Single Lymphocytes Research Articles

Well-devised quantification analysis for duplication mutation of Duchenne muscular dystrophy aimed at preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) has been performed for deletion and point mutation type of Duchenne muscular dystrophy (DMD). Our aim was to develop a PGD technique, not yet established, to directly detect duplication mutation instead of substitute diagnosis similar to gender determination. Our method is based on comparative quantification using conventional duplex PCR, real-time PCR and gender determination. We evaluated this method in single lymphocytes from a duplication type of DMD patient and a normal male. There was a significant difference in the mean values of the ratios (the mutation locus/a normal reference): mean value +/- SE was 1.84 +/- 0.15 in the duplication patient, and 1.00 +/- 0.09 in the normal male (p < 0.001). It is suggested that our comparative quantification method could be a new option in PGD for carriers with duplication mutation who wish to have an unaffected son.

Determination of the Labile Iron Pool of Human Lymphocytes using the Fluorescent Probe, CP655

The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a “cytosolic” medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 ± 0.27 μM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.

Lysosomal membrane-associated glycoproteins are differentially expressed in acute and chronic human thymic involution

The lysosomal membrane-associated glycoproteins LAMP-1 and LAMP-2 are the major constituents of lysosomal membranes with still undefined biological functions. As autophagy is an alternative model of programmed cell death in which lysosomes play a crucial role, we hypothesize that LAMPs might participate in this phenomenon in the involuting thymus. Thymic glands from cases with acute (infection induced) and chronic (senile) involution were examined immunohistochemically for the expression of LAMPs. In acute involution LAMP-1 was localized mainly in medullary epithelial cells, in single macrophages and lymphocytes. Hassall's corpuscules were stained less intensely as compared to control specimens. The quantitative analysis showed a significantly elevated LAMP-2 expression compared to LAMP-1. LAMPs were detected with very slight reactivity in the senile thymus. The enhanced expression of LAMPs, and mainly of LAMP-2, in epithelial cells of incidentally involuted thymus might be an indicator of acute cell injury requiring autophagic degradation of damaged structures. The diminished expression of LAMPs in age-involuted thymus could be a sign of the morphological reorganization and the functional disregulation of the gland. In conclusion, we present novel evidence for differential expression of LAMP-1 and LAMP-2 in thymic involution suggesting their possible involvement in the process of accidental involution of the thymic gland.

Multiple displacement amplification improves PGD for fragile X syndrome

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.

Hyaluronate and its receptors in bone marrow

Cell-cell and cell-matrix interactions, which are mediated by cell adhesion molecules, play a fundamental role during many cellular processes including growth, differentiation, cell migration and cancer metastasis. One molecule playing a major role in these processes is the CD44 surface receptor, which is expressed in a wide range of cells including many cells of the hemopoietic system, where it mediates the interaction with its major ligand, hyaluronate. However, little is known about CD44 and hyaluronate in bone marrow and this was investigated immunohistochemically in trephine biopsies and in cultivated human bone marrow stromal cells. In biopsy specimens, patches of hyaluronate deposition were detected in the extracellular matrix (ECM). However, most of the areas of the ECM were devoid of hyaluronate. Single mast cells and lymphocytes scattered throughout the marrow were CD44 immunopositive. Marrow-derived stromal cells (MDSC) expanded in cell culture were immunopositive for CD44, hyaluronate synthase, and hyaluronate. Hence, a marked difference between CD44 immunolocalisation and hyaluronate deposition can be observed between in situ and under cell culture conditions. Since in normal marrow in situ the number of CD44 immunopositive cells was low, interactions of CD44 and hyaluronate would appear to not to play a major role in cell adhesion in the normal bone marrow.

Hypergravity speeds up the development of T-lymphocyte motility

The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1-11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.

Identification of a Novel Single Nucleotide Polymorphism of HADHA Gene at a Referred Primer-binding Site During Pre-diagnostic Tests for Preimplantation Genetic Diagnosis

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene disorder.

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90-98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene.

