 804-9.

5. Rose PG, Rubin RB, Nelson BE, Hunter RE, Reale FR: Accuracy of frozen-section (intra-operative consultation) diagnosis of ovarian tumors. Am J Obstet Gynecol 1994; 171:823-6.

6. Tempfer CB, Polterauer S, Bentz EK, Reinthaller A, Hefler LA: Accuracy of intraoperative frozen section analysis in borderline tumors of the ovary: A retrospective analysis of 96 cases and review of the literature. Gynecol Oncol 2007; 107:248-52.

7. Lechago J: The frozen section. Patholoy in the trenches. Arch Pathol Lab Med 2005; 129(12): 1529-31.

8. Park JY, Kim DY, Kim JH, Kim YM, Kim YT, Nam JH: Surgical management of borderline ovarian ovarian tumors. The role of fertility sparing surgery. Gynecol Oncol 2009; 113(1):75-82.

7

Doctor’s Profile

Dr. Chitra Madiwale, MD - Pathology

Consultant - Histopathology & Cytopathology

Professional Experience

Dr. Chitra Madiwale has been associated with Hinduja Hospital for almost four years. Prior to this she was Associate Professor

in Pathology at the Seth GS Medical College and KEM Hospital with a total tenure of 23 years. She is an accomplished nephro-

pathologist. She holds special interest in nephro-pathology and gynecologic pathology.

Research & Publications

Dr. Madiwale has published several articles in indexed journals and contributed two book chapters on lymphoproliferative

disorders following renal transplant. She has numerous paper presentations and has participated in many workshops at the

national and state level and has been part of the organizing committee for some of them.

She has also been invited as guest speaker and faculty member at various academic sessions, conferences and events. She

also conducts lectures for post-graduate Pathology students. She is actively involved in organizing workshops and scientific

sessions. In the past 5 years she has been faculty in 4 gynecologic pathology workshops of which two were organized by the

GGP, Mumbai.

Affiliations

Member of Mumbai Nephrology Group , Member of the Group of Gynecologic Pathologists (GGP), Mumbai.

Awards and Achievements

Dr. Madiwale was awarded Fellowship in Nephropathology with Dr. Jacob Churg and Dr. Surya Venkataseshan at the Mount

Sinai Institute of Medical Sciences, New York, USA; the leaders in the field of Nephro-pathology.

In association with GGP a project on inter-observer evaluation of frozen sections, which was presented at the Update in

Gynecologic Pathology, 2008 held at Hinduja Hospital.

dr_mchitra@hindujahospital.com || Ph: 022-24451515 Extn. 7152/7157

8

Utility of Lymph Node Flow Cytometryin the Diagnosis of Lymphomas

Dr. Tina Dadu, DNB

Hematology

Associate Consultant -

The nomenclature of lymphoid neo-

plasias has evolved over time and this

process is certain to continue. Until

recently, characterization of the lym-

phomas and lymphoproliferative

disorders was largely descriptive and

based exclusively on microscopic

observations. However, with more

meaningful biological properties for their

category definitions and extensive

descriptions of the immunophenotype of

the neoplastic cells by the World Health

Organizatio (WHO) classification, the

laboratory diagnosis of Non-Hodgkins

Lymphoma (NHL) is also accompanied by

immunological evaluation. This is by

either immunohistochemistry (IHC) or

flow Cytometry (FCM). In some cases,

other ancillary techniques may also be

employed, such as in-situ hybridization

and gene rearrangement studies for the

immunoglobulin heavy chain gene or the

T-cell receptor or chain genes. It should

be clear, however, that FCM and other

ancillary techniques serve an adjunctive

role in the diagnosis and classification of

hematopoietic neoplasms and should

always be reconciled with morphology

for such diagnoses. Although FCM offers

many advantages in comparison to IHC in

the diagnosis and classification of NHLs,

it should be used as a complementary

rather than a competitive technique for

this role.

Advantages of Using FCM

for Analysis of Neoplastic

Lymphoid Cells

FCM offers important advantages over

competing laboratory technologies in the

detection of lymphoid and plasma cell

neoplasias. In addition to being a fast 1procedure, FCM :

> can analyze a broader array of

antigens than those detectable by

conventional, fixed tissue-based

immunohistology

> facilitates the analysis of cells within

discrete subpopulations defined and

selected (gated) based on other

parameters

> allows a clear-cut correlation of

multiple measurements (antigen

expressions, DNA content, light

scatter) in individual cells

> has the ability to quantify both

population frequencies and level of

antigen expression in individual cells

Clinical Usefulness of Flow

Cytometry on Lymph Nodes

! Diagnosis and classification of

Lymphoma especially in small samples

! S-Phase Fraction

! Heavy Chain Analysis

9

FCM offers many

advantages in

comparison to IHC in

the diagnosis and

classification of NHLs,

it should be used as a

complementary rather

than a competitive

technique for this role.

Diagnosis of Lymphoma

Assessment of B-cell Clonality:

In normal lymph nodes or peripheral

blood, virtually every B-cell expresses

surface light-chain immunoglobulins,

and the fraction of kappa to lambda

immunoglobulin-expressing B-cells is

approximately 1.5. The lack of expression

of surface immunoglobulins in mature B

cells, or a significant increase or decrease

in this normal ratio (light chain

restriction), supports the presence of a 1monoclonal B-cell population.

Clonality analysis is most useful

when interpretation is done for

immunophenotypical ly abnormal

discrete populations (Ex: Figure 1), which

can be achieved by flow cytometry

Assessment of T-cell Clonality

In contrast to the straightforward B-cell

clonality determination based on single

immunoglobulin light chain expression,

clonality assessment of T-cells is not

possible with FCM using conventional

Figure 1: Flow Scattergrams from a lymph node showing two populations of B cells (one which is CD10 positive and one which is CD10 negative)

A) Gating the CD20+, CD10- cells show the polyclonal nature of the cells

B) Gating the CD20+, CD10+ cells shows the monoclonal nature of the cells

10

marker analysis. Molecular analysis of T-

cell receptor (TCR) gene rearrangements

is indicated in cases of suspected T-cell

malignancy to determine the clonal

status of the T-cells. However, many

antibodies against V domains of TCR

molecules have been developed, and it is

now possible to recognize approximately

70% of all individual V domains. Thus,

FCM analysis of the V repertoire of

TCR molecules may be useful in the

detection of T-cell clonality. Although the

requirement for a large antibody panel to

cover the majority of the V repertoire

makes it costly and thereby not feasible

to do the test in the Indian scenario.

Classification of Lymphomas

In the current World Health Organization

classification, all NHLs are distinct

clinicopathologic entities defined by

their morphology, immunophenotype,

genetic alterations (in several cases), and

clinical features. Thus, determination of

immunophenotype is an integral

component for proper classification

except in those cases where morphology

alone can be diagnostic of a particular

lymphoma type, such as follicular

lymphoma and small lymphocytic

lymphoma. The use of FCM permits

simultaneous evaluation of several

surface antigens, with clonality analysis

providing objective and quantitative

results even on very small samples.

The immunophenotypes of lymphomas

are widely known, but atypical patterns

do occur and pose significant diagnostic 2difficulties. It should be remembered

that different pieces of information carry

Clonality analysis is

most useful when

interpretation is done for

immunophenotypically

abnormal discrete

populations

11

different weights for different diagnostic

expression of cyclin D1 is

much more specific than the expression

pattern of CD5 or CD23 antigens by FCM

for mantle cell lymphoma (MCL). Also,

iden

sification, it is

by no means the most definitive modality

for this purpose in all cases.

>

Growth fraction measurements in

lymphoma and myeloma have proven

to be of prognostic value. In a study

of Flow Cytometric S-Phase Fraction

(SPF) as a complementary biological

parameter for the cytological grading 3of NHL , the mean SPF values varied

from 6.5% in indolent NHLs, to 20.4%

in aggressive NHL (NOS), and to 35.3%

in Burkitt's lymphomas. Nearly all

aggressive NHLs have an SPF >15%,

while the vast majority of indolent

NHLs show an SPF <10%. The mean SPF

value in the reactive nodes is 6.6%.

>

As concerns for cost and invasiveness

of surgical biopsies intensify,

procedures for obtaining samples

of potential lymphomas using

fiberscopes or needles for tissue

categories; for example, the immuno-

histochemical

tification of specific translocations

rules out certain entities, irrespective of

the immunophenotype. Thus, although

FCM immunophenotyping is an integral

component of proper clas

Diagnosis of Lymphoma in Small

Samples, especially FNA (Fine

Needle Aspirates) from Lymph

Nodes

S-Phase Fraction

aspirations and biopsies are becoming

commonplace. As a consequence

of this practice, pathologists are

increasingly confronted with small

amounts of tissue samples, or just

cytological preparations for diagnosis,

which limits the interpretative

capabilities of microscopic obser-

vations. In these situations, the added

phenotypic information provided by

modern FCM instruments may allow

an accurate recognition and classi-

fication of neoplastic cells, even

in specimens with extremely low 4

numbers of cells. Likewise; accurate

diagnosis may be attained on fluids

that contain small numbers of cells,

such as vitreous humor, cerebral spinal 1fluid, or pericardial fluid.

