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PubMedDate not supplied

Single- and double-color oligonucleotide primed in situ labeling (PRINS): applications in pathology.

Molecular cytogenetics is mostly performed by fluorescence in situ hybridization using long DNA probes that are generated by vector cloning. Oligonucleotide primed in situ labeling (PRINS) is a recent method that has been established for the detection of the centromeric or telomeric region in metaphase chromosomes. In this overview, we demonstrate the possible applications of PRINS and provide elaborated protocols for its use in intact interphase cells of routine cytological preparations, e.g., cell smears, touch preparations, and cytospins of non-neoplastic and neoplastic tissues. Moreover, the various modifications of the PRINS method, such as multi-color PRINS for targeting different chromosomes within one cell or the enzymatic detection of the PRINS product instead of the more commonly used fluorochromes, are discussed.

Centromere↗
PubMed2002

Primed in situ labeling (PRINS) for evaluation of gene deletions in cancer.

Rearrangements involving the 13q14 and 17p13 chromosomal regions are often observed in leukemias and lymphomas. These rearrangements are not always identifiable cytogenetically. In more than 50% of cases, deletions occur at the submicroscopic level and the karyotypes appear normal. Molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) have accordingly contributed to the identification of a variety of subtle rearrangements such as those involving submicroscopic deletions. However, FISH is expensive, time consuming, technically burdensome, and requires cloned DNA probes. A newer technique, primed in situ labeling (PRINS), has been tested as a possible alternative to FISH. To assess the utility and efficiency of the PRINS method in the detection of RB1 and p53 deletions, we evaluated 10 patients with hematological disorders and known rearrangements, i.e., deletions involving 13q14 and 17p13 regions. The data in these cases were validated against data obtained with standard FISH probes. Our results indicate that PRINS could be used with relative ease in cytogenetics laboratories and could serve as an alternative to conventional FISH for defining deletions involving unique sequences.

Base Sequence↗
PubMed1997

Applications of single-color and double-color oligonucleotide primed in situ labeling in cytology.

Interphase cytogenetics is mostly performed with use of fluorescence in situ hybridization (FISH), using long DNA probes of several hundred or thousand base pairs in length. Recently, oligonucleotide primed in situ labeling (PRINS) was established for staining centromeres and telomeres of chromosomes in metaphase spreads by Taq-polymerase-mediated incorporation of labeled nucleotides. We investigated the use of PRINS in intact interphase cells of various cytologic preparations, targeting chromosomes 1, 8, and 9. Examining cell smears (n = 3), touch preparations (n = 20), and cytospins (n = 11) of non-neoplastic and neoplastic tissues, PRINS was as sensitive and reliable as the FISH method in assessing the exact chromosome number. Aneuploidy in tumor cells was confirmed by double-color PRINS in a part of the specimens. The PRINS reaction, which requires heating of the cell preparations to as high as 96 degrees C, did not affect the cytomorphologic details. Because PRINS is much faster and approximately 10 times less expensive than FISH, this method allows an increased application of interphase cytogenetics in diagnostic cytopathology.

Aneuploidy↗
PubMed1994

Pseudodicentric chromosome 18 diagnosed by chromosome painting and primed in situ labelling (PRINS).

We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.

Abnormalities, Multiple↗
PubMed2001

Trisomy 8 and monosomy 7 detected in bone marrow using primed in situ labeling, fluorescence in situ hybridization, and conventional cytogenetic analyses. A study of 54 cases with hematological disorders.

Trisomy 8 and monosomy 7 are the two most frequent aneuploidies found in hematological disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, primed in situ labeling (PRINS), fluorescence in situ hybridization (FISH) and conventional cytogenetic approaches were used to investigate 54 cases of hematopoietic disorders. Of these cases, there were 22 cases of trisomy 8, 2 cases of tetrasomy 8, 14 cases of monosomy 7, and 16 cases with two copies of both chromosomes 7 and 8. PRINS was carried out in interphase nuclei of bone marrow samples using primers that can specifically detect alpha-satellite DNA sequences of chromosomes 7 and 8. In parallel, using the alpha-satellite probes for chromosomes 7 and 8, FISH was performed for all the cases. PRINS and FISH techniques showed similar specificity and sensitivity. In comparison to FISH, PRINS is more advantageous since it is a faster, easier, and more cost-effective technique. Additionally, for most of the cases, a higher proportion of aneuploidy was detected in metaphases using conventional cytogenetics, as compared to the one found in interphase nuclei identified with PRINS and FISH techniques. In other words, for these cases, the cells with trisomy 8 or monosomy 7, had a distinct proliferative advantage compared to the disomic cell population. Therefore, to better quantify the proportion of aneuploid cells in bone marrow, we recommend to investigate the frequency of aneuploidy in nuclei using PRINS, rather than studying only the dividing cells as detected by conventional cytogenetic techniques.

Adult↗
PubMed1995

Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting).

In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primed in situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting).

Centromere↗
PubMed1997

Clinical applications of primed in situ labelling (PRINS) rapid identification of a marker chromosome in a fetus.

