Staining For Placental Alkaline Phosphatase Research Articles

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

A histamine-independent itch pathway is required for allergic ocular itch

Non-histaminergic TRPA1 neural pathway is required for the development of allergic ocular itch. Pharmacological inhibition of TRPA1 channel can lead to novel therapeutic strategies to treat ocular itch in severe allergic conjunctivitis.

Intracranial Germinoma Manifesting as Cavernous Sinus Syndrome

An 11-year-old girl presented with a rare case of neurohypophyseal germinoma manifesting as cavernous sinus syndrome with left abducens nerve palsy. Magnetic resonance (MR) imaging showed a mass located in the left cavernous sinus. Biopsy revealed an inflammatory reaction with lymphocytic infiltration. Postoperative MR imaging showed regression of the mass. Her eye movements were also restored. However, after 5 months, she developed right oculomotor and abducens nerve palsy, and MR imaging showed a large suprasellar mass invading the bilateral cavernous sinuses. The transcranial biopsy sample showed positive staining for placental alkaline phosphatase and c-kit, and the diagnosis of germinoma was confirmed. Chemotherapy and radiotherapy were administered, and the patient has been in complete remission for over 4 years.

Pineal Germinoma With a Prominent Epithelioid Cell Granuloma Component -Case Report-

A 20-year-old man presented with a rare case of germinoma with a large component of epithelioid cell granuloma manifesting as oscillopsia. Magnetic resonance imaging demonstrated a mass in the pineal region with homogeneous enhancement with gadolinium. Craniotomy was performed, ending in biopsy. The initial histological diagnosis was epithelioid cell granuloma, but systemic investigation detected no evidence of granulomatous disorder. A revised diagnosis of germinoma was based on positive immunohistochemical staining for placental alkaline phosphatase (PLAP) and c-kit. Histological diagnosis is sometimes incorrect if granulomatous reaction is dominant. Immunohistochemical staining for PLAP and c-kit should be performed if germinoma is clinically suspected.

Placental Alkaline Phosphatase (PLAP) Staining and Human Chorionic Gonadotropin (hCG) Production in Cultures of Fresh and Cryopreserved Cytotrophoblasts Isolated by CD9/MHC Class I/MHC Class II Immunoelimination

We have further characterized villous trophoblasts isolated by trypsinization and purified by elimination of CD9/MHC class I/MHC class II expressing cells. The cells isolated were >99.99% cytotrophoblasts by criteria of cytokeratin (positive) and vimentin (negative) expression. Purified cells directly after isolation (fresh) were compared with cryopreserved and thawed cells (frozen) for production of human chorionic gonadotropin (hCG) and expression of placental alkaline phosphate (PLAP) after 4 h of culture. We found that fresh cells may adhere slightly more strongly than frozen cells, contained approximately 8-fold more PLAP-positive cells (indicating syncytial fragments) after adherence but neither preparation would secrete hCG until day 4 of culture. We conclude that the cells isolated by cell elimination were cytotrophoblasts with only a small number of PLAP (<0.2% of the fresh population plated) positive cells and that both populations shared the property of very low hCG production until cultured past day 4. We speculate that cells isolated by other methods (accompanying paper) may be contaminated by even more syncytial fragments, detectable by PLAP staining and by production of hCG in the first 48 h of culture.

Two cases of pineal germinoma with granulomatous inflammation

We report two cases of pineal germinoma with remarkable chronic granulomatous inflammation. In the first case, the pineal mass was totally removed via an occipital transtentorial approach as symptoms were due to direct mass effect. In the second case, endoscopic third ventriculostomy and tissue biopsy was performed to alleviate worsening hydrocephalus. Pathological examination of specimens of both cases showed chronic granulomatous inflammation associated with a few germ cell tumor nests, which demonstrated positive staining for placental alkaline phosphatase. Both patients received post-operative craniospinal irradiation with no subsequent neurological deficits. Follow-up magnetic resonance imaging (MRI) of the second case showed an asymptomatic, shrunken residual tumor mass. MRI of the first case showed no residual or recurrent disease. Thus, a pineal mass with unusual features on MRI and chronic granulomatous inflammation on histopathology, should raise the suspicion of germinoma. In cases with symptomatic mass effect, open resection can be considered. In cases with lesser mass effect, conventional therapeutic modalities without resection can achieve a good outcome, as for other germinomas.

