Deoxyribonucleic Acid Amplification Research Articles

Identification of human papillomavirus types 16 and 18 deoxyribonucleic acid sequences in bulky cervical cancer after chemotherapy

OBJECTIVE: The objectives of this study were to evaluate the effect of chemotherapy on the continual presence of human papillomavirus deoxyribonucleic acid sequences in bulky cervical cancer tissues and the relationship between the presence of human papillomavirus and the response of these patients to chemotherapy. STUDY DESIGN: Multiple tissue sections obtained from 33 patients with bulky cervical cancer both before and after chemotherapy were analyzed for the presence of human papillomavirus types 16 and 18 by deoxyribonucleic acid amplification. RESULTS: The cytotoxic effects of chemotherapy did not significantly alter the continual presence of human papillomavirus deoxyribonucleic acid sequences in these tissues ( p = 0.8048). The presence of human papillomavirus type 16 deoxyribonucleic acid in tumors treated with neoadjuvant chemotherapy was significantly associated with favorable tumor response compared with type 18-positive patients and type 16/18-negative patients (94.7% vs 42.9%, p = 0.0059 and 94.7% vs 44.4%, p = 0.0004, respectively). Additionally, patients with type 18 deoxyribonucleic acid had a significantly higher risk of recurrence than did type 16-positive patients ( p = 0.0123). CONCLUSIONS: These results seem to suggest that the presence of human papillomavirus deoxyribonucleic acid sequences may serve as a marker to predict the response of bulky cervical cancer to chemotherapy and may be useful in reassessing neoadjuvant treatment for those patients who are free of human papillomavirus or those with type 18 deoxyribonucleic acid. (Am J Obstet Gynecol 1997;176:865-9.)

Detection of human papillomavirus DNA in primary squamous cell carcinoma of the male urethra

Objectives. The prevalence of human papillomavirus deoxyribonucleic acid (DNA) in primary squamous cell carcinoma of the male urethra and corresponding control tissue was studied. Methods. The technique of polymerase chain reaction DNA amplification was used to detect specific humanpapillomavirus DNA sequences in archival pathologic and control tissue. We analyzed 16 cases of primary squamous cell carcinoma of the male urethra and 22 specimens of normal male urethra stratified by location within the urethra. Results. Primary squamous cell carcinoma of the pendulous urethra was significantly associated with humanpapillomavirus type 16 DNA (6 of 6 cases), but the control, tissue from the pendulous urethra was not (0 of 12, P<0.001). Primary squamous cell carcinoma of the bulbous and posterior urethra was not associated with human papillomavirus infection. Conclusions. Infection of the male urethra with human papillomavirus type 16 may have a role in the pathogenesis of primary squamous cell, carcinoma of the pendulous urethra, for which it has a strong predilection, vis-à-vis the bulbous and posterior urethra.

A blind study of the polymerase chain reaction for the detection of mycobacterium tuberculosis DNA

Setting: Nine French laboratories routinely involved in mycobacterial work. Objective: To assess the detection of Mycobacterium tuberculosis in experimental samples by polymerase chain reaction (PCR) using the insertion sequence IS 6110 as a target for deoxyribonucleic acid (DNA) amplification. Design: Nine laboratories participated in a blind study of the detection of M. tuberculosis by PCR in 20 coded samples containing either a definite number of M. tuberculosis complex (positive samples) or environmental mycobacteria (four samples) or no mycobacteria (five samples). Results: Five laboratories reported false-positive PCR results, with an average rate of 7%. All laboratories except one reported positive PCR results for samples containing 10 5 cfu/ml or more. M. tuberculosis DNA was detected in two thirds of samples containing 10 4 and 10 3 cfu/ml, and in one third of the samples containing 10 2 cfu/ml. Conclusion: The results of the study suggest that PCR using IS 6110 as a target for DNA amplication is neither very sensitive nor really specific for the detection of M. tuberculosis.

