Molecular Diagnostics

Molecular Diagnostics

LAB PROFILE

The Molecular Biology Laboratory at Amrita Institute of Medical Sciences offers services for Histocompatability and Immunogenetics, Thrombophilia genetics, Cancer genetics, Pharmacogenomics, and Molecular testing of infectious diseases along with facilities for DNA isolation and banking.

LAB SERVICES

Using molecular diagnostic techniques, we at Amrita Institute of Medical Sciences do HLA typing (where we compare the allele profiles between the patient and the donors for organ / stem cell transplantations), perform genetic disease screening and detect the presence of pathogens causing various infectious diseases. We also provide facility for DNA isolation and Banking.

  • Molecular Biology lab at Amrita Institute of Medical Sciences is the pioneer in molecular tissue typing for solid organ transplantation programs in Kerala.
  • Our lab is the NABL accredited lab in Kerala to perform serological crossmatch testing for solid organ transplantations.
  • It is the only lab in Kerala to offer Luminex platform based tests for Histocompatability and Immunogenetics.
  • Our laboratory services are accreditated by the National Accreditation Board for Testing and Calibration Laboratories (NABL) (ISO 15189: 2012) since 2010.
  • We have dedicated areas for testing transplantation compatibility, gene testing and infectious disease diagnosis.
  • We process about 350 – 400 samples per month.

HISTOCOMPATABILITY AND IMMUNOGENETICS TESTING AT AIMS

Both cell based and solid phase immunoassays are used to determine if patients are sensitized to HLA antigens, to determine the strength and specificity of HLA antibodies and to determine the strength of reactivity with specific donors.

Anti-HLA antibodies can arise following sensitization events such as pregnancy, particularly multiple pregnancies, blood or platelet transfusions, or previous transplant(s). If detected, HLA antibodies can prove detrimental to the graft as it might target the donor tissue/organ as part of immune response.

  • To know if your patient has any anti-HLA antibodies send for

    HLA Antibody Detection (HLA Class I &II antibody PRA screen)

Purpose: Detects the presence of anti-HLA IgG antibodies. Differentiates whether the antibodies are of the Class I type or Class II.

Limitations: Detects only IgG antibody isotype; IgM antibody will not be detected. This test will not give the information about the specific antigen against which the antibodies will react.

Test Results: Results are reported as HLA Class I and Class II IgG antibodies presence/absence but does not provide specificity.

  • To know if your patient has anti-HLA antibodies against a particular donor send for

    HLA Donor Specific Antibody detection assay (DSA assay)

Purpose: Detects the presence of IgG antibodies against donor’s HLA antigens.

This test is performed after a potential donor has been identified. It helps to determine if the intended recipient has any preformed HLA IgG antibodies (Class I & Class II) that could target the donor’s organ/tissue.

Test Indications:

  • Pre-transplant antibody detection
  • Reprospective antibody detection
  • Pre and Post transplant immunologic monitoring

Donor Specific Antibodies (DSA) have been shown to be involved in both chronic and acute rejection of transplanted allografts.DSA assay is used before and after transplantation to monitor patients for presence or absence of anti-HLA class I and class II antibodies or to monitor the appearance or disappearance of antibodies over time.

Limitations: Antibodies against HLA-DP and HLA-DQ are not detected. Detects only IgG antibody isotype; IgM antibody will not be detected

Test Results: Results are reported as HLA Class I and Class II IgG antibodies negative/positive but does not provide specificity.

  • To know if your patient has any preformed complement fixing antibodies against a particular donor using cell based assay, send for

    Serological crossmatch – Allo (Complement Dependent Cytotoxicity assay)

Purpose: Complement-dependent cytotoxicity test identifies preformed antibodies in the patient’s serum against the donor’s cells. These antibodies are responsible for hyper acute rejection of the grafts. If a patient contains circulating antibodies in the serum that act against the donor’s HLA antigens, they may experience rapid and irreversible destruction of the graft upon transplantation i.e., hyperacute rejection.

Limitations: This test cannot distinguish between class I and class II HLA antibodies. Complement fixing non- HLA antibodies may also be detected. Viable cells are required for the test.

Test Results: Results are reported as the percentage of the cross- reaction between the donor HLA antigens and preformed antibodies in the patient’s serum in the presence of complement and a vital dye. The results of this test are reported as negative (<10%), marginal positive (11-20%) or positive cross-reaction (>20%).

  • To know if your patient has any auto antibodies, send for

    Serological crossmatch - Auto (Complement Dependent Cytotoxicity assay)

To establish if auto-antibodies are responsible for a positive crossmatch reaction, an auto-crossmatch should be performed.