Marker chromosomes pose a serious problem in clinical cytogenetic diagnosis since the conventional banding analyses are often not useful in identifying their origin or composition. In the absence of information, counseling as to the clinical significance and prognosis is difficult, especially in prenatal diagnosis. With the introduction of fluorescence in situ hybridization (FISH) marker identification has became feasible. However, FISH is relatively time-consuming and expensive. In an effort to overcome these disadvantages, we have used primed in situ labelling (PRINS) technique as an alternative. Presented here is one case in which PRINS was useful in rapidly identifying the origin of a marker chromosome detected on amniotic fluid chromosome analysis. Based on our experience with this case and others, we propose that PRINS can become a viable and cost effective alternative to FISH and is as reliable as FISH in terms of accuracy, specificity, and sensitivity.

Adult↗
PubMed1997

The monosomy 7 clone in interphase and metaphase cell population: a combined chromosome and primed in situ labeling study.

Loss of a chromosome 7 is associated with a poor prognosis in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Some studies have shown higher frequencies of monosomy 7 (-7) in dividing than nondividing myeloid cells, which might indicate that -7 confers a proliferative advantage on the host cell. As other groups have not been able to confirm this, we compared the -7 frequencies in bone marrow metaphases as studied with conventional cytogenetics and in interphase cells using primed in situ (PRINS) labeling. We found significantly higher -7 frequencies in metaphase than in interphase cells irrespective of diagnosis and presence or absence of additional chromosome aberrations. Further, we found a significant correlation between the -7 percentages in resting and dividing cells. Finally, as our material showed a clear male preponderance, Mitelman's Catalog of Chromosome Aberrations in Cancer was searched for -7. Of 815 cases with AML or MDS, 491 (60.3%) were found to be men. To our knowledge, this is the first observation of a clear deviation from the 1:1 sex ratio in -7 patients.

Acute Disease↗
PubMed1994

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS).

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.

Base Sequence↗
PubMed1997

Detection of karyotype changes in interphase cells: oligonucleotide-primed in situ labelling versus fluorescence in situ hybridization.

Interphase cytogenetics is a rapidly developing technique which is usually performed by fluorescence in situ hybridization (FISH). Recently, oligonucleotide-primed in situ synthesis (PRINS) has become established as a method of labelling centromeric regions of chromosomes in metaphase spreads. We tested the suitability of PRINS in detecting the exact copy number of chromosomes 1, 3, 7 and 8 in intact interphase cells of 17 cytological preparations derived from normal and neoplastic tissues. Control procedures consisted in preparation of metaphase spreads of lymphocytes of healthy donors, conventional cytogenetics in some of the specimens, and omission of the primers or Taq polymerase from the reaction mixture. All specimens were additionally examined by FISH and analysed blind by two experienced observers. Both PRINS and FISH revealed a corresponding distribution of hybridization signals for all chromosomes examined in specimens of normal bone marrow (n = 5), normal liver cells (n = 5), three samples of acute nonlymphocytic leukaemia in which conventional chromosome analyses had shown monosomy 7 or trisomy 8, and in four hepatocellular carcinomas that displayed trisomy 1. Overall, statistical analysis revealed no significant difference in the signal distribution between the two techniques. Our results demonstrate that PRINS is as reliable as FISH for detecting chromosome copy numbers in interphase nuclei of intact cells. The PRINS method, however, is easier to perform, faster and less expensive, holding great potential for future applications in diagnostic pathology.

Biopsy↗
PubMed2001

Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21.

The centromeric alpha-satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence. So far it has proven difficult to discriminate between sequence variations in the chromosome 13 and 21 alpha-satellite regions using in situ techniques. To analyze whether the method of modified single-color and double-color PRINS could be used to detect single nucleotide polymorphisms within this region, we used previously published primers D13Z and D21Z that differ in the terminal 3'-nucleotide and an additionally constructed primer "D13/21-test" lacking the final nucleotide at the 3' end. The results show that a one-base pair mismatch at the 3' end is sufficient to be detected by PRINS. Surprisingly, only about 35% of our samples exhibited the expected combination of two chromosomes 13 specifically labeled with only primer D13Z and two chromosomes 21 specifically labeled with only primer D21Z. The rest of the samples showed a polymorphic distribution of the target sequence for the primers, therefore these primers are not suited for routine detection of chromosomes 13 and 21 during interphase. Our data indicate that an interchromosomal exchange of alpha-satellite DNA takes place between chromosomes 13 and 21, possibly due to a concerted evolution process.

Base Sequence↗
PubMed1991

Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS).

A new method for the detection of RNA in situ is presented. It is based on sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and subsequent chain elongation catalyzed by reverse transcriptase. Under the conditions described, biotin-labeled nucleotides can be incorporated and the cDNA synthesized in situ can thus be detected using fluorescein-conjugated avidin. Compared to traditional in situ hybridization the use of short oligonucleotide primers has the potential advantage of being better to discriminate between closely related RNA transcripts. Compared to in situ transcription with radioactive precursors we find it more attractive to use fluorescein-conjugated avidin as detection system because it allows a more detailed study of cell and signal simultaneously.

Animals↗
PubMed1995

Staining of human telomeres with primed in situ labeling (PRINS).

As described, the PRINS method is a very rapid and reliable way of staining human telomeres. To obtain the maximum frequency of stained telomeres, the primer (CCCTAA)7 should be used, although the average frequency never quite reaches 100%. The frequency is strongly dependent on the age of the individual, being significantly higher in children and newborns than in adults. A difference between the (CCCTAA)7 primer and the complementary primer is demonstrated and a possible explanation is proposed, namely, that gaps in the C-rich strand cause chain elongation termination after the addition of only one dTTP molecule.

Adult↗