Early Identification of Candiduria by Polymerase Chain Reaction in High Risk Patients

Purpose Polymerase chain reaction amplification of Candida albicans deoxyribonucleic acid was evaluated as a diagnostic tool for candiduria. Materials and Methods Urine from 120 patients was tested for C. albicans 158 base pair deoxyribonucleic acid by polymerase chain reaction. The study groups included 30 patients with positive urine cultures, 60 critically ill patients and 30 healthy volunteers. Results All patients with proved candiduria had a positive polymerase chain reaction. Of the 60 critically ill patients 5 (8.3 percent) had a positive polymerase chain reaction for C. albicans 24 to 48 hours before identification with routine fungal culture. No healthy volunteer had a positive polymerase chain reaction. Conclusions Polymerase chain reaction amplification is an effective laboratory tool for the early diagnosis of candiduria.

Deletion polymorphism of the angiotensin-converting enzyme gene is independently associated with left ventricular mass and geometric remodeling in systemic hypertension

An insertion/deletion ( I D ) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with myocardial infarction, cardiomyopathy, and left ventricular (LV) hypertrophy. LV mass and geometry are related to cardiovascular morbidity and mortality. Two-dimensional directed M-mode echocardiograms and 24-hour ambulatory blood pressure monitoring were performed in 67 hypertensive subjects. Echocardiographic measurements were assessed in blinded fashion. LV mass index and relative wall thickness were calculated. ACE genotypes were determined by polymerase chain reaction amplification of deoxyribonucleic acid prepared from leukocytes, using primers that encompass the polymorphic segment. Systolic ambulatory blood pressure was higher in subjects with the II genotype. All other patient characteristics were similar across genotype groups. After adjustment for other covariables, the DD and ID genotypes were associated with significantly higher LV mass index than was the II genotype. Adjusted relative wall thickness was also higher in subjects with the DD genotype than in subjects with the ID and II genotypes. On multiple regression analysis, systolic ambulatory blood pressure, gender, body mass index, and the ACE genotype were each independently related to LV mass index (R 2 = 0.53). Systolic ambulatory blood pressure, race, and ACE genotype were each independently related to relative wall thickness (R 2 = 0.34). The ACE genotype explained an additional 3% and 4% of the variations of LV mass index and relative wall thickness, respectively. In conclusion, ACE polymorphism accounted for a small but statistically significant proportion of the variation in LV mass and geometry in our hypertensive subjects.

RhD genotype determination by single sperm cell analysis

OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of her Rh-positive partner. Currently, a reliable technique for an absolute determination of RhD genotype is not available. This study was initiated to develop an accurate method for RhD genotyping in men. STUDY DESIGN: RhD genotype was determined by deoxyribonucleic acid amplification of a D-specific sequence in single sperm cell samples. Micromanipulation techniques were used for sampling of single sperm cells, which were further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in all of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately determined in 10 of 10 donors. A total of 132 single sperm cells were analyzed (8 to 17 samples per donor), of which 96 were successfully amplified as assessed by an internal control. As expected, the specific region of the RhD gene was amplified in all, some, and none of the signal-positive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh-negative subjects, respectively, allowing accurate determination of the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the management of Rh isoimmunization. (A M J O BSTET G YNECOL 1996;174:1300-5.)

Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program: Gibbons W.E.; Gitlin S.A.; Lanzendorf S.E. Am J Obstet Gynecol 1995 172/4 I (1088–1096)

Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program: Gibbons W.E.; Gitlin S.A.; Lanzendorf S.E. Am J Obstet Gynecol 1995 172/4 I (1088–1096)

Accuracy of prenatal determination of RhD type status by polymerase chain reaction with amniotic cells

OBJECTIVE: Our purpose was to determine the accuracy of RhD typing by use of amniocytes obtained at amniocentesis in RhD-negative women. STUDY DESIGN: One hundred thirty-five RhD-negative women undergoing amniocentesis for management of suspected alloimmunization ( n = 95) or routine second-trimester cytogenetic indications ( n = 40) were studied. Amniocytes were then used as template to amplify specific portions of the Rh D and Rh CcEe genes by polymerase chain reaction. The fetal RhD type was confirmed by serologic techniques either after fetal blood sampling or cord blood samples at birth. RESULTS: Thirty-six fetuses were serologically type as RhD negative and all 36 were typed RhD negative by polymerase chain reaction. Ninety-eight fetuses were serologically typed as RhD positive; of these, 96 were correctly typed as RhD positive and two were incorrectly typed as RhD negative, with an overall error rate of 1.4%. Both of the errors occurred in a single batch of six samples tested at the same time. In one of these cases the fetus had mild Rh alloimmune disease and required exchange transfusion at birth. In the second case the fetus had severe hydrops fetalis and died in utero at 28 weeks. Deoxyribonucleic acid isolated from fetal blood was tested with the same polymerase chain reaction technique after delivery, and in both cases the fetus was correctly typed as RhD positive. Deoxyribonucleic acid amplification repeatedly failed in one case. CONCLUSION: Prenatal fetal RhD typin gby polymerase chain reaction with amniotic fluid cells is accurate and reliable. In sensitized pregnancies it allows early management of Rh disease and avoids invasive procedures in RhD-negative fetuses. In nonsensitized pregnancies it avoids the use of anti-RhD immunoglobulin after invasive procedures or during pregnancy. To eliminate the possibility of genetic and laboratory sources of errors, we suggest using different sets of primers at two different loci (e.g., exon 4 to 5 and exon 10).

