Kristi J. Smock

Gene expression profiling reveals intrinsic differences between T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma

T‐cell acute lymphoblastic leukemia (T‐ALL) and T‐cell lymphoblastic lymphoma (T‐LL) and are often thought to represent a spectrum of a single disease. The malignant cells in T‐ALL and T‐LL are morphologically indistinguishable, and they share the expression of common cell surface antigens and cytogenetic characteristics. However, despite these similarities, differences in the clinical behavior of T‐ALL and T‐LL are observed.

Proliferative nodules in congenital melanocytic nevi: a clinicopathologic and immunohistochemical analysis.

Congenital melanocytic nevi (CMN) occur in 1% to 2% of newborns, and the risk of malignant melanoma is increased in patients with large CMN. Appearance at birth or later of a nodular or hyperpigmented area within a CMN simulates malignant melanoma and prompts biopsy. Although their clinical and pathologic features seem ominous, proliferative nodules (PNs) typically are benign and may regress, although atypical features cause greater concern. Here we report clinical and pathologic findings with outcome in 10 children who had multiple biopsies of large CMN with PNs. We reviewed 78 separate samples from the 10 patients and classified the 60 PNs according to published criteria. A subset of 30 samples containing both the CMN and a PNs was analyzed for immunohistochemical reactivity for melanocytic (S-100 protein, HMB45, melan-A), lymphocytic (CD45), cell-cycle/proliferative (Mib-1, p16, p21, p27, c-Myc), apoptotic (p53, Bax, c-kit, CD95), and anti-apoptotic (bcl-2) markers. Both CMN and PNs had similar expression of melanocytic, lymphocytic, and most cell-cycle/proliferative and apoptotic markers, including Mib-1, p16, p21, p27, c-Myc, Bax, CD95, and bcl-2. A greater proportion of PNs than CMN were reactive for p53 (67% vs. 30%, P < 0.0098) and c-kit (97% vs. 3%, P < 0.0001). p53 and p21 expression in CMN and all types of PNs were inversely correlated. When ordinary and atypical PNs were compared, the atypical PNs more frequently expressed p53, Mib-1, Bax, and bcl-2, but less frequently expressed p21. The c-kit expression in nearly all PNs and its absence in nearly all CMN is potentially useful for recognition of PN, suggests a delayed melanocytic maturation process in proliferative nodules, and may be likely indicative of their benign nature. p53 reactivity in concert with a lack of p21 up-regulation by immunohistochemistry suggests that a p53 mutation may be present in PN, although the immunohistochemical findings alone cannot exclude possible overexpression of wild-type p53. Regressive, involutional, or maturational changes were observed in sequential samples from 4 patients. No patient developed malignant melanoma or another melanocytic nevus-associated malignancy during the follow-up period. These findings underscore the similarities between PNs and the underlying CMN and suggest that maturational, proliferative, and apoptotic processes are involved in their clinical evolution.

Polycythemia vera-associated acquired von Willebrand syndrome despite near-normal platelet count†

