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!

753 lflt€ruutiolrt! .lournol tlf t rl!ttboi"clit'.: ltc:;t'rri('h ott !tt:t'(tlul frlctlic'ine & Puhlit: Ilealth

Ilifferentiati*ffi ol''X'tr xut{i$-$ 1,? i{h $Pe q:itic }ted Cell Adherence

{5{kt.A}t*:st

Dr. Abhishck A Vlangaonkar o, Dr. A (l Valand

' Intern. Crant Meclical Collcgc ancJ Sir.l..l. Croup o1'l-lospitals. lVlrrmbai" tndia

2professor, Dept. of Patlrolog-v. Crant Metlical College and Sir.l.J. Groupof Hospitals. Mumbai,

lnd ia

_ Corresponcling Author: L)r'. r\lrhishek A Mangnonliat'. ltttcrn. Cjrant Medical College and Sir

J.J. Group of l-{ospitais. MLrrnbai. India. [:-rnail ll): abhiTT8gtrr,grtrail.com. Phone Nurnber: *91

9870578452

A[3$l']t,,\{-l'l'

Introduction: The A & tJ blootl groLrp antigens present on various body tissues are lost

following a malignant transfbrmation. Tlrrrrugh this study. u,e have made an attempt to

differentiate benigrr & maligna|rt tulrors by the usc of this corrcept. The technique used was

Specific Red Cell Aclherence (SRCA) based on the principle of Mixed Cell Agglutination on

Fine-Needle Aspirat iorr (FN A ) stttnp les'

Objective: Our airn nas to pertirrrl tlre SR( A test on FNA samples of various human solid

tulnors and compare rhern with histopi.tthology (Gold Standard).

Method: A total of 35 FNA sanrplcs of srvellings suspected to be tumors were collected & the

SRCA test was perforrned on thern.'flrc results \^/ere conlpared with histopathology.

Result: Tlre sensitivity o{'SttcA tc.>t u,as Iburrcl to he 88.23%: spcciflcity was72-22o/o; positive

prediotive value was 759,0: negative prcclictivc valttc rvas 86.6696 (p<0.05).

\inl.4No.5(2012)

754 Internutionul Journul o.f'(itlluhrtt'r,rire ltLr,scurt'lt ott lrttcrnul fuIctlicine & Puhtit: Iteatth

Conclusion: SRCA test is an easy & cost-eftective technique that can be used for differentiation

of benign & rnalignant tlll'l'lors on FNA samples'

Keyrvords: Specific

Agglutination

Red Cell Adherence (SRCA), FNA (Fine-Needle Aspiration), Mixed cell

Introduction

The blood group antigens (A & B) are expressed in normaltissues of the human body otherthan

red blood cells 1 . Such antigens are also known as isoantigens. Some of these are lost in cells

undergoing a maliglant transformation. This property differs in different tissues l-15- Majority

of these isoantigens are expressed on epithelial

tissues & most human callcers tend to originate fiom these cells.

Solid turnors are a grollp o1'cells which have properties of invasiveness & metastasis, but at

varying degrees. It is very diftlcult to correlate phenotypic characteristic of tumor cells with

malignant biological beliavior l. The loss of 4 & B isoantigen expression has been shown in

malignapt epithelial neoplasnts of the lung l, 2, gastrointestinaltract 3, oral, cavity 4. cervix uteri

5, bladder 6-10, &. prostate ll, thyroid neoplasm 12, epithelial ovarian cancer 13 & laryngeal

cancer 14.1n these turnor systems, the loss of isoantigens has been used to demonstrate

transformation from benign to a rnalignant process 4; it's loss has also been used for prognosis 5,

g, 10. Studies on carcinomas of breast have shown no significant correlation between malignant

transformation & loss of A & B isoantigert expression l5'

Fine needle aspiration cytology (FNAC) errtails using a narrow gauge (25-22G) needle to collect

a sample of a lesion fbr microscopicexarnirration. lt allows a rninirnally invasive' rapid diagnosis

of tissue but does not preserve its histological architecture. ln some cases this limits the ability to

make a definitive diagnosis. As with any invasive procedure there are risks, and as with all

diagnostic tests involving sarlpling and interpretation. important diagnoses can be missed'

Immunohistochemical techniques are used in some centers. but are expensive' Hence there is a

\zol.4No"5(2012)

7SS Itierfifilionrtl Jtturnttl tt7'Cttllttlxtrutira Rescarch ott Intcrrrul llledic'ine & Pultlic Ilealth

need to develop cheaP & affordable

morphological study. We have tried

malignant tumor cells through an

rnodality.

wa),s to diagnose tnalignant tumors to supplement routine

to do the satne by determining A & B isoantigen loss on

altbrdable & easy technique & using it as a diagnostic

We used atechnique based on the principl. of "Mi*.,1 Agglutination of Erythrocytes" 4.