The Detection of Human Papillomavirus Deoxyribonucleic Acid in Intraepithelial, in Situ, Verrucous and Invasive Carcinoma of the Penis

Purpose We study the prevalence of human papillomavirus deoxyribonucleic acid (DNA) in squamous cell carcinoma and control tissue of the penis. Materials and Methods The technique of polymerase chain reaction DNA amplification was used to detect specific human papillomavirus DNA sequences in archival pathological and control tissues. We analyzed 42 cases of invasive squamous cell carcinoma, 13 of carcinoma in situ, 12 of penile intraepithelial neoplasia, 3 of verrucous carcinoma and 25 of balanitis xerotica obliterans, as well as 29 routine neonatal circumcision specimens and 32 adult circumcision specimens. Results Overall, the detection rates for human papillomavirus DNA in the study and control tissues were 55 percent (23 of 42 cases) for invasive squamous cell carcinoma, 92 percent (12 of 13) for carcinoma in situ, 92 percent (11 of 12) for penile intraepithelial neoplasia, 0 percent (0 of 3) for verrucous carcinoma, 4 percent (1 of 25) for balanitis xerotica obliterans, 0 percent (0 of 29) for neonatal circumcision and 9 percent (3 of 32) for adult circumcision. In all groups human papillomavirus type 16 was the most common genotype identified. Conclusions The prevalence of human papillomavirus DNA is significantly greater in carcinoma of the penis than in control tissue. Moreover, the prevalence is greater in noninvasive lesions (carcinoma in situ and penile intraepithelial neoplasia) than in invasive carcinoma.

Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program

OBJECTIVES: Our purpose was to identify and evaluate practical methods within a preimplantation genetic diagnosis program that will increase the percentage of embryos for which a genetic diagnosis can be obtained, including clinical responses after failure of deoxyribonucleic acid implantation has occurred. STUDY DESIGN: Known human lymphoblast cell lines and human embryo blastomeres were evaluated in a single-cell, nested primer polymerase chain reaction system with primer sequences for the specific locus surrounding the four base pair insertion mutation on exon 11 of β-hexoaminidase A—Tay-Sachs disease, the ΔF508 mutation of cystic fibrosis, and the sex-determining region on the Y chromosome. Reamplification polymerase chain reaction with standard polymerase chain reaction and primer extension preamplification was performed in deoxyribonucleic acid preparations after previous polymerase chain reaction amplification attempts had resulted in failure of amplification. RESULTS: The amplification efficiency of Tay-Sache disease, 51% ( 97 187 ), was significantly lower than that for cystic fibrosis, 85% ( 87 107 ), and for the sex-determining region on the Y chromosome, 85% ( 77 90 ). Tay-Sache disease polymerase chain reaction amplification occurred in 51% of one-cell lymphoblasts, 89% of two-cell lymphoblasts, and 94% of samples when more than two cells were processed together. When previous amplification failure had occurred, standard Tay-Sachs disease polymerase chain reaction resulted in an amplification efficiency of 16% (three of 19), whereas primer extension preamplification polymerase chain reaction for Tay-Sachs disease resulted in amplification of 52% ( 31 59 ) lymphoblasts and 54% ( 13 24 ) of polyspermic human blastomers. Four of six human blastomeres in which amplification failure occurred in a Tay-Sachs disease preimplantation genetic diagnosis cycle amplified by primer extension preamplification polymerase chain reaction, which increased the diagnostic information obtained from four to six of the seven embryos on which biopsy was performed. CONCLUSIONS: We suggest that practical approaches for consideration within a clinical preimplantation genetic diagnosis program to limit the net effect of amplification failure (i.e., reduced embryo transfer number) include increasing the deoxyribonucleic acid content in the polymerase chain reaction tube by using more than one blastomere and by using primer extension preamplification when the initial attempt at amplification fails.

