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Background: The distinction between dermatofibroma (DF), dermatofibrosarcoma protuberans (DFSP), and other benign and malignant cutaneous spindle cell lesions frequently requires immunohistochemical staining. CD34 and factor XIIIa are the most commonly used immunostains; however, they may exhibit aberrant expression and introduce the potential for misdiagnosis. There is some data supporting that p75 and S100A6 may be additional helpful immunohistochemical markers. Methods: We undertook a large case series examining the use of CD34 and factor XIIIa as well as p75 and S100A6 in DF, cellular DF, DFSP, indeterminate fibrohistiocytic lesion, and scar. Results: As expected, CD34 stained DFSP, although it was usually negative in DF. Factor XIIIa was generally positive in DF and negative in DFSP. There were exceptions in both cases of DF and DFSP. S100A6 was routinely negative in all entities studied. P75 was negative in all cases except DFSP, approximately half of which showed weak and/or patchy positivity. Conclusions: We conclude that to date, CD34 and factor XIIIa remain the most reliable immunohistochemical markers for DF and DFSP.

IgG4-related disease is a syndrome which involves lymphoplasmacytic infiltrates and soft tissue sclerosis, elevated serum IgG4 titer, and increased IgG4-positive plasma cells in a variety of tissues. Scleroderma is also characterized by fibrosis and lymphoplasmacytic infiltrates. To our knowledge, the presence of IgG4-positive cells has not been well characterized in scleroderma. A retrospective review of scleroderma and related disorders (calcinosis, raynaud's syndrome, esophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, progressive systemic sclerosis, morphea) was performed. Thirty-four cases of scleroderma and related disorders were identified; IgG4-positive and IgG-positive plasma cells were counted in 10 HPF and an IgG4:IgG ratio determined. A cutoff ratio of 0.3 was used to define significant elevation. Three of the scleroderma cases had IgG4:IgG greater than 0. Only 1 case had a significant elevation. Of the 3 cases with elevated ratio, IgG4-positive cells ranged from 2 to 64 (median = 14), with an IgG4:IgG ranging from 0.06 to 0.34 (median = 0.22). Similar results were produced with the other sclerosing disorders. These results suggest that scleroderma is not part of the IgG4-related disease spectrum.

Background Fontana-Masson (FM) staining is a histopathology technique used to identify dematiaceous fungi. The result often guides initial species identification and antifungal treatment; however, there is evidence that nondematiaceous fungi might react with this stain. Few studies in the current literature address this issue. Objective Our aim was to classify the FM staining patterns of common fungal pathogens for use by pathologists attempting to accurately identify fungi on histopathologic examination. Methods In total, 132 cases of culture proven mycoses were identified. We stained tissue with 2 different FM protocols and recorded the intensity and distribution of results. Results There was variability in staining, and many nondematiaceous fungi showed positivity, including Zygomycetes, Aspergillus, and Fusarium spp. Limitations The study was limited by the number of cases. Of 132 cases identified, 112 cases had adequate tissue left after sectioning deeper into the block. Conclusion Nondematiaceous fungi frequently stained positive with FM. The course of treatment should not be based on the result of this stain alone. Histopathology should be examined in multiple tissue sections, and therapy should be determined on the basis of clinical context and culture results.

