Horatiu Olteanu

Clopidogrel anti-platelet effect: An evaluation by optical aggregometry, impedance aggregometry, and the Platelet Function Analyzer (PFA-100™)

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4–30% of patients. Traditionally, optical aggregometry is used to assess clopidogrels anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100™). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 µM) and collagen (1 and 2 µg/mL). For PFA-100™ analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71 ± 34% vs. 34.2 ± 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100™ ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100™ was unable to detect clopidogrel effect in the majority of the subjects.

CD200 expression in plasma cell myeloma

Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., Scott, M.A., Erber, W.N., Green, A.R. & Cancer Genome Project (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365, 1054–1061. Bellanné-Chantelot, C., Chaumarel, I., Labopin, M., Bellanger, F., Barbu, V., De Toma, C., Delhommeau, F., Casadevall, N., Vainchenker, W., Thomas, G. & Najman, A. (2006) Genetic and clinical implications of the Val617Phe JAK2 mutation in 72 families with myeloproliferative disorders. Blood, 108, 346–352. Bernardi, M., Ruggeri, M., Albiero, E., Madeo, D. & Rodeghiero, F. (2009) Isolated erythrocytosis in V617F negative patients with JAK2 exon 12 mutations: report of a new mutation. American Journal of Hematology, 84, 258–260. Jäger, R. & Kralovics, R. (2010) Molecular basis and clonal evolution of myeloproliferative neoplasms. Haematologica, 95, 526– 529. Landgren, O., Goldin, L.R., Kristinsson, S.Y., Helgadottir, E.A., Samuelsson, J. & Björkholm, M. (2008) Increased risks of polycythemia vera, essential thrombocythemia, and myelofibrosis among 24,577 first-degree relatives of 11,039 patients with myeloproliferative neoplasms in Sweden. Blood, 112, 2199–2204. Lee, F.S. (2008) Genetic causes of erythrocytosis and the oxygen-sensing pathway. Blood Reviews, 22, 321–332. Review. Oh, S.T., Simonds, E.F., Jones, C., Hale, M.B., Goltsev, Y., Gibbs, Jr, K.D., Merker, J.D., Zehnder, J.L., Nolan, G.P. & Gotlib, J. (2010) Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms. Blood, 116, 988–992. Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele, J. & Vardiman, J.W. (Eds) (2008) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer, Lyon. Tefferi, A. (2010) Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia, 24, 1128–1138. Villar, D., Vara-Vega, A., Landázuri, M.O. & Del Peso, L. (2007) Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains. Biochemical Journal, 408, 231–240.

The Specificity of Immunophenotypic Alterations in Blasts in Nonacute Myeloid Disorders

Data regarding flow cytometry (FC) in nonacute myeloid disorders is confounded by variable gating strategies and controls limited to normal bone marrow (BM) samples. Blasts in diagnostic BM samples of myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and chronic myelomonocytic leukemias (CMMLs) were compared with 20 nonneoplastic cytopenias/cytoses (CCs) and negative staging BM samples using 4-color FC. Blasts in 10 of 20 CCs showed immunophenotypic differences vs control samples. Immunophenotypic alterations were identified in 18 of 21 MDSs, 11 of 14 MPNs, and 7 of 7 CMMLs vs control samples and 13 (62%) of 21 MDSs, 7 (50%) of 14 MPNs, and 3 (43%) of 7 CMMLs vs CCs. Neoplastic-specific blast immunophenotypic changes included expression of CD7, CD11b, CD15, CD36, and CD56; CD34 overexpression; HLA-DR variability; lack of CD13 and CD33; underexpression of CD13, CD33, CD45, and HLA-DR; and partial loss of CD13, CD33, CD38, and CD117. In all cases, blasts were CD34+. Several blast immunophenotypic alterations are shared in neoplastic and nonneoplastic BM samples. Approximately 40% to 60% of neoplastic BM samples exhibited aberrancies not seen in reactive BM samples.

The Unique Immunophenotype of Double-Hit Lymphomas

We read with interest the article “‘Double-Hit’ Mature B-Cell Lymphomas Show a Common Immunophenotype by Flow Cytometry That Includes Decreased CD20 Expression” by Wu et al.1 We have similarly noticed distinct immunophenotypes, in addition to decreased CD20 expression, in double-hit lymphomas (DHLs) analyzed by flow cytometry at our institution. Because few data exist in the literature,2,3 we chose to test the specificity of this distinct immunophenotype by comparing the immunophenotypic characteristics of patients with DHL with those of patients with Burkitt lymphoma (BL) and CD10+ diffuse large B-cell lymphoma (DLBCL) by using 4-color flow cytometry with the following antigens: CD5, CD10, CD19, CD20, CD22, CD23, CD38, FMC-7, and surface monoclonal and polyclonal κ and λ light chains, when possible. Antigen expression patterns were compared with those of normal, polytypic B cells. Decreased expression encompassed negative, dim, and partial patterns, with antigen positivity defined as more than 20% expression compared with an isotypic control threshold. We analyzed 12 specimens from 9 patients with DHL (3 cerebrospinal fluid samples, 3 tissue samples, 3 peripheral blood samples, 2 bone marrow samples, and 1 peritoneal fluid sample), 7 specimens from 6 patients with BL (5 tissue samples, 1 bone marrow sample, and 1 peripheral …

