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Original Research |

Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure Syndromes

Daniel E. Dunn, MD, PhD; Patcharin Tanawattanacharoen, MD; Piernicola Boccuni, MD; Shoichi Nagakura, MD, PhD; Spencer W. Green, BS; Martha R. Kirby, BA; Mysore S. Anil Kumar, MD; Stephen Rosenfeld, MD; and Neal S. Young, MD
[+] Article, Author, and Disclosure Information

From the National Heart, Lung, and Blood Institute, Bethesda, Maryland; and MCP Hahnemann University Hospitals, Philadelphia, Pennsylvania.

Note: Drs. Dunn and Tanawattanacharoen contributed equally to this work.

Grant Support: By the National Institutes of Health, National Heart, Lung, and Blood Institute, Hematology Branch.

Requests for Reprints: Daniel E. Dunn, MD, PhD, 309 East Osceola Street, Suite 208, Stuart, FL 34994-2240.

Current Author Addresses: Dr. Dunn: 309 East Osceola Street, Suite 208, Stuart, FL 34994-2240.

Dr. Tanawattanacharoen: Department of Medicine, Division of Hematology, Siriraj Hospital, Mahidol University, 2 Prannok, Bangkok 10700, Thailand.

Dr. Boccuni: Via Aniello Falcone 153, Napoli 80127, Italy.

Dr. Nagakura: The Second Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto City, Kumamoto, Japan 860-0811.

Dr. Anil Kumar: MCP Hahnemann University, Broad and Vine Streets, Philadelphia, PA 19102.

Mr. Green, Ms. Kirby, and Dr. Young: National Heart, Lung, and Blood Institute, 9000 Rockville Pike, Bethesda, MD 20892-1652.

Ann Intern Med. 1999;131(6):401-408. doi:10.7326/0003-4819-131-6-199909210-00002
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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 (56).

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Grahic Jump Location
Figure 1.
Protocol for identification of paroxysmal nocturnal hemoglobinuria (PNH)phenotype granulocytes. A.B.C.+D.+E.−−

Forward and side scatter (size and granularity) gates used to define polymorphonuclear cells. Expression of CD15 by gated cells from panel A. Staining of CD15 granulocytes with isotype controls for CD16 and CD66b. Staining of CD15 granulocytes (defined in panel B) with CD16-PECy5 and CD66b-FITC in a healthy participant. Staining of cells from a patient with PNH with CD16-PECy5 and CD66b-FITC. A normal population is seen in the upper right quadrant, and a distinct pan-glycosylphosphatidylinositol-anchored protein-negative population is evident in the lower left quadrant. Results of several Ham tests in this patient ranged from negative to borderline (3%). Values in boxes (0.1% and 50%) are the proportion of cells exhibiting the PNH phenotype: CD16 /CD66b .

Grahic Jump Location
Grahic Jump Location
Figure 2.
Comparison of the percentage of hemolysis detected by the Ham test with results of flow cytometric analysis of erythrocytes in patients with paroxysmal nocturnal hemoglobinuria.GPIn

Forty-eight independent samples from patients with glycosylphosphatidylinositol ( )-anchored protein-deficient granulocytes (≥ 1%) were analyzed concurrently for GPI-anchored protein-deficient erythrocytes by using flow cytometry and the Ham test. All events stacked at the far left of the graph ( = 31) represent samples with a negative result on the Ham test (<1% hemolysis) and a population of GPI-anchored protein-deficient erythrocytes (≥ 0.2%).

Grahic Jump Location
Grahic Jump Location
Figure 3.
Percentage of glycosylphosphatidylinositol (GPI)-anchored protein-deficient granulocytes plotted as a function of time after immunosuppressive therapy among patients with aplastic anemia.Pr2

Each patient is represented only once, at the latest time point of analysis. The solid line represents the least-squares regression of the data, and the dotted lines represent the 95% CI of the regression. This CI includes a horizontal line, showing no correlation. The probability that the slope of the regression differed from zero was not significant ( = 0.088; = 0.14).

Grahic Jump Location




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