Flow cytometric analysis of DNA content in human bladder tumors and irrigation fluids

Flow cytometry (FCM) was used to study the DNA distribution of 99 tumor biopsy specimens and 41 bladder irrigation samples from patients with transitional cell carcinoma of the bladder. For tumor biopsy and cystectomy specimens, the frequency of aneuploidy increased with advancing tumor stage and grade. All T0 tumors were diploid. Twenty-seven percent of T1, 71.4% of T2, and 75% of T3 and T4 tumors were aneuploid. All Grade I tumors were diploid. Thirty percent of Grade II and 76.9% of Grade III tumors were aneuploid. The frequency of aneuploidy of tumors in the early stages (Ta, T1) is similar to the incidence of subsequent progression by these tumors described in the literature. For irrigation fluids, the relationship between grade and stage and the frequency of aneuploidy was similar to the relationship seen with tumor specimens. All four patients with only carcinoma in situ had aneuploid cells in their irrigations. The comparison of FCM data of bladder biopsy and bladder irrigation from the same cystoscopic evaluation suggests adequate representation of tumor cells in the irrigation fluids for almost all cases. The authors conclude that DNA ploidy analysis by FCM appears useful in a clinically important group of patients with aneuploid superficial tumors of moderate or high grade. Bladder irrigation analysis appears useful in the follow-up of patients with a history of carcinoma in situ and those with aneuploid tumors.     

Flow cytometric determination of breast tumor heterogeneity.

Flow cytometric analysis was done on the DNA content of nuclei obtained from different sites of small breast tumors. Although specimens for analysis were obtained within a few millimeters of each other, dramatic differences were occasionally observed in the DNA histograms. In a limited study involving 141 consecutive breast specimens submitted for flow cytometry, 52% (74) were found to have at least one DNA aneuploid population. In 18% of DNA aneuploid tumors, one or more specimens from areas grossly identified as tumor had no DNA aneuploid population. Because of the proposed correlation of aneuploidy with a poorer prognosis and possible responsiveness to chemotherapy, multiple sites should be assayed when flow cytometric DNA analysis is done.     

Flow and image cytometric DNA analysis in rhabdomyosarcoma

Rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, with an overall 3-year disease-free survival of 73%. DNA content is known to correlate with prognosis and therapy response in many cancers. To determine the role of DNA content in rhabdomyosarcoma, 23 tumor samples were studied retrospectively: 18 primary tumors and 5 post-chemotherapy recurrences or specimens obtained at second-look surgeries. The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow and image cytometry and correlated with the histology and clinical history. Of the primary tumors 4 were diploid, 4 polyploid, and 10 aneuploid (9 with a single aneuploid G0G1 peak and 1 multiploid) by flow cytometry. The concordance rate between flow and image cytometry was 19 of 23 (83%); one case did not have flow cytometry available. Most embryonal rhabdomyosarcomas were aneuploid (10 of 12; 83%), and they had a high incidence of recurrence in Stages III and IV (4 of 12; 33%). Although aneuploidy in pediatric cancers may predict a therapeutic response and good prognosis, this was not supported by our findings in rhabdomyosarcoma. The tumor DNA content correlated with the clinical stage but not with the patient's clinical course or tumor histopathological type. DNA content did not appear to be as important a prognostic tool as tumor stage.     

Flow cytometric analysis of the nuclear DNA content of hepatoblastoma.

The nuclear DNA content of 15 hepatoblastoma cases was determined in paraffin-embedded tissues by flow cytometry. The DNA index (DI) was calculated, and the ploidy pattern of nuclear DNA was estimated. The correlation between the ploidy pattern and clinicopathologic findings was studied, and the prognostic significance of the ploidy pattern was investigated. An aneuploid pattern was seen in 50% of the lesions with histologic embryonal and anaplastic types. It was not seen in the fetal type. In the tumors with combined epithelial components, the fetal-type component had a diploid pattern in all five cases. The embryonal-type component was associated with aneuploidy in two of five cases. In aneuploid tumors, vascular invasion (tumor emboli in the vessels) was observed more frequently. The prognosis of the patients with an aneuploid tumor was significantly poorer. These results indicate that nuclear DNA ploidy pattern analysis might be useful in investigating the prognosis of hepatoblastoma.     

