**4. The past and present of HLA typing**

HLA genes contain 5–8 exons ranging in length from 4 to 17 kb. Most high-resolution fourdigit HLA typing technologies, mainly sequence-based techniques (SBT) or probe-based hybridization techniques, primarily focus on deciphering the sequence of the antigen-binding groove only. This is due to the high cost of complete HLA genotyping and the limited time interval before HSCT. Thus, only exons 2 and 3 (540 bp) for class I molecules and exon 2 (270 bp) of class II molecules are typically analyzed, providing intermediate-resolution typing.

Before these techniques came into light, less informative serology methods were acquired for HLA typing. On the other hand, the future seems very promising thanks to the evolution of sequencing technologies [20].

Next, we will provide a brief review of evolution of the most widely applied techniques for HLA typing, frequently utilized by clinical laboratories.

#### **4.1. Serological methods**

#### *4.1.1. CDC (complement-dependent lympho-cytotoxicity) technique*

Lymphocytes incubated with polyclonal sera in the presence of complement, was the first attempt for determining patient-donor compatibility. CDC utilizes sera from multiparous alloimmunized women, whose HLA specificity (reactivity against a particular HLA type) is determined using a panel of lymphocytes of already known HLA type.

From a population of peripheral blood lymphocytes (PBLs), T lymphocytes are used for determining class I antigens, while B-lymphocytes (professional APCs) are separately isolated for determining class II antigens. The cells are incubated along with the characterized serum and complement. Their reactivity is determined based on the lysis of Ab-covered lymphocytes, from complement components, as shown in **Figure 2**.

**Figure 2.** Example of a serological reaction utilizing the CDC technique. The lysis of the lymphocytes by a serum containing anti-HLA B35 Abs denotes the HLA-B35 positivity of the cells.

The low-resolution two-digit serologic typing this technique offers is further limited by the availability of sera containing various HLA specificities. However, this method has value in confirming the presence or absence of an antigen in case of mutations in promoter regions or genes not otherwise analyzed [5, 18, 21].

A variation of the above technique involves incubation of the donor's cells with serum from the patient in the presence of complement. The results are interpreted the same way as previously described.

Both techniques rely on cell viability in order to be successful and accurate. Also, cell populations need to be lymphocyte-specific so that the results be interpretable. Samples contaminated by other lymphocytes and/or precursor cells lead to inaccuracies [22].

#### *4.1.2. Other serological techniques*

**3.6. Seeking for permissive (relatively well-tolerated) HLA mismatches**

52 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

related mortality, delayed immune reconstitution, but not disease relapse.

sequences, regarding the protein's function.

**4. The past and present of HLA typing**

HLA typing, frequently utilized by clinical laboratories.

from complement components, as shown in **Figure 2**.

*4.1.1. CDC (complement-dependent lympho-cytotoxicity) technique*

determined using a panel of lymphocytes of already known HLA type.

sequencing technologies [20].

**4.1. Serological methods**

It is well understood that even single nucleotide substitutions might impact the course of transplantation significantly either on the aspect of GVHD, engraftment success and transplant

Of course the extent of their impact is driven not only by the kind of genetic alteration (SNP, CNV, InDel, inversion), the effect it exerts on the final protein product (synonymous versus nonsynonymous polymorphisms) and the gene it appears at (HLA class I or II), but also the exact nucleotide position it is located, since this might affect more or less important amino acid

With the advent of novel sequencing techniques allele level typing provides extensive nucleotide sequence data, which in correlation with previously available clinical data, is going

HLA genes contain 5–8 exons ranging in length from 4 to 17 kb. Most high-resolution fourdigit HLA typing technologies, mainly sequence-based techniques (SBT) or probe-based hybridization techniques, primarily focus on deciphering the sequence of the antigen-binding groove only. This is due to the high cost of complete HLA genotyping and the limited time interval before HSCT. Thus, only exons 2 and 3 (540 bp) for class I molecules and exon 2 (270 bp) of class II molecules are typically analyzed, providing intermediate-resolution typing.

Before these techniques came into light, less informative serology methods were acquired for HLA typing. On the other hand, the future seems very promising thanks to the evolution of

Next, we will provide a brief review of evolution of the most widely applied techniques for

Lymphocytes incubated with polyclonal sera in the presence of complement, was the first attempt for determining patient-donor compatibility. CDC utilizes sera from multiparous alloimmunized women, whose HLA specificity (reactivity against a particular HLA type) is

From a population of peripheral blood lymphocytes (PBLs), T lymphocytes are used for determining class I antigens, while B-lymphocytes (professional APCs) are separately isolated for determining class II antigens. The cells are incubated along with the characterized serum and complement. Their reactivity is determined based on the lysis of Ab-covered lymphocytes,

to provide significant information in the context of retrospective studies [8, 12, 19].

