The latest addition to the chain of centers of excellence at Piramal Diagnostics, the Center of Excellence in Molecular Biology provides esoteric tests in the fields of PCR, Real Time PCR, and Flowcytomery meeting all international quality standards, in Laboratory Medicine.
In accordance with our principles of providing quality and reliable diagnostic tests; every test is validated meticulously before being made available. We also strive to verify if normal ranges for the test compare with that of the Indian population, as most of the commercially available testing kits are manufactured abroad. Therefore, we have established new reference ranges covering Indian population, which allows the clinicians to interpret lab results in proper way.
To further strengthen the QA based approach, Piramal Diagnostics Pvt Ltd is a regular participant in both external and internal proficiency testing programs of the College of American Pathologists (CAP).
Accreditations
The Center of Excellence: Molecular Biology is accredited by the National Accreditation Board for Laboratory Testing and Calibration (NABL) and the College of American Pathologists (CAP).
We offer the following tests at present through our center of excellence in molecular biology:
DNA/RNA based Tests
At Piramal Diagnostics Pvt Ltd we provide DNA/RNA based diagnostics testing for detection and quantitative assessment of the viral load of infectious agents, including HIV and associated organisms like HCV, HBV, TB, and CMV using PCR, PCR RFLP and Real Time PCR.
Viruses
- HBV Real time DNA PCR
- HBV YMDD Mutation Detection
- HCV Real time RNA PCR
- HCV Genotyping
- HIV Proviral DNA PCR
- HIV Real time RNA PCR
- CMV DNA PCR
- EBV DNA PCR
- HSV Real time PCR
- HPV 16-18 DNA PCR-RFLP
- Dangue rt-PCR
- Chikungunya Real time PCR
HBV: Hepatitis B virus (HBV) is a member of the Hepadnaviridae family having circular, partially double-stranded DNA. The virus is blood-borne and is transmitted efficiently by exposure to blood. HBV is also commonly transmitted sexually and through prenatal transmission. Because of the potentially severe consequences of chronic HBV infection, monitoring the disease's progression is prudent. At Piramal Diagnostics HBV DNA is quantified by FDA approved Abbott Real Time HBV assay. The highly conserved region in the “S” gene is used for the detection of genotype A-H An unrelated DNA sequence is simultaneously amplified by PCR, and serves as an internal control to demonstrate that the process has proceeded correctly for each sample. The assay results can be reported in copies/ml or International Units/ml (IU/ml). The analytic measurement range of this assay is 51 to 3,410,000,000 copies/ml.
HCV: Hepatitis C virus is one of the most common causes of post transfusion hepatitis, and it is associated with acute and chronic liver diseases and hepatocellular carcinomas. The FDA approved Abbott Real Time HCV assay uses rt-PCR to generate amplified product from the RNA of HCV in clinical specimens, with homogenous real time fluorescent detection. The target sequence is in the 5’ utr region of the HCV genome. This region is highly conserved and hence can detect genotype 1 to 6.An unrelated RNA sequence is simultaneously amplified by rt-PCR, and serves as an internal control to demonstrate that the process has proceeded correctly for each sample. The assay results can be reported, as IU/ml. The analytic measurement range of this assay is 30 to 100,000,000 IU/ml.
HIV: Human immunodeficiency virus (HIV) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). AIDS is transmitted by sexual contact, exposure to blood (including sharing contaminated needles and syringes) or certain blood products, or transmitted from an infected mother to her fetus or child during the pre-natal period. Additionally, transmission of this virus can occur through tissue transplantation. The FDA approved Abbott Real Time HIV-uses rt-PCR to generate amplified product from the RNA genome of HIV-1 in clinical specimens, with homogenous real time fluorescent detection. The target sequence for the assay is in the pol region of the HIV-1 genome. This region is highly conserved and hence can detect diverse group M subtypes and group O isolates. An unrelated RNA sequence is simultaneously amplified by rt-PCR, and serves as an internal control to demonstrate that the process has proceeded correctly for each sample. The assay results can be reported as IU/ml or copies/ml. The analytic measurement range of this assay is 127 to 17,400,000 IU/ml or 75 to 10,000,000 copies/ml.
HBV YMDD Mutation Detection: The mutation in the YMDD motif of the Polymerase gene of HBV is known to cause resistance to Lamivudine therapy. On the basis of detection of Tyrosin (Y), Methionin (M), and Aspartate (D): YMDD mutation the clinician can decide treatment protocol for the patient.
