Additional mutation in PROKR2 and phenotypic differences in a Kallmann syndrome/normosmic congenital hypogonadotropic hypogonadism family carrying FGFR1 missense mutation.

Congenital hypogonadotropic hypogonadism (CHH) is a genetically and clinically diverse disorder encompassing Kallmann syndrome (KS) and normosmic CHH (nCHH). Although mutations in numerous genes account for nearly 50% of CHH cases, a significant portion remains genetically uncharacterized. While most mutations follow the traditional Mendelian inheritance patterns, evidence suggests oligogenic interactions between CHH genes, acting as modifier genes to explain variable expressivity and incomplete penetrance associated with certain mutations.In this study, the proband presented with nCHH, while his son exhibited KS. We employed whole-exome sequencing (WES) to investigate the genetic differences between the two, and Sanger sequencing was used to validate the results obtained from WES.Genetic analysis revealed that both the proband and his son harboured a mutation in FGFR1 gene. Notably, an additional rare mutation in PROKR2 gene was exclusively identified in the son, which suggests the cause of the phenotypic difference between KS and nCHH.

A case of microsatellite instability-high clinically advanced castration-resistant prostate cancer showing a remarkable response to pembrolizumab sustained over at least 18 months.

Defective DNA mismatch repair genes can lead to microsatellite instability (MSI)-high status in prostate cancer (PC). Accumulation of replication errors in DNA leads to the production of abundant neoantigens, which could be targets for immune checkpoint inhibitors (CPIs). However, the incidence of MSI-high PC is low, and not all patients show a satisfactory therapeutic response to CPIs. Here, we present the case of a patient with MSI-high castration-resistant PC who showed a remarkable and durable response to pembrolizumab. The patient was resistant to abiraterone, docetaxel, and cabazitaxel and was suffering from multiple tumor-associated or treatment-related complications, such as urinary tract infection, infective endocarditis, and uncontrollable prostatic hemorrhage. Soon after the start of pembrolizumab therapy, the patient showed a dramatic decrease in prostate-specific antigen from 35.67 ng/mL to an undetectable level and a remarkable reduction in the size of a massive prostate mass and lymph node metastases, with an absence of treatment-related complications. Specimens from the transurethral resection of prostate cancer during cabazitaxel treatment for control of prostate bleeding and also that from the prostate biopsy at initial diagnosis revealed MSI-high status. Immunohistochemistry showed loss of MSH2 and MSH6, and whole-exome sequencing revealed an approximate tumor mutation burden of 61 mutations/Mb as well as biallelic loss of MSH2 Pembrolizumab could show a significant effect even in a heavily treated patient with MSI-high advanced PC. Accumulation of detailed clinical and genomic information of cases of MSI-high PC treated with pembrolizumab is necessary for optimal patient selection.

Barriers for sperm cryopreservation in advanced germ cell tumor patients: a 20-year experience.

BACKGROUND: This study aimed to investigate barriers to sperm cryopreservation for patients with advanced germ cell tumors. METHODS: We reviewed data collected from patients who underwent chemotherapy for advanced germ cell tumors in our institutions from 1996 to 2016. We divided sperm cryopreservation procedures into three steps: offering information about sperm cryopreservation, patients' decision making and sperm collection, and investigating the barriers in each step. RESULTS: Of the 91 patients, 43 (47%) successfully completed sperm cryopreservation. Thirty (33%) patients were not offered information about sperm cryopreservation from the doctors. Nine patients (9.9%) were offered but declined preservation. Nine patients (9.9%) were not able to provide sperm because of azoospermia (5) and anejaculation (4). Among 43 patients who successfully provided sperm, 10 (23%) had their cryopreserved sperm used for 21 cycles of in vitro fertilization. Ten pregnancies and 7 fatherhoods were achieved. Thirteen patients (30%) had their cryopreserved sperm discarded without use, whereas 20 (47%) continued preserving their sperm for a median 12.5 years. CONCLUSIONS: Not only offering proper information about sperm cryopreservation, but also shared decision making prior to chemotherapy, and considering fertility preservation before orchiectomy are imperative. The rate of use of preserved sperm was relatively high, but decision making for sperm disposal should also be supported.

Sperm cryopreservation: Clinical and fertility outcomes in male oncological patients with germ cell tumors or hematological disorders.