FACS overcomes two additional obstacles of the CDC method. The first refers to the positive reactions mediated through cytotoxic Abs directed against non-HLA molecules (lower specificity). The second regards positive reactions that are driven only by the complementactivating Abs, thus failing to detect complement-independent acting donor-specific Abs (lower sensitivity).

However cell viability dependence is still an obstacle. Combined with its high cost, this technique is prohibitive for HLA-typing in a routine use by a clinical laboratory.

Solid phase assays, such as ELISA (enzyme-linked immunosorbent assays) and the Luminex technology, the last utilizing fluorescent dye impregnated beads bound to HLA molecules, have also been developed for HLA typing. These have mainly, but not exclusively, been studied in solid organ transplantation studies. In HSCT studies, these techniques mainly focus on HLA-Ab screening due to the renewed interest on donor-specific Abs (DSA) and their importance in graft failure [22–27].

Since molecular methods have proven to be more reliable, we will focus on them for a much more comprehensive analysis [28].

#### **4.2. Molecular methods**

The development and extensive usage of molecular methods soon substituted serologic techniques, for determination of individuals' HLA type.

Molecular methods provided higher resolution typing, without the need for preserving characterized sera of all HLA types, even those not very common ones, and without the prerequisite of cell viability for test success.

Molecular methods of typing are still the main approaches to HLA typing resolution of low (two-digit) or intermediate (partial four-digit) level. The four-digit resolution these techniques offer for analysis of limited but HSCT important loci, contributed to the identification of serologically indistinguishable variants of HLA class I and II molecules with few, but detrimental, amino acid changes.

High-resolution typing at the four-digit level for all HLA loci is an unrealistic goal with these techniques. Molecular methods mainly focus on identifying polymorphisms in exons 2 and 3 of the class I locus and exon 2 of the class II locus, which are crucial for HSCT, as mentioned before [5, 18, 29].

#### *4.2.1. PCR/SSP (sequence-specific priming PCR)*

Genomic DNA (gDNA) is isolated from the under investigation sample and HLA regions of interest are amplified using PCR technology.

PCR utilizes in vitro prepared small oligonucleotide sequences (primers or oligos). These oligos bind to an exact location of a DNvA molecule, according to complementarity rules, acting as starting points for the production of multiple complementary copies of the intermediate region between a pair of them (amplification). More primers per reaction or more pairs of primers may be included in a single PCR, depending on the purpose of each protocol [29–32].

Sequence specific PCR (PCR/SSP), whether characterized as allele-specific amplification PCR (PCR/AS), amplification refractory mutation system PCR (PCR/ARMS) or multiplex PCR, exploits PCR for the amplification of specific HLA regions with minor alterations in primer design each time.

The amplification primers are polymorphic-specific, meaning that they only extend and form a product if the targeted polymorphism exists. The primers are designed in such a way, so that their 3' end nucleotide is complementary to the investigated genomic alteration. Thus products of specific length are produced depending on the polymorphism and the primer design. Afterward, these are visualized using gel electrophoresis.

This technique might result in no amplification at all if none of the polymorphisms analyzed exists, thus another set of primers is coamplified. This corresponds to a monomorphic target sequence that produces an extra fragment of distinguishable length. The last determines the quality of DNA and the validity of the technique (successful reaction) [24].

PCR/SSP may become a time and labor intense technique, when not used in a multiplexed format for the analysis of many polymorphic sites. If multiplex format is preferred, the conditions for a successful PCR need to be very stringent. Also, this technique is prone to falsepositive bands and false-negative results, especially for degraded samples.

However, this method is especially useful if applied in conjunction with PCR/SSOP (sequence specific oligonucleotide probes) hybridization typing, providing higher resolution, since it allows the separate amplification of the two alleles in a heterozygote [28, 33–35].

