Laboratory for Viral Immune Pathogenesis-Clinical Monitoring (LVIP-CM)

The Laboratory for Viral Immune Pathogenesis (LVIP) studies the pathogenesis of viral infections, in particular HIV infections.

 We perform both fundamental and patient-related research under supervision of Dr. N.A. Kootstra (Head LVIP).

Within our Clinical Monitoring division we apply and develop laboratory tests for the diagnosis and follow up of acquired immunodeficiency disorders (mainly HIV/AIDS) . We also provide high quality laboratory support for pharmaceutical studies and vaccine trials. This includes viable freezing and cryopreservation of peripheral blood mononuclear cells.

Over more than 25 years we have acquired vast experience in immunology and virology projects and have an excellent track record for storage and retrieval of cryopreserved samples as part of the Amsterdam Cohort Studies on HIV infection and AIDS and for third parties when additional immunology and/or virology research is required after completion of a trial. We are imbedded in an academic setting allowing direct interaction with highly reputable experts in the fields of virology and immunology.

The laboratory where the samples are handled

Sample processing and cryopreservation

  • Peripheral blood mononuclear cells (PBMC) are isolated from whole blood samples by Ficoll gradient density centrifugation. PBMC are cryopreserved using a Sy-Lab IceCube controlled rate freezer and subsequently stored in liquid nitrogen vapor phase in CryoSolutions MVE 1800 series cryostats.
  • Plasma and/or serum is isolated from whole blood by centrifugation and stored at -80°C in Sanyo MDF series upright freezers.
  • Cerebrospinal fluid and urine samples are centrifuged to remove cell debris and stored at -80°C in Sanyo MDF series upright freezers.
  • Stool samples are homogenized in RNAlater® and stored at -80°C in Sanyo MDF series upright freezers.

Immunological tests

  • Enumeration of mature human T, B, and natural killer (NK) lymphocytes. Using a Becton Dickinson FACSCanto II we enumerate mature human T (CD3+), B (CD19+), helper T (CD3+CD4+), cytotoxic T (CD3+CD8+), and NK (CD3-CD16+ and/or CD56+) lymphocytes.

    • CD3+ T lymphocytes are a subset of lymphocytes defined by their development in the thymus and by heterodimeric receptors associated with the proteins of the CD3 complex. T cells carry out a variety of functions in an immune response, acting always by interacting with another cell in an antigen-specific manner.
    • CD3+CD4+ helper T lymphocytes activate macrophages and help B cells produce antibody.
    • CD3+CD8+ cytotoxic T lymphocytes kill cells infected with viruses and other intracellular pathogens.
    • CD19+ B lymphocytes are one of the 2 major types of lymphocytes. The antigen receptor on B lymphocytes, usually called the B-cell receptor, is a cell-surface immunoglobulin. On activation by antigen, B cells differentiate into cells producing antibody molecules of the same antigen specificity as this receptor.
    • CD3-CD16+ and/or CD56+ NK cells are large granular, non-T, non-B lymphocytes, which kill virus-infected and some tumor cells. They bear a wide variety of invariant activating and inhibitory receptors, lack antigen-specific receptors. NK cells are important in innate immunity to viruses and other intracellular pathogens, and in antibody- dependent cell-mediated cytotoxicity (ADCC).

Reference value (5-95%) of lymphocyte subpopulations in HIV negative controls with high risk behaviour

absolute number *10 E9/LTotal lymphocytes1,22 - 3,28CD3+ (T cells)0,83 - 2,49CD4+ (T helper cells)0,49 - 1,54CD8+ (cytotoxic T cells)0,21 - 1,02ratio CD4/CD80,89 - 3,99CD19 (B cells)0,09 - 0,50CD3-CD16+CD56+ (NK cells)0,09 - 0,50

percentage (%)CD3+ (T cells)62 - 84CD4+ (T helper cells)32 - 60CD8+ (cytotoxic T cells)14 - 40CD19 (B cells)5 - 21CD3-CD16+CD56+ (NK cells)5 - 22

  • Enumeration of activated human T lymphocytes. CD38 and HLA-DR monoclonal antibodies are used for enumeration of activated (CD38+ and/or HLA-DR+), helper (CD3+CD4+) and cytotoxic (CD3+CD8+) T lymphocytes.

  • Enumeration of naïve and memory human T lymphocytes. CD45RA and CD27 monoclonal antibodies are used for enumeration of naive memory T lymphocytes. Four subsets can be distinguished based on expression of CD45RA and CD27 antigens:

    • Naïve cells:CD45RA+/CD27+

    • Cytotoxic effector-type cells:CD45RA+/CD27-

    • Memory-type cells:CD45RA- /CD27+

    • Memory/effector-type cells:CD45RA-/CD27-

  • Analysis of human mononuclear cells for a custom set of differentiation, activation, and exhaustion markers. A combination of 8 different custom membrane markers can be analyzed using the Becton Dickinson FACSCanto II. Assays for custom analysis of membrane markers are developed for large cohort studies and will be validated according to CCKL standards.