Flow cytometrically determined DNA content of breast carcinoma and benign lesions: correlations with histopathological parameters

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p less than 0.01) were also observed for the percentage of S cells between normal tissues (6.2 +/- 3.2 SD) and benign lesions (11.1 +/- 6.6 SD), normal tissues (6.2 +/- 3.2 SD) and aneuploid tumors (19.7 +/- 10.3 SD), benign lesions (11.1 +/- 6.6 SD) and aneuploid tumors (19.7 +/- 10.3 SD), and diploid (7.9 +/- 4.0 SD) and aneuploid tumors (19.7 +/- 10.3 SD).     

Association of DNA content and proliferative activity with clinical outcome in patients with diffuse mixed cell and large cell non-Hodgkin's lymphoma

Formalin-fixed and paraffin-embedded lymph node biopsy specimens from 52 untreated patients with newly diagnosed diffuse large cell (n = 48) or mixed cell (n = 4) non-Hodgkin's lymphoma (NHL) were analyzed for DNA content and proliferative activity (PA) by flow cytometry. The results obtained by flow cytometry were compared with the results of cytogenetic studies performed on 28 of the specimens. The median age of the patients was 65 years (range, 15-84 years) and the male to female ratio was 3 to 2. All patients were uniformly staged and uniformly treated with cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, and prednisone. The flow cytometric results were compared statistically by univariate analysis with the rate and duration of complete remission and survival. Tumors with low PA (greater than or equal to 80% of cells in G0/G1 phase) were found in 65% of the patients; 74% of those with low PA versus only 44% of those with high PA achieved an initial complete remission (P less than 0.02). DNA aneuploidy was detected in tumors of 56% of the patients and was associated with a significantly longer duration of complete remission (P less than 0.01). Both low PA and aneuploidy independently predicted longer survival. The predicted 2-year actuarial survival for patients with tumors with low PA was 68% versus 10% for those with high PA (P less than 0.01). Similarly, the 2-year survival of patients with aneuploid tumors was 60% versus 36% for those with diploid tumors (P less than 0.01). The combination of PA and DNA content categorized the patients into four groups with decreasing 2-year survivals: low PA/aneuploid (n = 20), 77%; low PA/diploid (n = 14), 57%; high PA/aneuploid (n = 9), 32%; high PA/diploid (n = 9), 0%. The flow cytometric results correlated well with those of the cytogenetic studies. We conclude that low PA and DNA aneuploidy, both separately and in combination, predict a favorable clinical outcome for patients with diffuse mixed cell and large cell NHL.     

Prognostic value of static cytometry in transitional cell carcinoma of the bladder: recurrence rate and survival in a group of patients at 10 years follow-up

Many studies have indicated that nuclear DNA content evaluation can be used to predict biological behavior of transitional cell carcinoma (TCC) of the bladder. Some authors also indicated that static cytometry is more useful in DNA content analysis than flow cytometry. The aim of the present study was to evaluate the prognostic significance of DNA ploidy in TCC of the bladder, performed by using static cytometry with an image analyzer, and monitoring patients at 10 years follow-up. Thirty-one consecutive patients underwent transurethral or open surgery for bladder tumors, and neoplastic tissue samples taken from each patient were imprinted on glass slides and sent for histopathological and DNA content evaluation. DNA ploidy evaluation was performed using a CAS 200 image analyzer. Nuclear DNA content evaluation was compared to patient follow-up on recurrence, progression or survival performed 10 years after surgery. Pathological evaluation demonstrated the presence of superficial TCC in 23 patients, while 8 had an invasive bladder tumor. Twenty-nine tumor samples were adequate for DNA content measurement, with 13 showing diploid DNA content and 16 with aneuploid DNA content. At 10 years follow-up, all patients with aneuploid DNA content demonstrated a lower survival time (p=0.049) and higher recurrence rate (p=0.0346). A log-rank test demonstrated that stage, grade and nuclear DNA content are the most useful prognostic parameters for predicting the biological behavior of TCC of the bladder. These results confirm that static cytometry is a good and reliable method to evaluate DNA tumor content and considered a useful prognostic parameter for predicting recurrence rate, disease progression or survival in patients affected by bladder tumors.     

