Spencer W. Green

Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure Syndromes

Aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) are rare hematologic diseases that often appear in the same patient. Patients with aplastic anemia have severe thrombocytopenia, neutropenia, and anemia accompanied by absent hematopoietic precursors in an empty bone marrow (1). In contrast, the classic evidence of PNH is the intermittent appearance of dark urine due to excretion of hemoglobin, the result of intravascular hemolysis (2). The knowledge that this peculiar form of erythrocyte destruction resulted from increased susceptibility of the PNH erythrocyte to complement led to the development of laboratory assays, such as the Ham and sugar hemolysis tests. Modern clinical studies have shown that patients with PNH experience serious morbidity and mortality, mainly from venous thromboses and, especially in younger patients, pancytopenia (3). On the basis of results of the Ham test in several patients, Lewis and Dacie (4) formalized the overlap between the two diseases as the aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome. With improved survival in aplastic anemia, many patients show laboratory and clinical evidence of PNH, often months or years after completion of successful immunosuppressive therapy (5, 6). In aplastic anemia, hematopoietic cells appear to be destroyed by the patients own immune system (7). In contrast, the basis of PNH is a somatically acquired mutation in a hematopoietic stem cell. Almost all patients with PNH have molecular lesions in the PIG-A gene, which is located on the X chromosome (8). The PIG-A gene product is required at an early step in the synthesis of a glycosylphosphatidylinositol (GPI) structure, which serves as an anchor for a group of proteins that are linked to the cell surface by this greasy foot rather than by the more typical transmembrane configuration. As a result, affected cells are globally deficient in GPI-anchored proteins. Deficiency in one of these proteins, CD59, which inhibits late-acting complement component activity on the erythrocyte surface, accounts for the hemolytic component of the disease (9). The pathophysiologic basis of the clinical relation between PNH and aplastic anemia is unknown. Lewis and Dacie (4) postulated the development of an abnormal clone of haemopoietic cells in a regenerating, previously aplastic marrow as a cause, and Rotoli and Luzzatto (10, 11) speculated that an insult leading to a hypocellular environment or some feature of marrow failure might lead to either aplasia or PNH. Observations of patients with lymphoma who received monoclonal antibody treatment directed against a GPI-anchored protein have demonstrated the appearance of PNH-like lymphocytes as an inadvertent result of therapy (12). These results confirmed the in vivo plausibility of a third hypothetical mechanism: that preexisting PNH clones might be selected as a result of their relative insusceptibility to autoimmune attack (6, 10). To test these hypotheses, we used a sensitive flow cytometric method to analyze blood cells from patients in various states of marrow failure for evidence of PNH clones. Methods Patients We developed a rigorous three-color flow cytometric protocol for identification of GPI-anchored protein-deficient granulocyte populations and applied this method over 1.5 years to patients presenting to our clinic at the National Heart, Lung, and Blood Institute with newly diagnosed or previously treated bone marrow failure syndromes. Most of the patients with previously treated bone marrow syndromes had received therapy at the National Institutes of Health up to 11 years before sampling for this study. Samples from 254 participants were analyzed from April 1997 to October 1998. Of these participants, 115 had aplastic anemia, 39 had myelodysplasia, 28 had recently undergone bone marrow transplantation, 20 were controls who had undergone renal transplantation and had received antithymocyte globulin as treatment for graft rejection, 13 had large granular lymphocytosis, 18 had undergone multiple cycles of chemotherapy for cancer, and 21 were healthy controls. All clinical research samples were obtained after informed consent was given under protocols approved by the institutional review board of the National Heart, Lung, and Blood Institute. Other specimens were collected during routine phlebotomy procedures and were studied without unique patient identifiers. Aplastic anemia was defined as bone marrow cellularity of 30% or less and two of the following three laboratory abnormalities: absolute neutrophil count less than 0.5 109 cells/L, platelet count less than 20 109 cells/L, and reticulocyte count less than 60 109 cells/L. Myelodysplasia was defined by the standard morphologic criteria of either dysplastic myeloid or megakaryocytic (but not solely erythroid) bone marrow elements in patients who required 2 or more units of red blood cell transfusions per month for 2 or more months, with or