Preimplantation genetic diagnosis for Tay-Sachs disease: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction

To determine the ability to apply preimplantation genetic diagnostic techniques to screen for and prevent Tay-Sachs disease (TSD). A couple, both carriers for the 4 base pair (bp) insertion in exon 11 of the beta-hexosaminidase A gene, which results in TSD, underwent IVF, pre-embryo biopsy, polymerase chain reaction (PCR) DNA amplification of the biopsied blastomeres, and pre-embryo transfer. One to two blastomeres were aspirated using a biopsy pipette that was inserted through an opening in the zona formed with acidified phosphate buffer. Polymerase chain reaction was performed on the individual blastomeres for 20 cycles followed by an additional 30 cycles using nested primers. This yielded amplified DNA products of 272 and 276 bp for the normal and mutant gene, respectively. Heteroduplex formation was used for identification of normal, homozygous affected, and heterozygous pre-embryos. Seven of 13 oocytes fertilized normally and were biopsied at the four- to eight-cell stages. Deoxyribonucleic acid amplification occurred in four of seven pre-embryos (one homozygous affected and three homozygous normal pre-embryos). The three normal pre-embryos that continued to cleave after biopsy were transferred on the evening of day 3 after retrieval. Subsequently, a single gestational sac was observed and the genetic diagnosis was confirmed at amniocentesis. A successful pregnancy and birth were accomplished after preimplantation genetic diagnostic screening for the prevention of TSD.

Pre-Implantation Genetic Diagnosis: A Model of Progress and Concern

Pre-implantation genetic diagnosis (PGD) consists of in vitro fertilization (IVF) and embryo biopsy to remove one or two blastomeres, typically from an eight-cell embryo, followed by single cell diagnosis by deoxyribonucleic acid (DNA) amplification or fluorescent in situ hybridization (FISH). The first pregnancies following testing for X-linked recessive diseases were reported in 1990 from the Hammersmith Hospital, London, U.K. In the past four years, capabilities have greatly expanded to include disease-specific and chromosomal methods. At least eight clinical programmes worldwide are now underway, with a recent (June 1994) global estimate of 149 PGD treatment cycles completed and 29 children born. Although PGD may be a welcome alternative to the currently employed methods of prenatal diagnosis, especially for women who are already undergoing IVF, or for whom therapeutic abortion is morally unacceptable, discussion of the ethical and societal issues must not fall behind the rapidly advancing genetic technologies. We must appreciate that these new technologies will inevitably bring greater pressures to bear on the beneficiaries, the practitioners, and society. We must heed the warnings of societal advocates reinforcing the unforeseen dangers when science moves more rapidly than society can comprehend. We must always remember to consider ‬Just because we can, should we?” and determine in a research setting “For what?” and “For whom?” this technology is appropriate and, “What benefits?” and “What harm?” exist, before any new technology becomes part of our clinical armamentarium.

The allograft valve in heart transplantation and valve replacement: Genetic assessment of the origin of the cells by means of deoxyribonucleic acid profiles

Assessment of the cellular origin of allograft valves is essential in comprehending their biologic behavior and is improving preparation methods. In this study we retrospectively analyzed 10 allografts obtained form patients who underwent valve replacement or heart transplantation. Histologic evaluation and deoxyribonucleic acid amplification by polymerase chain reaction technology with fluorescence labeled primers was performed on different parts of the valve leaflets. Automated analyses of the obtained amplifiers showed in the heart transplantation group the presence of receptor cells intersperses with native donor cells in three cases. Preliminary results for the valve replacement group are inconclusive as yet. ( J THORAC CARDIOVASC SURG 1995;109:218-23)

Single-cell analysis of the RhD blood type for use in preimplantation diagnosis in the prevention of severe hemolytic disease of the newborn

