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Molecular Diagnostics – A Revolutionary System to Improve Healthcare

Molecular testing systems enable precise detection of major infectious diseases; monitoring of treatment efficacy; and selection of targeted, individualized treatment options. Our real-time PCR based molecular diagnostics system is applicable for a wide range of clinical sample types and can analyze both RNA and DNA and can detect and quantify a broad range of infectious diseases pathogens which is not available in any other laboratory in the country. PCR (polymerase chain reaction) is a Nobel prize winning technology that gives highest sensitivity and specificity than any other methods. Our goal is to improve patient care by developing molecular diagnostics that translate an individual’s status into clinically actionable information.

Flow Cytometry is a technology that simultaneously measures multiple characteristics of a single cell at a rapid rate. Flow Cytometric Immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasm.

These services are the first standard routine practice in the country and will serve as essential service for stem cell transplantation. Molecular Lab Consultant was familiar in Flow Cytometric Immunophenotyping while was in Japan for 14 years and was in charge of Flow Cytometer Unit of Equipment Center of Yamagata University Faculty of Medicine, Japan for 2009-2011

On Going Molecular Tests

Flow Cytometric Immunophenotyping

  • Immunophenotyping – Acute Leukemia full Panel (CD45, CD34, HLA-DR, CD13, CD33, CD117, MPO, CD10, CD19, CD79a, CD3, CD4, CD5, CD7, CD8, cyCD3, CD14, CD64)
  • Immunophenotyping – Acute Leukemia basic Panel (CD45, CD34, HLA-DR, CD13, CD33, CD117, CD10, CD19, CD20, CD3, CD5, CD7)
  • Immunophenotyping – Follow up Panel (MPO, cyCD3, cfCD79a, CD45)
  • Immunophenotyping – Multiple Myeloma (CD45, CD19, CD117, CD38, CD56, CD138, Anti-Kappa, anti-Lambda)
  • Immunophenotyping – CLL/SLL/HCL Panel (CD3, CD5, CD10, CD19, CD20, CD23, CD25, C D34, CD45, CD103, CD200, FMC-7, Kappa, Lambda)
  • Immunophenotyping – HIV (CD45, CD3, CD4, CD8)
  • Immunophenotyping –Any 4 marker
  • Stem Cell (CD34+) Count-absolute
  • T and B Cell Ratio
  • T and B Cell Count – absolute
  • CD4/CD8 Ratio
  • CD4 and CD8 Count- absolute
  • PNH Clone RBC, WBC by Flow Cytometry

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hematopoietic stem cell disease that is caused by a somatic mutation of PIG-A gene. Since the PIG-A protein is involved in the initial stage of synthesis of the glycosylphosphatidyl-inositol (GPI) anchor, these defects result in partial or absolute deficiency of all GPI-linked proteins/glycoproteins in a clone of hematopoietic stem cells. The ultimate effect is damage of RBC, WBC and release of hemoglobin in the circulation and then in the urine. PNH is a progressive, destructive, and life-threatening disease and can cause thrombosis, end-organ damage, and early mortality.

A flow cytometric-based assay can detect the presence or absence of these GPI-linked proteins in RBC and WBC (Neutrophils and Monocytes), thus avoiding the problems associated with red cell-based diagnostic methods (Ham’s test) in which recent hemolytic episodes or recent transfusions can give false-negative results. Ham’s test also cannot detect PNH clone on WBC.

Specimen should be tested within 48 hours of blood drawn, however, it is best to be tested by 24 hours. Those who are sending samples to India for this test will definitely get incorrect result as it takes more than 48 hours during transport to India.

Leukemia Associated Translocation PCR

  • Real Time PCR for 7 translocation – t(9,22) minor, major,and micro; t(1,19); t(12,21); inv16; t(15,17) S, V and L; t(8,21) and t(4,11)
  • Real Time PCR for (BCR-ABL1) (Major, minor, micro), t(8;21) (RUNX1-RUNX1T1), inv16(CBFB-MYH11)
  • Real Time PCR for BCR-ABL1 (3 variants), t(4;11)(MLL-AFF1), , t(1;19)(TCF3-PBX1), t(12;21)(ETV6-RUNX1)
  • Real Time PCR for PML-RARA (3 variants)
  • BCR-ABL p210 Quantitative PCR
  • BCR-ABL p210 Qualitative PCR

JAK-2 mutation PCR in Myeloproliferative Disorders

The JAK2 V617F is present in 95% to 98% of polycythemia vera, 50% to 60% of primary myelofibrosis (PMF), and 50% to 60% of essential thrombocythemia (ET). It has also been described infrequently in other myeloid neoplasms, including chronic myelomonocytic leukemia and myelodysplastic syndrome. This mutation is not seen in chronic myelogenous leukemia (CML) or in reactive conditions with elevated blood counts. Detection of the JAK2 V617F is useful to help establish the diagnosis of MPN. Our adopted technology can also detect Non-V617F mutations (V617I, V617-C618>FR, V617V) along with V617F.