Flow cytometric DNA analysis of parathyroid tumors. Implication of aneuploidy for pathologic and biologic classification

The previous cytometric studies on parathyroid tumors have provided conflicting data regarding the relationship between DNA content and histopathology, resulting from differences in technical methods and data analysis. This study measured nuclear DNA of parathyroid tumors by flow cytometry in fresh material and determined whether DNA aneuploidy really assists in making a pathologic diagnosis of carcinoma or not. From May 1987 through April 1989, 65 consecutive patients operated on for primary hyperparathyroidism had DNA analysis of the freshly excised parathyroid tumors. Three of the patients had metastatic lesions of parathyroid carcinoma in the lung, cervical lymph nodes, and lung and mediastinal lymph nodes, respectively. Pathologic classifications of the lesions from the other 62 patients were 54 adenomas, four carcinomas, and four hyperplasias. In all the latter patients, hyperplasia was associated with a multiple endocrine neoplasia syndrome. Unequivocal evidence of aneuploidy was found in all of the metastatic lesions and 60% of the primary lesions of the carcinomas, in 9% of the adenomas and in 50% of the hyperplasias. Therefore, parathyroid carcinomas were more apt to be aneuploid than were adenomas (P = 0.0015, both-sided testing). In each of the cases of aneuploid hyperplasia, a small aneuploid peak was found. The high incidence of aneuploidy in patients with multiple endocrine neoplasia type 1 may indicate the presence of clonal heterogeneity of hyperplastic glands and the presence of an abnormal subset of cells that have malignant potential. Cell distribution analysis did not provide any significant information beyond ploidy level. In conclusion, DNA flow cytometric analysis of DNA ploidy patterns is a valuable adjunct to the histopathologic diagnosis of parathyroid neoplasms.     

Evaluation of DNA ploidy and degree of DNA abnormality in benign and malignant melanocytic lesions of the skin using video imaging

BACKGROUND: Making a morphologic distinction between benign and malignant melanocytic tumors of the skin is frequently difficult, especially because "gray zones" between these lesions often exist. DNA image cytometry as an adjuvant method for the diagnosis and prognostic prediction of premalignant lesions and malignant tumors of many other organs is already well established. The aim of this study was to determine whether DNA image cytometry is helpful in distinguishing benign from malignant melanocytic lesions and whether cytometry would give valid information with which to predict the prognoses associated with malignant melanomas. METHODS: DNA image cytometry was performed on 127 benign and 58 primary maligant melanomas of the skin as well as 11 metastatic melanomas, using an enzymatic single cell solution according to a method described by Heiden et al. in Cytometry (1991;12:614-21). RESULTS: DNA aneuploidy was graded by DNA index (DI) and a 2c deviation index (2cDI). In contrast to benign melanocytic lesions (with 16% DNA aneuploidy), primary and metastatic malignant melanomas had significantly higher frequencies of DNA aneuploidy (86% and 73%, respectively). In the degree of DNA aneuploidy, significant differences between benign and malignant melanocytic tumors could be observed. The mean 2cDI of aneuploid benign lesions was 1.0, whereas the primary malignant melanomas had a mean 2cDI of 2.92 and the metastatic melanomas a mean of 6.9. The frequency of DNA aneuploidy increased with Breslow thickness. Twenty-one patients with primary malignant melanoma developed metastases. All metastasizing primary tumors were aneuploid and showed a significantly higher grade of DNA aneuploidy than nonmetastasizing malignant melanomas. Moreover, none of the diploid malignant melanomas developed metastases. CONCLUSIONS: This study reveals that DNA image cytometry is prognostically and diagnostically relevant to the evaluation of melanocytic lesions of the skin. Nevertheless, it cannot be relied on alone to provide enough information for a diagnosis.     

