Clinical Immunogenetics Laboratory

Clinical Immunogenetics Laboratory Services

HLA typing of patients, family members, and potential related and unrelated or cord blood donors prior to allogeneic hematopoietic stem cell transplant (HSCT) under research protocols.
Request HLA Typing at Fred Hutch
HLA Requisition
HLA Specimen Drawing and Storage Requirements
Chimerism Testing
Evaluation of patient alloimmune status and antibody sensitization prior to HSCT.

Evaluation of previous HLA typing for the Fred Hutchinson Cancer Center Clinical Coordinator Office prior to consult appointments (includes review of CIL and outside lab typing).
Review and advice on HLA linkage for the Unrelated Donor Program to define efficient search strategies for unrelated donor/patient workups.
Chimerism testing of patients after HSCT (and for certain specific protocols before transplant) to determine recipient/donor origin of nucleated hematopoietic cells.

Description of HLA

HLA stands for “Human Leukocyte Antigen.” These antigens are protein molecules we inherit from our parents. Together, these molecules make up your HLA type. We currently know about more than 2500 different HLA molecules. It is very important to determine your HLA type before you have a stem cell transplant, and we can do this by taking a sample of your blood or other body tissue cells.

HLA testing will also determine the HLA type of anyone who may donate stem cells to you. It is important in stem cell transplants to see how closely the HLA type of the transplant patient matches the HLA type of the stem cell donor. The HLA “match” is the number of HLA molecules that any two people have in common for stem cell transplantation. HLA matching is usually based on 10 HLA molecules. The more molecules two people share, the better the match. When two individuals share the same HLA molecules, they are said to be a good “match.” That is, their immune systems will not see each other as “foreign” and are less likely to attack each other.

The most likely place to find an HLA match between two people is among siblings (that is, brothers and sisters who have the same mother and same father). If two siblings inherit the very same HLA molecules from both parents, they are said to be an “HLA identical match.”

You have a 25 percent (1 in 4) chance of being an HLA identical match with your sibling. Why? Because there is a basic rule in HLA inheritance: you have a 25 percent chance of inheriting the same HLA molecules as your sibling, a 25 percent chance of inheriting none of the same HLA molecules as your sibling, and a 50 percent chance of inheriting half of the same HLA molecules as your sibling.

However, two unrelated people can just happen to be a good HLA match, too. Although it is less likely, it is possible that you could have some of the same HLA molecules as someone you don’t even know.

When a doctor decides that a stem cell transplant is the best treatment for a patient, the patient, all of his or her siblings, and sometimes their parents will have samples collected for HLA typing. If one of the family members is an HLA identical match, the lab will do further testing to be absolutely sure that they are the best match possible.

If none of the siblings are a good HLA match, the doctor will sometimes ask to have additional family members tested. Since your HLA type is inherited from your parents and passed on to your children, among your relatives, your parents and children have the next best chance of being closely HLA matched with you. If it happens that there are no close HLA matches within the patient’s family, an unrelated search can be initiated in order to find an unrelated volunteer donor with the same HLA molecules as the patient.

HLA antibodies are proteins that may be present in the patient’s blood, which could interfere with the success of the transplant. If the stem cell donor is not an absolutely perfect match, HLA antibodies can attack the donated stem cells and may make the patient’s body reject them. The patient may also need to have transfusions of platelets during the recovery period. HLA antibodies can interfere with platelet transfusions by killing the donated platelets before they have a chance to work. That is why patients are tested for HLA antibodies before a transplant.

Some people do not develop HLA antibodies at all, while others do. It is not clearly understood why this is so, but people are most likely to develop HLA antibodies from pregnancies, prior blood or platelet transfusions, or organ transplants.

The HLA antibody level is referred to as the PRA (Panel Reactive Antibody). The PRA tells us what percentage of the general population the patient has HLA antibodies to, and can give us a good idea about how easy or difficult it will be to find a compatible platelet donor for this patient. In addition to measuring how much or how little PRA a patient has, we can also test if a patient has antibody to a particular HLA molecule. Some patients have antibody to one or two HLA molecules, while others have antibodies to many HLA molecules and therefore have a higher PRA.