Synthesis and immunosuppressive activity of l-rhamnopyranosyl flavonoids

Astilbin, a flavonoid isolated from different plants, shows diverse biological activities. This paper reports the synthesis and immunosuppressive activity of seven analogues of astilbin, which may shed light on the structure-activity relationship of the compounds. The following glycosyl flavonoids, 6-alpha-L-rhamnopyranosyloxyflavanone (20), 3-alpha-L-rhamnopyranosyloxyflavone (22), 3-alpha-L-rhamnopyranosyloxyflavanone (24), 3-alpha-L-rhamnopyranosyloxychromanone (26), 4-alpha-L-rhamnopyranosyloxychromanol (27), 7-hydroxy-3-alpha-L-rhamnopyranosyloxyflavanone (30) and 4'-hydroxy-3-alpha-L-rhamnopyranosyloxyflavanone (32) were prepared respectively by glycosylation of 6-hydroxyflavanone (1), 3-hydroxyflavone (2), 3-hydroxyflavanone (5), 3-hydroxychromanone (8), 4-chromanol (9), 7-benzyloxy-3-hydroxyflavanone (12), 4'-benzyloxy-3-hydroxyflavanone (15). Among them, compounds 5, 8, 12 and 15 were synthesized from flavanone (3), 4-chromanone (6), 7-hydroxyflavanone (10) and 4'-hydroxyflavanone (13) respectively. Similar to astilbin (4), compounds 22, 24, 26, 30 and 32 significantly inhibited the single mixed lymphocytes reaction (sMLR) and enhanced the apoptosis of spleen cells isolated from mice with sheep red blood cell-induced delayed-type hypersensitivity respectively. However, compound 20 only showed a slight tendency to inhibit sMLR at higher concentration. Both compounds 20 and 27 did not influence the cell apoptosis. These data suggest that the following factors play essential roles in determining the biological activity of the flavonoids: the position at which the sugar is linked to the flavone, the presence of carbonyl on C-4 and phenol hydroxyl group in A or B ring. However, the presence of a B ring is unfavorable for the biological activity and the double bond at C2-C3 in C-ring shows little effect on the activity.

Live birth following the first mutation specific pre-implantation genetic diagnosis for haemophilia A

Haemophilia A is an X-linked, recessive, inherited bleeding disorder which affects 1 in 5000 males born worldwide. It is caused by mutations in the FactorVIII (F8) gene on chromosome Xq28. We describe for the first time two mutation specific, single cell protocols for pre-implantation genetic diagnosis (PGD) of haemophilia. A that enable the selection of both male and female unaffected embryos. This approach offers an alternative to sexing, frequently used for X-linked disorders, that results in the discarding of all male embryos including the 50% that would have been normal. Two families with a history of severe haemophilia. A requested carrier diagnosis and subsequently proceeded to PGD. The mutation in family 1 is a single nucleotide substitution c.5953C > T, R1966X in exon 18 and in family 2, c.5122C > T, R1689C in exon 14 of the F8 gene. Amplification efficiency was compared between distilled water and SDS/proteinase K cell lysis (98.0%, 96/98 and 80%, 112/140 respectively) using 238 single lymphocytes. Blastomeres from spare IVF cleavage-stage embryos donated for research showed amplification efficiencies of 83.3% (45/54) for the R1966X and 92.9% (13/14) for the R1689C mutations. The rate of allele dropout (ADO) on heterozygous lymphocytes was 1.1% (1/93) for R1966X and 5.94% (6/101) for R1689C mutations. A single PGD treatment cycle for family 1 resulted in two embryos for transfer but these failed to implant. However, with family 2, two embryos were transferred to the uterus on day 4 resulting in a successful singleton pregnancy and subsequent live birth of a normal non-carrier female.

The “Spanning Protocol”: A new DNA extraction method for efficient single-cell genetic diagnosis

We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Successful preimplantation genetic diagnosis for alpha-thalassemia using fluorescent polymerase chain reaction

To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia. Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles. The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD. This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.