The frequency of intratubular embryonal carcinoma: implications for the pathogenesis of germ cell tumours.

To define the frequency and distribution of intratubular embryonal carcinoma (IEC) in an attempt to shed light on the pathogenesis of non-seminomatous germ cell tumours (NSGCTs). Intratubular germ cell neoplasia of unclassified type (IGCNU) is common in NSGCT; however, IEC is rarely described. Sixty-two germ cell tumours were reviewed. Immunochemistry for CD30, placental alkaline phosphatase (PLAP) and c-kit was performed. The distribution, immunohistochemistry and morphology of the intratubular neoplasia were noted. All cases showed widespread IGCNU with PLAP and c-kit staining. CD30 showed strong focal intratubular positivity in 20/31 NSGCTs, 1/29 seminomas and 1/4 mixed seminomas/NSGCTs. In 17 of these cases, the CD30+ tubules were not easily identified as IEC on routine stains. These tubules were scanty in number and c-kit was negative, though some showed patchy PLAP staining. The cells within these tubules differed morphologically from IGCNU. IEC defined by CD30 positivity is not always easily identified on haematoxylin and eosin staining. We suggest that IEC is a common intermediate step between IGCNU and NSGCTs. The patchy and focal distribution of IEC suggests it may evolve quickly to invasive disease.

Clinicopathological significance of plap, NSE and WT1 detection in ovarian dysgerminoma

Objective: To evaluate the expression of placental alkaline phosphatase (PLAP), neuron-specific enolase (NSE), prolactin (PRL) and Wilms’ tumor gene (WT1) protein in ovarian dysgerminoma and its Clinico-pathological significance. Methods: Clinicopathological data were retrospectively reviewed in a total of 31 patients with pure dysgerminoma who were treated at College Clinical Medicin, Chongqing Medical University from January 1983 to October 2002. Immunohistological staining for PLAP, NSE, PRL and WT1 was performed in all 31 tumor tissues. Results: The age of the patients ranged from 12 to 42 years old (average 25 yrs). According to the clinical staging, eighteen patients (58.1%) had stage I disease, 5 (16.1%) had stage II, 6(19.4%) had stage III, and 2 (6.4%) had stage IV disease. Of the 31 cases, the positive expression rates of PLAP, NSE, PRL and WT1 were 100% (31/31), 70.9%(22/31), 3.2%(1/31) and 12.9(4/31) respectively. There was a significant relation between NSE expression and clinical stages (P<0.05) and 5 years survival rate (P<0.05). The positive expression rate of WT1 was significantly related to histological types (P<0.05). Conclusion: Dysgerminomas in earlier stages (stage I/II) are associated with a favorable prognosis. NSE positive expression is closely related with advanced tumor stage (stage III/IV). PLAP and NSE can be considered as important markers of dysgerminoma tissues, and the tumor tissues are the main resources of serum PLAP and NSE. WT1 expression correlates with poorer differentiation of dysgerminoma. The importance of PRL in dygerminoma was not certified in this study, and remains to be defined.