Introduction: The classification of spitzoid melanocytic tumors can be difficult, and pathologists rely on both histological features and clinical information to arrive at a diagnosis. We proposed that an immunohistochemical panel could be useful in classifying these neoplasms and designed a study to test the independent contribution of the panel to the final diagnosis. Methods: We identified 121 cases previously signed out either as (1) Spitz nevus, (2) atypical spitzoid neoplasm, favor Spitz nevus, (3) atypical spitzoid neoplasm of uncertain malignant potential, (4) atypical spitzoid neoplasm, favor melanoma, and (5) spitzoid melanoma. The slides were reveiwed in random order by 4 pathologists. For the first review, the pathologists received only hematoxylin and eosin sections and patient age. Subsequently, the same pathologists interpreted the immunohistochemically stained slides (S-100A6, HMB-45, and MIB-1) on the same cases in randomized order without the benefit of either hematoxylin and eosin sections or patient age. The original diagnosis (based on a combination of clinical information, hematoxylin and eosin-stained sections and immunohistochemical stains) was the gold standard used for statistical analysis. The primary aim of the study was to determine the level of agreement between interpretions based on hematoxylin and eosin sections and age, the immunostains alone, and the gold standard, thus providing a measurement of the degree to which each of these elements contributes to the final diagnosis. The agreement between the gold standard and external review was also determined for those cases sent for external review. Results: The generalized kappa statistic was 0.95 for both the hematoxylin and eosin-stained slides alone and the immunohistochemical stains alone, implying a high level of agreement among the 4 pathologists. The combined weighted kappa statistic for the comparison of hematoxylin and eosin sections and patient age to the gold standard was 0.49, and for the immunohistochemically stained slides to the gold standard 0.48, indicating that a diagnosis based on hematoxylin and eosin sections alone or immunostains alone show only a moderate and similar level of agreement with the gold standard diagnosis. Only the most controversial cases were sent for external review. The weighted kappa statistic estimate was 0.30 for the gold standard diagnosis on those cases and the external review. Conclusions: Spitzoid neoplasms remain a difficult area in dermatopathology and experts frequently disagree on the most challenging cases. An immunohistochemical panel contributes to the diagnosis of spitzoid tumors, and the contribution is statistically similar to that of hematoxylin and eosin sections and age. Interpretation remains subjective, as evidenced by the comparison of the gold standard and external review.

We aim to report that skin biopsy, a non-invasive test by neurological standards, may lead to a diagnosis. A 4-year-old male presented with a 2-year history of epilepsy and progressive developmental regression. The patient had a mildly elevated ammonia level; however, evaluation for the accumulation of excess serum amino acids and evaluation of urine for organic acids was negative. MRI revealed cerebral atrophy, and an electroencephalogram demonstrated multifocal sharp and slow waves. Due to the progressive degenerative neurologic presentation, a neurologic storage disease was favored. An axillary skin biopsy was performed, revealing eosinophilic intra-cytoplasmic inclusions within the eccrine glands. A periodic acid-Schiff stain also highlighted these inclusions. Electron microscopic studies demonstrated characteristic multiple membrane-bound inclusions within the eccrine epithelial cells, containing curvilinear inclusion material characteristic of neuronal ceroid lipofuscinosis. The clinical, histological, electron microscopic and enzymatic studies were diagnostic of late-infantile onset neuronal ceroid lipofuscinosis.

Background Nevoid basal cell carcinoma syndrome (NBCCS) is associated with multiple basal cell carcinomas, odontogenic cysts, craniofacial anomalies, and childhood medulloblastomas. In addition, it has been associated with irradiation-induced neoplasms including, meningiomas, sarcomas, and gliomas. Methods We present a 19-year-old man with NBCCS who presented with a sinonasal carcinoma 17 years after receiving craniospinal irradiation for treatment of medulloblastoma. Results To our knowledge, this is the first report of a sinonasal tumor after irradiation in a patient with NBCCS. Conclusions With this case, the authors examine the genotype of NBCCS patients and their propensity for radiation-induced tumors. In addition, the management of neoplasms in these tumor-sensitive patients is reviewed.