Benign Lichenoid Keratosis

Benign lichenoid keratosis, otherwise known as lichen planus-like keratosis, is a common, cutaneous entity that is often confused with cutaneous malignancy. Few studies have examined the multiple clinical and pathologic guises of this entity, particularly within the context of clinical pathologic correlation or magnitude of this study. We examined the epidemiologic, clinical, and pathologic attributes of 1040 consecutive cases of benign lichenoid keratosis referred for pathologic examination at a busy laboratory over an entire year. Clinical parameters assessed included the age, anatomic location, gender, and multiplicity of the lesions. Pathologic attributes were assessed yielding discernment of five different subtypes that included a classic type, bullous type, atypical type with cytologically atypical lymphocytes, an early or interface type, and a late regressed or atrophic type. The results yielded an average age at presentation of 59.5 years with an age range of 36 to 87 years. The gender frequency was 76% female, 24% male. The trunk was the most common location (76%), followed by the extremities (33%) and head and neck (7%); 8% of patients presented with two lesions and less than 1% with three lesions prompting consideration of lichen planus. The classic, atypical, and bullous forms of the disease clinically presented with erythematous papule/plaque(s). The early or interface type showed erythematous to hyperpigmented brown macules and the regressed or atrophic type presented as violaceous papules or irregularly distributed macular pigmentation; 81% of the lesions showed the classic histology consisting of epidermal acanthosis with a band-like lichenoid lymphocytic infiltrate. Variable numbers of plasma cells, eosinophils, and neutrophils were identified as well as epidermal parakeratosis distinguishing these lesions from typical lichen planus. The bullous variant showed intraepidermal or subepidermal bullous cavities with a dense associated lymphocytic infiltrate and increased numbers of necrotic basilar layer keratinocytes. The atypical variant showed features of the classic type with scattered enlarged CD-3, CD-30 (+) lymphocytes possessing hyperchromatic, irregular nuclei. The early interface type showed single lymphocytes aligned along the dermoepidermal junction without epidermal acanthosis and adjacent lentigo. The regressed or atrophic variant showed epidermal atrophy with papillary dermal scarring, patchy lymphocytic infiltrates and melanin incontinence. The clinicopathologic spectrum of benign lichenoid keratosis is broad and encompasses several unrelated entities. An awareness of its expanded presentation is essential to avoid misdiagnosis and may serve as an important forerunner of pathogenic discernment.

Application of High Density Array-CGH in Detecting Embryo Chromosomal Abnormalities

ObjectiveDetection of chromosomal abnormalities at the embryo level as a model for preimplantation genetic diagnosis applications using high density array-CGH on amplified embryos by multiple displacement amplification.DesignAbnormalities in chromosomes of human embryos could reach up to 50%. Such high percentage urges the establishment of a technique capable of detecting chromosomal aneuploidy in addition to deletions and duplications.Materials and methods30 blocked embryos were amplified by multiple displacement amplification (MDA) and analysed by BACs DNA microarray with 1MB resolution. The amplified embryos were compared to XX normal karyotype single lymphocytes amplified with the same technique and using the same protocol.ResultsThe analysis of the blocked embryos showed a high percentage of aneuploidies mainly in chromosomes 19, 18, 22, 8 and 21 in addition to sex chromosomes. Furthermore, deletions and duplications were detected in chromosomes 1 and 8.ConclusionThe high percentage of abnormalities in blocked embryos might explain the cause of embryo growth failure. Furthermore, the detection of deletions show the need for high density Array-CGH able to detect such deletions in addition to aneuploidy and other chromosomal gain or loss. Our results would open a new area in embryo diagnosis for better pregnancy outcome in preimplantation genetic diagnosis since any abnormality could be detected by our technique. ObjectiveDetection of chromosomal abnormalities at the embryo level as a model for preimplantation genetic diagnosis applications using high density array-CGH on amplified embryos by multiple displacement amplification. Detection of chromosomal abnormalities at the embryo level as a model for preimplantation genetic diagnosis applications using high density array-CGH on amplified embryos by multiple displacement amplification. DesignAbnormalities in chromosomes of human embryos could reach up to 50%. Such high percentage urges the establishment of a technique capable of detecting chromosomal aneuploidy in addition to deletions and duplications. Abnormalities in chromosomes of human embryos could reach up to 50%. Such high percentage urges the establishment of a technique capable of detecting chromosomal aneuploidy in addition to deletions and duplications. Materials and methods30 blocked embryos were amplified by multiple displacement amplification (MDA) and analysed by BACs DNA microarray with 1MB resolution. The amplified embryos were compared to XX normal karyotype single lymphocytes amplified with the same technique and using the same protocol. 30 blocked embryos were amplified by multiple displacement amplification (MDA) and analysed by BACs DNA microarray with 1MB resolution. The amplified embryos were compared to XX normal karyotype single lymphocytes amplified with the same technique and using the same protocol. ResultsThe analysis of the blocked embryos showed a high percentage of aneuploidies mainly in chromosomes 19, 18, 22, 8 and 21 in addition to sex chromosomes. Furthermore, deletions and duplications were detected in chromosomes 1 and 8. The analysis of the blocked embryos showed a high percentage of aneuploidies mainly in chromosomes 19, 18, 22, 8 and 21 in addition to sex chromosomes. Furthermore, deletions and duplications were detected in chromosomes 1 and 8. ConclusionThe high percentage of abnormalities in blocked embryos might explain the cause of embryo growth failure. Furthermore, the detection of deletions show the need for high density Array-CGH able to detect such deletions in addition to aneuploidy and other chromosomal gain or loss. Our results would open a new area in embryo diagnosis for better pregnancy outcome in preimplantation genetic diagnosis since any abnormality could be detected by our technique. The high percentage of abnormalities in blocked embryos might explain the cause of embryo growth failure. Furthermore, the detection of deletions show the need for high density Array-CGH able to detect such deletions in addition to aneuploidy and other chromosomal gain or loss. Our results would open a new area in embryo diagnosis for better pregnancy outcome in preimplantation genetic diagnosis since any abnormality could be detected by our technique.