OBJECTIVE: Our purpose was to develop a molecular assay to determine the fetal RhD blood type on single diploid cells, including blastomeres. STUDY DESIGN: Polymerase chain reaction amplification of a 99 bp deoxyribonucleic acid fragment of the RhD gene or a 113 bp fragment from the RhCE gene was performed from 20 venous blood samples and 20 amniotic fluid samples and from 60 single-cultured lymphoblasts and 12 media blanks mixed in a blinded fashion. This reaction was similarly tested after whole-genome amplification on 10 lymphoblasts and seven human blastomeres. RESULTS: Deoxyribonucleic acid amplification was successful and correct from all genomic deoxyribonucleic acid samples. Ninety-seven percent of single cells amplified: correct diagnosis was made in 96%. Five blastomeres successfully amplified. No media blanks produced amplified, contaminating deoxyribonucleic acid. CONCLUSIONS: The RhD blood type can be determined reliably from single cells and can be used for preimplantation genetic diagnosis for the prevention of rhesus hemolytic disease.

A Novel Mutation of the Androgen Receptor Gene Ligand-Binding Domain (V734P) Associated with the Complete Androgen Insensitivity Syndrome

We report a patient who had the complete androgen insensitivity syndrome (CAIS) associated with a novel point mutation in the ligand-binding domain of the androgen receptor (AR) gene.The patient was 12 years old with complete female external genitalia, inguinal testes, and a 46, XY karyotype. In endocrine tests, basal serum levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were elevated and the FSH and LH responses to luteinizing hormonereleasing hormone (LH-RH) administration were exaggerated. The basal testosterone level and response to human chorionic gonadotropin (HCG) were compatible with those of a normal boy.The AR gene was examined and a single nucleotide exchange from G to T at position 7, 362 was detected, which resulted in the substitution of phenylalanine for valine at position 734. This amino acid is located on exon E within the ligand-binding domain of the AR. Screening for the G to T mutation at nucleotide position 7, 362 by allele-specific amplification of genomic deoxyribonucleic acid (DNA) did not reveal the mutant DNA in normal subjects. The valine residue at position 734 is highly conserved in the ligand-binding domains of the steroid receptor superfamily. Thus, this novel mutation of exon E (V734P) may be associated with the phenotype of CAIS.

Schistosomiasis in Dogon country, Mali: identification and prevalence of the species responsible for infection in the local community

The prevalence of schistosomiasis amongst the Dogon people in 4 villages and one school of the Bankass district of Mali was determined during 2 surveys in 1992; 1398 urine and 1199 stool samples were examined. The most common schistosome was Schistosoma haematobium, with an overall prevalence of 51.3%; S. mansoni had a prevalence of 12%. No S. intercalatum egg was seen in the stools. Biomphalaria pfeifferi and Bulinus truncatus were found in pools at the base of the Dogon cliffs; Bulinus forskalii was found in smaller numbers in brick pits. Two isolates from urine samples of children were identified as S. haematobium in the laboratory using an α-glycerophosphate marker, restriction enzyme analysis of ribosomal deoxyribonucleic acid (rDNA) and random amplification of polymorphic DNA. The isolates did not develop in Bulinus forskalii or B. crystallinus of the B. forskalii group. Some evidence for past hybridization of S. haematobium and S. intercalatum is provided by the enzyme and rDNA results as well as the positive Ziehl-Neelsen staining of polymorphic eggs in urine samples. The findings are discussed in relation to the published observations concerning schistosomiasis in travellers returning from this region of Mali.

The use of polymerase chain reaction to determine fetal RhD status

Objective: Our purpose was (1) to establish the accuracy of a deoxyribonucleic acid amplification method in determination of RhD status in adult blood samples, including weak D variants (previously referred to as Du) and a D mosaic, and (2) to apply the method to determine fetal RhD status in alloimmunized pregnancies.Study Design: Twenty-five adult blood samples, including five weak D variants and one D mosaic, were analyzed with a polymerase chain reaction to determine RhD type. The method was then applied to amniotic fluid samples obtained by amniocentesis from three RhD-negative women with known RhD sensitization.Results: RhD type determined by polymerase chain reaction for all adult blood samples agreed with serologic typing results. All weak D variants and the D mosaic gave results consistent with RhD positivity. Fetal RhD status was determined in each of the three alloimmunized pregnancies, and obstetric management decisions were made on the basis of these results.Conclusions: This polymerase chain reaction method allows rapid and accurate determinations of fetal RhD status by amniocentesis. Fetal blood sampling or serial amniocenteses may be avoided when the fetus is RhD negative, and plans for surveillance and intervention can be confidently made if the fetus is RhD positive. However, before the widespread use of this assay, its sensitivity and specificity must be established. Because weak D variants and a D mosaic demonstrated RhD-positive status by polymerase chain reaction, the method described is applicable to these RhD variants.