Tuberculosis Diagnosis by MTB/NTM Real Time PCR

Tuberculosis (TB) is one of the world’s most common severe infectious diseases. Approximately 2 million deaths per year are TB-related. A single patient with an active TB may infect 10 to 15 other people each year. In the fight to stop the spread of TB the most important milestone is early diagnosis. However, diagnosis of TB is a challenge. Apollo Molecular Lab is pioneer in detecting TB from any kind of specimen. Moreover, we can simultaneously detect non-tuberculous mycobacterium (NTM). We already have detected TB/NTM by real time PCR from sputum, BAL, any aspirate, body fluids, CSF, urine, tissue, tissue block (paraffin block), pus and are accepting these specimens for test.

Detection of Atypical Pneumonia (Mycoplasma/Chlamydia Pneumoniae)

Only we are doing multiplex PCR for simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae which causes atypical pneumonia. Most of the atypical pneumonia in this country remains undetected due to lack of any sensitive test.

Community associated pneumonia (CAP): Identification of bacteria by multiplex PCR

Pathogen causing community associated pneumonia (CAP) and hospital acquired pneumonia (HAP)  are different. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella spp are most common in CAP pneumonia. Among these Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella spp are very slow growing bacteria and cannot be detected in culture and always missed in conventional culture.  Utilizing Multiplex Real Time PCR it is possible to detect and differentiate all of these bacteria simultaneously from the same specimen.

Targets:

  • Streptococcus pneumoniae
  • Staphylococcus aureus
  • Haemophilus influenzae
  • Moraxella catarrhalis
  • Legionella spp.
  • Mycoplasma pneumoniae
  • Chlamydia pneumoniae
  • Internal control

Specimen: throat/nasal swabs, bronchoalveolar lavage, sputum

Neonatal Meningitis

Bacteria causing meningitis are age dependent. In case of neonates the culprits are Group B Streptococcus (S. agalactiae), Listeria monocytogenes, E. coli. Isolation of these bacteria from CSF is very difficult due to low bacterial load. PCR is certainly much more sensitive and specific than culture and by utilizing Multiplex Real Time PCR it is possible to detect and differentiate all of these bacteria simultaneously from small volume (0.2 ml) CSF.

Non-Neonatal Meningitis

Bacteria causing meningitis in pediatric and adult are Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenza. Multiplex real Time PCR can detect and differentiate these bacteria from small volume of CSF (0.2 ml) more reliably and rapidly than culture due to its higher sensitivity and specificity.

Viral Meningitis: Identification of Viruses by multiplex PCR

Diagnosis of viral meningitis is still going on by traditional CSF cytology and biochemical study which is neither specific nor sensitive and often misleading. Isolation of virus by culture is not going in routine lab due to its difficulty and complexity, on the other hand, there is no reference lab in the country where virus isolation and detection is going on! As Apollo Molecular lab is already familiar with multiplex real time PCR we are now offering 11 viral targets (mentioned below) detection and differentiation by multiplex real time PCR from CSF.

  • cytomegalovirus (CMV)
  • Epstein-Barr virus (EBV)
  • adenovirus (AV)
  • herpes simplex virus 1 (HSV1)
  • herpes simplex virus 2 (HSV2)
  • varicella-zoster virus (VZV)
  • enterovirus (EV)
  • parechovirus (PV)
  • human herpes virus 6 (HHV6)
  • human herpes virus 7 (HHV7)
  • parvovirus B19 (B19)

We are using high standard PCR kits from Europe which is CE-IVD marked

Virus Detection and Quantification by Real Time PCR

Precise and accurate detection of viral load and its genotypes ensures confidence in results. Our virological test systems includes hepatitis B, C for hepatitis, CMV/EBV/HHV-6, and HIV for immunocompromised patient, HPV for cervical cancer and HSV 1/2 for CNS, eye and genital infections by Real Time PCR enable you a reliable detection of virus which is available only in Apollo Hospitals Dhaka.