Retrograde, transneuronal tracing with Bartha's strain of pseudorabies virus was used in rats to identify spinal cord, brainstem and hypothalamic loci of uterine-related neurons that could function in the regulation of uterine activity. Based on the premise that estrogen might influence such uterine-related neurons, the existence of estrogen receptors in neurons in these same loci was examined. Viral injections were made into the uterine cervix, body and cervical end of the uterine horns, and the rats allowed to survive for four to six days. After four days, mainly the spinal cord, medulla and pons contained virus-infected neurons. After longer survival times, progressively higher levels of the neuraxis contained viral-labeled neurons, so that by six days hypothalamic uterine-related neurons were identified. First-order virus-infected neurons were visualized by immunohistochemistry in the pelvic paracervical parasympathetic ganglia and in inferior mesenteric sympathetic ganglia. Preganglionic and putative interneurons were labeled in the lumbosacral spinal cord and thoracic spinal cord mainly in the lateral horn area (sacral parasympathetic nucleus and intermediolateral nucleus), lateral aspect of the dorsal horn, intermediate gray, lamina X and dorsal gray commissural area. In the brainstem, labeling was most evident and consistent in the nucleus tractus solitarius, ventrolateral medulla, raphe magnus and pallidus nuclei, parapyramidal area, A5 cell group, Barrington's nucleus of the pons and periaqueductal gray of the midbrain. In the hypothalamus, virus-infected neurons were most marked in the paraventricular nucleus, with fewer in the medial preoptic area and ventromedial hypothalamic nucleus. Estrogen receptor-immunoreactive neurons were most often present among the virus-labeled uterine-related neurons of the spinal cord, nucleus tractus solitarius, ventrolateral medulla, periaqueductal gray, medial preoptic area and ventromedial hypothalamic nucleus. These results identify a multisynaptic pathway of neurons whose eventual output is involved in uterine functions, whose distribution is similar to that revealed by pseudorabies virus tracing from other visceral organs, and which are often mixed among estrogen-responsive neurons.

Verruciform xanthoma (VX) is an uncommon mucocutaneous lesion of uncertain etiology. Originally thought to be limited to the oral mucosa, its occurrence in other mucosal and nonmucosal sites also has been documented. Histologically, VX is characterized by subepithelial foamy histiocytes associated with papillomatosis, parakeratosis, and dyskeratosis. Subepithelial foamy cells are lipid-containing, non-Langerhans cell histiocytes. A variety of etiologies have been proposed without much consensus, including infectious (bacterial, viral, and fungal), degenerative, reactive/ reparative, inflammatory, metabolic, reactive/ multifactorial, and immunosuppressive factors. Verruciform xanthoma of the external ear is exceedingly rare. Herein, we report a rare case of VX occurring on the earlobe at a piercing site in an immunosuppressed patient and provide a discussion of the possible pathogenetic mechanism(s).

The subepidermal hormonally sensitive tissue of the vulva is anatomically unique and may give rise to a wide variety of vascular tumors. As a consequence, classifying vulvar vascular lesions has been challenging due both to the wide variety of lesions that may be encountered and the heterogeneity in reporting across several disciplines. The purpose of this study is to present an institutional experience of vulvar vascular lesions. Overall, 85 patients were identified over a 26-year period. Vascular lesions belonging to the following classes included (n, %total) benign vascular tumors (32, 38%), dilatations of preexisting vessels (31, 36%), hyperplasia/reactive (7, 8%), tumors with significant vascular component (11, 13%), malformations (3, 4%), and malignant vascular tumors (1, 1%). Two reaction patterns based on vulvar lymphatic pathology were identified: one is a stromal dominant pattern and the other is a vascular dominant pattern. Vulvar vascular malformations and true vascular malignancies, although rare, may have associated high morbidity. To accurately classify vulvar lymphatic lesions, the pathologist must carefully consider the patient's clinical history taking into account features such as preexisting lymphedema.

Background Multicentric reticulohistiocytosis (MRH) is a rare disease of uncertain etiology that most commonly presents as a papulonodular cutaneous eruption accompanied by erosive polyarthritis. Although MRH is considered a systemic disorder in that it targets skin and joints, involvement of thoracic and visceral organs is uncommon. Observations A woman presented with diffuse cutaneous nodules, and skin biopsy findings revealed classic features of MRH. However, she also manifested severe pulmonary symptoms. A lung biopsy specimen showed prominent histiocytic infiltrates exhibiting the same characteristic morphologic features as those seen in her skin. Furthermore, the lung biopsy findings were significant for a pattern of usual interstitial pneumonia accompanied by notable lymphoid aggregates, a pattern of interstitial lung disease typical of systemic autoimmune and inflammatory conditions. Conclusions These findings are notable because a histiocytic pulmonary infiltrate suggestive of direct pulmonary involvement by MRH is a rare event. In addition, presentation of MRH in the setting of usual interstitial pneumonia is unique. These observations document a new clinical and histopathologic presentation of MRH that is significant for expanding the idea of MRH as a systemic disease while supporting the notion that MRH is promoted by an inflammatory milieu.