>

The diagnostic use of heavy chains can

provide valuable information in

selected cases. One important use

is in excluding the diagnoses of

Lymphoplasmacytic Lymphoma or

Burkitt's lymphoma with a reasonable

degree of certainty if IgM is not

expressed. The expression level of

heavy chains can help differentiate

a benign from a malignant lympho-

proliferative disorder in atypical cases.

Most cases of benign lymph nodes do

not express any heavy chains in

amounts greater than 25%. In

contrast, level of expression of a heavy

chain usually exceeds 50% in 5lymphomas .

Heavy Chain Analysis

Growth fraction

measurements in

lymphoma and

myeloma have proven to

be of prognostic value.

Take Home Message:

Flow cytometry on lymph nodes currently enjoys widespread use and a well established role in lymphoma diagnosis in most clinical laboratories throughout the United States and many European countries, but in India it still has a long way to go. Immunophenotyping is one of the integral components in the current World Health Organization lymphoma classification scheme, and FCM has proven value in the diagnosis and proper classification of most categories, even with very small sample size. The multiple roles that FCM plays in lymphoma diagnosis, classification and pro-gnostication serve the current management plans and treatment protocols. The unique attributes of flow cytometry, therefore, gives it a leading edge in lymphoma.

12

Diagnostic Limitations

With all the uses and advantages that

FCM offers, it is equally important to

acknowledge the limitations of this

technique as well.

>

Tissue: One of the main drawbacks of

immunophenotyping by FCM is the

requirement for fresh, unfixed tissue

for analysis.

> Retrograde study using archival

tissue not possible: Although

prospective handling of specimens

for lymphoma diagnosis often

yields ample tissue for immuno-

phenotyping, such analy cannot be

performed on archival fixed tissues.

> Limitation in Hodgkin's and T-cell

Rich B cell lymphomas: Flow

cytometry has had little success in the

evaluation of Hodgkins lymphoma.

Likewise the diagnosis of T-cell-rich B-

cell lymphoma, which is often similar

to Hodgkin lymphoma, is frequently

made based on morphology and IHC.

Cases of T-cell-rich B-cell lymphoma

evaluated with FCM may only show a

large number of reactive T-cells and

may not detect the clonality in B-cells.

Requirement For Fresh, Unfixed

sis

One important

diagnostic use of heavy

chains is in excluding

the diagnoses of

Lymphoplasmacytic

Lymphoma or Burkitt's

lymphoma

References:1. Braylan RC. Impact of Flow Cytometry on the Diagnosis and

Characterization of Lymphomas,Chronic Lymphoproliferative Disorders and Plasma Cell Neoplasias. Cytometry Part A. 2004; 58A:57-61.

2. Kaleem Z. Flow Cytometric Analysis of Lymphomas Current Status and Usefulness. Arch Pathol Lab Med.2006;130: 1850-1858.

3. Pinto A, Cabec¸adas J, No´brega SD, et al. Flow Cytometric S-Phase Fraction as a Complementary Biological Parameter for the Cytological Grading of Non-Hodgkin's Lymphoma. Diagnostic Cytopathology.2003; 29: 194-199.

4. Jeffers MD, Milton J, Herriot R, et al. Fine needle aspiration cytology in the investigation on non-Hodgkin's lymphoma. J Clin Pathol. 1998; 51:189-196.

5. Kaleem Z, White G, Vollmer RT. Critical analysis and diagnostic usefulness of limited immunophenotyping of B-cell non-Hodgkin lymphomas by flow cytometry. Am J Clin Pathol. 2001; 115:136-142.

13

Doctor’s Profile

Dr. Tina Dadu, DNB

Associate Consultant - Haematology

Professional Experience

Dr. Tina Dadu has done DNB (Pathology) from Sir Ganga Ram Hospital, New Delhi and MNAMS from National Academy of

Medical Sciences, New Delhi. She was Jr. Consultant, Hematology Dept., at Sir Ganga Ram Hospital & B.L. Kapur Memorial

Hospital. She has pursued her Fellowship and Post Graduate Training in 2008 as short term scholar, Hematopathology

Section, Dept. of Pathology & Laboratory Medicine at the University of Florida College of Medicine.

Expertise

With special interest in neoplastic hematopathology and flow cytometry, she holds expertise in detection, diagnosis and

classification of hemopoeitic malignancies (in blood, bone marrow and lymph nodes) using multi-parametric flow

cytometry; analysis of DNA content and TCR V beta in lymphoid proliferations for T cell clonality.

Research & Publications

Her research experience encompasses the following projects:

> Utility of Immature Platelet fraction in Dengue patients

> Comparison of platelet counts by Sysmex XE 2100 and LH 750 with the International Flow Reference Method in

thrombocytopenic patients

> Clinicopathological classification of the tumours of the central nervous system and the utility of immunohistochemistry in

their diagnosis

Dr. Dadu also has several articles published in indexed journals, newsletters and also has contributed chapter in a book

“Utility of flow cytometry in the diagnosis of lymphoma: Dr. M.B Aggarwal, Hematology Year book 2010”. She has also been

invited as guest speaker and faculty member at various academic sessions, conferences and events.

Affiliations

Awards and Achievements

Dr. Dadu was selected by the American Society of Hematology for training in flow cytometry at United States under Dr. Raul

Braylan, one of the pioneers in the field of flow cytometry .

The Cytometry Society (TCS), Indian Association of Pathologists and Microbiologists and Delhi Society of

Haematology, New Delhi.

dr_tina.dadu@hindujahospital.com || Ph: 022-24451515 Extn. 7145

Why estimate Neurotransmitters?

Neurotransmitters are the chemical

messengers in the brain cells that

TRANSMIT thoughts from one cell to the

next, allowing brain cells to talk to each

other. We now know that there are

various types of transmitters that change

constantly in the brain cells to make

neurons talk to each other at the ''proper

volume”. It is therefore critical that all the

major neurotransmitters be present in

amounts, sufficient enough for the brain

to be chemically balanced, letting the

neurons neither talk ''too loud'' or ''too

soft'' to each other.

With

the advent of newer markers and

sophisticated equipments, i140 is now

possible to quantitate neurotransmitter

imbalances in blood or urine samples.

Laboratory analysis can now provide

precise information on brain neuro-

transmitter deficiencies or over-loads as

well as detect hormonal and nutrient co-

factor imbalances which influence

neurotransmitter production.

Besides, laboratory analysis can also

provide information as to which of

Insufficient or excess of any of the

neurotransmitters can upset the balance

resulting in various clinical symptoms

like depression, moods, irritability,

sleeplessness, anxiety, panic etc.

r these are excitatory or

The exact quantitative information on

the circulating levels of important

neurotransmitters being done in our

hospital can thus help the neuro-

physician to target imbalances, correlate

with the clinical symptoms and

customize supplemental therapy.

Where do imbalances come from?

Imbalances in neurotransmitters could

be due to:

> High levels of stressful lifestyles,

causing the body to lose neuro-

transmitters rapidly leading to very

low levels over time.

> Poor dietary habits, thereby depleting

the body of essential amino acids, the

building blocks.

> Environmental toxins such as

cells

containing neurotransmitters.

the neurotransmitters are imbalanced,

whethe inhi-

bitory or whether the total excitatory/

inhibitory balance is disrupted.

indus-

trial cleaners, air and water pollution,

solvents that can affect brain

> Genetics plays an important role in

maintaining proper levels and

functioning of neurotransmitters.

Dr. Vipla Puri, Ph.DConsultant - RIA Laboratory

14

Insufficient or excess of

any of the

neurotransmitters can

upset the balance

resulting in various

clinical symptoms like

depression, moods,

irritability, sleeplessness,

anxiety, panic etc.

15

Types of Neurotransmitters

Neurotransmitters can be broadly categorized into two groups - the classical 'small'

molecule neurotransmitters and relatively 'larger' neuropeptide neurotransmitters.

Type Small Molecule Neurotransmitters

Post Synaptic Effect

Biogenic Amines Glutamate*AspartateGamma Amino Butyric Acid (GABA) Glycine

ExcitatoryExcitatoryInhibitoryInhibitory

Aminoacids AcetylcholineDopamine*Adrenalin / Epinephrine*Nor adrenalin / Nor-Epinephrine*Histamine* Serotonin*

ExcitatoryExcitatoryExcitatoryExcitatoryExcitatoryInhibitory

Large Molecule Neurotransmitters

Corticotropin Releasing Hormone (CRH)Corticotropin (ACTH)*Beta EndorphinSubstance PSomato statin Vasopressin*

Others Brain Derived Neurotropic Factor (BDNF)*Melatonin*

We discuss here some of the important neurotransmitters being done at our hospital,

which can help the clinician to rule-in or rule-out any critical imbalances in these

chemicals that might be of direct relevance to patient specific symptoms.