Purpose: Auto antibodies are those directed against self antigens and are typically of the IgM isotype. These arise mainly due to autoimmune disorders, viral infection, SLE, connective tissue diseases and/or certain medications. These auto antibodies not only bind to the patient’s own cells, but also sometimes to the donor cells, causing cell lysis in the presence of complement, and therefore can be detectable in antibody screens and crossmatches. IgM antibodies are generally not considered as detrimental to the graft.

Limitations: This test cannot distinguish between class I and class II HLA antibodies. Complement fixing non- HLA antibodies may also be detected.

Test Results: Results are reported as the percentage of cross- reaction between the antigens of the patient’s lymphocytes and preformed antibodies in the patient’s serum in the presence of the complement.

Serological crossmatch - Final

This test includes both serological crossmatch-allo and auto. It is performed ideally one week prior to transplantation. In case of a blood transfusion the test has to be repeated after 2 weeks.

Serological crossmatch with cadaveric donor

This “out of hours service” is available to deal with urgent testing requirements. This test is performed to screen compatible organ recipients from cadaveric donors.

  • To know what are the specific antigen(s) against which the patient has antibodies and which donors to avoid send for

    Single antigen bead assay

Purpose: This test is performed to determine unacceptable HLA antigen mismatches as a preventive measure to avoid donor-specific HLA antibody-mediated damage.

This is useful especially in the case of highly sensitized patients who may have antibodies against many different HLA alleles. The Luminex-supported single antigen bead test allows the precise characterization of HLA antibody specificities and provides information regarding the unacceptable antigens in the donor.

HLA Typing

Purpose: Compatibility of the HLA antigens between the donor and recipient are carried out.

  • HLA - A, B, DR Loci tissue typing (SSP PCR)
  • HLA - ABC Loci tissue typing (SSP PCR)
  • HLA - DQ , DR Loci tissue typing (SSP PCR)

One of the major problems in transplantation is immunoreaction by the recipient against the alien tissues. This is caused by differences in the HLA types of recipient and donor. Human Leucocyte Antigens (HLA) are integral cell membrane glycoproteins that bind peptide fragments and present them to the lymphocytes. Their main function is concerned with the immunity and self-recognition. Compatibility of the HLA of the donor and the recipient increases the chance for a successful engraftment. Matching is determined by comparing alleles. This test is performed by using HLA sequence specific primers which amplify the genes encoding the HLA antigens.

HLA Disease associations:

Certain disease states have been found to be associated with particular HLA antigens, such as ankylosing spondylitis which is associated with HLA-B*27, Behcet’s syndrome is associated with HLA-B*51. HLA typing may also be important in preventing reactions to certain medications as side effects have been noted with certain drugs and specific HLA types.

  • HLA - B Locus typing (SSP PCR)
  • HLA - DQ Locus typing (SSP PCR)
  • HLA - DR Locus typing (SSP PCR)

HLA Disease Associations

Disease condition

HLA allele(s) associated

Ankylosing Spondylitis

HLA-B27

Behcet’s

HLA-B51

Coeliac Disease

HLA-DQ2 or HLA-DQ8

Colitis

HLA-DRB1*01:03

Graves Disease

HLA-DRB1*03:01-DQA1*05:01-DQB1*02:01) or DRB1*04:01- DQA1*03:01-DQB1*03:02 (DR4, DQ8)

Insulin Dependent Diabetes Mellitus

HLA-DRB1*03:01, DQB1*02:01 (DR17, DQ2) or DRB1*04:01, DQB1*03:02 (DR4, DQ8)

Juvenile Idiopathic Arthritis

Oligoarthritis: HLA-A2, DRB1*08 and DRB1*11

Polyarthritis: HLA-DRB1*04

Multiple Sclerosis

HLA-DRB1*15

Myasthenia Gravis

HLA-DR3

Narcolepsy

HLA-DQB1*06:02

Systemic Lupus Erythematosus

HLA-DRB1*03:01, DQB1*02:01 and the DRB1*15:01, DQB1*06:02

Uveitis

HLA-B27

Abacavir hypersensitivity

HLA-B*5701

Carbamazepine hypersensitivity

HLA-B*1502

LIST OF TESTS AT THE MOLECULAR BIOLOGY LABORATORY

HISTOCOMPATABILITY AND IMMUNOGENETICS

S. No.

TEST NAME

1

SEROLOGICAL CROSSMATCH - ALLO

2

SEROLOGICAL CROSSMATCH - AUTO

3

SEROLOGICAL CROSSMATCH WITH CADAVERIC DONOR

4

SEROLOGICAL CROSSMATCH - FINAL*

5

DONOR SPECIFIC ANTIBODY (DSA) TESTING

6

HLA ANTIBODY DETECTION (HLA Class I &II antibody PRA screen)

