Immunologic monitoring of the cardiac transplant patient.

Peripheral blood lymphocyte morphology and cell surface markers were repeatedly evaluated in 49 orthotopic cardiac transplant patients over a period of three years to determine the utility of these parameters in predicting an episode of acute cardiac rejection. Lymphocytes were measured with a calibrated microscope and termed "activated" if greater than 10 mu in diameter. If a patient demonstrated greater than 30 activated lymphocytes/cu mm, the same lymphocytes were stained with monoclonal antibodies directed to T-cell subsets and B cells. These findings were retrospectively correlated with endomyocardial biopsy results. Absolute numbers of T-cell subsets and B cells were analyzed via Student's t test to identify significant differences during acute cardiac rejection. Of 347 biopsy specimens, 47 demonstrated histologic features of acute cardiac rejection. Simultaneous immune activation of lymphocytes occurred with 33 of 47 samples, while another 51 episodes of lymphocyte activation were detected without acute rejection. No statistically significant differences in absolute numbers of T-lymphocyte subsets were noted at the time of acute rejection.

DNA analysis of atrial myxomas.

The atrial myxoma is a primary tumor of the heart which may have an uncertain clinical course. In this study, we performed flow cytometric DNA analysis of 15 paraffin-embedded atrial myxomas and correlated DNA ploidy status and proliferative fraction with clinical findings. Twelve of 15 cases (80 percent) were diploid and the remaining three cases (20 percent) were aneuploid. Two patients with aneuploid histograms were free of tumor at the time follow-up; the third patient experienced local tumor recurrence and metastases. Five patients with diploid myxomas demonstrated an elevated (greater than or equal to 17 percent) proliferative cell cycle fraction; four of these patients experienced embolic phenomenon or tumor recurrence. This pilot study suggests that an atrial myxoma with either aneuploid DNA content or elevated proliferative fraction may be associated with aggressive biologic behavior.

Potential role of hematopoietic stem cell transplantation in children with secondary acute lymphocytic leukemia.

Secondary acute lymphocytic leukemias (ALL) are uncommon events in the pediatric patient population. There are few detailed reports on the laboratory characteristics and clinical course of patients with secondary lymphocytic leukemia. Historically, these patients have had a poor outcome. We report two patients treated at one institution who developed treatment-related secondary ALL. Both patients underwent hematopoietic stem cell transplantation, one with a compatible unrelated donor cord blood unit and one with an HLA-matched sibling donor bone marrow. One of the two patients survives disease-free 3 years after transplantation.

DNA ploidy, cell cycle kinetics, and low versus high grade atypia in endometrial hyperplasia.

There have been few studies of DNA ploidy and cell cycle kinetics in endometrial hyperplasia. The authors studied archival cases of proliferative endometrium, simple, complex and atypical endometrial hyperplasia and well, moderately, and poorly differentiated endometrial adenocarcinoma by flow cytometry and also evaluated the significance of the degree of cytologic atypia (low versus high) in endometrial hyperplasia relative to the occurrence of carcinoma. All proliferative endometria, all types of hyperplasia and well and moderately differentiated carcinomas were diploid. Two-thirds of poorly differentiated adenocarcinomas were aneuploid. Neither S-phase fractions or proliferative fractions (S+G2M) could distinguish among the different types of hyperplasia or predict which hyperplasias were associated with carcinomas. The degree of cytologic atypia in atypical hyperplasia was not predictive of the occurrence of carcinoma. Poorly differentiated carcinomas showed significant differences in DNA ploidy, S-phase, and proliferative fractions from endometrial hyperplasia and lower grade carcinoma. These results support the concept that there are two fundamentally different types of endometrial carcinoma.

Subcutaneous phaeohyphomycosis of the finger caused by Exophiala spinifera.

A patient with severe rheumatoid arthritis treated with prednisone had a painless soft tissue nodule develop on the dorsal aspect of the ring finger. She denied any history of hand trauma, animal exposure, or systemic symptoms such as fever or malaise. Fungal cultures performed on an aseptically obtained aspirate of this lesion demonstrated dark, olive-black creamy colonies on Sabouraud's agar. Slide cultures made from mold colonies produced slender conidial forms with annellations and spine-like conidiophores, features characteristic of Exophiala spinifera. The lesion was surgically excised, and the patient was successfully treated with a course of oral itraconazole. This nodular lesion has not recurred at the time of this writing. Exophiala species are difficult to differentiate, and E. spinifera may be confused with Exophiala jeanselmei. A literature review will consider Exophiala species and clinical manifestations produced by these dematiaceous fungi.

Rapid analysis of lymphocyte subsets in cord blood.