HLA: Human Leukocyte Antigen, proteins we inherit from our parents, found on the surface of cells, also called histocompatibility or tissue antigens.

HLA Matching: Comparing the number of HLA molecules (antigens) two people have in common.

Haplotype: a set of HLA antigens that are inherited from each parent.

Molecular HLA typing: an HLA typing that was determined by using DNA to look at the gene that codes for the protein found on the surface of the cells.

Serologic HLA typing: an HLA typing that was determined by looking at the reactivity of the HLA molecule on the surface of the cells with known antibodies.

HLA Antibodies: proteins in the patient’s blood directed against the donor’s HLA that could attack transplanted or transfused cells.

PRA: Panel Reactive Antibody: a measure of how much HLA antibody the patient has.

Crossmatch: a laboratory test that looks for patient antibodies directed against a potential donor’s cells.

Stem Cell: cells responsible for generating red blood cells, white blood cells, and platelets.

Bone Marrow: the inner spongy portion of large bones, where most stem cells reside.

Graft versus Host Disease (GVHD): an immune attack by transplanted donor cells against the patient’s body.

Host versus Graft (HVG): an immune attack by the patient’s cells (host) against the donated (transplanted) cells (graft), can lead to the patient's rejection of the transplant.

Request HLA Typing

We typically receive HLA typing requests from treating physicians, for example, your primary oncologist. While we can certainly discuss HLA typing with you directly, we recommend that your physician be involved to work with us.

Upon receiving your request, our Intake department will work with your local physician to determine three things:

What is the clinical urgency? This is based on your clinical condition.
What is your treatment plan?
Is searching for an alternate donor being considered if there is no family match?

After determining the items above:

We contact you to explain the HLA typing process and collect insurance and family member information.
We contact your insurance carrier for financial clearance and communicate financial issues to you to ensure you are aware of any financial implications from HLA typing.
If we need to discuss altering the HLA typing strategy based on any of the information collected, our Clinical Coordinator Physician will contact your local physician and/or you.
Our Clinical Immunogenetics Laboratory (CIL) will send instructions on how to collect and send specimens for HLA typing to your local physician.

If you want to discuss HLA Typing, feel free to contact our Intake department. The Intake department hours of operation are Monday through Friday from 8:30 am to 5:00 pm (PST) and our main phone number is 800.804.8824 or 206.606.1024.

Tests We Offer

The primary purpose of HLA testing is to identify suitable donors for patients being considered for hematopoietic cell transplantation (HCT). List of histocompatibility tests performed (PDF).

Potential Donors

The primary purpose of HLA testing is to identify suitable donors for patients being considered for hematopoietic cell transplantation (HCT). Potential donors can be categorized as follows:

HLA Identical Sibling (“Genotypic Match”)

The ideal donor is a healthy sibling who has inherited the same HLA determinants as the patient. The HLA genes reside on chromosome 6 within the major histocompatibility complex (MHC). Five HLA genes are defined for matching purposes (A, B, C, DRB, and DQB) and are usually inherited as a block or “haplotype.” HLA-A, B, and C are referred to as Class I genes and HLA-DRB and DQB are referred to as Class II genes. Any two full siblings have a 25% chance of inheriting the same HLA genes (genotypic match) because they have inherited the same two haplotypes from their parents.

HLA Phenotypic Match

Because some HLA alleles are common in certain populations, there may be a family member who has the same HLA alleles as the patient, despite having inherited one different haplotype. In this case, patient and the phenotypically matched donor would have the same HLA type, but may differ for other so called minor histocompatibility antigens.

HLA Haplotype Match

A patient’s parent or child will be a haplotype match with the patient because they will share one haplotype due to inheritance. The other unshared haplotype may, due to chance, carry one or more HLA alleles in common with the patient, but usually the unshared haplotype will carry different HLA alleles.

HLA Matched Unrelated

An unrelated donor (URD) who has the same HLA alleles as the patient at HLA-A, B, C, DRB, and DQB. If a patient cannot find a suitable family donor, an unrelated search will attempt to identify a fully matched URD for 10 out of 10 HLA alleles.

HLA Mismatched Unrelated

An unrelated donor who is mismatched for one, two, or even three HLA alleles.