Background Dermatitis herpetiformis is an autoimmune disease typically characterized by pruritic vesicles located on the extensor surfaces. Classic disease consists of neutrophils in the dermal papillae. Additional histopathologic findings include fibrin deposition and edema within the dermal papillae. Subepidermal vesicles also may be present. Direct immunofluorescence demonstrates granular IgA in the dermal papillae. Observations A 58-year-old man with tender and pruritic erythematous macules and papules ranging from 2 to 6 mm in diameter had bilateral knee, elbow, forearm, scalp, and neck involvement. Petechiae also were present on the hands, thigh, knee, and ankle. A biopsy specimen initially demonstrated leukocytoclastic vasculitis. The results of workup for systemic vasculitis were negative. Subsequent biopsy specimens and direct immunofluorescence showed histologic evidence of dermatitis herpetiformis and leukocytoclastic vasculitis in the setting of an elevated serum IgA antitissue transglutaminase level. Marked improvement of the lesions was observed with a reduction of gluten in the patient's diet. Conclusions Physicians should consider the possibility of dermatitis herpetiformis in patients with petechiae and leukocytoclastic vasculitis because leukocytoclastic vasculitis may be a prominent feature of dermatitis herpetiformis.

T-cell lymphomas have a broad spectrum of cutaneous involvement. Several subtypes of T-cell lymphomas are associated with Epstein-Barr virus (EBV)-driven lymphoproliferative processes. We present a case of a composite, primary, cutaneous, EBV-associated, diffuse, large B-cell lymphoma and mature T-cell lymphoma occurring in a patient with Klinefelter karyotype (47, XXY). The patient had a characteristic clinical course of a systemic mature T-cell lymphoma before the presentation of the composite, primary, EBV-associated, diffuse, large B-cell lymphoma. Although similar cases have been described in extracutaneous locations, we believe that this is the first description with a primary cutaneous presentation.

Background Several forms of ichthyosis are associated with neurologic manifestations, including Sj ogren-Larsson syndrome, Refsum disease, and mental retardation –enteropathy-deafness-neuropathy-ichthyosis-keratoderma (MEDNIK) syndrome. We report a case of X-linked steroid sulfatase deficiency, ichthyosis, seizures, abnormal hair banding pattern, and unilateral polymicrogyria. Observations A 3-year-old Caucasian male with a history of ichthyosis since birth presented with generalized tonic seizures. Findings from a physical examination were remarkable for thin hair, retinitis pigmentosa, and poor dentition. Polarized light microscopic examination of all the hair samples demonstrated a banding pattern. Magnetic resonance imaging of the brain revealed left hemispheric polymicrogyria with decreased sulcal pattern and stable asymmetric dilation of the left lateral ventricle. Constitutional microarray revealed the typical approximately 1.5-Mb deletion of the steroid sulfatase gene. Conclusions Steroid sulfatase deficiency is a cause of X-linked ichthyosis; however, our patient also had retinitis pigmentosa, seizures, and abnormal hair findings. The presence of abnormal hair with a banding pattern on polarized microscopy may be helpful for diagnosis; however, this pattern is not specific to this disease. In addition, to our knowledge, the presence of a malformation of cortical development has not been previously reported in patients with steroid sulfatase deficiency.