Genetics plays an

important role in

maintaining proper

levels and functioning

of neurotransmitters.

16

Functions of Neurotransmitters

>

neurotransmitter to be discovered

and shown to be responsible for

stimulating muscles by activating

motor neurons that control skeletal

muscles. It has been shown to

regulate the activities in certain areas

of brain associated with attention,

arousal, learning and memory. People

with Alzheimer's disease are usually

found to have a substantially low level

of acetylcholine.

> Dopamine:

concentrated

in very specific groups of neurons

collectively called the basal ganglia.

emotions. Drugs like

cocaine, heroine, nicotine, opium and

alcohol are known to increase the

level of dopamine for which the users

of such drugs feel good. Decreased

level of

Parkinson's disease while patients of

schizophrenia are usually found to

have excess dopamine in the frontal

lobes of the brain.

> Adrenaline:

It

regulates attention, mental focus,

arousal and cognition. It is also known

to inhibit insulin excretion and

Acetylcholine: It is one of the first

Dopamine is a mono-

amine neurotransmitter

It

controls voluntary movements of

the body and is associated with

pleasurable

dopamine is associated with

Also known as Epine-

phrine, released from the adrenal

glands, is an excitatory neuro-

transmitter and hormone essential for

metabolism and reflective of stress.

increasing the amount of fatty acids in

the blood.

Elevated levels of epinephrine have

been linked to sleep problems, anxiety

and ADHD like symptoms while low

levels of this neurotransmitter are

associated with fatigue, lack of focus

and difficulty losing weight.

>

> Histamine: Histamine is an organic

nitrogen compound involved in

local immune reponses as well as

regulating physiological function

in the gut and acting as a neuro-

transmitter. It helps in the regulation

of sleep and wake cycle and ability

to concentrate, control the mecha-

nisms by which memories and

learning are forgotten. Increased

levels of histamine metabolites have

been reported in CSF of patients

with schizophrenia with decreased

efficiency of H (1) receptor binding

sites.

> Serotonin: Serotonin is an important

inhibitory neurotransmitter with a

Nor-adrenaline: Also known as

Nor-Epinephrine is an excitatory

neurotransmitter. Elevated levels

of this hormone are strongly

associated with bringing our nervous

system into ''high alert'', increases

in heart rate and blood pressure

while low levels are linked to lack

of energy, decreased focus and

motivation and sleep cycle problems.

A significantly low level

of serotonin has been

found to be associated

with conditions like

depression, suicidal

thoughts and obsessive

compulsive disorder.

Many anti-depressant

drugs work by affecting

the levels of this

neurotransmitter.

17

very important role in a range of brain

functions. It is synthesized from the

amino acid tryptophan. Within the

brain, serotonin is localized mainly in

nerve pathways throughout the

brainstem, the cerebral cortex and the

spinal cord.

In the central nervous system, sero-

tonin is involved in fear and flight

responses-an activity which is

opposed by the aggression stimulating

hormone adrenaline and neuro-

transmitter dopamine. In addition to

mood, emotion and anxiety, serotonin

is involved in the regulation of sleep,

pain, perception, body temperature,

blood pressure and hormonal

activities.

Outside brain,

serotonin exerts a number of

important effects particularly in-

volving the gastrointestinal and

cardiovascular systems.

> Glutamate is a major

excitatory neurotransmitter known to

be associated with learning and

memory. It is the most common

neurotransmitter in the central

nervous system and it is now

documented that excess of it can

actually be toxic to neurons and may

be related to Amyotrophic Lateral

Sclerosis (ALS) or Lou Gehrig's disease.

A significantly low level

of serotonin has been found to

be associated with conditions like

depression, suicidal thoughts and

obsessive compulsive disorder.

Many anti-depressant drugs work

by affecting the levels of this

neurotransmitter.

Glutamate:

Dysfunction in glutamate levels are

involved in many neuron degenerative

diseases such as Alzheimer's, Parkin-

son's, Huntington's and Tourette's.

High levels of glutamate are linked to

depression, obsessive compulsory

disorder and autism.

>

(BDNF): BDNF is part of cascade of

proteins produced in the brain that

promotes neuron growth and stops

neurons from degenerating.

It has been implicated in the patho-

physiology of mood disorders

including major depressive disorders

and bipolar disorder. BDNF has also

been shown to protect against stress

induced neuronal damage and affect

neurogenesis in the hippocampus,

which is thought to be involved in the

pathogenesis of mood disorders and in

the activity of therapeutic agents in

patients with major mood swings.

BDNF is a mediator involved in

neuronal survival and plasticity of

dopaminergic, cholinergic and

serotonergic neurons in the central

nervous system.

> Melatonin: Melatonin is a ubiquitous

natural neurotransmitter like com-

pound produced primaily by the

pineal gland in the brain. Its

production is regulated by light

through retino hypothalamic tracts. It

controls the sleep circadian rhythm

which is controlled by circulating

levels of melatonin that are 3 times

Brain Derived Neurotropic Factor

Dysfunction in

glutamate levels are

involved in many

neuron degenerative

diseases such as

Alzheimer's, Parkinson's,

Huntington's and

Tourette's.

18

higher at night than during the

day. Low levels of melatonin are

known to cause insomnia while

high levels can cause drowsiness.

Besides controlling sleep patterns,

melatonin is also involved in the

modulation of mood, sexual behavior,

reproductive alterations and immu-

nological functions, in various neuro-

psychiatric

nt, epilepsy, attention deficit

hyper-activity disorder (ADHD) and

Alzheimer's disease.

Since the effects that neurotrans-

mitter abnormalities have on

disorders including mental

retar-dation, autism, visual impair-

me

behavior

form the basis for pharmacologic

interventi

The complex nature of neuronal

activity in the brain makes it difficult to

implicate any one neurotransmitter

system in a particular behavioral

problem. This paradigm does how-

ever give a

on, medications used to

modify behavioral disturbances may

be chosen based upon the neuro-

transmitter class on which they are

believed to exert their major effects.

rational starting point

by estimating quantitatively the

circulating neurotransmitter levels

and to choose a pharmacologic agent

for management and to strategies

patient specific treatment centered on

the improvement of symptoms.

Doctor’s Profile

Dr. Vipla Puri, Ph. D

Consultant - RIA Laboratory

Professional Experience:

Dr. Vipla Puri has over 21 years of experience at Hinduja Hospital in the Department of

RIA and her area of expertise lies in developing newer cost effective diagnostics for

various disorders.

> She developed and standardized 3 new assays for the diagnosis of Movement

Disorders, Pure Red Cell Aplasia and Multiple Sclerosis. Relevant documents for filing

'PATENT' have been submitted.

> She also established newer technologies of Microarray to diagnose Food Intolerance

and Chemiluminescence to diagnose various allergies.

The complex nature of

neuronal activity in the

brain makes it difficult

to implicate any one

neurotransmitter system

in a particular

behavioral problem.

Her experience in the radioimmunoassay lab has given her international recognition and

has been invited to numerous international and national conferences in various

capacities.

> The UNDP / UNFA / WHO / World Bank Special Programme of research, Development

and Research training in Human reproduction, Geneva as advisor and member of task

force on laboratory methods.

> Research guide in the University of Mumbai.

> Invited to review multicentre trial results on “Screening for Pre-eclampsia; evaluation

of predictive abilities of angiogenic factors for pre-eclampsia. WHO - Geneva.

> Visiting Research Assistant Professor, Department of Obstetrics and Gynaecology,

Washington University School of Medicine, St Louis, MO, USA.

Awards and Achievements

Dr. Vipla Puri has numerous publications to her credit in both international and national

journals and most recently the “Evaluation of Osteopathy in Thalassemia by Bone

Mineral Densitometry and Biochemical Indices” published in the Indian Journal of

pediatrics.

Dr. Puri has been awarded by the WHO fellowship to work as a visiting scientist at

Karolinska Hospital Stockholm, Sweden and at the Department of Biochemistry,

University of Paris, Bicetre. She has been the recipient of ICMR - Research Grant to work

on Foam Cell Formation in Leprosy and has received best paper award for my work on

Hyperhomocystenemia an independent risk factor for Osteoporosis in men.

dr_vpuri@hindujahospital.com 24451515 Extn. 7148/7149|| Ph: 022-

19

20

Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: A Review

Paroxysmal Nocturnal Hemoglobinuria

(PNH) is an acquired hematopoietic stem

cell disease that is caused by a somatic

mutation of the X-linked phosphatidyl-(1,2)inositol glycan (PIG-A) gene . Expansion

of PNH-l ike clones can also be

detected in

/myelodysplastic syndromes caused

by an immune-mediated failure of

normal hematopoiesis or defect in (3)hematopoietic stem cells . Typical

clinical features of PNH are: bone marrow

failure of variable severity, thrombosis

in unusual sites, chronic intravascular

hemolytic anemia that leads to

hemoglobinuria, iron deficiency anemia,

and increased incidence of acute myeloid (2)leukemia .