To the editor: The pathogenesis of bleeding diathesis associated with myeloproliferative neoplasms (MPN) is not well understood; implicated mechanisms include acquired von Willebrand syndrome (AVWS), platelet storage pool deficiency, and defective aggregation [1]. Because MPN is also associated with abnormal clotting, antithrombotic measures including use of aspirin exacerbate the underlying bleeding diathesis. AVWS also occurs in the setting of other unrelated conditions including lymphoma, plasma cell dyscrasia, cardiovascular disease, and autoimmune disorders [2]. Implicated pathogenetic mechanisms include targeting and selective clearance of large von Willebrand factor (VWF) multimers by clonal cells, abnormal vessels/valves, and autoimmune antibodies [3,4]. VWF antigen (VWF:Ag) assays measure VWF monomer content but not its multimeric composition; in other words, they are helpful in detecting VWF deficiency but not dysfunction. Decreased VWF:Ag is usually accompanied by decreased factor VIII coagulant activity (FVIII:C) since the former carries and stabilizes the latter in the circulation. Laboratory assays used to assess VWF function include ristocetin cofactor activity (VWF:RCoA), which measures binding of VWF to platelet GP Ib, and VWF multimer analysis, which measures multimer distribution. In AVWS, because of selective loss of the largest VWF multimers, VWF:RCoA is decreased despite normal levels of VWF:Ag and VWF:RCoA/VWF:Ag ratio is usually <0.7 (normal ratio is 1.0). In a recent study, none of the above-listed assays, other than VWF multimer analysis, had adequate sensitivity to detect the presence of a clinically relevant (i.e., associated with bleeding symptoms) AVWS [5]. More than 50% of patients with polycythemia vera (PV) or essential thrombocythemia (ET) display laboratory evidence AVWS, which is sometimes accompanied by clinically overt mucocutaneous bleeding [6,7]. The underlying mechanism is believed to include an abnormally increased VWF proteolysis that results in selective depletion of the largest multimers [8]. There is evidence to support the role of platelets in this regard, especially in ET and reactive thrombocytosis, and therapeutic lowering of the platelet count has been shown to correct the laboratory abnormality in some patients [9,10]. Herein, we describe a patient with PV who experienced bleeding complications associated with AVWS despite a platelet count of <500 3 10/L. A 61-year-old patient with JAK2V617F-positive PV presented with a hemoglobin level of 21 g/dL (hematocrit 66%), white blood cell count (WBC) of 13.6 3 10/L, and platelet count of 481 3 10/L, in October 2009. During treatment with phlebotomy in November 2009, the patient displayed several areas of ecchymoses and localized bleeding despite optimal venipuncture techniques and absence of treatment with aspirin. Laboratory studies at the time showed complete absence of the large VWF multimers (Fig. 1, lane 4): FVIII:C (89%; normal range 56–191), VWF:Ag (166%; normal range 52–214), VWF:RCoA (92%; normal range 51–215), and VWF:RCoA/VWF:Ag 0.6. On that same day, his WBC was 13.6 3 10/L, hemoglobin 17.9 g/dL (hematocrit 56%), and platelet count 488 3 10/L. After aggressive phlebotomy and treatment with hydroxyurea, resolution of bleeding symptoms and correction of VWF multimer distribution was documented by January 2010 (Fig. 2, lane 8): hemoglobin 16.4 g/dL (hematocrit 50%), WBC 8.4 3 10/L, platelets 276 3 10/L; FVIII:C 166%, VWF:Ag 157%, VWF:RCoA 109%, and VWF:RCoA/VWF:Ag 0.7. Increased clearance of the larger VWF multimers in ET has been proposed by some investigators to be the result of enhanced proteolytic degradation of VWF as a direct result of platelet excess [8,9], which may facilitate the interaction between platelet surface receptor GP Ib and VWF, thus inducing the appropriate conformational changes in VWF that allows ADAMTS13 to gain access to its cleavage site. In our patient with PV, despite the near-normal platelet count, a similar pathogenetic contribution from platelets cannot be excluded and the platelet-VWF complex-induced accelerated proteolysis might have been enhanced by high shear stress from increased blood hyperviscosity [11]. Regardless, there are two important practical points that are illustrated by our case report: (i) clinically relevant AVWS can occur in PV despite near-normal platelet count and (ii) VWF multimer analysis is essential for accurate diagnosis of AVWS.

Effects of EDTA on routine and specialized coagulation testing and an easy method to distinguish EDTA-treated from citrated plasma samples

Apoptosis deregulation is important for cancer development, chemotherapy response, and prognosis. Survivin and X-linked inhibitor of apoptosis protein (XIAP) are 2 members of the inhibitor of apoptosis proteins family (IAP). We used semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to determine the levels of expression of survivin and XIAP in 30 patients with de novo acute myeloid leukemia (AML) and 20 age- and sex-matched healthy volunteers. Survivin and XIAP overexpression were detected in 36.7% and 43.3% of cases, respectively. Patients with overexpression of either survivin or XIAP showed unfavorable response to chemotherapy in 81.2% and 91.7%, respectively. Also, these cases showed shorter median survival time (30 days) compared to patients with normal expression of either survivin or XIAP (150 days and 180 days). Patients with overexpression of both survivin and XIAP showed unfavorable response to induction therapy in 100% of the patients and the shortest median survival (30 days). These findings suggest that survivin and XIAP may have a role in leukemogenesis and provide prognostic information.Coagulation testing is performed with citrate-treated plasma. Samples submitted in other anticoagulants, such as EDTA, should not be tested. We aimed to evaluate the effects of EDTA on routine and specialized coagulation tests and to establish sodium tetraphenylborate testing as a quick and reliable method to identify EDTA-treated plasma samples. We performed the following measurements on citrateand EDTA-treated plasma samples from 10 healthy volunteers: sodium tetraphenylborate testing, prothrombin time (PT), partial thromboplastin time (PTT), potassium concentration, and functional assays for factors II, V, VII, VIII, IX, X, XI, XII, proteins C and S, and antithrombin. Mean values for citrate- and EDTA-treated plasma were most different for PT, PTT, factors V and VIII, and proteins C and S. Sodium tetraphenylborate testing correctly classified 100% of citratetreated and EDTA-treated samples. We confirm that EDTA has effects on coagulation assays. Sodium tetraphenylborate testing is a quick, simple, and inexpensive method for coagulation laboratories to identify samples erroneously submitted in EDTA.

Thrombocytopenia: an update

Thrombocytopenia is a common clinical problem with numerous potential causes including decreased bone marrow platelet production, increased peripheral platelet destruction, increased splenic sequestration, and dilution. Investigation of the etiology of thrombocytopenia requires careful consideration of clinical history and laboratory features. A complete blood count and peripheral smear review are essential components of the diagnostic work‐up, and physicians should be knowledgeable about appropriate selection and interpretation of more specialized tests, including bone marrow examination, to assist with diagnosis. This review article aims to summarize and address appropriate work‐up of the major and/or life‐threatening causes of thrombocytopenia and some of the better‐characterized congenital thrombocytopenias.