Mixed agglutination reaction u,ers originalty described as a result of investigations on the

phenomenon of agglut inatiorr. Sonre of the workers described agglutination of two

morphologically distirrguishable cell types when they were mixed with antiserum to one of the

cell types 4. If two differerrt cells (for exarnple, a red blood cell (RBC) & a tumor cell), are

mixed with antiserum to one of the cell types (the RBC), the two cells will agglutinate if they

share the same antigen (the A & B antigens in or:r case)'

Retrospective studies have been done on paraffin-embedded sections of established malignant

lesions to determine the loss ol'AtsO isoantigens. 1'he techniques used in these studies are

variations of the mixed cell agglLrtinatiott techrrique'

No studl'could be fitunrl t.lentgn,struting tlte use of'this technique on Fine-Needle-Aspiration

(FNA) smears.

The aim of this study is to develop a test using the principle of mixed cell agglutination to

determine A & B isoantigeps on turror cells seen on "Fine-Needle-Aspiration (FNA)".samples

of mass lesions or solid tumors & to detennine whether it can be used as a diagnostic modality.

Material and Methods

l. Time-period : 6 nronths

2. Inclusion criteria:

a. Patients with rnass lesions suspected to be tunlors

b. Age grouP 12-60 Years-

Vol.4No.5(2012)

756 Intcruutionu! Journul tl't'rtllufutt'utiv llt saurt;lt ort Inlcrnul ilfcdic'ine & Puhlit'Ilealth

3. Exclusion criteria:

a. Patients suffering frorn HIV & Hepatitis

B

b. Patients with suspected inflamrnatory

swellings.

c. Bloody FNA sarriple.

Methodology

. FNA samples were collected fi'om patients when they were undergoing routine FNAC

procedure after obtaining a valid written informed consent. A rninimum of two slides were

prepared extra for every patierrt.

. The slides were imnrediately fixed in95 o/o ethyl alcohol'

. The slides then wele tested for determination of A & B isoantigens by a technique called

"Specific Red Cell Aclherence Test (Stl,CA)" by the technique of Kovarik et al 4. The

procedure is described in the subseqLlent steps"

o The first slide was treatdd with antibodies fbr btood group A (using the anti-sera routinely

used for blood-group cross matching) & incLrbated in a moist chamber at room temperature

for a period of 20 minutes.

. The excess antiserum was then washed of usirrg normal saline'

o The slide was then treated with 5% solution of Red Blood Cells (RBCs) of group A.

. The same procedure was repeated fbr the second slide but tliis time the antiserum for blood

group B & 5% solution of RBCs of the B group was used.

o The slides were then kept in a vertical position in 95Yo ethyl alcohol for a period of 15

minutes. This ensured that the unattached RBCs & other RBC clumps settle down &

simultaneous fixirrg took place for: stairrirrg.

. Then, the routine Hernatoxylirr & Eosirr (H&E) staining was done, and the slides were

exanrined under the microscope by a qualified pathologist-

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7s1 Intenuilionul Journsl o.f'Collubat'utiw llesesrclt ort Intenml frledicine & Puhlic lleatth

r More than or equal to five red blood cells adhering to a tumor cell was considered a

positive result.

. The results were compared to routine morphological examination

histopathological results of the sarne case.

Results

The technique was used on 35 samples of huuran solid tumors. Of thern, l4 were breast tumors,

5 were lipomas, 6 were lymph node swellings, 3 were ganglion cysts, 3 were metastatic tumors

& 4 others (1 bepign spindle cell turnor. I epidennal cyst, I pleomorphic adenoma, 1

neurofibroma) Figure l. The serrsitivity of the test was fourid to be88.23Vo, specificity 72.22Vo,

positive predictive value 75%o, negative predictive value 86.66%o & accuracy was 80%. The

likelihood ratio of a positive tesr was 3.17 & negative test was 0.16 Table l. Significance was

calculated-by the yates chi-square test. corrected tbr continuity (degree of freedom was one).

The p value was calcr.rlated to be 0.001 I (p<0.05).

Discussion

Our study is a prelinrinary one to attempt the use o1'the SRCA technique on FNA samples. No

study could be found demonstratirrg the use of this test on FNA samples. he SRCA technique

based on mixed cell agglutination was first described by Kovarik et al 4. The malignant

transformation of lung 2. cervix 5, bladder 6-10. prostate ll, gastrointestinal tract 3, thyroid 12,

laryngeal cancer l4 & epithelial ovarian cancer l3 correlate strorrgly with A & B isoantigen loss,

most of these turnors being epithelial neoplasms. Orrr study does not study the tumors

individually.