7

SINGLE ANTIGEN BEAD ASSAY

8

HLA – A, B, DR (SSP PCR) Tissue typing

9

HLA – DR (SSP PCR) Tissue typing

10

HLA – DR, DQ (SSP PCR) Tissue typing

11

HLA – DQ (SSP PCR) Tissue typing

12

HLA – A (SSP PCR) Tissue typing

13

HLA – B (SSP PCR) Tissue typing

14

HLA – A, B, C (SSP PCR) Tissue typing

Specimen: Allo Crossmatch – Recipient: Serum - 2ml & Donor: Whole blood (Heparin) - 10 ml

Auto Crossmatch – Patient : Serum (2ml) and Whole blood (Heparin) - 10 ml

*All Final crossmatches include auto crossmatch also

DSA Testing - Recipient: Serum - 2ml & Donor: Whole blood (ACD) - 10 ml

HLA Antibody Detection - Patient : Serum (2ml)

Single antigen bead assay - Patient : Serum (2ml)

Tissue typing - Recipient and Donor: Whole blood (EDTA) - 10ml each

THROMBOPHILIA GENETICS, CANCER GENETICS, PHARMACOGENOMICS & OTHER GENETIC TESTS

S. No.

TEST NAME

1

HLA B27 Gene Detection

2

Factor V Leiden Gene Mutation

3

Prothrombin Gene Mutation

4

Factor V Leiden & Prothrombin Gene Mutation

5

Methylenetetrahydrofolate reductase (MTHFR) Gene Mutation Analysis

6

Venous thrombosis risk assessment panel (Factor V Leiden, Prothrombin & MTHFR mutation analysis)

7

Spinal muscular atrophy (SMA) Deletion Analysis

8

b - Thalassemia – 5 common mutation analysis

9

HFE gene (H63D, S65C, C282Y) mutation analysis

10

JAK2 Gene Mutation

11

JAK2 Exon 12 mutation analysis

12

Bcr - Abl Translocation - Qualitative

13

BRAF (V600E) Mutation analysis*

14

IDH 1 (R132H) gene mutation analysis*

15

IDH 1 (R132H) & IDH 2 (R140Q, R140W, R172G) gene mutation analysis*

16

TPMT genotyping

17

Dihydro Pyrimidine Dehydrogenase (DPD) Gene Mutation

18

Warfarin dosage test (CYP2C9 & VKORC1) mutation analysis

Specimen: Whole blood (EDTA) – 10 ml, * Tissue FFPE block / fresh tissue in Normal Saline

MOLECULAR TESTING OF INFECTIOUS DISEASES

S. No.

TEST NAME

1

Hepatitis B (HBV) - DNA PCR - Qualitative

2

Hepatitis B (HBV) - DNA PCR - Quantitative

3

Hepatitis C (HCV) - RNA PCR - Qualitative

4

Hepatitis C (HCV) - RNA PCR - Quantitative

5

Hepatitis C (HCV) Genotyping

6

Cytomegalovirus (CMV) DNA PCR – Qualitative*

7

Cytomegalovirus (CMV) DNA PCR – Quantitative*

8

Leptospira PCR – Qualitative*

9

Mycobacterium tuberculosis (MTB) PCR - Qualitative

10

Human Papilloma Virus (HPV) Detection PCR

Specimen: Whole blood (EDTA) – 8 ml * Whole blood (EDTA) – 8ml Or Urine: 10-50 ml

For MTB PCR- Sputum/ Body fluids / Whole blood (EDTA) – 8 ml/ Urine: 10-50 ml / FFPE block / fresh tissue in Normal Saline.

Contact Us:

Laboratory address

Molecular Biology Laboratory
Amrita Institute of Medical Sciences and Research Center
Ponekkara P. O

Kochi
Kerala, 682041

Hours of service

Normal Opening Hours: Monday to Saturday: 9.00 a.m. - 6.00 p.m.

Out of Hours Service

An “out of hours service” is available to deal with urgent testing requirements for screening of solid organ cadaveric donors

General Contact Details

Telephone: 0484-2851234 Extn: 6327 / 6329 / 6345

E-mail: molecularbiology@aims.amrita.edu

 

Doctors / Faculties-Molecular Diagnostics