Tissue Microarrays Are an Effective Quality Assurance Tool for Diagnostic Immunohistochemistry

There has been considerable variability in the reported results of immunohistochemical staining for some diagnostically relevant antigens. Our objectives in this study were to (1) use a multitumor tissue microarray with tissue from 351 cases received in our department, representing 16 normal tissues and 47 different tumor types, to compare immunohistochemical staining results in our laboratory with published data, using a panel of 22 antibodies; (2) assess interlaboratory variability of immunohistochemical staining for S-100 using this microarray; and (3) test the ability of hierarchical clustering analysis to group tumors by primary site, based on their immunostaining profile. Tissue microarrays consisting of duplicate 0.6-mm cores from blocks identified in the hospital archives were constructed and stained according to our usual protocols. Antibodies directed against the following antigens were used: B72.3, bcl-2, carcinoembryonic antigen, c-kit, pankeratin, CD 68, CD 99, CK 5/6, CK 7, CK 8/18, CK19, CK 20, CK 22, epithelial membrane antigen, estrogen receptor, melan-A, p53, placental alkaline phosphatase, S-100, synaptophysin, thyroid transcription factor-1, and vimentin. Staining results on the array cases were compared with published results, and hierarchical clustering analysis was performed based on the immunohistochemical staining results. Unstained slides of the multitumor tissue microarray were sent to five other diagnostic immunohistochemistry laboratories and stained for S-100 protein. The staining results from the different laboratories were compared. Staining results using our current methods and samples from our laboratory were compatible with those described in the literature for most antigens. Placental alkaline phosphatase staining was not specific with our protocol, showing staining of a broad spectrum of different tumors; this finding initiated a review of our recent requests for placental alkaline phosphatase immunostaining and revealed two instances in which placental alkaline phosphatase positivity was incorrectly interpreted as evidence of a germ cell tumor. S-100 staining was less sensitive but more specific for the diagnosis of melanoma or neural tumor in our laboratory, compared to some published reports. Assessment of interlaboratory variability of S-100 immunostaining showed that there was more frequent staining of carcinomas in some laboratories, resulting in decreased specificity of S-100 staining in distinguishing melanoma from carcinoma. Hierarchical clustering analysis showed a strong trend for tumors to cluster by tissue of origin, but there were significant exceptions. We conclude that multiple-tumor microarrays are an efficient method for assessing the sensitivity and specificity of staining with any antibody used diagnostically. As a tool for quality assurance, they offer the advantage of taking into account local differences in tissue fixation, processing, and staining. They also allow cost-effective assessment of interlaboratory variability in immunohistochemical staining. Results of hierarchical clustering analysis show the potential for panels of immunohistochemical stains to identify the primary site of metastatic carcinomas but also confirm the limitations of currently available antibodies in giving unequivocal tissue-specific staining patterns.

The value of the biopsy of the contralateral testis in patients with testicular germ cell cancer: the recent German experience.

Testicular intraepithelial neoplasia (TIN; so-called carcinoma in situ of the testis), the precursor of testicular germ cell neoplasms can be detected by testicular biopsy many years before the clinical manifestation of the tumour. This study looked at the prevalence of contralateral TIN in patients with testicular germ cell cancer. The purpose was to evaluate this new approach of early detection of testicular cancer and to evaluate the current management strategies. 1954 consecutive patients with unilateral testicular germ cell tumour underwent contralateral biopsy. All specimens were examined immunohistologically with staining for placental alkaline phosphatase. Patients with TIN were usually submitted to low-dose radiotherapy of the testis. A rebiopsy was performed after 3 months. Endocrinological evaluations were done before, during and after treatment. TIN was observed in 4.9% (95% confidence intervals 3.95%-5.91%). Testicular atrophy constitutes a 4.3 fold increased risk of having contralateral TIN. 64% of the cases with TIN were found in clinically normal testes. Patients with TIN were significantly younger than those without (p < 0.017). No case with TIN was found in patients older than 50 years. Three patients developed a second testicular tumour during follow-up despite a negative biopsy. After radiotherapy, all of 23 patients had complete disappearance of TIN in the rebiopsy. After chemotherapy, 3 of 10 patients had persistent TIN histologically. After radiotherapy, 12 of 41 patients required testosterone replacement. The prevalence of contralateral TIN accords well with the known prevalence of bilateral testicular tumours. Testicular atrophy is a strong indicator for the presence of TIN but about 60% of TIN-cases occur without atrophy. Local radiotherapy to the testis with 18-20 Gy is efficaceous in eradicating TIN, but it causes significant damage to almost one quarter of these patients. Chemotherapy is an unsafe treatment for TIN. This study shows the feasibility of early detection of testicular cancer in a high-risk population by means of searching for TIN. Although the management of the condition still needs refinement, the TIN-concept offers an avenue for the early detection of testicular cancer and early conservative management.