A 57-year-old man status post several myocardial infarcts and heart transplantation presented with a slowly growing violaceous plaque on his lateral left knee at the site of prior minor trauma. A biopsy revealed a suppurative dermatitis with associated pseudocarcinomatous epithelial hyperplasia. There were multiple non-pigmented eosinophilic organisms with clear cytoplasmic halos within the infiltrate. A methenamine silver stain showed round to ovoid organisms of slightly variable size. Rare uni-polar budding, some of which was broad based, was apparent. A few short hyphae with indeterminate septa were also noted. Fontana-Masson, mucicarmine, Alcian blue and Fite stains were all negative. These findings suggested a diagnosis of blastomycosis. However, a fungal culture grew colonies of Alternaria species. Alternariosis has been previously shown to possess morphologic characteristics that can simulate other fungal infections. To our knowledge, a striking similarity to blastomycosis, as seen in our case, has not been previously reported. Dermatopathologists should be aware that alternariosis may mimic blastomycosis, especially when hyphal forms are rare or absent in tissue specimens. Culture is necessary for definitive classification. Osmond GW, Walters RW, Puri PK. Cutaneous alternariosis microscopically mimicking blastomycosis.

Lesions of the lacrimal caruncle vary widely due to its unique composition. Rarely, dermatologists and dermatopathologists encounter biopsies taken from this location. The aims of this study were to (1) retrospectively review caruncular lesions of the eye examined at Duke University Medical Center and (2) review the literature associated with caruncle pathology with emphasis on oncocytoma. A retrospective search of all lesions of the caruncle examined at Duke University Medical Center from 1991 to 2009 was performed. Fifty-nine lesions of the caruncle were identified. Nevi (40.7%) were the most common lesions observed, although squamous papillomas were the second most common entity (8.5%). Three oncocytomas were identified (5.1%), and were the next most common lesion present at this body site. These 3 entities represented 54.3% of the lesions found in the caruncle. Fifteen percent of the caruncular biopsies contained malignant or borderline malignant lesions, whereas the remaining 85% were benign entities. Most lesions present in the caruncle are benign. Although relatively rare, oncocytomas may be encountered by the dermatopathologist and may be confused for other neoplasms. Oncocytomas should be considered in the differential diagnosis based on this location in addition to histopathologic features.

Background: Spitz nevi typically show strong diffuse staining with S100A6, whereas staining in melanomas is commonly patchy and weak. To our knowledge, S100A6 has not been studied in pigmented spindle cell nevus (PSCN), considered by many to be a variant of Spitz nevus. Methods: Forty-six archived PSCNs were stained with S100A6 and then categorized by predominant cell size and staining pattern. Results: Eighteen (55%) of the small cell predominant nevi showed patchy staining, eight showed diffuse staining and seven were negative for S100A6. Two predominantly large-celled ‘PSCNs’ were diffusely positive and had many histopathological attributes of classical Spitz nevi. On review, these two cases were reclassified as Spitz nevi and excluded from the remainder of this study. Of the nevi with mixed cell size, one had no expression of S100A6. In the remaining tumors, the small cells showed patchy staining in eight (80%) and diffuse staining in two (20%). The large cells showed patchy staining in four (40%) and diffuse staining in six (60%). Conclusion: In contrast to the strong diffuse S100A6 staining typical of Spitz nevi, the small spindle cells of PSCN commonly show patchy staining or fail to stain completely. In melanocytic neoplasms composed of small spindle cells, patchy S100A6 staining should not be interpreted as evidence of supporting a diagnosis of melanoma. Puri PK, Elston CA, Tyler WB, Ferringer TC, Elston DM. The staining pattern of pigmented spindle cell nevi with S100A6 protein.

Background Studies examining head and neck (H&N) melanoma in the pediatric population are scarce. Objective The goal of this study is to describe pediatric H&N melanoma with the intent of increasing understanding of the course of disease. Methods The Duke Melanoma Database and Duke Tumor Registry Database were searched for patients with a diagnosis of melanoma occurring on the H&N before age 18 years, with exclusion of ocular/mucosal/aerodigestive melanomas. Results Queries yielded 39 Caucasian pediatric patients, 24 (61.5%) of them male. The mean age at diagnosis was 14.2 years (15 years, median). The primary sites were represented as follows: cutaneous auricular (1/39, 2.6%), facial (15/39, 38.5%), and scalp/neck (23/39, 59%). The follow-up time ranged from 2 months to 23 years with a median of 9.9 years (95% confidence interval: 6.2-13 years). At the time of follow-up, there were 12 (12/39, 30.8%) melanoma-associated deaths. The anatomic distribution of primary melanoma for these 12 patients follows: 4 (33.3%) facial and 8 (66.7%) scalp/neck. Histologic data revealed 24 (61.5%) tumors classified as superficial spreading melanoma with nodular melanoma (12.8%) a distant second. The mean Breslow depth for patients with melanoma-related mortality was 2.4 mm, compared with 1.8 mm for those who were alive at last follow-up. Limitations Small sample size limited this study. Conclusion This study found that the majority (59%) of H&N melanomas presented as scalp or neck lesions with a predilection for adolescents and boys. Those who experienced melanoma-related mortality had thicker lesions. Superficial spreading melanoma was the most common subtype.