  • CMV Real Time PCR
  • EBV Real Time PCR
  • CMV/EBV Real Time PCR
  • CMV/EBV/HHV-6 Real Time PCR
  • CMV Real Time PCR Quantitative
  • EBV Real Time PCR Quantitative
  • HSV1/2 Real Time PCR
  • HPV Real Time PCR(High Risk Group)
  • HPV Real Time PCR (low risk group)
  • HPV 16/18 typing
  • HBV Real Time PCR, Quantitative
  • HCV Real Time RT-PCR, Qualitative
  • HCV Real Time RT-PCR, Quantitative
  • HCV Genotyping
  • HIV-1 Real Time RT-PCR, Quantitative

Detection of Sexually Transmitted Infections

1. PCR for N.Gonorrhoea, Chlamydia Trachomatis, Mycoplasma Genitalium, and Trichomonas vaginalis

Neisseria gonorrhoeae, Chlamydia trachomatis, mycoplasma genitalium, Trichomonas vaginalis are the most common pathogens that cause sexually transmitted bacterial infection (STI). The organism causes genitourinary infections in women and men and may be associated with dysuria and vaginal, urethral, or rectal discharge and many times do not produce any symptoms initially. In women, complications include pelvic inflammatory disease, salpingitis, and infertility. Approximately 25% to 30% of women who develop acute salpingitis become infertile. Complications among men are rare, but include epididymitis and sterility. PCR is the most sensitive and specific method for the detection of these pathogens and is now recommended for diagnosis.

 

Sample Materials

  • Urethral/Vaginal swab (after removing excess mucus)
  • Scrapings of epithelial cells
  • Saliva
  • Semen
  • Prostatic fluid
  • Urine (Patient should not have urinated for at least 1 hour prior to specimen collection. Collect random first part of stream.)

 

Test Code: 9739

 

2. HIV-1 Real Time RT-PCR

 

Specimen Type: Whole blood/Plasma EDTA (when transportation time >6 hours centrifuge whole blood with EDTA and separate plasma and send to us only plasma)

Collection Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

 

Additional Information: This test can be used for detection and diagnosis of HIV-1 infections, including in children <18 months of age when serologic tests are not useful (due to presence of maternal HIV antibodies).Currently, there are 2 types of HIV; HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1 has been isolated from patients with AIDS, AIDS-related complex, and asymptomatic infected individuals at high-risk for AIDS. Accounting for >99% of HIV infection in the world, HIV-1 is transmitted by sexual contact, by exposure to infected blood or blood products, from an infected pregnant woman to fetus in utero or during birth, or from an infected mother to infant via breast feeding. HIV-2 has been isolated from infected patients in West Africa and it appears to be endemic only in that region. However, HIV-2 also has been identified in individuals who have lived in West Africa or had sexual relations with individuals from that geographic region. HIV-2 is similar to HIV-1 in its morphology, overall genomic structure, and ability to cause AIDS.

Test Code: 7256

 

 

3. HSV 1/2 Real Time PCR

 

Specimen Type: Genital (for CMV only)

Sources: Cervix, vagina, urethra, anal/rectal, other genital sources

Container/Tube: Culture Swab

 

Specimen Type: Fluid

Sources: Spinal, body, amniotic, ocular

Container/Tube: Sterile container

Specimen Volume: 0.5 mL

 

Test Code: 6353

Cervical Cancer Screening

Cervical cancer is the third most prevalent cancer in women around the world, and the fourth highest cause of women’s cancer-related deaths. The link between cervical cancer and human papillomavirus (HPV) has become clear over the past few decades—today we know that persistent infection with specific types of HPV account for nearly all cases of cervical cancer.
Because cervical cancer rarely causes overt symptoms in its early stages—when treatment is most effective—screening for HPV infections at the greatest risk of progressing to cervical pre-cancer and cancer is imperative. 14 high-risk HPV types are detected in nearly 100% of cervical cancers.Of these, two types are associated with 70% of cervical cancers. Type 16 causes approximately 55% to 60% of cervical cancer cases, and type 18 accounts for 10% to 15%. Real time PCR is the most sensitive and specific method for the detection of HPV.

  • HPV Real Time PCR(High Risk Group)
  • HPV 16/18 typing
  • HPV 6/11 Real Time PCR for wart

Diagnosis of Immunodeficiency at cellular level by Flow Cytometry

These services are mainly focused for diagnosis of Immunodeficiency at cellular level which was not possible before due to not use of flow cytometry.
Perhaps, primary or even secondary immunodeficient patient in the country is not negligible as like India. You will be surprised to know about Institute of Immunohematology, Bombay where huge work is going on in this field and they are pioneer to utilize these kind of services. Certainly, these services are available in all developed country.