A flow cytometric analysis of 23 carcinoid tumors

Twenty-three carcinoid tumors were investigated from paraffin-embedded tissue with flow cytometry (FCM) in order to correlate the DNA ploidy with clinical variables. DNA aneuploidy was found in ten tumors (45%) and one tumor was classified as tetraploid. Diurnal urinary excretion of 5-hydroxy indolic acetic acid (5-HIAA) was known to be elevated in seven of eight diploid tumors and in four of seven aneuploid carcinoids with distant metastases. Six (55%) of the diploid tumors had not given rise to metastases at the time of diagnosis, compared with three (30%) of the aneuploid tumors. Six of seven patients with an aneuploid tumor and three of five patients with a diploid tumor, observed for at least 10 years, died of the disease. It was concluded that, unlike in earlier studies with static DNA cytometry, DNA aneuploidy is common in human carcinoid tumors and may occur in tumors secreting biogenic amines.     

Flow cytometric analysis of nuclear DNA content in esophageal cancer. Aneuploidy as an index for highly malignant potential

Flow cytometric analysis of nuclear DNA content was performed on excised malignant tissue of the esophagus. The DNA ploidy pattern was compared with a variety of histologic parameters and the subsequent clinical course to determine whether or not this pattern is associated with the mode of malignant potentiality. Of the 31 patients, eight had the diploidy DNA pattern and 23 the aneuploid DNA pattern. Tumors with the aneuploidy DNA pattern had a significantly higher frequency of lymph node metastasis than did those with the diploidy DNA pattern (P less than 0.01). Mitotic rates in the aneuploid tumors were significantly higher than was the case in diploid tumors (P less than 0.0005). The incidence of recurrence within 12 months after surgery was higher in patients with aneuploid tumors (83.3%) than in those with diploid ones (16.7%), with a statistical difference (P less than 0.05). Thus, the DNA aneuploidy based on flow cytometry closely correlates with the high frequency of nodal involvement and high mitotic rates, factors generally indicative of the aggressive behavior of the malignant tumors. DNA aneuploidy based on flow cytometric analysis is a pertinent index for determining the highly malignant potential in esophageal carcinoma.     

Significance of DNA abnormalities in primary malignant melanoma and nevi, a retrospective flow cytometric study

DNA ploidy of melanocytic skin tumors from 87 patients (53 primary melanomas, 34 nevi) was determined by flow cytometry from routinely prepared paraffin blocks. Ploidy data correlated strongly with conventional morphological parameters. Only 1 of 34 nevi, but 13 of 53 melanomas were aneuploid. Among the melanomas, none of 21 levels I-III melanomas was aneuploid, but 13 of 32 levels IV and V melanomas were aneuploid. There was also a significant correlation between increasing Breslow thickness and the presence of DNA aneuploidy. For 33 melanoma patients with over 2 yr of follow-up (average, 7.1 yr), only 4 of 23 diploid tumors have recurred, but 9 of 10 aneuploid tumors have recurred. We conclude that the biological potential of melanocytic skin tumors is strongly linked to DNA aneuploidy. Since this parameter can be conveniently determined from paraffin blocks, determination of ploidy abnormalities in these tumors may be clinically useful.     

Comparison of flow cytometric DNA content analysis in fresh and paraffin-embedded ovarian neoplasms: a prospective study.

DNA ploidy analysis was performed on both fresh and paraffin-embedded preparations from each of 54 malignant ovarian neoplasms. Aneuploidy was detected in both the fresh and the paraffin-embedded tissue in 19 out of 54 (35%) malignant cases. In addition, aneuploidy was detected exclusively in fresh tissue in seven of the malignant cases, and exclusively in paraffin-embedded tissue in one of the malignant cases, yielding a total of 27 out of 54 (50%) aneuploid cases. The correlation coefficient (r-value) for fresh and paraffin-embedded tissue ploidy analysis in the malignant specimens was 0.91. Although the frequency of recurrence was higher and overall survival lower in the malignant aneuploid specimens of both types, the combined analysis of DNA and survival rates indicated superior prognostic significance of fresh tissue. Of the seven patients in whose specimens aneuploidy was detected exclusively in fresh tissue, all died of recurrent disease during the follow-up period. Our finding indicates that data generated by flow cytometry analysis of formalin-fixed tissue should be interpreted with caution before the data can be used to draw clinical inferences.     