PURPOSE: There is insufficient understanding of the effects of malignant diseases themselves and chemotherapy on semen quality and final fertility outcomes. Here, the authors focused on the patients with malignant diseases who cryopreserved sperm pre- or post-chemotherapy for future fertility, and revealed how clinical settings can affect semen quality and final outcomes. METHODS: The authors reviewed the records of 257 patients with malignant diseases who cryopreserved sperm. Among 257 cases, 113 men with germ cell tumors (GCTs) and 111 men with hematological disorders (HDs) were included in this study. Twenty-five patients who achieved successful outcomes using cryopreserved sperm were also analyzed. RESULTS: In the men with GCTs and HDs, respectively, differences were observed in age (28 vs 27 years), sperm concentration (32.6 vs 46.1 million/mL, P < 0.05), motility (42.2% vs 41.0%), and the rate of cryopreservation before chemotherapy (90% vs 59%, P < 0.0001). For successful pregnancies and deliveries, age at cryopreservation (30.0 vs 35.3 years, P < 0.05) and disease type (12/16 vs 3/9, P < 0.05) were significant factors. CONCLUSIONS: Compared to patients with GCTs, those with HDs have a lower pregnancy and delivery rate, even though semen quality is higher. Disease type and age at cryopreservation are significant factors for successful outcomes.

Obstructive azoospermia as an unusual complication associated with herniorrhaphy of an omphalocele: a case report.

INTRODUCTION: Iatrogenic damage to the seminal tract is one of the causes of obstructive azoospermia, which can be an indication for reconstruction surgery. We present a case of obstructive azoospermia as an unusual complication after neonatal herniorrhaphy of an omphalocele. CASE PRESENTATION: A 30-year-old Japanese man was diagnosed with obstructive azoospermia. He had undergone herniorrhaphy of an omphalocele immediately after birth. Reconstruction surgery of both seminal tracts was performed to pursue the possibility of naturally achieved pregnancy. Intra-operative findings demonstrated that both vasa deferentia were interrupted at the internal inguinal rings, although the abdominal end of the right vas leading to the seminal vesicle was found in the abdominal cavity. The discharge from the stump of the testicular end had no sperm, although the right epididymal tubules were dilated with motile sperm. Therefore, we performed right-sided vasovasostomy in the internal inguinal ring and ipsilateral epididymovasostomy simultaneously. CONCLUSION: To the best of our knowledge, this is the first report describing obstructive azoospermia as an unusual complication of herniorrhaphy of an omphalocele. It is important to pay attention to the existence of seminal tracts in such surgery as well as in inguinal herniorrhaphy.

Dicoumarol enhances doxorubicin-induced cytotoxicity in p53 wild-type urothelial cancer cells through p38 activation.

OBJECTIVE: To investigate the effectiveness of a combined treatment of 3-30-methylene-bis[4-hydroxycoumarin] (dicoumarol) with doxorubicin for the treatment of urothelial cancer, as doxorubicin is a common chemotherapeutic agent but its therapeutic efficacy is limited. MATERIALS AND METHODS: The synergistic effect of dicoumarol with chemotherapeutic agents such as cisplatin, doxorubicin and paclitaxel was evaluated in RT112 urothelial cancer cells. Then, dicoumarol-mediated enhancement of doxorubicin-induced cytotoxicity was screened in urothelial cancer cell lines with different p53 statuses or RT112 stable transfectants with a dominant-negative mutant of p53 (p53DN). To clarify the importance of the modification of p53 function by dicoumarol to enhance doxorubicin toxicity, the change in the p53-p21 pathway and mitogen-activated protein kinase (MAPK)-mitochondria pathway by the combined treatment were elucidated by Western blot analysis. Finally, the effect of p21 knockdown in the susceptibility to doxorubicin was examined with RT112 stable transfectants with short hairpin RNA (shRNA) of p21. RESULTS: Dicoumarol significantly increased the susceptibility of RT112 cells to cisplatin and doxorubicin, but not to paclitaxel in RT112 cells. Dicoumarol (100 microm) also enhanced the cytotoxicity of doxorubicin in other bladder cancer cell lines with wild-type p53 (wt-p53; three times in 253J and 13 times in KK47), but not in those with mutant-type p53 (TCCsup, J82 and EJ) or in RT112 p53DN. The combined treatment with dicoumarol suppressed p53/p21 induction by doxorubicin and resulted in sequential p38 MAPK activation, myeloid cell leukaemia 1 suppression and caspase cleavage. The synergistic effect of doxorubicin/dicoumarol was suppressed by the p38 MAPK inhibitor SB202190 and, furthermore, p21 knockdown with shRNA transfection made RT112 cells six times more susceptible to doxorubicin with p38 MAPK activation. CONCLUSION: These results suggest that concomitant use of dicoumarol could enhance the cytotoxicity of doxorubicin in urothelial cancer cells with wt-p53 through the p53/p21/p38 MAPK pathways. This combined treatment may provide a new therapeutic option to overcome chemoresistance in bladder cancer.