Ectopic placental site reaction in the Fallopian tube

Although decidual change in the Fallopian tube following either intra-uterine or ectopic pregnancy is not uncommon, ectopic placental site reactions from prior unrecognized ectopic pregnancy are extremely rare. We describe the occurrence of an ectopic placental site reaction in a Fallopian lube, previously occluded by a Falope ring, which was subsequently re-anastomosed at salpingoplasty. The patient, a 37 year old women, subsequently had a partial salpingectomy performed a year after an intra-uterine pregnancy. Bela-HCG levels had consistently remained in the normal range. However, histological examination of the excised segments of Fallopian tube revealed an ectopic placental site reaction in the right Fallopian tube. Large mononuclear cells with bizarre atypical nuclei were present in the plicae, arranged in a nodular fashion, resembling placental site reactions as seen in the uterus. Immunohistochemical staining revealed negative staining for beta-HCG but focal positive staining for placental alkaline phosphatase and generalized positive staining for low molecular weight cytokeratin, (CAM 5.2) in keeping with intermediate trophoblast. Some suture material was present on the serosal surface of the Fallopian tube, surrounded by concentric layers of fibrosis and a focal foreign body type giant cell reaction; a marker of the previous surgery. It is possible that the previous surgery on the Fallopian tube may have played a role in the occurrence of the placental site reaction. The differential diagnosis of these histological appearances includes degenerate decidua and metastatic carcinoma. The possibility of a placental site trophoblastic tumour should also be considered but this lesion was too small to produce a tumour like mass and no mitoses were identified. The bizarre appearance of the cells may give rise to an erroneous diagnosis of malignancy, especially on small laparoscopic biopsies of the Fallopian tubes.

Placental site nodule: a clinicopathologic study of 38 cases.

This report describes the clinical, histological, and immunohistochemical features of 38 cases of placental site nodule (PSN), a recently described lesion of intermediate trophoblast (IT). The patients ranged in age from 20 to 47 years (mean, 31.1 years). PSNs were diagnosed in endometrial (30 cases), endocervical (seven cases), and both endometrial and endocervical specimens (one case). The majority of biopsies were prompted by an abnormal Pap smear (13 cases) or complaints of menorrhagia (21 cases). All PSNs were microscopically detected, lobulated nodules composed of acellular, hyalinized material admixed with IT. Mitotic activity was noted in seven cases. Immunohistochemically, the IT expressed human placental lactogen (hPL) in 78% of cases and human chorionic gonadotropin (hCG) in 42% of cases, but their expression was weak and focal in contrast to uniform, strong staining for placental alkaline phosphatase (PLAP) (100%), cytokeratins (96%), and epithelial membrane antigen (EMA) (84%). Type IV collagen outlined the IT and stained the extracellular material in the cellular areas. Vimentin-positive cells within the lesions were fewer in number and in a different distribution than those expressing PLAP, CK, and EMA. Two consecutive PSNs occurred in one patient, but no patient developed gestational trophoblastic disease or a significant gynecologic neoplasm.

Epidermoid cyst of the testis: a review of clinical and histogenetic considerations