Laboratory evaluation of clopidogrel responsiveness by platelet function and genetic methods

Clopidogrel is a widely used antiplatelet agent that irreversibly inhibits platelet P2Y12 ADP receptors after conversion to an active metabolite. There are a number of laboratory tests capable of detecting clopidogrel‐induced platelet inhibition and published literature correlates suboptimal clopidogrel response to adverse cardiovascular outcomes. Genetic polymorphisms are thought to affect conversion of the prodrug to the active metabolite, and the FDA has recently added a black‐box warning to clopidogrel to highlight the effects of these polymorphisms on drug bioavailability and to inform prescribers about the availability of genetic testing. For these reasons, there is growing interest in the use of laboratory tests to monitor patients treated with clopidogrel. This article summarizes the currently available laboratory testing, including platelet function tests and genotyping for CYP2C19 variants. Am. J. Hematol., 2011.

Discard tubes are not necessary when drawing samples for specialized coagulation testing.

Discard tubes have traditionally been obtained when drawing samples for coagulation testing to avoid potential tissue factor activation of coagulation in the first tube that may lead to inaccurate results. Although discard tubes are no longer required for prothrombin time and partial thromboplastin time, the practice is still recommended for other coagulation studies due to lack of sufficient evidence that discard tubes are not needed. The objective of the present study was to determine whether the first citrate tube drawn can be used for special coagulation testing. We performed testing for fibrinogen, D-dimer, factors VIII, IX, XI, proteins C and S, and antithrombin on 30 healthy individuals and factors II, VII, IX, X, and proteins C and S on a second group of 30 healthy individuals and 30 individuals receiving warfarin. Testing was performed on two consecutive samples to evaluate the level of agreement between the two tubes. Paired t-testing showed no statistically significant differences between tube 1 and tube 2 for any of the tests performed. Most data pairs (tube 1, tube 2) agreed within 10% difference or less, and no positive or negative biases were observed. To our knowledge, this study is the first to evaluate the need for discard tubes in a variety of coagulation tests using both normal and abnormal samples. Our data suggest that discard tubes are not necessary when drawing samples for specialized coagulation testing.

Characterization of childhood precursor T-lymphoblastic lymphoma by immunophenotyping and fluorescent in situ hybridization: a report from the Children's Oncology Group.

T‐lymphoblastic lymphoma (T‐LBL) accounts for 25–30% of childhood non‐Hodgkins lymphoma and is closely related to T‐lymphoblastic leukemia (T‐ALL). Recently, we demonstrated distinct differences in gene expression between childhood T‐LBL and T‐ALL, but molecular pathogenesis and relevant protein expression patterns in T‐LBL remain poorly understood.

Assessment of orally dosed commercial silver nanoparticles on human ex vivo platelet aggregation

Abstract Enhanced in vitro human and ex vivo rat platelet aggregation from direct exposure to silver nanoparticles is previously reported. Given the increasing human use of engineered silver nanoscale products, platelet aggregation prompted by silver nanoparticles may contribute to human cardiovascular events. To understand how direct washed platelet exposure to silver nanoparticles translates to ex vivo platelet aggregation, the authors conducted a placebo-controlled, single-blind, dose-monitored, cross-over study design in 18 healthy human volunteers. After 2 weeks of daily oral silver nanoparticle ingestion, platelet aggregation was evaluated by light transmission aggregometry in response to collagen and ADP agonists, both at baseline and after silver nanoparticle or placebo diluent oral dosing. Final percent aggregation (PA) and the changes in PA were determined using a paired design (i.e., active and placebo solutions). Enhanced ex vivo platelet activation was not detectable at peak serum silver concentrations <10 µg/L. Further studies of colloidal silver nanoparticles on human platelet activities are warranted.

Factor XIII Assays and associated problems for laboratory diagnosis of factor XIII deficiency: an analysis of International Proficiency testing results.

We analyzed results from the External quality Control of diagnostic Assays and Tests program to assess current clinical laboratory practice and performance of different methods for factor XIII (FXIII) testing internationally. FXIII proficiency testing data from all eight surveys conducted in 2010 and 2011 were analyzed (1,283 results), comparing the three available methods for detecting FXIII deficiency, thus including clot-solubility qualitative activity, quantitative activity, and antigen. Clot-solubility qualitative assays detected a deficiency in only 16% (11/69) of samples with less than 2% FXIII. Assays using added thrombin detected more deficiencies (33%) than did assays without added thrombin (11%). The most commonly used quantitative activity method tended to produce higher results for low FXIII samples than other quantitative activity methods. Antigen results generally showed good accuracy compared with expected levels. The mean interlaboratory coefficients of variation showed wide variability, especially for samples with less than 10% FXIII activity. Laboratory self-classification of results (as normal vs. abnormal) was good, and was slightly better for specimens with ≤ 25% FXIII than for specimens with 26 to 70% or those with >70% FXIII. We conclude that quantitative activity assays perform better for detecting FXIII deficiency than clot solubility assays, although some quantitative activity assays overestimate low FXIII levels.