without thrombocytopenia or neutropenia. Monoclonal Antibodies We used CD55-PE (clone 143-30, mouse IgG1 [Research Diagnosis, Inc., Flanders, New Jersey]) and CD59-PE (clone MEM 43, mouse IgG2a [Research Diagnosis, Inc.]) for analysis of GPI-anchored protein expression on erythrocytes. Glycophorin-A-FITC (clone D2.10, mouse IgG1 [Immunotech, Westbrook, Maine]) was used as a nonanchored marker to positively identify erythrocytes. The isotypic control for erythrocyte phenotyping consisted of mouse IgG1-PE (clone X40 [Becton Dickinson, San Diego, California]) and mouse IgG2a-PE (clone X39 [Becton Dickinson]). For analysis of granulocyte GPI-anchored protein expression, we used CD66b-FITC (clone 80H3, mouse IgG1 [Immunotech]) and CD16-PECy5 (clone 3G8, mouse IgG1 [Caltag]), with CD15-PE (clone 80H5, mouse IgM [Immunotech]) as a non-GPI-anchored marker to positively identify granulocytes. The isotypic controls for granulocyte staining consisted of mouse IgG1-FITC (clone X40 [Becton Dickinson]) and mouse IgG1-PECy5 (clone MOPC-21 [Caltag]). Nonspecific Fc receptor-mediated binding of conjugated antibodies to granulocytes or erythrocytes was blocked by pre-incubating 1 mL of blood with 30 L of mouse IgG (Caltag). Antibody Staining and Flow Cytometry Analysis Blood was drawn by venipuncture into tubes containing EDTA. Samples were stained within 48 hours of collection; staining was usually done within 8 hours. (Pilot studies revealed no significant change in cytofluorometric results in samples stored for up to 48 hours.) Erythrocyte GPI-anchored protein expression was evaluated by incubating for 30 minutes at room temperature 50 L of a 1:20 dilution of whole blood with 20 L of Gly-A-FITC and either 10 L each of CD55-PE plus CD59-PE or the appropriate isotype controls. Samples were washed and resuspended in 1 mL of phosphate-buffered saline before flow cytometry. Granulocyte GPI-anchored protein expression was evaluated by incubating for 60 minutes at room temperature 100 L of whole blood with 10 L of CD15-PE and either 20 L of CD66b-FITC plus 5 L of CD16-PECy5 or the appropriate isotype controls. Erythrocytes in these samples were lysed by using a Q-Prep apparatus (Coulter, Fullerton, California) and were then fixed with paraformaldehyde. All of the samples were analyzed by using a Coulter XL flow cytometry machine equipped with a 488-nm argon laser and XL or XLII software. Strict criteria to distinguish cells lacking GPI-anchored proteins were applied for two reasons. First, like many cell surface transmembrane proteins, GPI-anchored protein can vary in expression according to the stage of cellular differentiationfor example, as a result of myeloid or erythroid maturation. In addition, in some hematologic diseases, differentiation may be abnormal, as reflected in abnormal structure and aberrant expression of specific proteins. Therefore, polymorphonuclear cells were identified on the basis of light-scatter properties that correlate with cell size and granularity and by staining with a specific antigranulocyte antibody (anti-CD15, conjugated to the fluorescent dye phycoerythrin). Two antibodies directed against distinct GPI-anchored protein (anti-CD16, conjugated to the fluorochrome PE-Cy5, and anti-CD66b, conjugated to fluorescein isothiocyanate) were used to determine the PNH phenotype. Erythrocytes were similarly analyzed by using appropriate antibody combinations (anti-glycophorin for identification of erythrocytes and anti-CD59 and anti-CD55 as labels for GPI-anchored protein). Interpretation of Flow Cytometry Data The gates used to define CD16 /CD66b granulocyte or CD55 /CD59 erythrocyte populations were set on the basis of the isotypic control analyses performed on the same day. The mean SD of double negative cells for 21 healthy participants was 0.129% 0.101% for granulocytes and 0.195% 0.116% for erythrocytes. Because many patients required red blood cell transfusion at the time of initial sampling, the classification of patients was based on granulocyte analysis; a population of GPI-anchored protein-negative granulocytes of 1% or more that was clearly separate from the wild-type CD16+/CD66b+ cells on a two-dimensional histogram (Figure 1) was required for categorization of a patient as harboring PNH cells. Figure 1. Protocol for identification of paroxysmal nocturnal hemoglobinuria ( PNH) phenotype granulocytes. A. B. C. D. E. Response Criteria Response to treatment in aplastic anemia was defined as failure to fulfill criteria for severity for at least 3 months (see above); this was previously shown to correlate with transfusion independence and absence of infections. Criteria for response to therapy in myelodysplasia were no need for transfusion for 8 or more weeks, hemoglobin values of 80 g/L or more, platelet counts greater than 20 109 cells/L, and absolute neutrophil counts of 0.5 109 cells/L or more. Statistical Analysis A Fisher exact test was used to determine two-tailed P values in 2 2 contingency tables. The chi-square test was used