To review the records of patients with epidermoid cysts and those with germ cell tumours to determine the most appropriate method of treatment. The records of the patients treated for epidermoid cysts and testicular germ-cell tumours in a period covering 22 years were analysed retrospectively. Nine specimens with an epidermoid cyst were examined by immunohistology for the presence of testicular intraepithelial neoplasia (TIN) (or carcinoma in situ of the testis). Ten patients with a simple epidermoid cyst and 481 patients with testicular germ-cell tumour were treated during the observation period. The relative incidence of epidermoid cysts in relation to the number of patients with testicular germ-cell tumours was 2.1% (95% confidence interval 0.8-3.35%). The mean age of the 10 patients with an epidermoid cyst was 24.1 years. The right testis was affected in seven patients and the left in three. Six of these patients received conservative surgery with excellent cosmetic results. No relapse had occurred after a median observation period of 30 months. Immunohistological staining for placental alkaline phosphatase disclosed the absence of TIN in the parenchyma surrounding the cysts. The absence of TIN calls into question the common assumption that an epidermoid cyst constitutes a monodermal teratoma. Two distinct entities of epidermoid cyst are proposed--one occurring in conjunction with a teratoma or a germ cell tumour (a 'complex' or 'mixed' epidermoid cyst) and the other a 'simple' epidermoid cyst without TIN in the adjacent tissue and thus representing a benign neoplasm that is not a teratoma. The absence of TIN near a simple epidermoid cyst justifies testis-sparing surgery.

Patterns of human gonodotrophin and placental alkaline phosphatase in complete and partial moles

A review of all cases of gestational trophoblastic diseases diagnosed histologically at the Department of Pathology, University Hospital, Kuala Lumpur between January 1989 and December 1990 revealed 24 cases of complete moles and 11 partial moles. The patterns of expression of human gonadotrophin (HCG) and placental alkaline phosphatase (PLAP) were studied immunohistochemically, using the standard peroxidase-antiperoxidase method. The pattern was defined as diffuse when almost all trophoblastic cells stained positively. When only few isolated collections of trophoblastic cells stained positively, the pattern was described as focal. A diffuse pattern of HCG staining was observed in 7 (63.6%) of the 11 partial moles; the rest were focal. In contrast, all 24 (100%) complete moles showed a diffuse pattern. With regard to PLAP staining, 6 (54.5%) partial moles expressed a diffuse pattern while the rest were focal. None of the complete moles expressed a diffuse pattern; all showed focal staining. It appears that the focal HCG and diffuse PLAP staining patterns are not consistent with a diagnosis of complete mole. However, the diffuse HCG and focal PLAP patterns are not reliable diagnostic clues in differentiating partial from complete moles.

Germ cell expression of placental alkaline phosphatase in male pseudohermaphroditism

We have reviewed testes removed from 14 individuals with male pseudohermaphroditism (13 with androgen insensitivity and one with 17 alpha-hydroxylase deficiency) and have studied staining for placental alkaline phosphatase in germ cells. Placental alkaline phosphatase positivity was identified in fetal and premature neonatal controls but not in cryptorchid males or normal autopsy control material from boys older than 6 months. It was present in autopsy testes younger than 6 months and cases of androgen insensitivity in boys younger than 8 months, suggesting immaturity. It was also expressed in two patients with male pseudohermaphroditism aged 5 years and 14 years, both of whom had intra-tubular germ cell neoplasia by morphological criteria. Our study confirms the use of placental alkaline phosphatase as a marker of germ cell neoplasia in this specific group who are at high risk of malignancy.

Immunocytochemical staining patterns of placentas associated with hydrops fetalis.

This study examined the morphology and immunocytochemical staining patterns of distribution of beta-human chorionic gonadotropin (beta-HCG), human placental lactogen (HPL), placental alkaline phosphatase (PLAP), and pregnancy-specific beta-1 glycoprotein (SP1) in 13 third-trimester placentas associated with hydrops fetalis and six normal control placentas matched for gestational age (+/- 2 weeks). Seven placentas were hydropic (540-1,080 g) and demonstrated histologic immaturity with large edematous chorionic villi showing few blood vessels, most of which contained immature hematopoietic elements. These placentas showed consistently increased staining for beta-HCG and decreased staining for PLAP when compared with control placentas, a pattern reminiscent of less mature placentas. HPL and SP1 staining were similar to those of controls. Six placentas were either sclerotic (four) or histologically unremarkable (two), and these did not differ in their immunocytochemical staining properties from control placentas. We conclude that third-trimester hydropic placentas, in addition to showing histologic immaturity, exhibit an immunocytochemical staining pattern associated with first-trimester placentas.

