New assay could prove useful in HSCT

Preparing for HSCT

Credit: Chad McNeeley

Researchers say they’ve developed an assay that allows for ultrasensitive DNA detection.

This haplotype-based assay could be used to detect relapse in patients who have undergone hematopoietic stem cell transplant (HSCT).

In fact, the researchers believe it would enable relapse detection earlier than existing microsatellite-based assays.

The new assay could also be used to detect microchimerism in solid organ transplants, in forensics, and for patient identification.

James Eshleman, MD, PhD, of the Johns Hopkins University School of Medicine in Baltimore, Maryland, and his colleagues described this assay in The Journal of Molecular Diagnostics.

The team noted that most bone marrow engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis.

“Repeat testing will only detect DNA that makes up at least 1% of a DNA sample, so it’s not great for situations in which results depend on small amounts of material within a larger sample,” Dr Eshleman said.

In these situations, evaluating single-nucleotide polymorphisms (SNPs) might seem like a better choice, but this method has a high error rate. Dr Eshleman and his colleagues found they could circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes.

To test their method, the researchers chose the HLA-A locus. They aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs. The team then tested a series of primers surrounding this region and selected the best pair on the basis of amplification efficiency and specificity.

They found it easy to differentiate some combinations of HLA-A alleles but not others. For instance, they discovered that 11 SNPs differentiate allele A*01 from A*02. But A*02 and HLA-A* 68:01:01:01 have a single SNP difference.

To test the possible cross talk between molecules that vary by 11 SNPs, the researchers sequenced 2 samples—one homozygous for A*01 and another homozygous for A*02—and analyzed each for the other allele. They found that, when there are enough discriminating SNPs between 2 individuals’ alleles, the haplotype assay is highly specific.

To evaluate the assay’s accuracy and limit of detection, the researchers generated various dilutions of 2 cell lines with known HLA-A genotypes. They made dilutions with cell mixes varying from 1 in 1 million (0.0001%) to 1 in 100 (1%), using 10 million cells for each dilution.

The team isolated DNA and performed PCR using 600 ng of DNA. And they sequenced each sample at least twice.

The assay proved highly precise at the 0.1% cell mix but less precise at the 0.01% cell mix.

“[Nevertheless,] we could detect cells when they made up just 0.01% of the mixture, which is a big improvement over the current method, which can only detect DNA that makes up 1% to 5% of a sample,” Dr Eshleman said.

The researchers also used their assay to test samples from 18 HSCT patients whose donor-patient HLA genotypes varied by at least 4 SNPs. All but 1 sample tested positive for some level of patient DNA, and the positives ranged from 0.001% to 1.47% patient DNA.

Finally, the team analyzed the human genome using the 1000 Genomes database and identified many additional loci that could be used with their assay.

Preparing for HSCT

Credit: Chad McNeeley

Researchers say they’ve developed an assay that allows for ultrasensitive DNA detection.

This haplotype-based assay could be used to detect relapse in patients who have undergone hematopoietic stem cell transplant (HSCT).

In fact, the researchers believe it would enable relapse detection earlier than existing microsatellite-based assays.

The new assay could also be used to detect microchimerism in solid organ transplants, in forensics, and for patient identification.

James Eshleman, MD, PhD, of the Johns Hopkins University School of Medicine in Baltimore, Maryland, and his colleagues described this assay in The Journal of Molecular Diagnostics.

The team noted that most bone marrow engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis.

“Repeat testing will only detect DNA that makes up at least 1% of a DNA sample, so it’s not great for situations in which results depend on small amounts of material within a larger sample,” Dr Eshleman said.

In these situations, evaluating single-nucleotide polymorphisms (SNPs) might seem like a better choice, but this method has a high error rate. Dr Eshleman and his colleagues found they could circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes.

To test their method, the researchers chose the HLA-A locus. They aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs. The team then tested a series of primers surrounding this region and selected the best pair on the basis of amplification efficiency and specificity.

They found it easy to differentiate some combinations of HLA-A alleles but not others. For instance, they discovered that 11 SNPs differentiate allele A*01 from A*02. But A*02 and HLA-A* 68:01:01:01 have a single SNP difference.