PNH has an estimated incidence of only

1.3 new cases per one million individuals

per year. Although PNH is rare, screening

of appropriate patients and correct

diagnosis are important, because PNH is

a chronic disease that persists for many

years and has a profound impact on

quality of life and survival for any

individual patient.

In the last few years, the development

and successful clinical trial of a

humanized mo ody against

cases of aplastic/hypoplastic

noclonal antib

the terminal complement protein C5

(Eculizumab) has improved the quality of

life for patients with hemolytic PNH by

reducing hemolysis, thrombosis, and (4,5)transfusion requirements . This has

made screening and accurate diagnosis

of PNH important priorities for many

clinical laboratories.

Diagnosing PNH

Various modalities have been used for

the diagnosis of PNH and these include

Complement based tests and sephacryl

gel card technique (GCT) (both of which

can detect PNH positive red cells), flow

cytometric based assays for red cells and

WBC's and molecular diagnostic tests for (6)detection of mutations in PIGA gene .

The Past-Hams Test And

Sucrose Lysis Test

Because PNH was initially recognized as a

type of hemolytic anemia, the initial

focus on red blood cells (RBCs) led to the

development of several RBC-based

assays. These included the Ham test and

the sucrose hemolysis test, both of which

demonstrated the increased sensitivity

of PNH RBCs to complement-mediated

Dr. Kunal Sehgal, MDAssociate Consultant -

Hematopathology

21

Although PNH is rare,

screening of appropriate

patients and correct

diagnosis are important,

because PNH is a

chronic disease that

persists for many years

and has a profound

impact on quality of life

and survival for any

individual patient.

play an important role in the control of

complement. While the absence or

partial expression of CD55 and CD59 is

specific for PNH, CD59 deficiency alone

appears to be responsible for hemolysis (2,7) and other clinical symptoms of PNH .

Gel Card Assay: The gel card assay is done

to evaluate the presence of CD55 and

CD59 on RBCs.

Principle of PNH Gel Card Assay

RBCs from patient's blood carrying CD55

and CD59 antigens bind the mouse

antibodies pre coated in the gel. After

centrifugation through the gel, cells

carrying antibodies, confirming the

presence of MIRL or DAF, will show a

positive reaction (RBCs will accumulate

at the top of the gel-Figure 1). This

denotes that the patient does not

have PNH.

If the patients cells don't express CD55

and CD59 there will be no antibody

binding and cells will go the bottom of

the tube as seen in the control tube in

Figure 1. This denotes absence of CD55

and CD59 and confirms the presence

of PNH.

The Present and Future: Flow

Cytometry - based assays

Diagnosis of PNH has been greatly

improved by the advent of flow

cytometry based assays. The initial offer

Figure 1: Gel agglutination assay for PNH via detection of DAF (CD55) and MIRL (CD59) by specific monoclonal antibodies. (Normal Peripheral Blood)

22

Severe neutropenia in

some patients makes

it difficult to identify

the target population

using light scatter

characteristics alone.

hemolysis under standard conditions.

Neither of these tests was specific, and

were cumbersome to perform. A more

specific test, the complement lysis

sensitivity test, measured the amount of

hemolysis at varying concentrations of

complement; this assay showed that PNH

cells lysed at lower concentrations than (5)did normal cells . This test also led to the

recognition that some PNH patients have

a population of cells with intermediate

complement sensitivity (Type II),

between normal RBCs (Type I) and the

mo st a b n o rma l PNH - t yp e R B C s (5)(Type III) .

The Present- Gel Card Assay

Activated serum complement was shown

to play an important role in the hemolytic

anemia and two GPI-linked structures

CD55 (Decay Accelerating Factor, DAF)

and CD59 (Membrane Inhibitor of

reactive lysis, MIRL) on red blood cells

were directed towards red cells using

CD55/CD59. The presence of recently

transfused normal RBCs significantly

reduces the ability to detect PNH RBCs.

As PNH is a rare disease, deficiency of at

least two GPI-linked structures on more

than one lineage, typically, neutrophils, is

an additionally required criterion to

establish a diagnosis. In situations of

prior hemolysis and/or recent red

cell transfusion, detection of PNH

granulocytes best reflects the size of the

PNH clone. However, severe neutropenia

in some patients makes it difficult to

identify the target population using light (2)scatter characteristics alone .

H een uni-

formity in selection of monoclonal

antibodies among laboratories. This can

largely be attributed to the fact that there

are monoclonal antibodies available

against many different GPI-anchored

proteins, all of which have some

capability of detecting PNH clones,

especially in frank PNH where such cells

are numerous. However, for example,

although CD55 and CD59 are widely used

for detecting granulocyte PNH clones,

there are data to suggest that these

reagents may not perform as well as

antibodies to other antigens in PNH (5)testing . Moreover, the increasing use of

flow cytometry to detect small clones has

magnified differences among reagents,

as some reagents suitable for detecting

large, obvious clones perform less well in

higher sensitivity analysis. There are

emerging data to suggest that one of best

istorically, there has not b

reagents available to study GPI-linked

antigens on leukocytes is the reagent

(Pine-

wood Scientific Services, Victoria, BC,

Canada). This is a fluorochrome-

conjugated inactive variant of the

bacterially derived channel-forming

prote in aero lys in , which b inds

specifically to GPI-anchors. FLAER may

offer significant advantages as a reagent

for PNH testing; in contrast with

antibodies against many of the GPI-

linked antigens normally studied, its

to the matu-

rational stage of the cells. FLAER can also

antibodies to GPI-linked and

non-GPI antigens for the detection of (2, 5, 8).PNH clones

As per recent international guidelines on

PNH Testing of RBCs alone in a routine

assay is not adequate for evaluation of

PNH patients, because hemolysis and

transfusion may greatly underestimate

the size of the PNH clone. For these

reasons, WBC clones are frequently

detected when RBC clones are not,

though significant RBC clones are never

seen without WBC clones. Nonetheless,

RBC testing is still important, both

because Type II cells are more readily

detected, and because comparison

of the relative sizes of RBC and

WBC clones may provide useful (5)clinical information . Sutherland et al

h ave re c e nt l y s h ow n t h at t h e

identification of PNH WBCs with the

fluorescent aerolysin or FLAER

binding is less sensitive

be used in multicolor combinations with

monoclonal

FLAER assay represents a far more

23

Hemolysis and

transfusion may greatly

underestimate the size of

the PNH clone.

more efficient and cost-effective primary

screening test for most clinical flow

laboratories that perform PNH testing

than RBC-based assays. This is most

significant for laboratories that perform

large numbers of tests for this rare (9)disease . The FLAER assay on WBCs more

reliably quantifies the size of the PNH

clone than does the CD59 RBC assay, a

result in keeping with other FLAER-based

and non-FLAER-based studies comparing (5, 9, 10, 11)granulocyte and RBC clone sizes.

Like in western countries the true

incidence of PNH in India is also not

known but it definitely is a rare disease.

However it is very essential to be certain

of the diagnosis in suspected patients

especially with the advent of targeted

drug therapy. There are only a handful of

laboratories in India doing PNH diagnosis

using flow cytometry and even fewer are

using FLAER. For the same reason,

Hematology laboratory at P.D. Hinduja

Hospital has an ongoing project for

establishing this protocol to standardize

the use of FLAER for the diagnosis of PNH

and that too by using a high sensitivity

assay so as to pick up small clones of PNH

cells even in cases of Aplastic Anemia or

MDS.

24

The true incidence of

PNH in India is also not

known but it definitely

is a rare disease.

[Fresh PNH sample stained with FLAER, CD33, Cd45, and CD14, Monocytes (71%) from R2 exhibit a CD14,

FLAER-PNH phenotype (lower middle)] Figure 2: PNH diagnosis using FLAER – PNH positive sample (2)

25

References

1. Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T. De.ciency of the GPI anchor caused by a

somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993; 73:703-711.

2. R Sutherland et al. Diagnosing PNH with FLAER and Multipoarameter Flowcytometry. Cytometry Part B 2007; 72B:167-177.

3. Dunn DE, Tanawattacharoen P, Boccuni P, Nagakura S, Green SW, Kirby MR, Kumar MS, Rosenfeld S, Young NS. Paroxysmal nocturnal

hemoglobinuria cells in patients with bone marrow failure syndromes Ann Int Med 1999; 131:401–408.