Intracranial mixed germ cell tumor with syncytiotrophoblastic giant cells and precocious puberty

A 9-year-old male patient developed a germ cell tumor in the right basal ganglia which secreted beta-human chorionic gonadotropin (beta-HCG) and caused precocious puberty. Histology and immunohistochemical staining for placental alkaline phosphatase (PLAP), alpha-fetoprotein (alpha-FP), and beta-HCG showed a mixed population of neoplastic germinocytes, embryonal carcinoma, and syncytiotrophoblastic giant cells (STGC). Immunohistochemical double-staining for alpha-FP and beta-HCG revealed that these two markers were produced by different subsets of cells. Expression of the proliferation marker Ki-67 showed a growth fraction of 53% for the neoplastic germinocytes and embryonal carcinoma cells, but only 21% for the STGC.

Placental Alkaline Phosphatase Immunoreactivity in Testicular Germ-Cell Neoplasms

We analyzed the sensitivity of a polyclonal antibody to placental alkaline phosphatase (PLAP) in labelling testicular germ-cell neoplasms, by utilizing the peroxidase-antiperoxidase technique. The immunoreactivity of 89 germ-cell tumors for PLAP was as follows: 98% of cases with seminomatous elements were PLAP-positive; 97% of embryonal carcinomas and 85% of endodermal sinus tumors also showed reactivity. Cytotrophoblastic cells were focally immunoreactive in one of two cases with choriocarcinomatous elements. Staining for PLAP was strongest and most diffuse in seminomas. Intratubular germ-cell neoplasia (ITGCN) or carcinoma in situ was present in 53 (84%) of 63 specimens that had adjacent seminiferous tubules available for evaluation; PLAP was demonstrated in 98% of these. In addition, the germ-cell elements in 11 gonadoblastomas were immunoreactive for PLAP. One of 17 cases of undescended testes had ITGCN that was also strongly immunoreactive, but the remaining 16 cases were negative. Five dysgenetic gonads without ITGCN were studied, and one was immunoreactive for PLAP. Three testicular biopsy specimens from infertile men without ITGCN were PLAP-negative. Our findings indicate that PLAP is a highly sensitive marker for seminomas, for the majority of embryonal carcinomas and endodermal sinus tumors of the testis, and for ITGCN.

Immunohistochemical study of placental alkaline phosphatase in primary intracranial germ-cell tumors.

Indirect immunoperoxidase staining by the peroxidase-antiperoxidase (PAP) technique was carried out on 23 human primary intracranial germ-cell tumors (17 germinomas, one embryonal carcinoma, one yolk-sac tumor, three teratomas, and one teratoma with embryonal carcinoma) and on six human primary pineal non-germ-cell tumors (one pineocytoma, two pineoblastomas, two astrocytomas, and one glioblastoma multiforme). The technique used specific rabbit antisera against placental alkaline phosphatase (PLAP), alpha-fetoprotein (AFP), and human chorionic gonadotropin (HCG). Thirteen of 17 intracranial germinomas (76.5%) showed positive staining for PLAP mainly on the tumor cell membrane. In six primary intracranial non-seminomatous germ-cell tumors, there was weak positive staining indicating the presence of PLAP in only a few cells of one embryonal carcinoma, and in some glandular epithelial cells of one teratoma; this staining was limited to the cytoplasm. None of the other six primary pineal non-germ-cell tumors showed any positive PLAP reaction. From these results, PLAP was shown to be very useful in histopathology as a diagnostic tumor marker of intracranial germinoma. Positive AFP staining was seen in several yolk-sac tumor cells and a few embryonal carcinoma cells. However, no intracranial germinomas and non-germ-cell tumors of the pineal region showed positive reaction. As for HCG, only one suprasellar germinoma and one pineal embryonal carcinoma among 29 specimens contained a few positive-staining tumor cells.