[Sperm cryopreservation for cancer patients: 5-year experience in a private hospital in Japan].

Private hospitals in Japan appear to play an important role in the fertility preservation in cancer patients. However, only a few university-related institutions have published their data about sperm banking. Here we report our experience in a private hospital. The database of 5 years of experience with sperm cryopreservation for male cancer patients was reviewed. We assessed the type of cancer, timing of collection, sperm quality, and utilization for reproductive purposes. There were a total of 88 oncology patients who underwent sperm banking at our institution during the study period. Types of cancer were various, with testicular cancer and hematological malignancies comprising the largest groups. Nearly 90% of the testicular cancer patients had their sperm preserved prior to the therapy, while only 53% of those with hematological disease did so. Evaluation of semen parameters for these groups revealed that oligozoospermia in testicular cancer patients, even prior to initiation of cancer therapy, was common. Five patients utilized their specimens for reproductive purposes. Of these, 3 patients successfully fathered a child. Our results suggest that sperm cryopreservation before initiation of cancer therapy in male oncology patients is under-utilized. Additionally, there is minimal use of cryopreserved specimens for reproductive purposes. This under-utilization is supposed to be due to physicians', especially hematologists', insufficient awareness toward chemotherapy-related infertility and the paucity of reports regarding reproductive outcome after freezing. More data should be compiled to help both physicians and patients who are considering sperm cryopreservation.

[Questionnaire survey in Japan for sperm cryopreservation before chemotherapy ].

Questionnaires were mailed to 104 urological departments in universities, colleges, and cancer centers in February 2008. Ninety institutions returned answers to the questionnaire. Sperm cryopreservation before chemotherapy was performed in 24 institutions (26.7%) mainly in the gynecological department, whereas the others introduced the candidates to other institutions including private hospitals and clinics. Fifty-eight institutes gave information about fertility preservation routinely to young patients with testicular cancer before chemotherapy. Urological doctors in 57 institutions (63%) had no experience in consultation of patients with hematological/pediatric malignancies for fertility-preservation. Among 24 institutions performing sperm cryopreservation before chemotherapy, 18 preserved motile sperm even if during chemotherapy, as well as before chemotherapy but 2 institutions replied that patients during chemotherapy were contra-indication for sperm cryopreservation. For the patients with azoospermia, 2 institutions replied they tried to preserve testicular sperm before chemotherapy. The fee for sperm cryopreservation was free in 10 institutions. This nation-wide questionnaire survey clarified that the systems and indications for sperm cryopreservation before chemotherapy were different among institutions.

Impact of retraction of vas deferens in postradical prostatectomy inguinal hernia.

OBJECTIVES: The incidence of inguinal hernia after radical retropubic prostatectomy (RRP) is high. We speculated that retraction of the vasa deferentia with a retractor might cause stretch injury of the myopectineal orifice and lead to inguinal hernia. In testing this hypothesis, we performed RRP with a modified technique and followed up patients prospectively. METHODS: From 1993 to 2002, 171 patients underwent RRP with a retrograde approach, in which the vasa deferentia were cut after the prostate and seminal vesicles were finally exposed. From 2003 to 2005, 150 patients were followed up prospectively who had undergone RRP with a modified technique, in which the bilateral vasa deferentia and surrounding tissues were dissected before placing a retractor so as not to retract them and injure the myopectineal orifice. The incidence rates of inguinal hernia in the two groups were compared. In addition, for the entire group of 321 patients, we determined the risk factors for inguinal hernia after RRP using multivariate analysis. RESULTS: Of the 150 patients in the modified method group, 22 (14.7%) developed an inguinal hernia during follow-up, and 42 (24.6%) of the 171 patients did so in the previous method group. No significant difference was noted between the two groups in terms of the hernia-free rate. Multivariate analysis revealed a body mass index of less than 23 kg/m2 and a history of previous inguinal hernia repair were significant risk factors for postoperative inguinal hernia. CONCLUSIONS: We found that retraction of the vasa deferentia with a retractor did not affect the high incidence of postoperative inguinal hernia after RRP.