Unrelated Umbilical Cord Blood

Frozen umblicial cord blood (UCB) units are an alternative source of hematopoietic cells. Cord blood units are typed to the allele level at HLA-DRB and to an intermediate (antigen) level at HLA-A and B but are not typed at HLA-C and DQB.

High Resolution/Allele Level Typing/Sequencing. The gold standard for HLA typing is high resolution molecular typing at the allele level. This test determines the exact DNA sequences at the HLA-A, B, C, DRB1, and DQB1 loci. DNA is isolated from nucleated cells (peripheral blood lymphocytes or, in some cases, buccal swabs) and the DNA within the HLA region is amplified using PCR (polymerase chain reaction) methods. This amplified DNA is then sequenced and analyzed using sophisticated software to identify the HLA alleles encoded. Allele typing is reported using four or more digits preceded by an asterisk (HLA-B*4501, for example). Depending on the HLA alleles involved, there may be ambiguous combinations of alleles. If this is the case, a second amplification and sequencing assay is required to remove the ambiguity.
Medium Resolution Level Typing. Medium resolution molecular typing also uses DNA and PCR methods, but does not determine an exact DNA sequence at HLA-A, B, C, DRB1, and DQB1. Instead it takes a more general approach using a panel of DNA probes to identify groups of HLA alleles. Such probes are called “sequence specific oligonucleotide probes” or SSOPs. A medium resolution result is usually expressed as a number followed by a letter code (B*45CKB, for example). The letter code is translated in the body of the report (e.g. B*45CKB = B*4501/*03/*07 means that the sample could be either a B*4501, a B*4503, or a B*4507). Medium resolution typing is faster and cheaper than sequencing, and thus is used to screen potential donors. Donors who are matched with the patient by medium resolution typing can then be tested by sequencing.
Serological Typing/Antigen Level Typing. Serology (or microcytotoxicity) is the original HLA typing method. This method requires intact viable cells (usually lymphocytes) that express HLA antigens on their cell surface. These cells are incubated with known HLA antisera and then treated with complement to create a pattern of cytotoxicity. By interpreting this pattern, the HLA antigens present on the cells can be deduced. Serological results are not preceded by an asterisk (“B45” in our example).
Antibody Screening (“PRA”). Another traditional test, PRA (for “Panel Reactive Antibody”) is a general measure of a patient’s antibody status regarding anti-HLA antibodies (or alloantibodies). Patient sera is tested against a panel of cells expressing a diverse set of HLA antigens. A patient who has never been pregnant and has never had a transfusion or transplant should not have any circulating alloantibodies; this patient’s serum would not react against any of the cells in the panel and would have a PRA of “0%.” Patients who have undergone multiple transfusions may have many anti-HLA antibodies in their circulation –- their sera will react with many of the cells on the standard panel and so could have a high PRA (70 – 100%).
Crossmatch Studies. Crossmatch tests are designed to identify specific alloantibodies directed against a particular donor. These donor specific antibodies may be directed against Class I molecules (detected in T or B cell crossmatches) or against Class II molecules (detected in B cell crossmatches). In addition, the alloantibodies may be further characterized as IgG or IgM in flow cytometry crossmatches.

Samples received in CIL must be accompanied with an Fred Hutch requisition. (PDF)

A. New Patient Initial Typing (No Prior Typing):

1) Patients without prior typing, their full siblings, or other potential family donors are typed in a two step process:

a) HLA-A, B, and DRB by medium resolution. If a family member is matched, then HLA-C and DQB by medium resolution and HLA-DRB1 by high resolution.

b) If no family match is found and the clinician plans to proceed with an alternate donor search, then full typing on the patient (HLA-A, B, C, and DQB by medium resolution and HLA-DRB1 by high resolution) may be requested.