4. Brodsky RA, Young NS, Antonioli E, Risitano AM, Schrezenmeier H, Schubert J, Gaya A, Coyle L, de CC, Fu CL, Maciejewski JP, Bessler

M, Kroon HA, Rother RP, Hillmen P. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients

with paroxysmal nocturnal hemoglobinuria. Blood 2008; 111: 1840-1847.

5. Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. Guidelines for the diagnosis

and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry Part B 2010;

78B: 211-230.

6. M Madkaikar et al. Paroxysmal Nocturnal haemoglobinuria: diagnostic tests, advantages, and limitations. European journal of

hematology 2009; 83:503-511.

7. Yamashina M, Ueda E, Kinoshita T, Takami T, Ojima A, Ono H, Tanaka H, Kondo N, Orii T, Okada N, Okada H, Inoue K, Kitani T. Inherited

complete de.ciency of 20-kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N

Engl J Med 1990; 323:1184-1189.

8. Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT, Borowitz MJ. Improved detection and characterization of

paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000; 114: 459-466.

[Normal peripheral blood sample stained with FLAER, CD33, CD45, and CD14. Cells gated in Region R2 exhibit the composite phenotype of normal monocytes (CD45+, CD33bright, CD14+, and FLAER+). Cells gated in Region R3 exhibit the composite phenotype of neurtrophills (CD45lo, CD33lo, CD14lo, and FLAER+). Cells gated in Region R4 exhibit the composite phenotype of lymphocytes (CD45hi, CD33-, CD14-, and FLAER+). Less than 0.5% monocytes and neutrophills display a CD14 negative, FLAER negative PNH phenotype.]Figure 3: PNH diagnosis using FLAER – Normal sample (2)

26

9. R Sutherland et al. Use of a FLAER-Based WBC Assay in the Primary Screening of PNH Clones. Am J Clin Pathol 2009; 132:564-572

10. Parker C, Omine M, Richards S, et al; for the International PNH Interest Group. Diagnosis and management of paroxysmal nocturnal

hemoglobinuria. Blood. 2005; 106:3699-3709.

11. Brodsky RA, Mukhina GL, Li S, et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using

fluorescent aerolysin. Am J Clin Pathol. 2000; 114:459-466.

Doctor’s Profile

Dr. Kunal Sehgal, MD

Associate Consultant - Hematopathology

Professional Experience

Dr. Kunal Sehgal is MD (Pathology) from PGIMER Chandigarh, India. He pursued his

Fellowship and Senior residency at Hemato-pathology lab, Pathology Dept., Tata

Memorial Centre, Mumbai, India. He underwent UICC ICRETT Research Fellowship. His

project work was on “Development and standardization of eight colour flowcytometric

antibody panels for immunophenotyping of AMLs and evaluating potential MRD

markers for AML”.

> Other activities as a research fellow involved:

> Training in multiparametric immunophenotyping of leukemias and lymphomas

> Minimal Residual Disease analysis for B cell ALL

> PNH diagnosis using FLAER

> CD34 Stem cell counts

Areas of Interest

Clinical Multiparametric Flow cytometric Immunophenotyping, MRD for Acute

Leukemias, Standardisation of FLAER based assay for PNH, Research Parameters on CBC

analysers

Research & Publications

Dr. Kunal has several flow cytometry related publications in journals. He has attended

and conducted various workshops and conferences related to flowcytometry.

Affiliations

T Grouphe Cytometry Society (TCS), ISA, Mumbai Hematology

Awards and Achievements

> International Union Against Cancer (UICC) - International Cancer Technology Transfer

Fellowship (ICRETT) at John Hopkins Hospital, Baltimore USA.

> Kataria Gold Medal - best overall student of PGIMER 2008.

dr_ksehgal@hindujahospital.com || Ph: 022-24451515 Extn. 7145

27

TRANSCON-2011 Update

> Second poster was in the Transfusion Transmitted

Diseases (TTI) category titled as “HGV-HCV/HBV Co-

Infection in Patients & HGV in Healthy Blood Donors: a

Pilot study” presented by Ms. Amruta Pathare. This was

one of the first studies in HGV infection from India which

showed HCV and HGV co-infection in one out of 40

HBV/HGV chronic liver disease patients. None of the

hundred healthy donors were positive for HGV infection.

The HGV 340/625IC kit was validated with the help of a

known HGV positive serum sample obtained from

National Institute of Health, Bethesda (U.S.). This poster

won First prize in Transfusion Transmitted Infections

(TTI) category.

> In this TTI category, the poster on “Correlation of nucleic

acid amplification testing & EIA results in tertiary care

hospital in India” presented our data for last 29 months

with total 15827 samples. 100% correlation was

observed between EIA and NAT results in case of HIV &

HCV test. In case of HBV, there was discordance between

EIA & NAT results in 10/169 (5.9%) cases which might be

due to very low number of viral copies inspite of strong

positive HBV EIA results or disappearance of viral

genome after antigen production. Our findings are

comparable with worldwide data available. A total of

three posters were presented under the category

Advances in Transfusion Medicine - Therapeutic red cell

exchange in a case of sickle cell anaemia; Review of

therapeutic plasmapheresis procedures in a tertiary care

hospital in India & a retrospective analysis of HLA allele

distribution in Indian population.

thThe 36 Annual Conference of Indian Society of Blood

Transfusion and Immunohematology (ISBTI) was held at th stPanchkula Chandigarh from 30 October to 1 November

2011.

The transfusion medicine team consisting of Dr. Anand

Deshpande Consultant Transfusion Medicine &

Hematology, Dr. Mangesh Kulkarni Clinical Associate,

Ms. Mamta Mishra Technical supervisor & Ms. Amruta

Pathare Research student were the attendees.

Six posters covering different aspects like Red Cell

Serology, d

Infections (TTI), and Advances In Transfusion Medicine

were presented from P. D. Hinduja National Hospital &

M.R.C. Mumbai.

? The poster under Immunohematology category was on

“ABO Incompatability”. The patient was a 3-days-old

female child who presented with low haemoglobin,

reticulocytosis & clinically insignificant jaundice.

Patient's blood group was B positive and DCT was weak

positive with presence of IgG while mother's blood

group was O positive and red cell antibody screening

was negative. Eluate was prepared from baby's cells

which gave negative red cell antibodies screening and

positive reaction with adult B cells indicating presence

of IgG anti B antibodies on the neonate's red cells.

Mother's serum showed presence of high titre of IgG

anti B antibodies (1:2048) with the help of 2

mercaptoethanol. For this poster presentation

Ms. Mamta Mishra was awarded Third prize in

Immunohematology category.

Immunohematology, Transfusion Transmitte

28

> Therapeutic red cell exchange was carried out using

automated cell separator COBE SPECTRA in a 22 years

old patient suffering from sickle cell crisis. Two

procedures were carried out at an interval of 9 weeks

during which the patient was asymptomatic. After the

exchange procedures Hb S percentage dropped down

from 60.3% to 14.1% and from 49% to 10.9%. Both the

procedures were completed within two hours without

any complications. The patient was hemodynamically

stable during and after the procedures. This poster

presented by Dr. Mangesh Kulkarni was selected by

judges for First prize in advances in transfusion

medicine category.

> The data related to 422 therapeutic plasmapheresis

procedures in 74 patients (17 pediatric & 57 adults) from

our hospital was analysed. Myasthenia gravis was the

commonest indication for TPE. On an average 1.5

volume plasma was exchanged with colloids &

crystalloids being used as replacement fluids. The

procedure related commonest complication was citrate

toxicity which was controlled by intravenous calcium

gluconate injection. In 95% of neurological cases the

results were satisfactory.

> A retrospective analysis of HLA allele distribution in 635

individuals belonging to various cast groups tested in our

hospital HLA laboratory was done. The HLA allele testing

was done by polymerase chain reaction-sequence

specific primers (PCR-SSP) method. All the posters were

highly appreciated by the delegates.

29

30

3rd CME - ‘Recent Concepts in Transfusion Medicine - Transfusion and Beyond’: held at 9th and 10th Oct 2010, Hinduja Hospital

Hematology Quiz

1. Which of the following intiates the extrinsic pathway of blood clotting?

a. Prothrombin

b. Plasmin

c. Thromboplastin (tissue factors)

d. Fibrinogen

2. Which organ plays a major role in absorption of vitamin B 12?

a. Esophagus

b. Small intestine

c. Large intestine

d. Oral mucosa

3. Which of the following statements does not apply to factor VIII and IX deficiencies?

a. They are also called haemophilia A and B.

b. They are the commonest inherited disorder of blood coagulation.

c. They are always hereditary. esult of several different mutations in factor VIII and Factor IX genes.