Antibiotic prophylaxis in radical prostatectomy: 1-day versus 4-day treatments.

OBJECTIVE: The standard protocol of antibiotic prophylaxis in radical prostatectomy remains to be established. We retrospectively compared the occurrence of perioperative infections following radical prostatectomy between two different protocols of antibiotic prophylaxis. METHODS: This study included 106 cases of radical retropubic prostatectomy managed on the clinical pathways. Two different protocols of antibiotic prophylaxis were used in otherwise identical pathways. Between January and December 2004, 50 patients received a second generation cephem, cefotiam, for 4 days, beginning 30 min before surgery (4-day group), whilst between December 2004 and July 2005, only two doses of cefotiam were given on the day of operation in 56 patients (1-day group). The incidence of surgical site infection (SSI) and remote infection (RI) was retrospectively investigated. RESULTS: Superficial incisional SSI occurred in one (1.8%) patient in the 1-day group, whereas no patient in the 4-day group developed SSI. No RI was observed in either the 1-day or 4-day group. Intravenous antibiotics were administered besides the pathway in a patient in the 1-day group because unexplained fever more than 38 degrees C continued postoperative day (POD) 2 through POD 4 without signs of SSI or RI. Excluding this case, postoperative more than 38 degrees C was rare and transient after POD 2. CONCLUSION: The incidence of SSI and RI was low and not significantly different between the 1-day and 4-day groups. Therefore, the 1-day protocol of prophylactic antibiotic treatment seems adequate for preventing perioperative infections in radical prostatectomy.

Lymphoepithelioma-like carcinoma of the ureter.

Lymphoepithelioma-like carcinoma is an undifferentiated epithelial tumor with a prominent lymphocytic infiltrate and is rarely seen in the upper urinary tract. Only 3 and 2 cases involving the ureter and renal pelvis, respectively, have been reported. We report the fourth documented case of lymphoepithelioma-like carcinoma of the ureter. Tumor cells were immunoreactive to cytokeratin and negative for Epstein-Barr virus. Our patient was disease free 30 months after nephroureterectomy. The prognosis is favorable for pure lymphoepithelioma-like carcinoma of the upper urinary tract.

Intraoperative electrophysiological confirmation of neurovascular bundle preservation during radical prostatectomy: long-term assessment of urinary and sexual function.

OBJECTIVE: We investigated the longitudinal recovery of urinary and sexual function after radical retropubic prostatectomy (RP) using an intraoperative electrophysiological test to confirm the functional preservation of the neurovascular bundle (NVB). METHODS: A total of 70 patients who underwent RP for localized prostate cancer were prospectively enrolled in our survey. During RP, electrophysiological testing was performed to confirm the NVB preservation. The NVB was electrostimulated and the responses were observed by monitoring the intracavernous or intraurethral pressure changes. All patients were classified into three groups according to the degree of nerve-sparing [a bilateral nerve-sparing group (BNS), a unilateral nerve-sparing group (UNS) and a non-nerve-sparing group (NNS)] based on the macroanatomical as well as the electrophysiological assessment. Both urinary and sexual function were measured before and 3, 6, 12 and 24 months after RP by a self-administered questionnaire. RESULTS: The concordance rate of nerve-sparing or non-nerve-sparing between the electrophysiological and macroanatomical assessment was 80%. According to the electrophysiological data, the BNS maintained significantly better urinary function at 3 months after RP than the NNS and UNS. After 6 months, each group had almost recovered continence. When considering sexual function, the BNS showed better sexual function scores than the NNS throughout the post-operative periods and the UNS at 2 years. According to the macroanatomical assessment, however, these differences were significant. CONCLUSIONS: Nerve-sparing RP as confirmed by intraoperative electrophysiological test may contribute significantly to the early recovery of continence and greater rate of sexual function after RP.

Quality of life after radical prostatectomy in Japanese men: 2 year longitudinal study.