Service Code Service Name Tariff
10525 T and B cell ratio, Flow Cytometry 5,000
10526 CD4/CD8 ratio, Flow Cytometry 5,500
10527 T and B cell count –absolute, Flow Cytometry 6,000
10528 CD4 and CD8 count -absolute, Flow Cytometry 6,000

USAGES
• Useful for diagnostic evaluation and follow-up of primary cellular immunodeficiency’s, including severe combined immunodeficiency
• Serial monitoring of CD4 T cell count in HIV-positive patients
• T-cell immune monitoring following immunosuppressive therapy for transplantation, autoimmunity, and other immunological conditions where such treatment is utilized
• Assessment of T-cell immune reconstitution post hematopoietic cell transplantation
• Early screening of gross quantitative anomalies in T cells in infection or malignancies
• Evaluating lymphocytoses of undetermined etiology

WHEN TO SUSPECT PRIMERY IMMUNODEFICIENCY (PI)
• Four or more new ear infections within 1 year
• Two or more serious sinus infections within 1 year
• Two or more months of antibiotics with little effect
• Two or more pneumonias within 1 year
• Failure of an infant to gain weight or grow normally
• Recurrent, deep skin or organ abscesses
• Persistent thrush in mouth or fungal infection on skin
• Need for intravenous antibiotics to clear infections
• Two or more deep seated infections including septicemia
• A family history of PI

SPECIMEN: Whole blood or Bone Marrow with EDTA/Heparin – single tube
TAT: 3 days (maximum)

HLA related tests

  • HLA B27 DNA Detection by Real Time PCR

HLA-B27 is present in approximately 89% of patients with ankylosing spondylitis, 79% of patients with Reiter syndrome, and 42% of patients with juvenile rheumatoid arthritis. However, lacking clinical features and other data, it is not diagnostic for these disorders.
Approximately 8% of the normal population carries the HLA-B27 antigen.

Chikungunya/Dengue Detection by RT-PCR

  • Chikungunya/Dengue Detection by RT-PCR

Dengue and Chikungunya are transmitted by the same type of mosquito (aedes) and have similar symptoms.Therefore it is very important to find out whether the suspected disease is Chikungunya or Dengue before beginning the treatment. To address this issue Apollo Hospitals Dhaka is pleased to announce the starting of “Detection and differentiation of Chikungunya virus and Dengue virus FROM THE FIRST DAY OF FEVER by reverse transcription polymerase chain reaction (RT-PCR)”. There is no need to wait for week to develop antibody which people check by ELISA or ICT method.

Specimen need: Whole blood in plain or EDTA tube
Turnaround time (TAT): 1-3 days Service
Name: Chikungunya/Dengue Detection by RT-PCR
Kit is from Europe, CE-IVD approved.
Tariff: 5,000 Taka

Frequently asked questions (FAQ)

What is PCR testing?
The polymerase chain reaction (PCR) is a biochemical technology used to amplify a specific DNA target in a test tube. In other words, it is a technology that both can find a “needle in a haystack” and then make billion of identical copies of the “needle”. After the PCR reaction, the amplified DNA can be visualized and analyzed by agarose gel electrophoresis. A standard thermal cycler instrument is needed to do the PCR test.

What is Real Time qPCR testing?
During quantitative PCR (qPCR) amplification, fluorescent light is generated and monitored by the PCR instrument. The PCR cycle number where the fluorescent light crosses a set threshold level correlates to the amount of gene target in the sample. Therefore, the qPCR reaction do not need an agarose gel electrophoresis analysis after the qPCR reaction. This makes qPCR tests easier and faster compared to conventional PCR tests. A real-time thermal cycler instrument is needed to do qPCR test.
Today, PCR and qPCR are common diagnostic techniques and they are used for a wide variety of applications.

What Is Flow Cytometry?
Flow Cytometry is a technology that simultenously measure multiple characteristics of a single cell at a rapid rate. Flowcytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasm. Myeloid lineage will be identified by CD13, CD33, CD117 and MPO. B lineage cells will be identified by CD10, CD19, CD20 and CD79a, T lineage cells will be identified by CD3, CD4, CD5, CD7, CD8. Monocyte will be identified by CD14, CD64 and NK cell by CD56. Blast cell (stem cell) will be identified by CD34. Activation status will be identified by HLA-DR. Plasma cell will be identified by CD38, CD138 and clonality will be by anti-light chain antibodies kappa, lambda. Pan-Leukocyte marker is CD45.
These services will be the first standard routine practice in the country and will serve as essential service for stem cell transplantation. Molecular Lab Consultant was familiar in flowcytometric immunophenotyping while he was in Japan and he was in charge of Flowcytometer Unit of Equipment Center of Yamagata University Faculty of Medicine for 2009-2011.

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