Trichothiodystrophy (TTD) is a rare group of autosomal recessive disorders of DNA repair unified by the presence of sulfur-deficient brittle hair. We report a 3-year-old boy with classic clinical features of TTD, including ichthyosis, alopecia, developmental delay, and tiger-tail banding of the hair shaft on polarizing microscopy. Brain MR imaging showed both diffuse dysmyelination and osteosclerosis, findings that, in combination, may be specific for TTD.

Pagetoid reticulosis is an indolent primary cutaneous T-cell lymphoma. It typically presents as a solitary and slowly growing patch or plaque on the extremity, histologically characterized by an acanthotic epidermis infiltrated with atypical lymphocytes. Here, we present histological, immunophenotypical and molecular findings of a 29-year-old Jamaican man with bilateral wrist plaques. Histology showed marked acanthosis, hyperkeratosis and an intraepidermal infiltration consisting of large atypical lymphocytes. Immunohistochemical stains showed CD3 and CD5 positive T cells with significant loss of CD7, double negative CD4 and CD8 and strong positive CD30. Molecular analysis showed a monoclonal T-cell receptor (TCR) gamma gene rearrangement. Review of the literature confirms that the immunophenotype of pagetoid reticulosis is variable with decreasing frequency of CD8+ cytotoxic/suppressor T cell, CD4+ helper T cell and least commonly CD4/CD8 double negative phenotypes. Although CD4/CD8 double negative phenotype appears to be associated with higher proliferation index, it does not appear to confer prognostic significance.

Cutaneous clear cell proliferations and degenerative change have been seen in a variety of entities including nevi, dermatofibromas, fibrous papules, atypical fibroxanthomas, basal cell carcinomas, and squamous cell carcinomas, to name a few. However, there have been no reports of clear cells within neurofibromas. We received a biopsy and excision from a 61-year-old man with a papule on his right lateral clavicle. The initial biopsy showed a proliferation of clear cells that stained positive with S-100 and focally with CD68. A clear cell granular cell tumor was favored. Subsequent excision showed the same population of clear cells as seen on the initial biopsy. Interestingly, a neurofibroma was also present immediately beneath the clear cells with areas of transition. A p75 stain highlighted both populations of cells. This is the first case of neurofibroma with clear cells reported in the literature. We postulate that the clear cells are due to degenerative change.

We report two cases of hydroxychloroquine-induced hyperpigmentation presenting in a 50-year-old Caucasian female (case 1) and a 78-year-old female (case 2), both receiving 400 mg per day. Case 1 had an arthritis predominant undifferentiated connective tissue disease, which was treated with hydroxychloroquine for 4-5 years. She presented with a mottled, reticulated macular gray pigmentation involving the upper back and shoulders. Case 2 had a history of systemic lupus erythematosus and rheumatoid arthritis, treated with hydroxychloroquine for 1.5 years. She presented to the hospital for treatment of constrictive cardiomyopathy and was noted to have a blue macular pigmentation involving the right temple. The biopsies from both patients showed superficial dermal, yellow-brown, non-refractile and coarsely granular pigment deposition. A Fontana-Masson stain highlighted some of these granules, while the Perl's iron stain was negative. Rare, previous reports of hyperpigmentation indicate the presence of both melanin and hemosiderin in patients being treated with antimalarial medication. To our knowledge, this staining pattern for hydroxychloroquine has not been previously reported in the literature and supports that hydroxychloroquine, in addition to chloroquine, binds to melanin.

