Morphologic and cytogenetic variables affect the flow cytometric recovery of plasma cell myeloma cells in bone marrow aspirates.

INTRODUCTION: It is widely recognized that plasma cells (PCs) are under-represented in flow cytometry (FC) studies, but the causes of this phenomenon are poorly understood. We sought to study potential variables that affect PC recovery by flow cytometry (FC) in the analysis of plasma cell myeloma (PCM). METHODS: We retrospectively performed PC differential counts and morphologic assessment on PCM peripheral blood (PB) smears, bone marrow (BM) aspirate smears and posterythrocyte lysis cytospins. PCs were enumerated by FC, excluding erythroid events/debris, and were defined as CD38(bright+), CD45(dim to negative) events. PC recovery was calculated as follows: cytospin/aspirate, FC/aspirate, and FC/cytospin. RESULTS: Sixty-four BM analyses from 42 patients showed a mean aspirate PC% of 32.9 ± 23.2%. The mean PC% decreased in both the cytospin (10.9%) and by FC (8.2%). The difference between PC% in the cytospin and by FC was statistically significant (P < 0.03). Mature PC morphology and lower aspirate PC% had poorer recovery (P < 0.05) but higher-risk cytogenetics (deletions of 13q and TP53) was associated with increased PC recovery. Immunophenotype, heavy chain type, and treatment did not affect PC recovery. PB specimens had superior recovery compared with BM samples. CONCLUSIONS: Similar to prior reports, the greatest loss of PC in BM evaluation occurs between the aspirate and postlysis specimens; however, a small amount occurs from further processing. Additional morphologic and cytogenetic factors also appear to influence recovery in addition to overall PC%.

High prevalence of Dapsone-induced oxidant hemolysis in North American SCT recipients without glucose-6-phosphate-dehydrogenase deficiency.

Dapsone (4-4'-diaminodiphenylsulfone) is commonly used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis in immunocompromised patients. Oxidant hemolysis is a known complication of dapsone, but its frequency in adult patients who have undergone a SCT for hematological malignancies is not well established. We studied the presence of oxidant hemolysis, by combining examination of RBC morphology and laboratory data, in 30 patients who underwent a SCT and received dapsone for PCP prophylaxis, and compared this group with 26 patients who underwent a SCT and received trimethoprim-sulfamethoxazole (TMP-SMX) for PCP prophylaxis. All patients had normal glucose-6-phosphate dehydrogenase (G6PDH) enzymatic activity. In SCT patients, dapsone compared with TMP-SMX for PCP prophylaxis was associated with a high incidence of oxidant hemolysis (87 vs 0%, P<0.001), and the morphological evaluation of oxidant hemolysis correlated well with laboratory evidence of hemolysis. Dapsone-induced oxidant hemolysis in SCT patients is 20-fold higher than the reported rate in the population of HIV-infected patients, and thus much higher than the prevalence of G6PDH variants in the general population. In our patients, it manifested clinically as a lower Hb that was not significant enough to result in increased packed RBC transfusions.

The immunophenotypic stability of plasma cell myeloma by flow cytometry.

INTRODUCTION: Flow cytometry (FC) has become increasingly utilized in the diagnosis and monitoring of plasma cell myeloma (PCM), though few studies have evaluated the longitudinal stability of antigen expression. METHODS: We studied 45 PCM patients by four-color FC for shifts in CD19, CD20, CD38, CD45, CD56, and cytoplasmic light chain expression, between diagnostic/first encounter and positive follow-up analyses. An immunophenotypic (IP) change was defined as gain, loss, or ½ log shift of antigen expression. RESULTS: An IP change was observed in 14/45 (31%) patients, with single IP changes in 9/14, two changes in 2/14, and three changes in 3/14. 3/14 reverted from an aberrant to a normal plasma cell IP, while remaining light chain-restricted. Changes in expression of CD45 occurred in 9/45 (20%), CD19 in 5/45 (11.1%), CD20 in 2/45 (4.4%), and CD56 in 5/45 (11.1%). CONCLUSION: Approximately 1/3 of PCM cases show IP changes over time, with CD45 the least stable antigen. Recognition of this relative instability is important to avoid narrow targeting of follow-up FC analyses, especially for minimal residual disease monitoring.

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 Tgamma PCR positive. Our results support that CD4(+) T-LGL lymphocytosis is a clonal disorder with clinicopathologic characteristics distinct from the more common CD8(+) variant.

Acute hemolytic transfusion reaction secondary to anti-Fy3.

A hemolytic transfusion reaction due to anti-Fy3 is reported in an African American patient with no history of sickle cell disease. This 82-year-old African American woman received two units of RBCs for anemia (Hab 7 g/dL) on admission for a left hip fracture. On hospital Day 7, the patient underwent left hip endoprosthesis surgery; she received two units of RBCs on the second postoperative day due to Hb of 6.1 g/dL. Her urine was dark during surgery and postoperatively. Her posttransfusion plasma was red. Her Hb dropped from 8.4 to 6.4 g/dL over 24 hours after the transfusion. Her total bilirubin rose to 4.0 mg/dL, with and LDH value of 1558 U/L and a haptoglobin of 10.9 mg/dL. Both the antibody detection test and the DAT were positive. An anti-Fy3 was identified in the serum and in the eluate. To the best of our knowledge, this is the first case of acute intravascular hemolysis due to anti-Fy3 in a patient without sickle cell disease.

Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal.

Methylation events play a critical role in the ability of growth factors to promote normal development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)- and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH-SY5Y human neuroblastoma cells, via a PI3-kinase- and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylation-sensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+) and Al(3+) were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation.

Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H. Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S., and Gravel, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3059-3064). In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 micromol min(-1) mg(-1) at 25 degrees C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is approximately 100-fold slower than that of P-450 reductase. The K(m) for NADPH is 2.6 +/- 0.5 microm, and the K(act) for methionine synthase reductase is 80.7 +/- 13.7 nm for NADPH-dependent activity of methionine synthase.