4. A 25 year old female presented with the following CBC report:

Hb- 10.5 g,

MCV- 65 fl,

MCH-23 pg,

RDW-14.5%

RBC count - 5 ml. The most likely diagnosis is

a. Iron deficiency anemia

b. Megaloblastic Anemia

c. B thalassemia trait

d. B thalassemia with concomitant Iron Deficiency Anemia

5. All the following are favorable cytogenetics in cases of Leukemias except

a. t(15.17)

b. t(8,21)

c. inversion 16

d. 11q23 translocations

d. They can r

.8 million/

Dr. Tina Dadu, DNB

Hematology

Associate Consultant -

31

Hematology Quiz

6. All the following vaccutainer colour coding combinations are correct except

a. EDTA-Lavender

b. Citrate-Green

c. Fluoride-Gray

d. Plain-Red

7. After starting a patient on a heparin drip for a pulmonary embolus, the earliest time to monitor the first set of PTT is:

a. 4-6 hrs

b. 24 hrs

c. 1 hrd. 12 hrs

8. Hypercellular marrow associated with pancytopenia is seen in all the following except

d. roblastoma infiltration

9. Microcytic hypochromic anemia is seen in

a. Malaria

b. Kala azar

c. Hookworm disease

d. Diphyllobothriasis

10. The granules of the basophiles contain all of the following expect

a. Histamine

b. Myloperioxidase

c. Heprin

d. Kallikrein

a. Megaloblastic anaemia

b. Fanconi anaemia

c. AML

Neu

Answers: 1-c, 2-b, 3-c, 4-c, 5-d, 6-b, 7-a, 8-b, 9-c, 10-b

32

TB LPA MOLECULAR ASSAYS FOR

MDR/XDR TB

Ms. Minal U. Paradkar,

M.Sc, DMLT,

Microbiology Dept.

Senior Officer-

Molecular Diagnostic,

Multidrug-resistant tuberculosis (MDR-

TB) poses a formidable challenge to TB

control due to its complex diagnostic and

treatment challenges.

Alarming

increases in MDR-TB, the emergence of

extensively drug resistant TB (XDR-TB),

mortality of MDR-TB and XDR-TB patients

with HIV co-infection, have highlighted

the urgency for rapid screening methods.

Conventional methods for mycobacte-

riological culture and drug susceptibility

testing (DST) are slow and require 4 weeks

in liquid culture (MGIT) to 3 months in

solid media (LJ) to grow. Moreover

culture technique is cumbersome and

requires sequential procedures for

isolation of mycobacteria from clinical

specimens, identification of Myco-

bacterium tuberculosis complex, and in

vitro testing of strain susceptibility to

anti-TB drugs. During this time patients

may be inappropriately treated, drug

resistant strains may continue to spread,

and amplification of resistance may

occur. Therefore rapid diagnosis and

identification of

The annual global

MDR-TB burden is estimated at around

4,90,000 cases, or 5% of the global TB

burden; however, less than 5% of existing

MDR-TB patients are currently being

diagnosed as a result of serious

laboratory capacity constraints.

MDR-TB strains are

prerequisites for the worldwide fight

against TB.

Novel technologies for rapid detection of

anti-TB drug resistance have therefore

become a priority in TB research and

development, and molecular line probe

assays (LPA) focused on rapid detection

of rifampicin (RIF) resistance [alone or in

combination with isoniazid (INH)] are

most advanced. The commercially ® available LPA GenoType MTBDRplus

test manufactured by Hain Lifescience is

a deoxyribonucleic acid (DNA) strip assay

which uses polymerase chain reaction

(PCR) and hybridization to detect

mutations in the inhA, katG, and rpoB

genes that confer INH and RIF resistance.

Mutations are detected by lack of binding

to wild-type probes, as well as by binding

to specific probes for the most commonly ®occurring mutations. The GenoType

MTBDRplus assay has been reported to

show sensitivity and specificity of 98.1%

and 98.7% respectively for detection of

Rifampicin resistance. The assay showed

sensitivity and specificity of 84.3% and

99.5% respectively for detection of ®Isoniazid resistance. The GenoType

MTBDRplus enables a rapid result from

smear-positive specimens and from

culture isolate. Also for diagnosing

patients after treatment failure and

relapse, with unknown anamnesis and

originating from high prevalence areas of

MDR-TB as well as for diagnosing

patients in high prevalence TB countries

and high burden MDR-TB regions the use ®of GenoType MTBDRplus is reasonable.

33

The annual global

MDR-TB burden is

estimated at around

4,90,000 cases, or 5% of

the global TB burden.

Dr. Camilla Rodrigues, MD

Microbiology

Consultant Microbiologist

Finally the test can also be applied for

screening purposes to develop country-

specific TB action plans.

The emergence and spread of (MDR-TB)

and XDR-TB are a major medical and

public problem threatening the global

health. XDR-TB emerges through

mismanagement of MDR-TB treatment. ®The GenoType MTBDRsl gives you the

possibility to diagnose patients with

MDR-TB to receive information on

further antibiotic resistances, to test

patient after therapy failure and relapse

as well as to diagnose patients from

countries with high usage of the

respective drugs. The identification of

resistance to fluoroquinolones is enabled

by the detection of the most significant

mutations of the gyrA gene (coding for

DNA gyrase). For the detection of

resistance to aminoglycosides/cyclic

peptides, the 16S rRNA gene (rrs) and for

detection of resistance to ethambutol

the embB gene (which, together with the

genes embA and embC, codes for

arabinosyl transferase) are examined. ®The GenoType MTBDRsl assay showed

sensitivity of 90.2%, 83.3% and 59% for

detection of ofloxacin, aminoglycosides

and ethambutol resistance respectively.

The assay has reported specificity of

100% for detection of ofloxacin,

aminoglycosides and ethambutol

resistance.

A notable advantage of LPA test is the

rapid turnaround time, which has

implications for patient management

and transmission of drug-resistant TB.

The turn-around time for the LPA assay is

2 days which is substantially faster than

conventional drug susceptibility testing ®(DST). Both GenoType MTBDRplus and

®GenoType MTBDRsl assays are

currently performed at PDHNH in

Microbiology department on Tuesday ®and Friday. The test code for GenoType

MTBDRplus is TB LPA molecular MDR

plus Hain for detection of Rifampicin and

Isoniazid resistance. The test code for ®GenoType MTBDRsl is TB LPA molecular

second line which detects resistance to

fluoroquinolones, aminoglycosides and

ethambutol. Both these LPA assays are

performed twice a week and are

performed on smear positive clinical

specimens and culture isolates. The cost ®of GenoType MTBDRplus and

®GenoType MTBDRsl is

The recommended use of line probe

assays is currently limited to culture

isolates and direct testing of smear-

positive specimens. New version is

expected to be launched by early 2012

for smear negative specimens.

1,700/- and

2,300/-.

A notable advantage of

LPA test is the rapid

turnaround time, which

has implications for

patient management

and transmission of

drug-resistant TB.

34

35

Dr. Camilla Rodrigues, MD- Microbiology

Under her leadership and guidance the Dept. of Immuno Serology was set up and the

Microbiology section of Lab Medicine was upgraded to a state of the art lab with subsections of

Bacteriology, Mycobacteriology, Mycology and Molecular infectious diseases. She also established the section of Infectious

Disease and assisted in obtaining post doctoral fellowship recognition for Infectious Diseases by National Board

Examinations, New Delhi.

Expertise

She has undertaken International collaborative projects with FIND (Foundation of Innovative New Diagnostics), Geneva,

Fondation Merieux, France, University of Oxford, UK, Translational Research Unit Infectious Disease, Italy, Imperial College,

UK etc. Additionally, in India she has conducted multi-centric trials with DBT – Department of Science and Technology,

Government of India and ICMR. She has been the microbiology coordinator of the yearly Infectious Diseases fellowship

program at Hinduja Hospital.

nce and MDR/XDR tuberculosis.

She has authored 132 publications in International, National journals and books and has conducted 59 research projects as

the chief/co-investigator with tuberculosis being area of focus. Under her guidance, a molecular test to identify Multi Drug

Resistance (MDR) in Mycobacterium tuberculosis was developed for which a patency has been filed by the hospital. To her

credit she also has 375 presentations, including orations, invited lectures as faculty, India and abroad.

Awards and Achievement

> SC Agarwal award for outstanding contributions in the field of Medical Microbiology, 2009.

>

Consultant Microbiologist

Professional Experience

Dr. Rodrigues did her MBBS & MD (Microbiology) from AFMC and served 5 years as a Medical

Officer in Indian Navy. She has been associated with Hinduja Hospital for more than 24 years.

She is also a recognized tutor for DNB Microbiology and is a guide for M.Sc/Ph.D at Mumbai

University. She served as a member of Task Force under DGHS, Govt of India, to assess, review and suggest measures for

antibiotic resistance in 2010.