Several investigators have enumerated cellular populations in neonatal cord blood with variable results. In this study, the authors established reference ranges for lymphocyte subsets in cord blood from healthy newborns using a whole blood lysis technique on the Coulter Immunoprep Epics Leukocyte Preparation System (Coulter Immunology, Hialeah, FL). All analyses were performed on a flow cytometer by gating on forward angle versus 90 degrees light scatter. Lymphocytes demonstrated all surface markers examined, including T4, T8, T3, T11, B1, NKH-1, I3, and 4B4; 2H4 suppressor inducer lymphocytes were prominent in neonatal blood. The authors think this standardized system may be suitable for use in neonatal and pediatric patients because it quickly processes small aliquots of whole blood with minimal sample manipulation.

Reference ranges for lymphocyte subsets in pediatric patients.

Peripheral blood lymphocyte subset reference ranges were examined in a large group (N = 130) of healthy pediatric patients ranging in age from 1 month to 17 years. All samples were stained with monoclonal antibodies, processed with a whole blood lysis technique, and analyzed on a flow cytometer. Data analysis demonstrated statistically significant changes in most lymphocyte subsets at age 3 years. The relative and absolute numbers of total lymphocytes, CD2, CD4, and CD19 cells; absolute numbers of CD3 and CD8 cells; and CD4/CD8 ratios were high at birth, decreased during early childhood, and closely approximated adult reference values after age 3 years. The relative numbers of CD8 lymphocytes were low in early childhood and then rose to adult values after 3 years of age. The relative percentage of CD3 cells remained stable over all ages studied. Although "adult" lymphocyte subset reference ranges may be similar to those in children older than 3 years, age-adjusted reference ranges should be used for the early childhood period.

Flow cytometric immunophenotypic characterization of pediatric and adult minimally differentiated acute myeloid leukemia (AML-M0).

We reviewed the clinicopathologic and immunophenotypic profiles of 7 pediatric and 11 adult minimally differentiated acute myelogenous leukemias (AML-M0). We also compared and evaluated myeloperoxidase in leukemic blasts using standard cytochemical and polyclonal antibody immunohistochemical stains. No distinctive clinical findings were noted in either patient group; however, thrombocytopenia typically was more prominent in adults. Adult AML-M0 also was associated with an immature myeloid profile (CD34+, terminal deoxynucleotidyl transferase positive, CD13+, and CD33+), in contrast with pediatric AML-M0, which usually lacked terminal deoxynucleotidyl transferase or CD34 but expressed bright CD33 with weak or negative CD13. Coexpression of the T-cell-associated antigen CD7 was observed in both groups. Antibody immunohistochemical stains were more sensitive than cytochemical stains for detection of myeloperoxidase activity and a useful adjunct for establishing a diagnosis of myeloid leukemia in paraffin-embedded marrow tissues.

Terminal deoxynucleotidyl transferase staining in acute leukemia and normal bone marrow in routinely processed paraffin sections.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear protein widely used as a marker for the diagnosis and classification of acute leukemia. The usual methods for detecting TdT require smears, imprints, or cryostat sections of unfixed tissue. A polyclonal rabbit anti-TdT serum was used to immunostain 54 routinely processed bone marrow sections from patients with acute leukemic disorders, using a recently described antigen-unmasking technique based on microwave oven heating. The specificity of this method of TdT analysis was confirmed by comparing the results obtained with conventional TdT analysis by indirect immunofluorescence. Terminal deoxynucleotidyl transferase reactivity was also evaluated in 44 nonmalignant and normal bone marrow specimens. All cases that were TdT-positive by immunofluorescence (41 of 42 "pre-B" and T-cell acute lymphoblastic leukemia, 2 of 5 acute myeloid leukemia, and 1 of 5 chronic myeloid leukemia in blast crisis) were also positive in paraffin sections. The percentage fluorescence positivity correlated with the percentage of immunoperoxidase stained cells in 44 of 45 cases. The remaining nonneoplastic and normal bone marrow biopsy specimens were TdT-negative. These results show that TdT immunoperoxidase staining of conventionally processed bone marrow specimens can be readily achieved by the use of a simple antigen-unmasking technique and may provide useful diagnostic information particularly in cases in which fresh tissue samples are unavailable.

Reference ranges for lymphocyte subsets. A comparison of standard vs rapid whole-blood lysis techniques.

Reference ranges for lymphocyte subsets may vary with processing techniques, monoclonal reagents, or analytic methods. We compared reference ranges obtained for T- and B-lymphocyte subsets by means of standard manual whole-blood lysis with a wash step vs a rapid, no-wash whole-blood lysis system. Both techniques demonstrated reference ranges similar to those in previous literature reports. The ranges established with standard and rapid lysis were similar when antibodies directed to the same cluster designation were used. Although slight statistical differences in relative percentages of CD2 and CD3 lymphocytes were observed, these differences were probably not clinically significant. These data indicate that the rapid technique provides a standardized method for enumerating T and B lymphocytes in peripheral blood.