B. Recommended Testing Following Review of Previous Typing (Prior Typing done by an outside laboratory)

If HLA testing if requested for a patient with prior HLA typing, test plans are determined on a case-by-case basis following a review of the existing HLA typing results and acceptability of the existing results for the treatment plan being considered at Fred Hutch (allo transplant, URD search, etc). Usual reasons for requesting HLA typing to be done by CIL:

To confirm reported HLA match between patient and reportedly matched family member(s)
To complete incomplete typing of patient (for example, only HLA-A and B typing results available) to be able to predict availability of potential unrelated donors
To confirm patient’s reported type and HLA type family members that have not yet had HLA typing completed
To update reported HLA typing results with molecular-based typing methods
To resolve reported discrepancies and inconsistencies with external HLA typing results

C. Modified Typing of Patients or Family Members

For some patients, circumstances may require modification of either the Standard Typing Plan (No Prior Typing) or the Recommended Typing Plan (Prior Typing).

Examples of circumstances include (1) expedited typing needed in cases of clinical urgency, (2) unusually large immediate family available, or (3) exceptional financial considerations present. For each patient, following the collection of family information and determination of HLA typing benefits, the CCO/Transplant Coordinator will determine if modified typing is needed. If modified typing is needed, details of typing to be completed are provided to CIL.

Chimerism Testing

The Clinical Immunogenetics Laboratory offers chimerism testing of patients after HSCT (and for certain specific protocols before transplant) to determine recipient/donor origin of nucleated hematopoietic cells.

Chimerism Testing/Engraftment Analysis

A Chimera was a creature in Greek mythology usually represented as a composite of a lion, goat, and serpent. Contemporary use of the term “chimerism” in hematopoietic cell transplant derives from this idea of a “mixed” entity, referring to someone who has received a transplant of genetically different tissue. A test for chimerism after a hematopoietic stem cell transplant involves identifying the genetic profiles of the recipient and of the donor and then evaluating the extent of mixture in the recipient’s blood, bone marrow, or other tissue.

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 tandemly 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 codominant 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 material 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 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 a fragment analysis software which sizes the DNA fragments using an internal lane standard run with the sample and assigns genotypes by comparison to an STR allele ladder included in the CE run. This provides distinct STR genotypic profiles for the donor and for the 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.

Samples tested may be from any material containing DNA, including bone marrow, peripheral blood, solid tumors, epidermal tissue, hair follicles, buccal swabs, and fractionated cell subsets. Since PCR amplification of a sample is routinely performed with less than 2 ng of genomic DNA (equivalent to approximately 300 cells), chimerism testing by this method can be successfully performed even for patients with graft failure, severe leukopenia, or from hematopoietic cell subset fractions. 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; as well as to confirm the genetic identity of putative identical twins, and to detect in-utero derived maternal cell engraftment among patients with a diagnosis of Severe Combined Immunodeficiency (SCID). STR/CE analysis is a rapid, reliable, accurate and reproducible procedure.

Limitations of the Assay

Sufficient DNA must be isolated from the test samples to allow robust PCR amplification. DNA isolation methods must be available to handle low cell count samples, such as might be encountered in recipients with graft failure and from Flow Cytometry lineage specific sorted white blood cell subsets. Samples may have concentrations too low to quantify by UV Spectrophotometry OD260. DNA samples from 5,000 to 30,000 isolated cells reliably provide acceptable amplification. The laboratory has guidelines that define when sample analysis should be repeated.
With the commercial kits, many STR loci have informative alleles for both donor and recipient in the unrelated donor setting. However, the number of informative STR loci may be limited to as few as 3 among related donor transplant pairs. Quantitative values representing the mean of as few as 3 STR loci have been found to be reproducible.
Patients with malignant diseases may have clonal mutations that impact certain STR loci. A patient allele may be absent at one or more STR loci when comparing the alleles identified in the patient pre-transplant sample versus the post-transplant sample. In rare circumstances, an extra patient STR allele may be detected at a specific locus that was not present in the pre-transplant sample. In most cases these anomalies are likely due to chromosome translocations. Missing alleles could also be the result of a mutation within the STR conserved flanking sequences where the PCR primers are located. The data from STR loci with missing or extra alleles is not used for quantification.
If patient pre-transplant cells are not available, samples such as buccal swabs, skin biopsy or hair roots may be collected post-transplant. However, it should be noted that buccal samples may contain significant amounts of donor cells.
The assay is not intended for the detection of minimal residual disease.