Memberships

National Working Group for Tuberculosis: Case Finding & Diagnostics for The National Strategic Plan (2012-2017), Central TB

Division and National Laboratory Committee (NLC) under revised National Tuberculosis Control Program, Govt of India.

Areas of Interest

Research & Publications

She has to her credit 16 awards/prizes and 400 faculty presentations internationally and regionally.

Rapid diagnosis of infectious diseases, Antibiotic resista

dr_crodrigues@hindujahospital.com|| Ph: 022-24451515 Extn no. 7155

Doctor’s Profile

Biochemical Genetic Testing for Inborn Errors of Metabolism

Dr. Alpa Dherai, Ph.D

Consultant Biochemistry

Inborn errors of metabolism (IEMs) are

monogenic disturbances causing

defective function or absence of an

enzyme or transporter protein resulting

in excess or lack of one or more

metabolites leading to a clinical

phenotype. Although individually rare,

the conjunct frequency in a high risk

group may be 200 times higher than that

identified in the general population. The

disorders present with an overlapping

clinical presentation, and hence in

these patients biochemical diagnosis

supercedes the clinical diagnosis.

al perspec-

tive; IEMs can be categorized into three

diagnostic groups:

! disorders involving small molecules

(eg. Amino and organic acidurias,

acidemias)

! disorders involving complex molecules

eg. lysosomal storage disorders,

peroxisomal disorders congenital

disorders of glycosylation and defects

of cholesterol synthesis and

! disorders associated with disruption of

cellular energy metabolism eg

mitochondrial respiratory chain

defects, disorders of carbohydrate

metabolism and disorders of fatty acid

oxidation

From the pathophysiologic

hyperammonemias and lactic

Metabolic defects involving small

molecules and cellular energy meta-

bolism may be acute and related to food

intake or metabolic states whereas

complex molecules are usually pro-

gressive and not related to food intake.

Early diagnosis of an IEM is important for

prognostication, early intervention,

treatment initiation, genetic counselling

and prenatal diagnosis.

Diagnostic approach

compre-

hensive chromatographic and mass

spectrometry techniques, enzyme assays

and DNA analysis. The tests are

technically demanding and usually

performed in specialised laboratories.

Small molecule disorders

In the setup of an acute illness, IEMs have

to be considered as a differential

diagnosis along with common disorders

related to infection and intoxication. The

blood and urine of patients with acute

neurological deterioration should be

examined for signs of acidosis, ketosis,

hypoglycaemia and hyperammonemia.

Based on these results, a further

evaluation of amino acid profile, organic

acid analysis etc. may be required. CSF

analysis may be required in case

of neurotransmitter defects, non ketotic

The technical approach ranges from

simple qualitative tests to

36

Early diagnosis of an

IEM is important for

prognostication, early

intervention, treatment

initiation, genetic

counselling and

prenatal diagnosis.

hyperglycinemia etc. The metabolite

profile in these patients with small

molecule disease may normalise during

stable periods or in samples obtained

after settling an acute episode. Thus

sampling during an acute episode is

required to obtain a true diagnosis.

> Amino acidopathies: Qualitative

estimation of amino acids was

traditionally done by thin layer

chromatography using ninhydrin as

color reagent. It was only later that

the analytical techniques such as

cation

(methodology used in amino acid

analyser-AAA), Reverse phase high

performance l iquid chromato-

graphy (HPLC) and recently Liquid

chromatography tandem mass

spectrometry (LC- MS/MS or TMS) are

being used for quantification.

AAA is considered as the gold standard

for quantification of physiological

amino acids. The amino acids are

separated on a cation exchange

column using a temperature and

Lithium buffer gradient followed by a

post column derivation using ninhydrin

reagent and determination at 570 and

440 nm. The high equipment and

operation cost of AAA resulted in

introduction of rapid,

fluorimetric applications

using reverse phase HPLC. Due to its

high sensitivity the method is suitable

for estimation from CSF while in case of

plasma and urine the analytical range

on HPLC varies from the clinical range

exchange chromatography

reproducible and

sensitive

and thus results in repeat dilution runs.

The fluorimetric derivatising reagents

are non-reactive with several physio-

logical amino acids resulting in the

method limitation to diagnose only a

few aminoacidopathies.

> Organic acidurias: Organic acide-

mias are a group of heterogeneous

disorders characterized by the

accumulation of intermediary meta-

bolites particularly in the urine.

chromatography/

Mass spectroscopy (GCMS) was first

undertaken by Dr. Kay Tanaka around

40 years ago. The method enabled

detection or around 20 disorders with

150 metabolites including acyl

glycines. The estimation is done by

liquid-liquid extraction followed by

oximation of 2 keto acids

Metabolite identification is further

done by comparing the mass spectra

with that of a referral library or an in

house library generated by the

laboratory over the years.

> Fatty acid oxidation defects: Carnitine

and its esters (acyl carnitines) are

physiologically present in all biological

fluids and play an essential role in fatty

acid oxidation and branched-chain

amino acid metabolism. Acyl carnitine

Their

detection using Gas

and

derivatisation either by methylation or

silylation. The metabolites are

separated using capillary GC column

and further detection by electron

impact ionisation mass spectrometry.

37

AAA is considered as

the gold standard for

quantification of

physiological amino

acids.

38

The diagnostic workup

of CDGs begins with

analysis of glycosylation

pattern of transferring

using isoelectric

focussing. Recently

HPLC and TMS have

been used for estimation

of glycosylated

transferrin.

profiling is exclusively performed by

Tandem Mass Spectromery (TMS) and

plays an essential role in new born

screening, evaluation of symptomatic

patients, prenatal diagnosis and post

mortem screening.

Acylcarnitine estimation is done

using a triple quadrupole analyser

combined with Electro spray ioni-

sation (ESI). The sample is mixed with

known amount of stable isotope

labelled standards followed by

derivatisation and injection in ESI. The

acylcarnitine profile generated is used

for identification and quantification of

the individual acyl carnitines.

Complex molecule disorders

The metabolism of complex molecules in

lysosomes and peroxisomes involves

different biochemical pathways from

those responsible for the processing of

dietary constituents.

> Clinically these

are a group of heterogeneous dis-

orders involving multiple organ

systems. The defects are caused either

by primary enzyme defect, co-

factor/activator defect. Peroxisomal

disorders include craniofacia l

abnormalities, encephalopathy,

neuronal migration, brain cortical

defects, limb malformations, ocular

a b n o r m a l i t i e s a n d h e p a t i c

and The

investi-gations are targeted either to

estimate an accumulated substrate

and are primarily used in screening

f

Lysosomal disorders:

intestinal dysfunction.

or urinary oligosaccharides (oligo-

saccharidosis), urine glycosami-

noglycans(GAGs),

various steps in glycoprotein bio-

synthesis that lead to a broad

spectrum of symptoms.

estimation of

markers which are proteins occurring

as a secondary change due to the

lysosomal enzyme defect or esti-

mation of enzyme activity from

different samples such as leuco-

cytes, fibroblasts, tissue biopsies,

prenatal samples etc. The assays use

artificial fluorigenic or chromogenic

substrates.

> Disorders of

oxidation are diagnosed by serum

estimation of very long chain fatty

acids (VLCFAs) using GCMS, disorders

of ether lipid biosynthesis by

estimation of plasmalogens using

GCMS, TMS etc., estimation of

phytanic acid & pristanic acid, bile

acids and enzyme assays etc.

> Congenital disorders of Glycosylation

(CDGs): CDGs are a group of disorders

characterized by disturbance of

The diag-

nostic workup begins with analysis of

glycosylation pattern of transferring

using isoelectric focussing. Recently

HPLC and TMS have been used for

estimation of glycosylated transferrin.

Disorders of energy metabolism

This group includes disorders of

mitochondrial energy metabolism,

gluconeogenesis, fatty acid oxidation

defects, disorders of ketogenesis and

ketolysis. The diagnosis of each defect is

done either by metabolite or enzyme

assays.

Peroxisomal disorders:

In addition to the above

disorders, IEM involve

disorders of several

other metabolic

pathways such as

purines & pyrimidines,

sterol, lipoproteins,

nuero-transmitters,

vitamins, metals &

trace elements etc.

39

In addition to the above disorders, IEM

involve disorders of several other

metabolic pathways such as purines &

pyrimidines, sterol, lipoproteins, nuero-

transmitters, vitamins, metals & trace

elements etc.

IEM diagnostic facilities at Hinduja

Hospital.

The laboratory offers a basic workup

in

pyruvate, ammonia,

ketones etc. S

homocysteine,

methylmalonic acids, keto acids and

sulphur containing aminoacids, Thin

layer chromatography for sugars, Muco-

polysaccharides etc. and quantitative

cluding the first line tests i.e. esti-

mation of lactate,

creening tests including

color reactions for urinary

estimation of plasma, urine & CSF amino

acids using HPLC.

Diagnostic tests to be introduced in the

year 2012

> Urinary organic acids and serum.