Proliferative activity and aneuploidy in pleomorphic adenomas of the salivary glands.

We used flow cytometry in a retrospective study of pleomorphic adenoma and carcinoma arising in pleomorphic adenoma, using paraffin-embedded tissue, to assess the relationship among proliferative activity, ploidy, and recurrence or malignant transformation. Twenty-four specimens obtained from 22 tumors were acceptable for analysis (co-efficient of variation, < or = 7.0), including multiple samples from two tumors. Fourteen tumors (13 benign and one malignant) were diploid. Six tumors were aneuploid: four benign pleomorphic adenomas and two carcinomas arising in pleomorphic adenoma. Two tetraploid tumors were malignant recurrences from the same patient. Of the recurrent tumors (nine benign and four malignant), 54% were aneuploid. The highest S-phase fractions were observed in recurrent and malignant pleomorphic adenomas. Immunostaining with p105, a nuclear proliferation antigen, revealed increased proliferative activity in a majority of pleomorphic adenomas. Increased proliferative activity and aneuploidy occurred in benign pleomorphic adenomas.

Flow cytometric DNA analysis of pediatric intracranial ependymomas.

OBJECTIVE: To examine the clinicopathologic features and perform flow cytometric DNA analysis of pediatric intracranial ependymomas to determine whether any of these parameters were predictors of clinical outcome. METHODS: Flow cytometric DNA analysis was performed on 17 paraffin-embedded tumors from patients aged 7 months to 16 years. RESULTS: Seven cases were aneuploid, while the remaining 10 were diploid. Proliferative fractions varied from 1% to 17%. CONCLUSIONS: No correlation between histologic features such as mitotic activity, cellularity, pleomorphism, vascular proliferation, and length of survival was observed. However, the presence of a diploid DNA stemline, elevated proliferative fraction, or young age were associated with a poor clinical outcome and shortened survival times (P < 0.05). Additional studies of larger patient groups with extended follow-up are necessary to confirm these findings.

Immunophenotyping of cytologic specimens by flow cytometry.

Immunophenotyping by flow cytometry is well established as an ancillary technique in the diagnosis of hematopoietic neoplasms. However, flow cytometry is rarely performed on cytologic specimens because most cytologist are more comfortable with direct microscopy and believe that there is inadequate cellularity for analysis. Paradoxically, cytologic material is usually cell suspensions making it ideal for flow cytometry. In order to evaluate the usefulness of immunophenotyping cytologic specimens by flow cytometry, we retrospectively reviewed all cytologic specimens submitted to our flow cytometry unit from 1988 to 1991. Thirty-one cerebrospinal fluid specimens were analyzed. There were inadequate cells for analysis in 15 cases. Five showed a monoclonal proliferation; 11 were nondiagnostic. A range (r) of one to six cell surface markers were performed. Thirty-two body cavity fluids were analyzed: 7 peritoneal, 19 pleural, 2 pericardial, and 4 bronchoalveolar lavage. There were cells to analyze in all cases. Seven had a monoclonal proliferation; 25 were nondiagnostic (r = 4-21 markers performed). One hundred eighteen fine needle aspirates (FNA) were reviewed; 58 FNA were radiologically guided, 60 were superficial lesions. There were inadequate cells for analysis in two cases. Sixty-one demonstrated a monoclonal proliferation; 55 were nondiagnostic (r = 1-22 markers performed). We conclude that immunophenotyping by flow cytometry is of limited value for cerebrospinal fluid analysis and that knowledge of previous immunophenotyping studies is essential for correct analysis; analysis of body cavity fluids is easily performed but less often demonstrates a monoclonal proliferation. Immunophenotyping by flow cytometry is a valuable adjunctive technique for FNA and yields adequate cells for analysis.

DNA analysis of neoplasia: an introduction for the family physician.

Pathologic evaluation of a neoplastic process has traditionally consisted of microscopic examination of a stained section of tissue. Although this method is generally reliable when performed by an experienced pathologist, the morphologic features of a lesion may not consistently predict biologic behavior. The DNA content of tumors can be studied with a flow cytometer to help determine the prognosis and risk of tumor recurrence. DNA analysis of a neoplasm may provide the clinician with important prognostic information and, at some future date, may help direct chemotherapy or other treatment protocols.

Flow cytometric DNA analysis of extramammary Paget's disease of the vulva.