HCV genotyping: Isolates of hepatitis C virus are grouped into six major genotypes. These genotypes are sub-typed according to sequence characteristics and are designated as 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. Reports suggest that patient prognosis and disease course may be genotype dependent. For example, hepatitis C virus type 1 and type 4 infections may be associated with more severe disease and decreased responsiveness to therapy. In addition, types 2 and 3 may be treated with shorter durations of therapy. At Piramal diagnostics HCV genotyping is done with the help of type specific sequence length amplified by Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
HIV DNA PCR: Human immunodeficiency virus (HIV) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). AIDS is transmitted by sexual contact, exposure to blood (including sharing contaminated needles and syringes) or certain blood products, or transmitted from an infected mother to her fetus or child during the pre-natal period. Additionally, transmission of these viruses can occur through tissue transplantation. The primer sets used are specific for the Indian isolates of HIV-1 as well as HIV-2 are from LTR-gag region of the virus.
CMV DNA PCR: CMV is an important pathogen in immunocompromised patients. It is a common congenital viral infection and the leading cause for mental retardation and also non-hereditary sensori-neural deafness. PCR, a rapid, sensitive test, is used to detect CMV in the blood and CSF of patients.
EBV DNA PCR: EBV, a lymphotropic virus, is associated with Burkitt’s lymphoma, nasopharyngeal carcinoma, and B-cell lymphoma. It can also manifest in to oral hairy leukoplakia in HIV infected patients. The virus is detected by PCR using specific primers. (TAT-3 days)
HPV 16-18 DNA PCR-RFLP: High risks HPVs are the established etiologic agents of cervical cancers. HPV type 16 & 18 are often associated with cervical cancers in India. Consensus primers are used to amplify distinct regions of the HPVs from cervical scrapings/biopsies. The PCR product is then digested with different restriction enzymes to yield the unique digestion to identify HPV 16/18.
Dengue rt PCR: Dengue virus is a member of the family Flaviviridae with a single-stranded positive sense RNA genome ,classified in to serotypes DEN1, DEN2, DEN3, and DEN4. Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) can be caused by infection with any of the four-dengue viruses (DENV). They are the most important mosquito-borne viral diseases affecting humans worldwide, and constitute a major public health problem in tropical and subtropical regions In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers from 3’UTR, which is the most conserved region of the viral genome to overcome the diversity of DENV genome. b-actin gene is simultaneously amplified and serves as an internal control to demonstrate that the process has proceeded correctly for each sample.
Chikungunya rt PCR: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus belonging to family Togaviridae It is an enveloped, positive-strand RNA virus capable of replicating in mosquito species. Chikungunya Quantification assay is developed for the specific amplification of Chikungunya Structural polyprotein gene, which covers gene sequences for both the African and Asian lineage strains. A part of the structural polyprotein gene is amplified for the direct detection of the specific & the reference gene amplicon. The analytic measurement range of the assay is 50 copies/ml to 25000000 copies/ml
Haematology
Factor V Leiden Mutation PCR-RFLP
PCR-RFLP is used for detection of blood coagulation Factor V Leiden mutation (Arg 506 Gln). The Factor V Leiden mutation is associated with increased risk for thrombosis.
Bacteria
Mycobacterium tuberculosis Amplified Detection by Transcription Mediated Amplification (Gen-Probe®): Gen-Probe's Amplified™ MTD Test detects Mycobacterium tuberculosis rRNA directly and rapidly while delivering the sensitivity of culture. It identifies the presence of Mycobacterium tuberculosis with sensitivity and specificity. Gen-Probe's test is the first FDA-approved direct test for helping to diagnose smear-positive and negative specimens. Recent CDC guidelines highlight the role of the Amplified MTD test in the diagnosis of a patient suspected of having TB
Mycobacterium tuberculosis DNA PCR Tuberculosis is a communicable disease with significant morbidity and mortality. It is the main opportunistic infection contributing to mortality amongst immunocompromised individuals, e.g. AIDS patients. It has become imperative to develop a system that not only ensures rapid and accurate detection of the organism but also distinguish between Mycobacterium tuberculosis complex (MTC) and Mycobacterium other than MtbC (MOTT ) as effective therapeutic regimens are different for patients infected with MTC and MOTT.. At Piramal diagnostics this is achieved by simultaneous amplification of two or more DNA targets, some specific to M. tuberculosis (insertion sequences IS6110and cfp32 protein (Rv0577) and others common to all mycobacteria (16S rRNA and heat shock protein 65 Kda complex). The PCR detects MTC and MOTT in clinical specimens with 100% specificity.