PCR for Detection of Mycobacterium tuberculosis in Experimentally Infected Dogs

The susceptibility of dogs to infection with Mycobacterium tuberculosis was studied through different ways of experimental infection. The examination shows, that in most cases the disease runs subclinically with pathological changes localized mainly in the lungs, lymph nodes, small intestines, liver, kidneys and spleen. Histological findings demonstrate granulomatous inflammation with caseosation and predominance of epitheloide macrophages and single lymphocytes. Tissue samples from internal organs of experimentally infected dogs as well as non-infected but contact animals were investigated by direct PCR. Specific PCR-products were obtained in 44 of 96 studied samples. Eighty-three (86.5%) of PCR results coincided with bacteriological finds, 82 (85.4%) with the pathological and 71 (74.0%) simultaneously with bacteriological and pathological results. The observed specific DNA products in tissue samples of infected and non-infected dogs demonstrate significant sensitivity of PCR method. It could be assumed that the transmission of M. tuberculosis infection is possible by close contact between ill and healthy dogs and that the naturally infected dogs or dogs suffering from tuberculosis may serve as a permanent source of infection to humans and other animals.

Suppression of tumor immunity by electromagnetic fields and glucocorticoids in mice with implanted Ehrlich carcinoma

The immune system plays a major role in the origin, growth and evolution of tumors; factors that decrease the immune response in any way can cause higher tumor incidence and its faster or uncontrolled growth and evolution. The research included 18 healthy male Han: NMRI mice, weighing between 25 and 30g, with ten-day-old tumor deposits, divided into three groups consisting of six mice each. The first group was continuously exposed to extremely low frequency electromagnetic fields (intensity 70-320 microT). The second group was treated with high doses of corticosteroids (dexamethasone). The control group was not treated with corticosteroids, nor was exposed to extremely low frequency electromagnetic fields. The exposure period lasted for ten days. The criteria used to evaluate tumor immunity were: histological findings of leukocyte infiltration around the tumor cells and white blood cell count. The control group presented with excellent immune response to tumor cells. Lymphoplasmacytic infiltrates widely surrounded the tumor. Numerous tumor cells showed signs of cell death. The results showed that exposure of animals to high doses of glucocorticoids resulted in extremely decreased leukocyte infiltration in the tumor tissue (single lymphocytes), while exposure to extremely low frequency electromagnetic fields significantly decreased leukocyte infiltration in comparison to the control group. Comparison of white blood cell count in treated groups revealed that the white blood cell count in both treated groups was decreased, compared with the control group. Extremely low frequency electromagnetic fields significantly suppress the immune response to tumor cells. Dexamethasone treatment resulted in almost complete absence of immune response to tumor cells. Electromagnetic fields and dexamethasone both decrease the white blood cell count.