> Very long chain fatty acids analysis

using GCMS.

> Amino acid estimation using cation

exchange chromatography with post

column derivatisation using lysosomal

enzyme assays.

quanti-

fication.

> Two dimensional electrophoresis for

mucopolysaccharidosis.

> Urinary glycosaminoglycan

Doctor’s Profile

Dr. Alpa J Dherai, Ph.D

Consultant Biochemist

Professional Experience & Expertise

Dr. Alpa has an experience of around 13 years in clinical chemistry and has a specific

interest in diagnosis of In-born errors of metabolism. She has to her credit the lead of

setting up first accredited biochemical genetics referral laboratory in the country during

her tenure at Sandor Proteomics Pvt. Ltd. Hyderabad (Sept 2007 – October 2011).

She underwent extensive training for diagnosis of In-born errors of metabolism, in Dept

of Neurochemistry Massachusetts General Hospital, Boston; Department of Clinical

Genetics, Erasmus Medical Centre, Rotterdam; Lab Genetic Metabolic Diseases &

Academic Medical Centre, Amsterdam.

Research & Publications

She has around 20 publications in refereed journals and several presentations in

National & International conferences.

Achievements

Dr. Alpa is a recipient of John Lawrence Fellowship & Travel training fellowship by European Research Network for Diagnosis

of Inborn Errors of Metabolism (ERNDIM).

She has been a co-investigator in ICMR-National task force multi centric study for newborn screening for congenital

hypothyroidism and congenital adrenal hyperplasia and High risk screening for In-born errors of metabolism. She has also

been a principal investigator for several other DBT funded and in-house projects during her tenure at Sandor Proteomics Pvt.

Ltd. Hyderabad.

dr_alpa.dherai@hindujahospital.com|| Ph: 022-24451515 Extn no. 7139

40

The Drug and Poison Information Center (DPIC), established in October 2007 at Hinduja Hospital, Mahim, is aimed at providing the complete range of emergency Drug & Poison decontamination, treatment, service information and to serve as a primary resource for poison education, prevention and treatment advisory.

> Only hospital in entire Maharashtra, offering free and confidential services to aid improvement in patient care.

> Receives queries on drug and poisoned cases and provides information on antidotes (including Indian antidotes) and various therapeutic drugs.

> Provides drug information on drugs including treatment, monitoring side effects, adverse drug reaction and the lab tests that are recommended using Micromedex Database (for poisons and drugs) which has been accepted all over the world.

> In the last three years successfully handled 5201 queries/interactions.

Contact details: Ms. Priyanka Mon - Sat 7.00 am to 7.00 pm || Ph. No. - 2446 4600|| Email: hindujapoisoncenter@hindujahospital.com

||

Galactomannan Antigen Assay

Dr. Anjali Shetty, MRCP

FRCP – Pathology

Consultant Microbiology

The platellia aspegillus EIA is an

immunoenzymatic sandwich micro plate

assay for the detection of aspergillus

galactomannan. Galactomannan is a

soluble antigen produced by the

budding aspergillus hyphae. Its detection

aids in the diagnosis of invasive

aspergillosis.

Samples tested - Serum, BAL fluid and

CSF

Sensitivity and Specificity: Sensitivity in

serum varies from 64% to 93% in

neutropenics and around 42% in non

neutropenics. BAL fluid galactomannan

is more sensitive than serum for

diagnosis of invasive pulmonary

aspergillosis. The sensitivity of BAL is

76% to 100% in neutropenics and 60% in

non neutropenics. Specificity ranges

from 81% to 98% in serum and 87% to

100% in BAL.

Indications

Serum Galactomannan is used for the

diagnosis of invasive aspergillosis. This

test is indicated in susceptible hosts (High

risk group) with corroborative clinical or

radiological suspicion of invasive

aspergillosis. The high risk group include

patients with -

> Hematological malignancies

> Allogenic stem cell transplant

> Solid organ malignancies with

chemotherapy induced prolonged

neutropenia

> Aplastic anaemia on treatment

> Post solid organ transplant on

immunosuppressants

> COPD patients

> Patients on prolonged steroids

> Liver cirrhosis

For patients with prolonged neutropenia

serum galactomannan is done as

screening test twice weekly for the early

diagnosis of invasive aspergillosis.

Causes of false positives

> Antibiotics - piperacillin-tazobactum

and amox-clav

> Other fungi Penicillium, Paecilomyces

and Alternaria species

> Dietary factors - Infant feeds as they

contain galactofuranose

> Compromised gut integrity

Causes of false negative

> Antifungal therapy

41

Serum Galactomannan

is used for the diagnosis

of invasive aspergillosis.

This test is indicated in

susceptible hosts (High

risk group) with

corroborative clinical or

radiological suspicion of

invasive aspergillosis.

Doctor’s Profile

Doctor’s Profile

Dr. Anjali Shetty, MRCP, FRCP – Pathology

Consultant Microbiology

Professional Experience

Dr. Anjali Shetty has more than 11 years experience both abroad and in India. Her special areas of interest are clinical

bacteriology and infection control.

Research & Publications

Dr. Shetty has numerous publications to her credit in both international and national journals and one of her recent

publications includes “Outbreak of Listeria Monocytogenes in an Oncology Unit Associated with Sandwiches Consumed in

Hospital”. The same was in association with Davies E, Mclaughlin J, Grant C, Ribeiro CD and was published in Journal of

Hospital Infection.

Awards and Achievements

She has been awarded the Travel Bursary Award by the Hospital Infection Society for lecture in South Africa on Infection

Control and for the best oral presentation at BIS in May 2005 for “A Case of Colonic Stricture in a Neonate” along with

Barnes RA.

42

dr_ashetty@hindujahospital.com || Ph: 022-24451515 Extn no. 7156

Conference Update

thThe 34 Annual conference of Mumbai Hematology Group

was held during 5 and 6 March 2011 at P. D. Hinduja

National Hospital and Medical Research Centre, Mahim,

Mumbai.

Dr. Shanaz Khodaiji, Dr. Kunal Sehgal and Dr. Tina Dadu were

the local organizers.

Around 225 delegates registered for this meeting. More

than 20 eminent hematologists of Mumbai were on the

faculty.

The meeting began with the trade-mark quiz which has

become hugely popular and attracts many students to this

meeting. 9 undergraduate and 10 post graduate teams of 3

members each participated in the quiz contest. Lively

debates and panel discussions were planned which were

extremely appreciated by the delegates because it gave

them an opportunity to interact with the top most

hematologists.

th th

Some of the topics included Newer parameters on CBC

analyzers, Interesting facts about BCR-ABL negative

myeloproliferative neoplasms, Tumour lysis syndrome and

es in

coagulation and pediatric hematology were presented and

discussed. The prestigious BGRC oration was awarded to

NIIH.

An overwhelming response was received for paper

presentations. There were 10 oral papers presented and 43

posters on display. All papers were of high quality research

work done by the presenters. Awards were given in each

category.

The logistic support by the Marketing, the Biomedical and

EDP departments was flawless. The FSD department

surpassed itself in providing sumptuous meals on both days

Overall, the conference was a resounding success.

nagging issues in ALL management. Interesting cas

Dr. Sumeet Gujral from TMH and the J.G. Parekh oration to

Dr. Kanjaksh Ghosh from

43

Annual Conference of the Mumbai Hematology Group held at Hinduja Hospital

44

Left to Right: Dr. Kunal Sehgal, Dr. Tina Dadu, Dr. Chitra Madiwale, Dr. Alpa J. Dherai, Dr. Tester Ashavaid, Dr. Anita S. Bhaduri,

Dr. Vipla Puri, Dr. R. B. Deshpande, Dr. Shahnaz Khodaiji, Dr. Camilla Rodrigues, Dr. Anjali Shetty and Dr. A. S. Deshpande

Old Building

Third FloorStat Lab – ReceptionStat Lab – Biochemistry.................................2444 7328Blood Bank....................................................2444 7308Donor Room..................................................2444 7306

Ground FloorOPD Blood Collection....................................2444 7079

S1 BuildingThird FloorBiochemistry Lab......................2444 7935 / 2444 7931

Fourth FloorHematology Lab............................................2444 7947Ria Lab..........................................................2444 7948

.....................................2444 7327

Fifth FloorMicrobiology/Serology Lab.......2444 7793 / 2444 7794Histopathology/Cytopathology Lab..............2444 7797

Eighth FloorChetna Patil, Lab Manager............2444 7943 Ext. 7143

Ninth FloorNAT Lab.....................................2444 7610 / 2444 7611

Home Collection Service.........3981 8181 / 6766 8181

Hinduja Poison Center................................2446 4600

Lab Medicine Fax Number..........................2444 2318

Telephone Numbers- Department Of Laboratory Medicine

Lab Medicine Team at Hinduja Hospital