#### *4.2.2. PCR/SSOP (sequence-specific oligonucleotide probes)*

#### *4.2.2.1. Reverse hybridization (reverse dot blot)*

Since molecular methods have proven to be more reliable, we will focus on them for a much

The development and extensive usage of molecular methods soon substituted serologic

Molecular methods provided higher resolution typing, without the need for preserving characterized sera of all HLA types, even those not very common ones, and without the

Molecular methods of typing are still the main approaches to HLA typing resolution of low (two-digit) or intermediate (partial four-digit) level. The four-digit resolution these techniques offer for analysis of limited but HSCT important loci, contributed to the identification of serologically indistinguishable variants of HLA class I and II molecules with few, but detri-

High-resolution typing at the four-digit level for all HLA loci is an unrealistic goal with these techniques. Molecular methods mainly focus on identifying polymorphisms in exons 2 and 3 of the class I locus and exon 2 of the class II locus, which are crucial for HSCT, as mentioned

Genomic DNA (gDNA) is isolated from the under investigation sample and HLA regions of

PCR utilizes in vitro prepared small oligonucleotide sequences (primers or oligos). These oligos bind to an exact location of a DNvA molecule, according to complementarity rules, acting as starting points for the production of multiple complementary copies of the intermediate region between a pair of them (amplification). More primers per reaction or more pairs of primers may be included in a single PCR, depending on the purpose of each protocol

Sequence specific PCR (PCR/SSP), whether characterized as allele-specific amplification PCR (PCR/AS), amplification refractory mutation system PCR (PCR/ARMS) or multiplex PCR, exploits PCR for the amplification of specific HLA regions with minor alterations in primer

The amplification primers are polymorphic-specific, meaning that they only extend and form a product if the targeted polymorphism exists. The primers are designed in such a way, so that their 3' end nucleotide is complementary to the investigated genomic alteration. Thus products of specific length are produced depending on the polymorphism and the primer design.

This technique might result in no amplification at all if none of the polymorphisms analyzed exists, thus another set of primers is coamplified. This corresponds to a monomorphic target

more comprehensive analysis [28].

techniques, for determination of individuals' HLA type.

54 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

prerequisite of cell viability for test success.

*4.2.1. PCR/SSP (sequence-specific priming PCR)*

interest are amplified using PCR technology.

Afterward, these are visualized using gel electrophoresis.

**4.2. Molecular methods**

mental, amino acid changes.

before [5, 18, 29].

[29–32].

design each time.

The PCR step of SSOP utilizes chemically modified (biotin labeled) primers nonpolymorphicspecific. Under normal circumstances, meaning the DNA is intact and no random polymorphism exists in the 3' end of a single primer, the biotinylated amplified products (amplicons) are produced by each primer pair. This way, the primer pair is designed in such a way in order to produce an amplicon that includes polymorphisms inside its sequence.

The PCR product is afterward incubated with a panel of already known polymorphic HLA sequence molecules (SSO probes), which are enzymatically poly-thymidine (poly-T) tailed. This enzymatic tailing enables their prestabilization on a solid surface, usually a nylon membrane.

The modified amplicon will cross react with only one of these probes (complete complementarity) during their incubation along with horseradish peroxidase (HRP)-conjugated streptavidin and a chromogenic or chemiluminescent substrate. HRP-conjugated streptavidin binds on the biotinylated product. In the presence of biotin, HRP enzyme is activated and metabolizes the substrate in order to emit light signal. This light is device detected and computer analyzed later on, to determine the polymorphism of the unknown sample, based on position analysis of the signal, since the stabilization position of each probe on the membrane is previously known.

As one can conclude the specific sequence of gDNA where the primers will bind, need to be known in advance. The polymorphisms under investigation also need to be already known, in order to prepare and stabilize the suitable set of probes.

Interpretation difficulties may arise from less intense, absent or dubious hybridization patterns, due to poor-quality and low-quantity DNA amplification or background signals due to poor membrane washing or temperature variation during hybridization.

Another limitation of this technique is the fact that the extremely polymorphic HLA alleles, especially those of class I, are impossible to analyze due to the very large number of probes such a design would require. However, as a general rule, wider number of probes for every HLA loci and larger number of unknown HLA regions investigated provides higher resolution level [29–31, 36].

#### *4.2.2.2. Direct hybridization (conventional dot-blot)*

An alternative to reverse dot-blot PCR-SSOP, is the conventional dot-blot technique, where the PCR amplified regions are the ones immobilized on the solid surface, and biotin-labeled SSO probes are incubated along with HRP-conjugated streptavidin and a substrate. The ones that bind the unknown amplicons emit light of specific wavelength identified in the same manner as previously described.

In addition to the previously described limitations this technique is also more cumbersome, since the number of SSOPs required for typing vastly increases, due to the high polymorphic state of HLA loci [30, 31].

#### *4.2.3. PCR/RFLP (restriction fragment length polymorphism)*

RFLP analysis involves digestion of gDNA with endonucleases, one at a time, which cleaved a specific nucleotide sequence motif, in order to produce fragments of various lengths. This variation can be detected by Southern blot analysis of the digested fragments. Suitable probes for detection are either cloned cDNA, or genomic DNA sequences, complementary to mainly HLA class II regions which were better studied. In fact, this technique was the first that revealed the incredible variation of HLA class II region.