Rapid diagnosis of cytomegalovirus lung infection by DNA amplification in bronchoalveolar lavages

We used polymerase chain reaction (PCR) to detect cytomegalovirus (CMV) deoxyribonucleic acid (DNA) in the bronchoalveolar lavages (BAL) of 16 CMV-infected patients with active disease. We also tested PCR on a control group of 20 patients including latently infected patients without evidence of active CMV infection. Results were compared with those of CMV culture and of a rapid method of diagnosis which detects CMV in BAL cells by nucleic acid hybridization. PCR allowed the diagnosis in 93% of the actively infected patients compared to 73% for the CMV culture. Among the 20 control patients without evidence of active CMV infection, PCR was negative in all the 24 BAL tested. Hybridization on BAL cells with the CMV probe detected nine out of 10 actively infected patients, but the specificity of the test was only 68·5%. In our experience, PCR appears to be at least as sensitive as CMV culture, it provides results faster and it performs better than the detection of the virus by hybridization on BAL cells. Only active CMV infection was detected with the PCR conditions used in our study. This suggests that the PCR can be applied to bronchoalveolar lavage fluids as a rapid method to detect CMV lung infection.

Clinical utility of fetal RhD typing in alloimmunized pregnancies by means of polymerase chain reaction on amniocytes or chorionic villi

Our purpose was to describe the clinical utility of a deoxyribonucleic acid amplification method for determining fetal RhD status in alloimmunized pregnancies Six RhD-negative women with alloimmunized pregnancies and heterozygous partners underwent amniocentesis (n = 5) or chorionic villus sampling (n = 1). Fetal RhD type was determined by polymerase chain reaction and results disclosed to the attending physicians. Knowledge of the fetal RhD status avoided further invasive procedures in two pregnancies and facilitated the timing or performance of intrauterine transfusions in the remainder. In alloimmunized pregnancies the ability to RhD-type the fetus in amniotic fluid avoids the risks of fetomaternal hemorrhage and increased sensitization associated with fetal blood sampling or chorionic biopsy. This allows more rational pregnancy management, avoiding invasive procedures in the presence of an RhD-negative fetus, or planning therapeutic interventions or offering termination of pregnancy in the presence of an RhD-positive fetus.

Immunoglobujin A in Graves’ orbital tissue: Deoxyribonucleic acid amplification by polymerase chain reaction

A role for IgA autoantibodies in Graves' ophthalmopathy is suggested by the presence of immunoglobulins of this class in Graves' orbital tissue, as detected by immunohistochemistry. We, therefore, investigated the possibility of using the polymerase chain reaction (PCR) to amplify IgA immunoglobulin genes from plasma cells infiltrating Graves' eye tissue. Template cDNA was reverse-transcribed from orbital muscle (M) mRNA of one patient (#7) and from orbital connective tissue/fat (F) mRNA of two patients (#1 and #7), both undergoing surgery for exophthalmos because of severe infiltrative ophthalmopathy. Preliminary studies to establish the PCR procedure were performed for kappa light chain DNA amplification. With the very small amount of orbital tissue template available, the sensitive "hot start" modification of the PCR was necessary to amplify significant amounts of kappa light chain DNA. Using this procedure, IgA heavy chain DNA was amplified from both connective tissue/fat (F7) and muscle (M7) cDNA of patient #7. The DNA yield was less for IgA than for IgG using the same template. There was no significant IgA (or IgG) DNA product using the connective tissue/fat cDNA of patient #1. While not implying that IgA-infiltrating plasma cells are specific for Graves' orbital tissue, our studies nevertheless demonstrate the feasibility of amplifying the genes coding for IgA antibodies from Graves' orbital tissue plasma cells. Expression of these immunoglobulin genes in future studies will make it possible to determine the antigen specificity of the antibodies expressed by Graves' orbital tissue plasma cells.