Conclusion

SRCA technique ot-fers the advantage of being highly economical, as compared to

immunohistochemical staining. With the elimination of the need for a detailed morphological

Vol.4No.5(2012)

75g ltfiertttrtionol ,ltturnal o.f'Colluhrtralive lll,s'tarch ttrt lntcnrul fuIedic'ine. & Puhlic l:{eulth

study, it also does not require extensive training. This not onty improves accuracy but also

reproducibility. lt has the potential of becoming a reliable & cost-effective alternative to other

techniqires of FNAC. More studies are needed to study individuat tumors by the use of this

technique & also to develop a standard statistical method for analysing the results.

Limitations:

l. Past studies demonstrating the use SRCA test

on histopathology slides demonstrate varied loss of A & B isoantigens in malignant

transformation of d iffere nt t issues'

2. Our study does not consider the tumors individually'

3. The test fails to give results on a bloody sample'

4. The blood group status of the patient was not considered'

Acknowledgement:

Indian Council of Medical Research (ICMR) fof supporting the study through a grant under the

STS-2008 progrilm.

Conflict of Interest: None declared.

References

l. Manju Sharma, SN Das. B Arora et al. Flowcytometric & Immunohistochemical observations

on Cell Cycle & Surface Antigens in Human Sotid Tumors. J Indian Acad Clin Med'

1999;5(2):130-34.

2. Davidsohn I, Ni LY. Loss of isoantigen A, B and H in carcinoma of the lung' Am J Pathol'

1969;57:307-34.

3. Davidshon I, Kovaric S, Lee CH. ABO substances in gastrointestinal carcinoma. Arch Pathol.

I 966;81 :3 8 I -90.

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759 Irttertrtrtiontt{ .lgurnal o/'Colluboratit'c llesaurch on lnternul Medicine & Puhlic l{eolth

4. Kovaric S. Davidsolip l, Stejskal R. ABO antigen in cancer. Detection with mixed cell

agglutinatio n. Arch Puthol. 968 86: 12-21

5" Gupta S, Gupta yN, Singh IJ et al. Tissue isoantigen A, B and H in carcinoma cervix uteri :

Their clinical significan ce- J Strg Oncol' 1981 ; I 6: 71-7'

6. Juhl BR. Blood group antigens in transitional cell tumours of the urinary bladder. An

immunohistochemical study. Dtrn Med Bull' 1994;' 4l( I ): l- I I '

7. De cenzo J M. Howard P. Irish CE. Antigenic detection and prognosis of patients with stage A

transitional bladder carcinoma. J Urol. 1975;l 14: 874'78'

g. Emmott RC, JanadpLrr N, Bergmarr SM et al. Correlation of cell surface antigens with grades

and stage of cancer of the bladder. .l Urol' 1979:121:37-9'

9. Lange PH, Limas C. Fraley EE. Tissue blood group antigens and prognosis in low stage

transitional cell carcinorna of the bladder. J Urol I 978; I I 9( I ):52-5'

10. Bergman S, Javadpour N: The cell surface antigen A, B or O (H) as an indicator of malignant

potential in stageA bladdercarcinoma: A preliminaryrepoft. .l tJrol.l978; 119(l): 49-51'

ll. Martenson S. Bigler.SA. Brown M et al. Sialyl-LewisX and related carbohydrate antigens in

the prostate . Human Putholog. 1995; 26(7): 735-9'

12. Conzalez-Campora R, Garcia-Sanatana JA, Jorda I et al' Blood group antigens in

differentiated thyroicl neoplasms. Arch Puthot Lab lried.l998;122(2):957-65.

13. Welshinger M, Firrstad CL, Venkatraman E et al' Expression of.A, B and H blood group

antigens in epithelial ovarian cancer : Ilelationship to tumor grade and patient survival. Gynecol

Oncol. 1996; 62(l): I 06- I 2.

14. Jiw, Feis, pan Z. Several blood group antigens expressions in laryngeal cancer tissue and

their clinical significance Chung Aua Erh P. Yen. Hon Ko Tsa Chih.D9a:29(6):330-2'

15. Teltem M. plotkin HR, Meranze DI{ et al. stuciies of Blood Group Antigens in benign and

Malignant Human Breast Tissue. canccr Res. 1963:23:1528-3 l.

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76lt ltfierrutiortul Jot;rncl o.f Co!lahorative Resaurtlt rttr lrtterrrul'Medicine & Public Heatth

Table 1: Table showing the paralneters of the SRCA test

Others11o/o

MetastaticSwellings

goh

GanglionCysts

9%

Lymph NodeSwellings

17%

Figure 1: Figure shoN,ing tlic t1,pe of tumors considered in the study

Parameters Value 957" Confidence IntervalLower limit Upper limit

Sensitivity 0.8823 0.6225 0.9793

Specificity 0.7222 0.4640 0.8928

Positive Predictive Value 0.7500 0.s058 0.9040

Negative Predictive Value 0.8666 0.s838 0.9765

Likelihood ratio Value

Positive test 3.17 64 1.4183 6.8252

Negative test 0. l 628 0.0424 0.6244

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Lipomas14%