  • Analysis of soluble marker of inflammation, immune activation or virus reactivation. A number of validated assays based on ELISA and PCR are available to test specific biomarkers associated with inflammation, immune activation or CMV reactivation in serum/plasma, cerebrospinal fluid and urine.

Virological tests

  • Determination of HIV-1 co-receptor use using the MT-2 cell line. MT-2 cells are of human origin, more specifically a CD4 positive T-cell lymphoma, which is continuously HTLV-1 infected. The express high levels of the C-X-C chemokine receptor 4 (CXCR4) and hence can be infected by HIV-1 variants that use this receptor alone (X4 variants) or in combination with CCR5 (R5X4 variants). Infection of MT-2 cells results in a cytopathic effect called syncytium formation (fusions between 2 or more cells) that can easily be detected microscopically. HIV-1 variants that infect MT-2 cells are thus scored as syncytium inducing (SI) variants. HIV-1 variants that do not infect MT-2 cells are scored as non-syncytium inducing (NSI) variants. The test is performed by direct co-cultivation of patient-derived peripheral blood mononuclear cells (PBMC) of with MT-2 cells. This culture is maintained for 4 weeks with twice weekly passage and microscopic observation after which the test result is reported.
  • Enumeration of the number of productively HIV-1 infected T cells using limiting dilution biological cloning. To determine the number of productively infected cells and to obtain a more complete picture of the diversity of the HIV-1 quasispecies present in an individual in vivo, a virus isolation protocol was developed that allows for the isolation of multiple variants from a single PBMC sample, avoiding overgrowth and loss of slowly replicating variants. The test is performed by limiting dilution co-cultivation of patient-derived PBMC with stimulated healthy donor PBMC in 96-wells plates. This culture is maintained for 4 weeks with weekly passage and testing of viral gag p24 production for each well. After passage, MT-2 cells are used to determine co-receptor usage for each virus producing well. This provides an exact estimate of the number of cells productively infected NSI and SI variants in the sample. Upon request, at the end of the culture individual clones can be expanded and cryopreserved for further characterization.

Host genetic testing

  • Determination of C-C chemokine receptor 5 (CCR5) genotype. HIV-1 susceptibility is most obviously determined by the CCR5 genotype and associated ß-chemokine production levels. The CCR5 gene encodes for the co-receptor for R5 HIV-1 variants. People who are homozygous for a 32 bp deletion in CCR5 - which results in a premature stop codon and the absence of functional CCR5 on the cell membrane - are more resistant to HIV-1 infection. HIV-1 infected individuals heterozygous for the 32 bp deletion experience a delayed disease progression compared to individuals carrying only the normal (wild type) alleles. The presence or absence of this 32 bp deletion is rapidly determined by polymerase chain reaction (PCR) amplifying the genomic region encompassing the 32 bp deletion and determining the length of the amplified region using agarose gel electrophoresis.

Sample Logistics

  • Sample submission forms: Each sample submitted for processing needs to be accompanied by a completed submission form with unique patient identification information, name and address of the person submitting the sample and the specific services requested. These forms can be provided upon request.

  • Sample collection and shipmentconditions:

    • Virological tests require whole blood with the anticoagulant heparin (7-10 mL, green cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 24 hours after blood draw.
    • Genetic testing requires whole blood with the anticoagulant EDTA (7-10 mL, purple cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 48 hours after blood draw.
    • Both anticoagulants can be used for whole blood samples submitted for immunological tests and cryopreservation, but samples must be kept at room temperature and arrive within 24 hours after blood draw.
  • Test results: Services can be performed on each work day. Turnaround times depend on the specific service in question, ranging from 1-2 work days for immunological tests to 4 weeks for virological tests. After quality checks and official review, the tests results are provided in writing to the person submitting the sample.
  • Privacy: Our Quality Assurance program includes privacy protection of submitted patient information.

Collaborations

The Laboratory of Viral Immune Pathogenesis has several longstanding national and international collaborations to study host and viral factors in the pathogenesis of HIV-1 infection. The diagnostics lab specifically interacts with:

Group Members

  • Dr. N.A. Kootstra (Head LVIP)

    • Ing. A.M. Harskamp (Manager LVIP-CM)

      • Ing. M.M. Mangas Ruiz (Technician)
      • Ing. I. Maurer (Technician)
      • Ing. A.F. Girigorie (Technician)
      • Ing. B.D.M. Boeser-Nunnink (Technician)