Congenital anaemia after transplacental B19 parvovirus infection.

We report three children with congenital anaemia after intrauterine infection with B19 parvovirus. All the fetuses developed hydrops fetalis that was treated by blood transfusion. After delivery the infants had hypogammaglobulinaemia. In all three, sera lacked B19 but viral DNA was found in bone marrow. All were treated with immunoglobulin. One child died and B19 was found in various tissues. In the other two cases, virus could no longer be detected after therapy but the patients remain persistently anaemic. Persistent B19 infection should be suspected in infants with congenital red-cell aplasia.

Indiscriminate activity from the B19 parvovirus p6 promoter in nonpermissive cells

B19 parvovirus is absolutely tropic for human erythroid progenitor cells. Among the untested mechanisms underlying this tropism is the possibility of cell-specific positive regulation of the promoter in permissive cells. Using the bacterial chloramphenicol acetyltransferase and firefly luciferase reporter genes, we detected strong activity from the B19 P6 promoter in transfected nonpermissive cells. Very high-level expression was seen in a T lymphoblastoid cell line, CEM. No transcriptional enhancement occurred in an erythropoietin-dependent semipermissive cell line. A putative second B19 promoter at map unit 44 (P44) was nonfunctional and unable to confer tissue specificity. Thus, tropism is unlikely to be regulated at the level of transcriptional initiation from either the P6 or P44 promoter.

Clinical Relevance of Parvovirus B19 as a Cause of Anemia in Patients with Human Immunodeficiency Virus Infection

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) of < or =24 and 4 (31%) of 13 HRV-infected patients with HCT of < or =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT of > or =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.

Upstream sequences within the terminal hairpin positively regulate the P6 promoter of B19 parvovirus

For the B19 parvovirus P6 promoter, a 96-nt minimal truncation mutant retained activity in transient reporter gene assays. Deletion of sequences further upstream from this minimal promoter markedly diminished reporter activity in certain cell lines. This upstream region lies within the terminal hairpin from -249 to -157 and contains a 14-nt sequence that is protected by DNase I footprinting. The exact sequence is directly repeated further within the hairpin, suggesting a regulatory role. The hairpin termini of parvoviruses were known to serve as origins of replication and to catalyze virion packaging. We now suggest that, in addition to these functions, they exert cis-acting effects on B19 P6-promoted gene expression.

Lack of evidence for parvovirus B19 viraemia in children with chronic neutropenia.

Cohen, B.J. & Buckley, M.M. (1988) The prevalence of antibody to human parvovirus B19 in England and Wales. Journal of Medical Microbiology, 2 5 , 151-153. Cohen, B.J., Field, A.M., Gudnadotti, S., Beard, S. & Barbara, J.A. (1990) Blood donor screening for parvovirus B19. Journal of Virological Methods, 30, 233-238. McClain, K., Estrov, Z., Chen, H. & Mahoney, D.H. (1993) Chronic neutropenia of childhood: frequent association with parvovirus infection and correlations with bone marrow culture studies. British Journal of Haematology, 85, 57-62. Ozawa. K., Kurtman, G. & Young, N. (1987) Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro. Blood. 70, 384-391.

A trial of recombinant human superoxide dismutase in patients with Fanconi anaemia

Summary Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia. The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage. Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA. Human SOD (rh‐SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast. We treated four FA patients with a 2‐week infusion of rh‐SOD (25 mg/kg d daily) to determine whether rh‐SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype. We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh‐SOD treatment in two patients and that bone marrow progenitors were increased in one patient.

Frequent HPRT mutations in paroxysmal nocturnal haemoglobinuria reflect T cell clonal expansion, not genomic instability

Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG‐A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI‐anchored proteins on the surface of the cells progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine‐guanine phosphoribosyltransferase (HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI‐anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI‐anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the Vβ2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity‐determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen‐driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH.

Congenital anemia after transplacental B19 parvovirus infection

middle (MCA) and anterior (ACA) cerebral arteries during the first 10 days of life. One hundred thirty-seven of these infants were non-acidotic at delivery and during the early neonatal period, and had normal cerebral ultrasound scans throughout the study period. These infants formed the reference group. In three gestational subgroups considered (~32 weeks, 33-34 weeks, ~35 weeks) from the reference group, the median RI for both the ACA and MCA was noted to fall significantly during the first 12 h of life (P < 0.01 for all groups). For infants delivering at ~33 weeks gestation, both MCA and ACA RI values reached a steady state with no significant change in the median value for the remainder of the study period. For infants delivering at sz 32 weeks, there was a further significant fall in both the MCA and ACA RI between 12 and 24 h of life (P C 0.05). after which a steady state value was reached. During the first 12 h of life the RI for both vessels was significantly higher in infants delivering at 5 32 weeks compared to the more mature infants (P < 0.01) but for the remainder of the study period, there were no significant differences in RI values between the gestational subgroups. Reference data for MCA and ACA in the uncomplicated pre-term infant during the neonatal period are reported. This study demonstrates that significant changes occur in measures of cerebral vascular resistance, during the first few hours of life in the pre-term infant, possibly as the result of changes in blood gas tensions with the onset of respiration, or as a consequence of alterations in blood flow with the change from a fetal to an adult circulation, but in infants with normal cranial ultrasound scans, after the first 12-24 h, very little change in cerebral vascular RI occurs. Gestational age at delivery or influences the degree and speed with which these changes occur, but after the first 24 h of life is not a significant influencing factor on vascular resistance. Comparison of resistance changes occurring in the cerebral circulation during the neonatal period of pre-term infants who develop cerebral pathology with the reference data reported for similar infants without pathology may improve our understanding of the pathophysiological events associated with the development of such pathology.