Clinical Indications for Chimerism Testing in Hematopoietic Cell Transplant

Routine post-transplant documentation of the donor/recipient origin of white blood cells in peripheral blood and/or marrow. Documentation of engraftment may include testing lineage-specific cell subsets, such as CD3 positive T-cells and CD33 positive myeloid cells.

Evaluate donor/recipient cells in patients with inadequate marrow function.
Define whether recurrent or new malignancy has originated from recipient or donor cells.
Assess prognostic risks of rejection and recurrent malignancy.
Document the persistence of donor cells post-transplant in patients with recurrent disease or prior to donor lymphocyte infusion (DLI).
Evaluate whether graft rejection has occurred in recipients that are candidates for a second transplant.
Differentiate the origin of donor cells in recipients who have received a second transplant with a different donor or a transplant with double cord blood units.
Detect the presence of maternal derived cells in patients diagnosed with Severe Combined Immuno-Deficiency (SCID).
Verify genetic identity of putative identical twins.

Frequently Asked Questions

It is essential to identify STR loci that show at least one unique STR allele for the patient and each donor.

Patients diagnosed with a Severe Combined ImmunoDeficiency (SCID) may have become engrafted with hematopoietic cells of maternal origin in-utero. Certain lineage specific cell subsets (especially CD3-positive cells) may be predominantly or entirely of maternal origin.

“Full” chimerism is used to refer to a patient which exhibits a post-transplant phenotype in hematopoietic cells that is all of donor origin.
“Mixed” chimerism refers to a patient which exhibits a mixture of patient and donor phenotype in hematopoietic cells after transplant.

Several options are available for obtaining a recipient sample post-transplant: buccal wipe, hair root, or skin biopsy samples can be used for a recipient baseline sample. Buccal wipe samples should be used with caution since donor cells may be present in significant quantities in buccal samples.

If the donor is living, obtain a new blood sample. If the donor is not living, post-transplant samples should be compared with the patient pre-transplant baseline to identify STR alleles that are detected but are not of patient origin.

Transplant donors are chosen to be as closely matched to the recipient as possible. There are no HLA markers differentiating the recipient and the donor when they are matched, and only one or two if they are mismatched. Additionally, other technologies are often more sensitive for this purpose than monitoring HLA mismatched alleles; consequently, other loci are used to provide unique profiles.

This is a locus with at least one allele unique to the recipient or the donor. To be useful for chimerism calculations, a locus must have alleles unique to both. There may be a large number of informative loci when an unrelated donor is used for the transplant, but very few if a matched sibling is used. Because of differences in amplification efficiencies of the alleles at each locus, it is preferable to get mean or median values from several loci to enhance accuracy. Results from individual loci are reliably reproducible, so even one locus can be used for trending sequential samples.

The amelogenin gene is located on the X and Y chromosomes. It is not an STR, but displays different sizes of products on the respective chromosomes. This makes it useful for determining gender. It is included in the primer panel by commercial kit vendors targeting the forensics community where X/Y differentiation is used extensively. It is generally not used for chimerism analysis since it can’t provide a unique female marker (a male sample always includes an X allele); nor is it helpful when evaluating samples after a sex-matched stem cell transplant.

“Genotype” refers to the particular alleles present in a specified locus. Genotyping is done to establish recipient and donor profiles in chimerism testing. When chimerism analysis is done after a stem cell transplant, the genotypic profiles of the recipient and of the donor are compared to the post transplant sample profile to evaluate how much of each component is present.

Genotyping is also used in other scenarios such as forensics and parentage testing. Forensic analysts use genotypes to identify the source of evidence in criminal cases and to exclude people from consideration as suspects. They also can identify human remains in “John Doe” cases and in mass disasters. Parentage determinations are done in a similar fashion to exclude someone as a possible parent by finding alleles present in the child that don’t match either the mother or the putative father.

Literature Cited/Recommended Reading

Bryant E, Martin PJ. Documentation of engraftment and characterization of chimerism following hematopoietic cell transplantation. In: Forman S, Blune K, Thomas ED, eds. Hematopoietic cell transplantation. 2nd ed. Malden, MA: Blackwell Science, 1998.

Bultler J. Forensic DNA typing. Elsevier Academic Press.

Clinical Immunogenetics Laboratory