CD23 Expression in Follicular Lymphoma: Clinicopathologic Correlations

Follicular lymphoma (FL) is typically a CD10+/CD5-/FMC-7+ B-cell lymphoma with variable CD23 expression. The clinical significance of CD23 expression in FL is uncertain. We studied the expression of CD23 by flow cytometry in 69 lymph nodes (LNs) and correlated it with pathologic and clinical parameters. Of 69 FLs, 48 (70%) were CD23+. Grade 3 FLs were CD23- more often (12/16 [75%]) than grade 1 and 2 cases (9/53 [17%]; P < .001). CD23 expression was more common in FLs in inguinal LNs than in other sites: 20 of 23 (87%) vs 28 of 46 (61%; P = .029). Overall survival (P = .002) and event-free survival (P < .0001) were longer in the CD23+ group than in the CD23- FLs. Our study shows that grade 3 FLs are more often CD23- than lower grade FLs and that FLs in inguinal LNs are more frequently CD23+ than in LNs from other sites. Furthermore, our findings also indicate that survival is significantly better in CD23+ FLs.

Flow Cytometric Analysis of Surface Light Chain Expression Patterns in B-Cell Lymphomas Using Monoclonal and Polyclonal Antibodies

Light chain (LC) expression by flow cytometry (FC) in B cell non-Hodgkin lymphomas (B-NHLs) can occasionally be detected with one anti-LC antibody but not with another. We retrospectively analyzed 564 four-color FC files from B-NHLs, assessing LC staining with monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs). Discrepancies in LC expression between mAbs and pAbs were present in 9.2% of cases, mainly in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 11.1%), diffuse large B-cell lymphoma (DLBCL; 10.2%), follicular lymphoma (9.5%), and mantle cell lymphoma (11.1%) and most frequently in body fluids. Equal proportions of cases were LC+ only with pAbs (4.8%) or mAbs (4.4%). Negative LC expression with both antibodies was present in 7.5% of cases, most frequently in DLBCL (21.6%) and body fluids (27.6%). Evaluation with both mAbs and pAbs increases the sensitivity for LC detection, with no single reagent outperforming the other, although CLL/SLL preferentially showed LC expression with pAbs.

Laboratory findings in CD4(+) large granular lymphocytoses

Large granular lymphocytic (LGL) leukemia is an uncommon disorder of mature T or natural killer (NK) cells. Most T‐LGL proliferations are CD3(+)/CD8(+), although rare CD4(+) clonal T‐LGL expansions have been reported. We report the clinicopathologic features of eight patients with aberrant CD4(+), cytotoxic T‐cell lymphocytoses. Median follow‐up was 29 months (range 8–100), during which all were alive without requirement for therapy. Four of eight patients had an additional malignancy; none had a history of rheumatoid arthritis, lymphadenopathy or hepatosplenomegaly. Morphologic expansions of granulated lymphocytes were evident in 6/8. All had immunophenotypically aberrant populations of CD4(+) T cells with uniform, moderate or bright CD56. Seven of eight expressed CD57, and four were CD8(partial dim +). Abnormal levels of expression of two or more T‐cell antigens were seen in all cases. All tested cases were Tγ PCR positive. Our results support that CD4(+) T‐LGL lymphocytosis is a clonal disorder with clinicopathologic characteristics distinct from the more common CD8(+) variant.

CD23 expression in plasma cell myeloma is specific for abnormalities of chromosome 11, and is associated with primary plasma cell leukaemia in this cytogenetic sub-group.