Helicobacter pylori CAG ‘A’ PCR: H. pylori is one of the most common bacterial infection in humans, and is the etiologic agent of chronic gastritis, gastric and duodenal ulcers .H. pylori also is associated with Gastric Cancer. The gene associated for this is identified by PCR, the current choice of a quick, reliable and specific test.
Oncology
Leukemia
Leukemia detection by RT-PCR of the major leukemia translocations has numerous advantages over conventional cytogenetics, including no requirement for dividing cells, grouping for related genes, rapid and efficient for test, detection of translocations that may be missed by conventional cytogenetics.
At Piramal Diagnostics after cDNA synthesis, type and variants of leukemia are detected by multiplex PCR with maximum specificity, sensitivity and accuracy.
BCR/ABL [t(9;22)(q34;q11)] : Detection of 3 types (b2a2, b3a2, e1a2) of BCR/ABL translocation found in CML(Chronic myelogenus leukemia), adult ALL(Acute lymphocytic leukemia) and child ALL.
PML/RARa [t(15;17)(q22;q21)] : Detection of bcr1, bcr2, bcr3 and other bcr variants of PML/RARa translocation found in most case of APL(Acute promyelocytic leukemia (M3)).
AML1/ETO [t(8;21)(q22;q22)] : Detection of AML1/ETO translocation found in AML.
Human Myeloproliferative disorders
Human myeloproliferative disorders form a range of clonal haematological malignant diseases, the main members of which are polycythaemia vera (PV), essential thrombocythaemia (ET) and myeloid metaplasia with myelofibrosis (MMM). A missensesomatic mutation in Janus kinase 2 (JAK2) tyrosine kinase gene has recently been reported in chronic myeloproliferative disorders, including PV, ET and MMM, strongly suggesting its role in the pathogenesis of myeloid disorders.
JAK2 Genotyping:
A single point mutation (V617F:1849G>T) in activation loop of JAK2 has been associated with chronic myeloproliferative disorders other than chronic myelogenous leukemia. Consequently, identification of the V617F JAK2 mutation is important for the diagnosis, classification and treatment of myeloproliferative disorders.
Flow cytometry
T-Cell Subsets (CD3, CD4,CD8 and CD45), Absolute Counts:
This assay is designed for enumerating the absolute cell counts of lymphocyte subsets in lysed whole blood. Lymphocytes are broken down mainly into CD4(+) cell and CD8(+) cells. The CD4 cells are Helper T-cells because they express both CD3 and CD4. The CD8 cells are Suppressor T-cells because they express both CD3 and CD8. Whole blood samples are mixed with antibodies against CD3, CD4, CD8, and CD45. The RBCs are lysed and latex beads are added for absolute count determination. Four-color flow cytometry is used to assess the absolute CD4 count (CD3+, CD4+, CD45+, CD8-) and the absolute CD8 count (CD3+, CD4-, CD45+, CD8+) per micro liter of whole blood.
Clinical Significance
- During HIV infection, antiviral therapy is often initiated when the absolute CD4 count drops below 500 cells/µL.
- When the absolute CD4 count drops below 200 cells/µL, therapeutic prophylaxis against PCP and other opportunistic infections may be initiated.
- When the absolute CD4 count drops below 100 cells/µL, prophylaxis against Mycobacterium avium complex and Pneumocystis carinii and other opportunistic infections is recommended.
- The Public Health Service (PHS) has recommended that Helper T-cell levels be monitored every three to six months in all HIV-infected persons.
HLA-B27
HLA antigens are the major histocompatibility antigens for tissue recognition. They are especially important in considering any type of tissue transplant, for example, kidney transplant or bone marrow transplant. Many HLA antigens exist, but some are of special interest since they are more common in certain autoimmune diseases. For example, HLA-B27 is found in 80 - 90% of people with ankylosing spondylitis and Reiter’s syndrome.
Piramal Diagnostics uses a FITC-labeled FD705 clone to identify HLA-B27 by direct immunofluorescence and whole blood lysis.
The advantages of flow cytometry over traditional microlymphocytotoxicity testing include:
- Rapid, simplified whole blood analysis
- Objective interpretation
- Reduced sample volume and viability requirements