OBJECTIVE: We performed a 2 year longitudinal survey of health-related quality of life (HRQOL) after radical retropubic prostatectomy (RP) in Japanese men with localized prostate cancer. PATIENTS AND METHODS: We measured 112 patients who underwent RP with SF-36 and University of California, Los Angeles Prostate Cancer Index before and 3, 6, 12, 18 and 24 months after surgery. RESULTS: Patients who underwent RP showed problems in some domains of general HRQOL, but these problems diminished over time. Mental health significantly improved throughout the follow-up period. The urinary function substantially declined at 3 months and continued to recover gradually but never returned to the baseline. Urinary bother at 3 months showed a significant decrease, but at 6 months it returned to baseline. The data of sexual function and bother showed a substantially lower score after RP. Patients lost their sexual desire significantly throughout the post-operative period. After 12 months, the nerve sparing group had significantly better improvement in sexual function than the non-nerve sparing group and this improvement continued up to 2 years after operation. CONCLUSION: Despite reports of problems with sexuality and urinary continence, general HRQOL was mostly unaffected by RP after 6 months. RP had a favorable impact on mental health. Although urinary function did not completely return to the baseline level even at 2 years after RP, recovery from urinary bother was rapid. RP had serious consequences on libido, erectile function and sexual activity. In the second year, the sexual function of those who underwent RP with bilateral nerve sparing procedure continued to improve.

Use of complementary and alternative medicine by patients with urologic cancer: a prospective study at a single Japanese institution.

OBJECTIVES: We prospectively evaluated the prevalence and predictors of complementary medicine (CAM) use among Japanese patients with urologic cancer 1 year after diagnosis. PATIENTS AND METHODS: A total of 349 patients with newly diagnosed urologic cancer answered a self-administered questionnaire on CAM use 1 year after diagnosis. General-health-related quality of life (GHQL) of the patients was also assessed at diagnosis and 1 year after diagnosis using the Medical Outcome Study Short Form-36 (SF-36). The overall prevalence, types of CAM used, and costs of CAM were assessed. The effects of several variables including GHQL at baseline and 1 year after treatment on the prevalence of use of CAM were evaluated. RESULTS: A total of 164 respondents (47%) admitted using some type of CAM, of which 73 (45%) had used multiple types. "Health food," in particular extract from Agaricus blazei, was the most common type of CAM used. CAM users had significantly lower scores for social function, general health perception, and vitality domains than CAM non-users 1 year after diagnosis. This tendency was more marked in users of multiple types of CAM. CONCLUSIONS: "Health food," including extract from A. blazei, was the most commonly used CAM in Japan. The prevalence of CAM use did not differ between patients with prostate cancer and those with urologic cancer other than prostate cancer. CAM users, especially those who used multiple types of CAM, had lower GHQL scores than non-users of CAM.

Risk stratification after radical prostatectomy in men with pathologically organ-confined prostate cancer using volume-weighted mean nuclear volume.

OBJECTIVE: We examined the impact of volume-weighted mean nuclear volume (MNV) on biochemical failure after radical prostatectomy (RP) in pathologically organ-confined prostate cancer (PC) and developed a prognostic factor-based stratification model for these patients. PATIENTS AND METHODS: We analyzed 141 patients with pathologically organ-confined PC treated solely with RP. Unbiased estimates of MNV were calculated from biopsy specimens based on a stereological method, and compared with other clinical and pathologic findings including patient age, pre-treatment PSA, biopsy and RP specimen Gleason score, pathologic stage, total cancer volume, index cancer volume, tumor differentiation, number of tumor foci, main tumor location, and surgical margin status, with regard to prediction of disease outcome after RP using Cox proportional hazard models. RESULTS: The median follow-up was 38.6 months (range 4--119 months). Twenty patients (14.2%) experienced biochemical failure. On multivariate analysis, MNV was demonstrated to be an independent prognostic factor, along with pre-treatment PSA and total cancer volume (P=0.0004, 0.0184, and 0.0285, respectively). All patients were stratified into three groups according to their prognostic scores developed on the basis of multivariate analysis, with statistically significant prognostic differences revealed for each of the between-group comparisons. CONCLUSION: The results demonstrated that estimates of MNV contribute most significantly to the prediction of biochemical control of pathologically organ-confined PC. The combination of MNV with other independent predictors such as pre-treatment PSA and total cancer volume provided a statistically verifiable basis for risk stratification, facilitating more accurate prediction of disease outcome.