Objective: Metastatic cutaneous neoplasms may be difficult to differentiate from primary cutaneous neoplasms. Herein, we report an unusual case of metastatic cutaneous carcinoid tumor mimicking an adnexal poroid neoplasm. Methods: A 53-year-old male man presented with a neoplasm on the vertex of the scalp, clinically resembling a pigmented basal cell carcinoma. Results: A shave biopsy was suggestive of an apocrine poroma, however, a metastatic carcinoma could not be excluded. After acquiring additional clinical information and the complete excision of the neoplasm, further immunohistochemical stains supported the diagnosis a metastatic carcinoid tumor. Conclusion: To our knowledge, this is the first case of metastatic carcinoid tumor reported that has mimicked a poroid neoplasm.

The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP. # 1 Fetal Lung Maturity Testing Clinical Chemistry No. CC 08-1 (CC-367) {#article-title-2} Neonatal respiratory distress syndrome (RDS) is a disorder of pulmonary immaturity with a high mortality characterized by low levels of pulmonary surfactant. Gestational age determines risk based on concentration of pulmonary surfactant, ie, as gestation progresses the concentration of pulmonary surfactant increases. As a result, newborns delivered at fewer than 28 weeks have a more than 60% risk of RDS, whereas those delivered at more than 34 weeks have a less than 5% risk of RDS. In situations where gestational age alone is not sufficient to determine RDS risk and preterm delivery is medically needed, amniotic fluid analysis can be performed to determine pulmonary surfactant concentration. Four common methods are the lecithin-sphingomyelin ratio, phosphatidylglycerol measurement, surfactant-albumin ratio, and lamellar body count. All of these tests have excellent negative predictive values but poor positive predictive values, ie, they are great at confirming maturity but poor at confirming immaturity. # 2 Alkaline Phosphatase Isoenzyme Analysis: Guiding the Differential Diagnosis of Elevated Serum Total Alkaline Phosphatase Clinical Chemistry No. CC 08-2 (CC-368) {#article-title-3} The measurement of alkaline phosphatase (ALP) has long been used in clinical medicine, primarily to aid the diagnosis and monitoring of liver and bone diseases. Its diagnostic utility is limited by the enzyme’s wide tissue distribution and by a variety of potential nonpathologic causes. Isoenzyme analysis is one of several tools available to the clinician hoping to further characterize an elevated total ALP. It provides differentiation among liver, bone, intestinal, placental, and tumor-related forms as the cause of an elevation. The case presents a patient with persistent elevated ALP and atypical bands in the isoenzyme electrophoresis gel. An atypical ALP may be the result of a macroenzyme, which, if present, complicates interpretation of the test result and may cause additional, unnecessary testing or procedures. Recognizing and reporting a macroenzyme is an important service the clinical laboratorian can provide a clinician. # 3 Indirect Phlebotomy: Is It Good Practice? Clinical Chemistry No. CC 08-3 (CC-369) {#article-title-4} Indirect phlebotomy, the collection of blood samples through vascular infusion devices, is a common practice adopted to minimize needle-stick pain and discomfort in hospitalized patients. However, this practice can lead to alteration of the specimen and medical errors associated with misrepresentation of the blood test values. Among the possible problems, dilution or concentration of a particular analyte may occur, depending on the contents of the line’s infusate. Although some studies may indicate that blood collection from lines can be acceptable for isolated laboratory tests, there are insufficient data to demonstrate the reliability of line draws as routine practice. Use of lines for specimen collection should be reserved only for those patients with truly poor vascular access or those patients who require multiple phlebotomies in a short time period. Samples should be collected from lines only under a direct physician order, with careful attention to technique and established protocols that take many variables into account, including the analytes, contents of the line, type of line, discard blood volume, time interval between interruption of infusion and sampling, and catheter size. Even with careful attention to these details, clinicians must remain wary of the potential for medical errors related to misinterpretation of laboratory test results based on such samples.