Nuclear DNA content and its prognostic value in lymphoma of the stomach

Nuclear DNA content of 27 primary lymphomas of the stomach was determined by flow cytometry from paraffin-embedded tissue. Thirteen (50%) of the 26 non-Hodgkin's lymphomas were aneuploid. The only case with Hodgkin's disease was diploid. The mean age of patients with aneuploid lymphoma was less than that of patients with diploid lymphoma (53 years versus 63 years, P = 0.02). DNA aneuploidy was more common in tumors with extragastric spread into the adjacent organs or the upper abdominal lymph nodes than in intragastric lymphomas (73% versus 17%; P = 0.003). Patients with aneuploid lymphoma had both inferior crude recurrence-free survival rate (P = 0.05) and survival rate corrected for known intercurrent diseases (P = 0.02) than patients with diploid lymphoma. Extragastric spread of lymphoma into the adjacent organs or the upper abdominal lymph nodes was, however, the most important prognostic factor by a multivariate analysis.     

DNA ploidy analysis of effectiveness of radiation therapy for cervical carcinoma

Cellular DNA content from 30 patients with cervical carcinoma was determined using flow cytometry before and after radiation therapy (RT). The authors attempted to correlate changes in DNA content, tumor response to RT, and post-RT pathologic findings. Before RT, tumors from eight of 30 patients (26.7%) were diploid or near-diploid; tumors from 22 patients (73.3%) were aneuploid. After RT, diploid or near-diploid tumors were found in 23 patients (76.7%), and aneuploid tumors were observed in seven patients (23.3%). Aneuploidy disappeared in 15 of the patient tumors, and complete tumor response (CR) was observed in 13 of these 15 patients (86.7%). Pathologic examinations were negative in 12 of 15 cases and suspicious in one of 15 cases. Of the seven patients whose tumor aneuploidy did not change after RT, CR was observed in only two (28.7%). Pathologic examinations were positive in five of seven cases and suspicious in one of seven cases. The CR for the 22 patients with pre-RT aneuploid tumors was 15 of 22 (68.2%); the CR for the eight patients with pre-RT diploid tumors was two of eight (25%, P less than 0.01). From these data the authors conclude there is a direct correlation between DNA content and radiosensitivity in cervical carcinoma. Aneuploid tumors from these patients were more radiosensitive than diploid tumors, and they patients had a better clinical tumor response and improved pathologic findings.     

Relationship between DNA ploidy, glandular differentiation, and tumor spread in human prostate cancer

DNA ploidy was evaluated by flow cytometry for 45 human prostate carcinomas (34 prostatectomy specimens and 11 biopsies). Twenty tumors (44.4%) contained a distinct aneuploid stem line. All 11 tumors confined to the prostate gland (pathological Stage B) were diploid. The frequency of aneuploidy increased with advancing stage, and most tumors with distant metastases were aneuploid. The degree of glandular differentiation was characterized by the Gleason score. One-third of tumors with a Gleason score of 5 to 6 were aneuploid, whereas over 70% of poorly differentiated tumors with a Gleason score of 9 to 10 were aneuploid. Among diploid tumors, 45.5% were localized carcinomas (Stage B), 36.4% were characterized by invasion outside the prostate (Stage C), and 18.2% formed pelvic nodal or distant metastases (Stages D1 and D2). In nearly two-thirds of patients with aneuploid tumors, pelvic nodal or distant metastases were found. When tumors were classified according to both DNA ploidy and degree of glandular differentiation, then subgroups of tumors with the highest and lowest degree of malignant potential became apparent. Only 7.1% of diploid tumors with a Gleason score of 5 to 6 formed metastases, but 80% of aneuploid tumors with a higher Gleason score (7 to 10) formed metastases. Diploid tumors with higher Gleason scores and aneuploid tumors with lower Gleason scores had intermediate frequencies of metastases. The presence of an aneuploid stem line in prostate carcinomas indicated that the tumor had spread outside the prostate gland or had metastasized. DNA ploidy may be an important prognostic factor for human prostate cancer. DNA ploidy and the degree of glandular differentiation considered together may improve prognostic evaluation of prostate carcinomas.     