To test the possible cross talk between molecules that vary by 11 SNPs, the researchers sequenced 2 samples—one homozygous for A*01 and another homozygous for A*02—and analyzed each for the other allele. They found that, when there are enough discriminating SNPs between 2 individuals’ alleles, the haplotype assay is highly specific.

To evaluate the assay’s accuracy and limit of detection, the researchers generated various dilutions of 2 cell lines with known HLA-A genotypes. They made dilutions with cell mixes varying from 1 in 1 million (0.0001%) to 1 in 100 (1%), using 10 million cells for each dilution.

The team isolated DNA and performed PCR using 600 ng of DNA. And they sequenced each sample at least twice.

The assay proved highly precise at the 0.1% cell mix but less precise at the 0.01% cell mix.

“[Nevertheless,] we could detect cells when they made up just 0.01% of the mixture, which is a big improvement over the current method, which can only detect DNA that makes up 1% to 5% of a sample,” Dr Eshleman said.

The researchers also used their assay to test samples from 18 HSCT patients whose donor-patient HLA genotypes varied by at least 4 SNPs. All but 1 sample tested positive for some level of patient DNA, and the positives ranged from 0.001% to 1.47% patient DNA.

Finally, the team analyzed the human genome using the 1000 Genomes database and identified many additional loci that could be used with their assay.

Preparing for HSCT

Credit: Chad McNeeley

Researchers say they’ve developed an assay that allows for ultrasensitive DNA detection.

This haplotype-based assay could be used to detect relapse in patients who have undergone hematopoietic stem cell transplant (HSCT).

In fact, the researchers believe it would enable relapse detection earlier than existing microsatellite-based assays.

The new assay could also be used to detect microchimerism in solid organ transplants, in forensics, and for patient identification.

James Eshleman, MD, PhD, of the Johns Hopkins University School of Medicine in Baltimore, Maryland, and his colleagues described this assay in The Journal of Molecular Diagnostics.

The team noted that most bone marrow engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis.

“Repeat testing will only detect DNA that makes up at least 1% of a DNA sample, so it’s not great for situations in which results depend on small amounts of material within a larger sample,” Dr Eshleman said.

In these situations, evaluating single-nucleotide polymorphisms (SNPs) might seem like a better choice, but this method has a high error rate. Dr Eshleman and his colleagues found they could circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes.

To test their method, the researchers chose the HLA-A locus. They aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs. The team then tested a series of primers surrounding this region and selected the best pair on the basis of amplification efficiency and specificity.

They found it easy to differentiate some combinations of HLA-A alleles but not others. For instance, they discovered that 11 SNPs differentiate allele A*01 from A*02. But A*02 and HLA-A* 68:01:01:01 have a single SNP difference.

To test the possible cross talk between molecules that vary by 11 SNPs, the researchers sequenced 2 samples—one homozygous for A*01 and another homozygous for A*02—and analyzed each for the other allele. They found that, when there are enough discriminating SNPs between 2 individuals’ alleles, the haplotype assay is highly specific.

To evaluate the assay’s accuracy and limit of detection, the researchers generated various dilutions of 2 cell lines with known HLA-A genotypes. They made dilutions with cell mixes varying from 1 in 1 million (0.0001%) to 1 in 100 (1%), using 10 million cells for each dilution.

The team isolated DNA and performed PCR using 600 ng of DNA. And they sequenced each sample at least twice.

The assay proved highly precise at the 0.1% cell mix but less precise at the 0.01% cell mix.

“[Nevertheless,] we could detect cells when they made up just 0.01% of the mixture, which is a big improvement over the current method, which can only detect DNA that makes up 1% to 5% of a sample,” Dr Eshleman said.

The researchers also used their assay to test samples from 18 HSCT patients whose donor-patient HLA genotypes varied by at least 4 SNPs. All but 1 sample tested positive for some level of patient DNA, and the positives ranged from 0.001% to 1.47% patient DNA.

Finally, the team analyzed the human genome using the 1000 Genomes database and identified many additional loci that could be used with their assay.