CD23 is a low-affinity Fc receptor for IgE and is pivotal in IgE homeostasis (Hibbert et al, 2005). CD23 exists in two isoforms; CD23a, present on mature B-cells, and CD23b, which requires induction by interleukin-4 for activation on T cells, monocytes, Langerhans cells, eosinophils, and macrophages. CD23 is also constitutively expressed on Epstein–Barr virus (EBV) transformed B-lymphoblasts, and is therefore also known as EBV surface antigen. The FCER2 gene on chromosome 19p13.3 encodes the CD23 45 kDa protein containing 321 amino acids. A multifunctional protein, CD23 has activities associated with cytokine modulation and B-cell growth factor function. The diagnosis of plasma cell myeloma (PCM) relies on the presence of an M-protein in the serum/urine, a clonal proliferation of plasma cells, and related organ/tissue damage, the latter defined as anaemia, hypercalcaemia, lytic bone lesions, renal insufficiency, hyperviscosity, amyloidosis, or recurrent infections (McKenna et al, 2008). Chromosomal abnormalities are identified in >90% of cases, often involving IGH@ on chromosome 14q32, as well as chromosome bands 11q13, 4p16.3, 16q23, 6p21, and 20q11 (Avet-Loiseau et al, 2007). Numerical and structural chromosomal irregularities are also frequent, the most common being 1q gains and 13q14 deletions. PCM usually shows monotypic expression of intracytoplasmic light chain, expression of CD79a, strong CD38, CD138, and VS38c. A majority of cases express CD56, but the cells usually lack CD19 and CD20. In contrast, normal plasma cells are mainly CD19 and CD56 (Lin et al, 2004). Expression of cyclin D1 and CD20, and lack of CD56 has previously been reported in association with the t(11;14)(q13;q32) (Hundemer et al, 2007). Primary plasma cell leukaemia (PCL) is immunophenotypically similar, with the exception being common negativity for CD56 (Garcia-Sanz et al, 1999). Although CD23 expression is well characterized in other haematological malignancies, to our knowledge evaluation of CD23 in plasma cell myeloma has not previously been reported. We examined 50 diagnostic PCM bone marrows (including three cases of primary PCL) for CD23 expression by immunohistochemistry. Five cases were also evaluated for CD23 expression by flow cytometry. Vector Laboratories CD23 mouse monoclonal antibody with Vector Antigen Unmasking Solution and high temperature antigen unmasking protocol was used for detection on paraffin sections from decalcified bone marrow biopsies when available and clot sections when no core biopsy was available. Immunophenotypes from each case were derived from routine flow cytometry performed at diagnosis using previously reported methods (Harrington et al, 2009). Assessment of CD19, CD20, CD45, CD56 and intracytoplasmic immunoglobulin light chain expression was done in most cases (49 out of 50). CD23 expression was correlated with presenting laboratory data, including: blood counts (white blood cell count, red blood cell count, Haemoglobin, mean cell volume, mean cell haemoglobin concentration, red cell distribution width, and platelet count), M-protein level, creatinine, beta-2 microglobulin, serum calcium, and cytogenetics. Cytogenetic analysis, including unstimulated culture, chromosome analysis and fluorescent in situ hybridization (FISH) of marrow cells, was performed using previously reported methods (Heerema et al, 2007). Commercially available DNA probes that detect characteristic clonal rearrangements in multiple myeloma [deletion p53, deletion 13q14, t(11;14), t(4;14) and t(14;16)] were obtained from Vysis (Des Plaines, IL) and used per the manufacturer’s instructions. For most cytogenetic analyses 20 metaphases were examined; for FISH, 200 interphase cells were analyzed for each probe in each case. Where t(11;14)(q13;q32) cytogenetics were identified, cyclin-D1 immunohistochemistry was also performed using the Thermo Scientific rabbit IgG protocol targeting the C-terminus of human cyclin-D1. Statistical analysis was performed using the Statistical Package for the Social Sciences software for Macintosh, version 11.0 (SPSS, Inc., Chicago, IL, USA). The study protocol was approved by the Medical College of Wisconsin internal review board. Of 50 PCM cases examined for CD23 expression, five (10%) showed uniform strong membranous positivity (Fig 1); three also showed golgi staining. CD23 expression was confirmed by flow cytometry in two of five cases. All five CD23 positive cases displayed abnormalities of chromosome 11 by conventional cytogenetics and/or FISH. Four of these cases contained a t(11;14)(q13;q32), while the fifth case showed a trisomy 11 by conventional cytogenetics. In total, t(11;14)(q13;q32) was identified in 10/50 PCM cases (20%). All t(11;14)(q13;q32) cases were positive for cyclin-D1 by immunohistochemistry (Bergsagel et al, 2005). No significant differences in presenting laboratory data or other immunophenotypic features were identified between the t(11;14)(q13;q32) cases that were CD23 positive and negative. Associations of immunophenotypic correspondence

Chronic lymphoproliferative disorder of natural killer cells: a distinct entity with subtypes correlating with normal natural killer cell subsets

Chronic lymphoproliferative disorder of natural killer cells: a distinct entity with subtypes correlating with normal natural killer cell subsets

Follicular lymphoma transformation to dual translocated Burkitt-like lymphoma: improved disease control associated with radiation therapy

Dual translocated (or “dual hit”) lymphomas are highly aggressive B cell neoplasms associated with an extremely poor prognosis. The optimal treatment for these lymphomas remains undefined. We present two cases of follicular lymphoma with transformation to Burkitt-like lymphoma. In both cases dual translocations involving both the bcl-2 and c-myc loci were present. Each patient underwent intensive induction immunochemotherapy followed by autologous stem cell transplantation and radiation therapy. The first patient received post-transplant mediastinal radiation and developed recurrence in multiple areas outside of the radiation field. The second patient received total body irradiation as part of the conditioning regimen, and is without recurrence 18 months after transplant, and 24 months after diagnosis of the dual translocated lymphoma. We review dual translocation B cell lymphoma in the setting of transformation from follicular lymphoma, and suggest a potential role for total body irradiation in the management of this highly aggressive non-Hodgkin lymphoma.