However, it is cumbersome, it requires a large amount of high molecular weight genomic DNA, and it can only be applied to regions that bear the respective restriction sites, which is not always the case and that is why it did not replace serological typing, but was rather used complementary.

PCR-combined RFLP (PCR/RFLP) analysis is an improvement to the previous method. The amplified HLA region is incubated with a restriction endonuclease that recognizes specific nucleotide sequence. The digestion reaction is performed whether a polymorphism exists or not, producing amplicon fragments of various lengths. The products are analyzed by gel electrophoresis.

However, like RFLP analysis, PCR/RFLP may lead to inconsistencies during HLA typing due to complex manipulation steps and possible incomplete digestion reactions. Also, this technique is unable to detect multiple recognition sites simultaneously [29, 33, 36, 37].

#### *4.2.4. PCR/SBT (sequencing-based typing)*

Another approach to high-resolution HLA typing is the PCR amplification and subsequent direct sequencing of previously described class I and II exons. Dideoxy-based Sanger sequencing, using capillary electrophoresis, provides increased reliability especially when applied after SSO or SSP.

The two alleles of heterozygous samples, which represent a substantial source of ambiguities, are usually sequenced separately following SSO or SSP typing, thus increasing the resolution of possible genotypes [29, 37].

This technique, although more automated, easier to implement and less prone to technical and interpretational errors, is less sensitive than others, like SSO, which when optimized provide more accurate results and less ambiguous results [24].

## *4.2.5. PCR/RSCA (reference strand-mediated conformational analysis)*

HLA loci and larger number of unknown HLA regions investigated provides higher resolution

An alternative to reverse dot-blot PCR-SSOP, is the conventional dot-blot technique, where the PCR amplified regions are the ones immobilized on the solid surface, and biotin-labeled SSO probes are incubated along with HRP-conjugated streptavidin and a substrate. The ones that bind the unknown amplicons emit light of specific wavelength identified in the same manner

In addition to the previously described limitations this technique is also more cumbersome, since the number of SSOPs required for typing vastly increases, due to the high polymorphic

RFLP analysis involves digestion of gDNA with endonucleases, one at a time, which cleaved a specific nucleotide sequence motif, in order to produce fragments of various lengths. This variation can be detected by Southern blot analysis of the digested fragments. Suitable probes for detection are either cloned cDNA, or genomic DNA sequences, complementary to mainly HLA class II regions which were better studied. In fact, this technique was the first that revealed

However, it is cumbersome, it requires a large amount of high molecular weight genomic DNA, and it can only be applied to regions that bear the respective restriction sites, which is not always the case and that is why it did not replace serological typing, but was rather used

PCR-combined RFLP (PCR/RFLP) analysis is an improvement to the previous method. The amplified HLA region is incubated with a restriction endonuclease that recognizes specific nucleotide sequence. The digestion reaction is performed whether a polymorphism exists or not, producing amplicon fragments of various lengths. The products are analyzed by gel

However, like RFLP analysis, PCR/RFLP may lead to inconsistencies during HLA typing due to complex manipulation steps and possible incomplete digestion reactions. Also, this

Another approach to high-resolution HLA typing is the PCR amplification and subsequent direct sequencing of previously described class I and II exons. Dideoxy-based Sanger sequencing, using capillary electrophoresis, provides increased reliability especially when applied

technique is unable to detect multiple recognition sites simultaneously [29, 33, 36, 37].

level [29–31, 36].

as previously described.

state of HLA loci [30, 31].

complementary.

electrophoresis.

after SSO or SSP.

*4.2.2.2. Direct hybridization (conventional dot-blot)*

56 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

*4.2.3. PCR/RFLP (restriction fragment length polymorphism)*

the incredible variation of HLA class II region.

*4.2.4. PCR/SBT (sequencing-based typing)*

Although PCR/SBT is able to detect unknown HLA polymorphisms, the problem of not being entirely able to resolve novel arrangements of known polymorphisms, also known as ambiguity, can be overcome by the PCR/RSCA technique [19].

PCR/RSCA can achieve high-resolution results without the ambiguities seen in the previously described methods. This technique is based on the principles that DNA fragments that differ in nucleotide composition exhibit different motilities after separation by nondenaturing polyacrylamide gel electrophoresis (PAGE). The amplified alleles under investigation are hybridized with a fluorescent labeled reference strand, forming a double stranded DNA with unique conformation (double strand conformation analysis; DSCA).

PCR/RSCA is capable of resolving even single base alterations and of course, identification of new mutations thanks to the different spatial structure of the newly formed DNA-probe duplexes [38].