Clinical application of multiple displacement amplification in preimplantation genetic diagnosis

Multiple displacement amplification (MDA) is a technique used in the amplification of very small amounts of DNA. MDA is reported to yield large quantities of high-quality DNA. The applicability of MDA to single cells was recently demonstrated as a potential technique for preimplantation genetic diagnosis (PGD). This paper shows the first clinical application of MDA in PGD. Two cycles of PGD were performed in two diseases, resulting in two pregnancies. All the diagnoses given on blastomeres were confirmed on the non-transferred whole embryos. The blastomere diagnosis was coupled with short tandem repeat (STR) analysis (16 loci) in all cycles. Allelic drop-out (ADO) assessment and amplification efficiency were evaluated on 40 single lymphocytes derived from parents of each disease. ADO and amplification failure were 10.3 and 2.2% for β-thalassaemia and 17.9 and 2.2% for cystic fibrosis respectively. HLA matching for A, B and DR was performed successfully on single cell for the β-thalassaemia family using similar methods to genomic DNA. The PGD protocol used in all diseases consists of MDA amplification, followed by a standard polymerase chain reaction protocol. Although HLA matching was not applied to embryos, its feasibility was shown on single cell DNA amplified by MDA. Altogether, these data show the simplicity and reliability of performing PGD in combination with HLA matching and STR analysis using MDA.

Expression of individual immunoglobulin genes occurs in an unusual system consisting of multiple independent loci.

Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B lymphocyte lineage. Haplotype or allelic exclusion restricts B lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus.

Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization.

The use of metaphase comparative genomic hybridization (CGH) to screen all human chromosomes for aneuploidy in preimplantation embryos is hindered by the time required to perform the analysis. We report in this paper a novel approach to manufacture a DNA microarray for CGH for the detection of aneuploidy in single cells. We spotted human chromosome-specific libraries on glass slides that were depleted of repetitive sequences and tested our array CGH method in 14 experiments using either single male and/or single female lymphocytes. For the autosomes, the mean normalized ratios were all close to the expected ratio of 1.0 with overall 300/308 (97%) of the normalized ratios falling within the range 0.75 to 1.25. It was possible to deduce the correct copy number of the X chromosome in 13/14 (92.9%) separate array CGH experiments but the Y chromosome in only 4/14 (29%). We tested our microarray CGH method on a single fibroblast from each of three cell lines containing a specific chromosome aneuploidy (trisomy 13, 15 or 18) and in each case our microarray analysis was able to obtain a diagnosis based on the fact that the aneuploid chromosome gave the highest ratio (1.32, 1.27 and 1.27 respectively) with the ratios of all other chromosomes falling within the range 0.75-1.25. Requiring just 30 h, our method may be more suitable for PGD aneuploidy screening than metaphase CGH.

Monitoring of apoptosis in intact single cells with photothermal microscope.

The methods for detection of apoptosis, cannot as a rule be applied to intact single cells and do not allow monitoring of the cell population. The photothermal (PT) method was evaluated for detection and monitoring of apoptosis in single intact cells--lymphocytes and blasts. PT microscopy is based on optical registration of cell response to the thermal impact that is induced in a cell due to absorption of a short laser pulse (532 nm, 10 ns) by cellular hem-proteins. With no pretreatment of cells, optical detection of cellular response with this method may allow monitoring of apoptosis. The PT method was applied for in vitro studies of lymphocytes and K562 blast cells during drug-induced apoptosis. Dexamethasone in 10 microM concentration was used and cell properties were monitored for 6 h. PT parameters of cells deviated from control after incubation with Dexamethasone. Control measurements with fluorescent microscopy (using the Annexin-V-FLUOS kit) verified the development of apoptosis in drug-treated cells, while there was no development of necrosis. Also necrotic cells had PT properties similar to those of control cells. The PT method allowed the detection of the influence of Dexamethasone at earlier stages (2-hr incubation) than the fluorescent technique (4 h). Results obtained confirmed the applicability of PT microscopy for detection and monitoring of apoptosis in single intact cells. The sensitivity of the suggested method may exceed the sensitivity of fluorescent methods.