Risk factors for subsequent development of bladder cancer after primary transitional cell carcinoma of the upper urinary tract.

OBJECTIVES: To determine the independent risk factors for intravesical tumor recurrence in patients with primary transitional cell carcinoma of the upper urinary tract, and to develop a risk-stratification model to allow more accurate prediction of recurrence risk. METHODS: Of 141 patients who underwent total nephroureterectomy for clinically localized transitional cell carcinoma of the upper urinary tract, the data from 89 patients were retrospectively reviewed. Patients with a previous history or concomitance of bladder cancer and/or a follow-up period of less than 1 year were excluded from this study. Multivariate analysis by Cox's proportional hazards model was used to determine independent risk factors for intravesical tumor recurrence. RESULTS: Of 89 patients, 37 (41.6%) experienced subsequent intravesical tumor recurrence during a median follow-up period of 39.7 months (range 12.0 to 186.6). On multivariate analysis, tumor multiplicity, pathologic stage, tumor size, and surgical modality had a statistically significant impact on the risk of intravesical tumor recurrence (P = 0.0075, P = 0.0221, P = 0.0377, and P = 0.0413, respectively). Pathologic stage and tumor size were inversely correlated to the risk. A scoring system for the risk of intravesical recurrence was developed from the proposed prognostic factors, and the patients were stratified into three groups according to their scores, with statistically significant prognostic differences between them (P = 0.0018). CONCLUSIONS: Tumor multiplicity, pathologic stage, tumor size, and surgical modality all had a significant impact on the incidence of intravesical tumor recurrence. A risk stratification model constructed from tumor biologic factors may be useful in the follow-up of patients with transitional cell carcinoma of the upper urinary tract.

Impact of superficial bladder cancer and transurethral resection on general health-related quality of life: an SF-36 survey.

OBJECTIVES: To assess the general health-related quality of life (GHQOL) of patients with superficial bladder cancer who underwent transurethral resection (TUR). METHODS: We assessed the GHQOL a total of 178 times for 133 patients with superficial bladder cancer before multiple TURs. The GHQOL was assessed using the Medical Outcome Study Short-Form 36-item survey (SF-36). Ninety-three patients answered the questionnaire at the first TUR, 34 at the second TUR, 17 at the third TUR, and 34 at the fourth or later TUR. RESULTS: Compared with age-matched and sex-matched Japanese norms, the general health perception was severely impaired in patients with superficial bladder cancer. Their mental health was also severely impaired at the first TUR, but gradually returned to normal as TUR was repeated. The scores of physical functioning, social functioning, and role-emotional demonstrated a nadir at the second or third TUR, and increased thereafter if TUR was repeated four or more times. Intravesical treatment for prevention of recurrence increased the score of bodily pain. CONCLUSIONS: Although superficial bladder cancer is not frequently lethal, patients with this disease believed their general health was much impaired. Only two domains of bodily pain and vitality had no negative impact from this disease. As urologists, we should notice the considerable affect of superficial bladder cancer on the GHQOL of patients.

Alteration of body configuration after retroperitoneoscopic nephrectomy and nephroureterectomy.

OBJECTIVE: To evaluate objective alterations of body configuration in patients who treated with retroperitoneoscopic nephrectomy (RN) and retroperitoneoscopy-assisted nephroureterectomy (RAN). PATIENTS AND METHODS: Twenty-six patients who had RN and 23 RAN were eligible for the study. Abdominal computed tomography films before and after surgery were evaluated. The intra-abdominal content surrounded by the vertebral bones and the muscles forming the trunk of the body were divided into four subspaces. The areas of these four portions were measured, and the ratio of occupation of each portion calculated before compared with that obtained after surgery. RESULTS: While RAN had little impact on body configuration, RN significantly affected it; after RN the total area and area of ventral subspaces decreased homogeneously throughout the L2-L4 levels. The ipsilatero-dorsal subspace had a marked cranial decrease in area and no caudal alteration. CONCLUSIONS: While RAN has little effect on body configuration, RN has a significant effect, as measured objectively. Continuing the skin incision between ports and muscle splitting to extract the specimen is probably responsible for the difference between the findings of RAN and RN.