The relationship of DNA ploidy to chromosomal instability in primary human colorectal cancers.

The aim of this investigation was to corroborate the relationship between DNA ploidy and chromosomal variation in surgically removed colorectal cancers. For 101 specimens from 21 advanced colorectal cancers, the numerical variations in chromosomes 7, 17, and 18 among cells were measured by fluorescence in situ hybridization using DNA probes specific for centromere of each chromosome, and DNA ploidy was determined by laser scanning cytometry or flow cytometry. DNA diploidy (DNA index = 1.0) was linked with minor variation in copy number of chromosomes 7, 17 and 18, whereas DNA aneuploidy (DNA index > or = 1.2) was found exclusively in tumors with large variations in centromere copy number for all chromosomes. There was a significant difference in the degree of intercellular variations in chromosome copy number between diploid and aneuploid clones for all chromosomes examined (P < 0.001). In near-diploid clones (1.0 < DNA index < 1.2), the numerical variation of chromosome 18 was significantly different from that in diploid clones (P < 0.002), but it was not different from that in aneuploid clones. These observations support the hypothesis that chromosomal instability is associated with DNA aneuploidy in colorectal cancers. Additionally, they suggest that near-diploid tumors are also unstable at a lower level than classic aneuploid tumors and that all chromosomes are not affected equally in near-diploid cases.     

Prognostic effect of DNA aneuploidy from bladder washings in superficial bladder cancer.

BACKGROUND: Superficial (papillary) bladder cancer is associated with progression and death from muscle-invasive bladder cancer, but no reliable predictors of the outcomes have been identified. METHODS: We analyzed the long-term prognostic effect of DNA flow cytometry in bladder washings from 93 subjects with previously resected T(a) and T(1) bladder tumors who participated in a chemoprevention trial of the synthetic retinoid fenretinide. Kaplan-Meier analysis and Cox regression were used to determine the prognostic effect of DNA aneuploidy on cancer progression and mortality in conjunction with conventional clinical factors after a median of 11.5 years (interquartile range, 9.5-11.7 years). RESULTS: Overall, 58 of 93 (62%) specimens were DNA aneuploid at baseline. Progression-free survival was significantly shorter in subjects with stage T(1) [hazard ratio (HR), 31.6; 95% confidence interval (95% CI), 2.6-386.1; P < 0.001] and in subjects with baseline DNA aneuploid washing (HR, 10.5; 95% CI, 1.1-126.1; P = 0.03). The risk of death was also greater for stage T(1) tumors (HR, 2.6; 95% CI, 1.04-6.7; P = 0.04). DNA aneuploidy was a significant prognostic factor also for overall survival (HR, 2.8; 95% CI, 1.0-9.0; P = 0.05). Fenretinide treatment had no significant effect on cancer progression and death. CONCLUSIONS: DNA aneuploidy in washings from endoscopically normal bladder is a significant predictor of progression and death in addition to tumor stage. This biomarker may help to identify and monitor a high-risk group who may benefit from a chemoprevention intervention.     

Early diagnosis of cutaneous T-cell lymphoma by DNA flow cytometry on skin biopsies

Seven patients with mycosis fungoides early plaque stage with nondiagnostic histology had single-cell DNA content measured by flow cytometry for estimation of clonal ploidy. A total of 63 skin specimens were examined by histology and DNA measurements concurrently during the course of the disease. In addition, six patients had blood samples studied. All seven patients demonstrated aneuploid DNA histograms when the specimens were obtained from skin lesions. In 36 specimens the aneuploid peaks were hyperdiploid. By sequential studies one patient demonstrated two different aneuploid cell clones, one located in the hyperdiploid region and one located in the hypotetraploid region. All patients developed mycosis fungoides which were histologically confirmed, and the time from first aneuploid DNA histogram until diagnostic histology varied from 5 to 21 months (median, 12 months). In six of the patients a normal diploid DNA histogram was found of peripheral blood lymphocytes. The finding of aneuploidy in patients with early mycosis fungoides who still have a nondiagnostic histology emphasizes the value of flow cytometry as a complementary diagnostic aid which facilitates an early diagnosis.