Methodologies

Complement-Dependent Lymphocytotoxicity

A complement-mediated assay commonly used in serum screening of anti-HLA antibodies and cross-matching which tests for cytotoxic antibodies in the serum of a potential recipient that may react with the lymphocytes of a potential donor. It is used to identify patients at risk for antibody-mediated damage to grafted organs or tissue.

Luminex Bead-based Multiplex Assay

Antibody profiling by solid phase assays is a bead-based, multiplexing technology designed for sensitive and accurate identification and monitoring of anti-HLA antibodies in patients’ serum samples.

Molecular HLA Typing

PCR-SSO

Reverse SSO hybridization is used to determine HLA-A, -B, -C, -DR, -DQ and -DP locus types at an intermediate level of resolution, somewhat higher than serological testing. Tests of this type are used when low or intermediate resolution typing is required or as a screening test to identify potential donors or individuals who may later require higher resolution testing.

PCR-SSP

PCR-SSP is used to determine HLA-A, -B, -C, -DRB1, DQB1 and DPB1 alleles at a high-resolution.

BD™ Stem Cell Enumeration Kit

The BD™ Stem Cell Enumeration Kit offers a single tube, single platform assay for accurate, reproducible and rapid enumeration of CD34+ hematopoietic and progenitor cells in line with ISHAGE-based protocol guidelines. The kit works with a wide range of stem cell specimens including normal and mobilized peripheral blood, fresh and post-thaw apheresis products, fresh and post-thaw cord blood, fresh and post-thaw bone marrow samples. The kit includes BD Trucount™ technology to improve test accuracy, eliminating the need for manual pipetting of counting beads. The kit uses one test tube per sample and does not require an isotype control, improving operational efficiency.

Flow Cytometry

Flow Cytometry measures properties of cells in a sample of bone marrow, lymph nodes, or blood. The sample is first treated with special antibodies and passed in front of a laser beam. If the antibodies attach to the cells, the cells will give off light. Looking for certain substances, or antigens, on the surface of cells helps us to identify the cell type.

In Vitro Culture

Using in vitro lymphocyte tissue culture, a heart biopsy fragment is placed into a sterile culture well in medium supplemented with recombinant IL-2 at5 units/ml.

Lymphocyte proliferations are examined visually at 24 and 48 hours with an inverted-phase microscope.

Short Tandem Repeat (STR)

Chimerism testing (engraftment analysis) by DNA employs methodology commonly used in human identity testing and is accomplished by the analysis of genomic polymorphisms called short tandem repeat (STR) loci. These loci consist of a core DNA sequence that is repeated a variable number of times within a discrete genetic locus. The term STR, also referred to as microsatellites, relates to the number of base pairs of a tandem repeated core DNA sequence which ranges from 2-8 base pairs in length. These loci exhibit alleles that may differ in length between individuals and are inherited as co-dominant Mendelian traits. STR loci have been identified throughout the human genome and some loci have more than 25 alleles. DNA sequence information within the conserved flanking regions of the loci is used to create oligonucleotide primer pairs for the STRs. These primers are used in PCR (polymerase chain reaction) amplification of test samples.

This technique can amplify the STR sequence as many as a billion times, providing PCR amplicons that can be separated with an electrophoretic gel or by capillary electrophoresis (CE). Genotyping is done by evaluation of the DNA fragment sizes. Reference to an allelic ladder may be used for exact identification of STR alleles.

The PCR-based STR/CE system has several advantages over other methods of analysis. The amplification of multiple STR loci can be combined (multiplexed) in a single tube, permitting analysis of up to sixteen loci in one reaction. Since minute amounts of DNA are required, samples with low cell numbers can be used, and the small size of the STR alleles even makes it possible to use degraded DNA samples. The digital data facilitates analysis and archiving, and the CE process is both fast and cost-effective. PCR amplification and analysis of STR loci provides a rapid and reliable method for the evaluation of engraftment status in the hematopoietic stem cell transplantation setting.

Currently used technology allows the co-amplification and three-color detection of sixteen loci which are subdivided into 3 sets of 5 or 6 loci that exhibit amplified fragments with non-overlapping size ranges.

During the PCR amplification step, the amplified fragments are labeled with fluorescent dyes. After PCR amplification, the samples are processed on a capillary electrophoresis (CE) system. Data analysis is facilitated by fragment analysis software which sizes the DNA fragments using an internal DNA fragment standard included in the sample CE run. This provides distinct STR genotypic profiles for the donor and pre-transplant recipient. STR loci that are polymorphic (i.e., informative) between these individuals are used to assess relative amounts of recipient and donor DNA in the post-transplant sample.

STR analysis has been used to evaluate the engraftment status of patients who have received a hematopoietic cell transplant including patients receiving double cord blood donor units or a second transplant from a different donor. STR-PCR /CE analysis is a rapid, reliable, accurate and reproducible assay.