Vulvar extramammary Paget's disease (EMPD) is an uncommon disease entity that occurs predominantly in postmenopausal white women. The clinical behavior of this neoplasm is extremely variable, reflecting the various histological patterns that have been reported with this lesion. Flow cytometry has been used as a method of obtaining prognostic information about a number of gynecological neoplasms, yet to date there have been no flow cytometric studies performed on this unusual neoplasm. We performed flow cytometric analysis of 14 cases of paraffin-embedded vulvar EMPD in patients ranging from 45 to 86 years of age. We correlated histological features and clinical recurrence risk with DNA analysis. Although we were unable to show a statistically significant correlation between DNA ploidy or S-phase and time to recurrence, we did show a statistically significant correlation between DNA ploidy and histological features. Aneuploidy appears to be associated with in situ sweat gland adenocarcinoma, invasive carcinoma, and lymphatic invasion. These results suggest that Paget's cells with aneuploid DNA stem lines may be associated with the potential for aggressive biologic behavior.

Flow cytometric analysis of DNA content and RAS P21 oncoprotein expression in ovarian neoplasms.

Flow cytometric analysis of DNA content and ras p21 expression were studied in paraffin-embedded normal ovary (NO, n = 10), serous cystadenoma (SA, n = 11), serous tumors of low malignant potential (LMP, n = 13), and papillary serous cystadenocarcinoma (SCa, n = 7). Tissue for DNA analysis was processed via a modified Hedley technique; a separate aliquot of the same sample was stained with a monoclonal antibody directed to oncoprotein ras p21 (clone Y13259). NO (10/10) and SA (11/11) demonstrated diploid DNA stemlines; 4/12 LMP and 5/7 SCa were aneuploid. S-phase fraction varied with significant differences (p less than 0.001) for SA (mean = 4.4%), LMP (mean = 9.0%), and SCa (mean = 12.7%). When all cases were evaluated, p21 expression was relatively lower in NO and SA (mean = 4.2%) in contrast to LMP and SCa (mean = 13.0% and 8.1%). Diploid cases were examined separately, and lesions with high p21 expression were associated with a diagnosis of LMP/SCa (p less than 0.001), whereas diploid tissues with low p21 expression were associated with a nonmalignant diagnosis (NO/SA). This study suggests that aneuploidy or diploidy with high p21 expression (greater than 10%) may be associated with LMP or frankly malignant ovarian tumors.

Flow cytometric DNA analysis of ovarian Brenner tumors and transitional cell carcinomas.

Flow cytometry has been previously used as a method of obtaining prognostic information about ovarian carcinomas using ploidy, DNA index, and S-phase fraction. DNA content has also been assessed in ovarian tumors of low malignant potential. Brenner tumor variants such as metaplastic, proliferating, and low malignant potential, recently designated as intermediate Brenner tumors, and malignant Brenner tumors are unusual tumors that present classification problems. Their histological appearance may not accurately reflect biological activity. We used flow cytometry to analyze paraffin-embedded tissue for DNA content and S-phase in 34 Brenner tumors, three ovarian transitional cell (urothelial) carcinomas (TCCs), and nine normal control ovaries. We correlated histological and clinical features with DNA analysis. Twenty-five Brenner tumors and three ovarian TCCs were acceptable for histogram analysis (coefficient of variation less than 7.0). Thirteen typical, three metaplastic (extensive mucinous or glandular metaplasia), and two proliferating (papillary formation with increased cellularity) Brenner tumors were diploid. One proliferating tumor was tetraploid. The single Brenner tumor of low malignant potential was diploid but had an increased S-phase. Four of five malignant Brenner tumors were aneuploid, and one was diploid. All the TCCs were aneuploid. S-phase was elevated in intermediate and malignant Brenner tumors and TCC. Limited numbers of cases available preclude prognostic prediction based on ploidy in malignant Brenner tumors or primary ovarian TCCs. DNA ploidy and S-phase reflect the intermediate status of metaplastic, proliferating, and low malignant potential Brenner tumors.

Flow cytometric DNA analysis of placental-site trophoblastic tumors.

The placental-site trophoblastic tumor is a rare form of gestational trophoblastic neoplasia. Although originally considered benign, it is now apparent that this lesion can be associated with aggressive clinical behavior. Our study examined the DNA ploidy status and clinicopathologic features of four new cases of placental-site trophoblastic tumor. Three cases demonstrated diploid DNA stemlines with S-phase fractions ranging from 6% to 16%. These patients were alive and well at follow-up and had low-serum human chorionic gonadotrophin (hCG) levels. A fourth patient, who had a large tumor, demonstrated a tetraploid DNA peak with a prominent S-phase fraction. This patient exhibited an elevated serum hCG at limited follow-up. Flow cytometric DNA analysis may be a useful adjunct for the identification of placental-site trophoblastic tumors with malignant potential.