new insights into the pathophysiology of acquired...

18 American Society of Hematology

New Insights into the Pathophysiology of Acquired Cytopenias

Neal S. Young (Chair), Janis L. Abkowitz, and Lucio Luzzatto

This review addresses three related bone marrowfailure diseases, the study of which has generatedimportant insights in hematopoiesis, red cellbiology, and immune-mediated blood cell injury. InSection I, Dr. Young summarizes the current knowl-edge of acquired aplastic anemia. In most patients,an autoimmune mechanism has been inferred frompositive responses to nontransplant therapies andlaboratory data. Cytotoxic T cell attack, with produc-tion of type I cytokines, leads to hematopoieticstem cell destruction and ultimately pancytopenia;this underlying mechanism is similar to otherhuman disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiplesclerosis, uveitis, colitis, etc.). The antigen thatincites disease is unknown in aplastic anemia as inother autoimmune diseases; post-hepatitis aplasiais an obvious target for virus discovery. Aplasticanemia can be effectively treated by either stem celltransplantation or immunosuppression. Results ofrecent trials with antilymphocyte globulins andhigh dose cyclophosphamide are reviewed.

Dr. Abkowitz discusses the diagnosis andclinical approach to patients with acquired pure redcell aplasia, both secondary and idiopathic, inSection II. The pathophysiology of various PRCAsyndromes including immunologic inhibition of redcell differentiation, viral infection (especially human

parvovirus B19), and myelodysplasia are dis-cussed. An animal model of PRCA (secondary toinfection with feline leukemia virus [FeLV], sub-group C) is presented. Understanding the mecha-nisms by which erythropoiesis is impaired providesfor insights into the process of normal red celldifferentiation, as well as a rational strategy forpatient management.

Among the acquired cytopenias paroxysmalnocturnal hemoglobinuria (PNH) is relatively rare;however, it can pose formidable managementproblems. Since its first recognition as a disease,PNH has been correctly classified as a hemolyticanemia; however, the frequent co-existence of othercytopenias has hinted strongly at a more complexpathogenesis. In Section III, Dr. Luzzatto examinesrecent progress in this area, with special emphasison the somatic mutations in the PIG-A gene andresulting phenotypes. Animal models of PNH andthe association of PNH with bone marrow failureare also reviewed. Expansion of PNH clones mustreflect somatic cell selection, probably as part of anautoimmune process. Outstanding issues intreatment are illustrated through clinical cases ofPNH. Biologic inferences from PNH may be relevantto our understanding of more common marrowfailure syndromes like myelodysplasia.

I. ACQUIRED APLASTIC ANEMIA

Neal S. Young, M.D.*

Aplastic anemia, the paradigm of bone marrow failuresyndromes, is defined as pancytopenia and an empty bonemarrow. Although not a common disease, aplastic ane-mia has a social impact disproportionate to its incidence.The presentation of the patient can be dramatic—often apallid, seemingly bruised young person with frighten-ingly low blood counts. The striking marrow pathologyhas invited speculation and later experimentation to de-termine a pathophysiology responsible for the complete

disappearance of the hematopoietic organ. Early asso-ciation of aplasia with benzene exposure led to heroicindustrial hygiene efforts to protect workers from toxic-ity and ultimately to the virtual disappearance of ben-zene as a causative factor in hematologic diseases in theUnited States. An epidemic of aplastic anemia appearedto follow the introduction of chloramphenicol in the1960s, and the disease has been linked by case reportsand formal epidemiologic studies to many classes of phar-

* National Institutes of Health, Building 10, Room 7C103, BethesdaMD 20892-1652

Hematology 2000 19

maceuticals widely used in medical practice. Becauseaplastic anemia is such a feared toxicity, even a few casescan have a profound impact on new drug developmentand on the pharmaceutical industry, as most recently il-lustrated by the fate of the antiepileptic felbamate. Epi-demiologically, aplastic anemia has a pattern of geo-graphic variation opposite to the leukemias, with higherfrequency in the developing world than in the industrial-ized West. Finally, and most gratifyingly, the success oftreatments, both stem cell transplantation and immuno-suppressive regimens, invite application to related hema-tologic syndromes and indeed to other medical diseasesthat share its underlying pathophysiology.

Clinical FeaturesBlood counts determine the presentation and the prog-nosis. Symptoms of anemia and mucocutaneous hemor-rhage usually prompt medical attention. Prognosis is di-rectly related to the reduction in peripheral blood counts,particularly the neutrophil number: < 200 granulocytes/µL defines the category of super-severe disease. In theearly twentieth century, patients often died within daysor weeks of congestive heart failure, profuse hemorrhage,or overwhelming infection; recurrent bacterial sepsis orfungal invasion of critical organs secondary to refractoryneutropenia are the usual causes of death in the modernera.

Reticulocytopenia and the absence of circulatingblasts suggests aplastic anemia, which is confirmed bythe fatty bone marrow biopsy. Despite the simple pathol-ogy, the differential diagnosis has become more difficultdue to a profusion of advanced laboratory assays and anas yet uncertain nosology. Constitutional marrow fail-ure, especially Fanconi anemia, can present in adulthoodand without typical physical stigmata; this diagnosis isestablished by examination of chromosomes from mi-totic peripheral blood cells after clastogenic stress. Some-times a constitutional etiology is suspected despite nor-mal chromosome studies, and a hereditary process mustbe inferred from a pedigree, physical signs such as theabnormal nails of dyskeratosis congenita, or the neuro-logic signs of the ataxia/pancytopenia syndrome. Amongacquired diseases, pancytopenia with a hypocellular bonemarrow can also result from aleukemic leukemia, lym-phoma with marrow invasion, or large granular lympho-cytic leukemia. The distinction between aplastic anemiaand hypocellular myelodysplasia may be nearly arbitrarywhen it depends on too subtle morphologic features, andeven abnormal marrow cytogenetics no longer excludean immune-mediated marrow failure syndrome. As willbe discussed below, paroxysmal nocturnal hemoglobin-uria now is most often diagnosed concurrently with aplas-tic anemia.

EpidemiologyLarge prospective studies indicate an annual incidenceof two new cases per million population in Europe andIsrael.1 The rate is much higher in the developing world,where aplastic anemia may rival acute myelogenous leu-kemia in frequency of diagnosis in hematology clinics;in formal studies in Thailand2 and China,3 the incidencehas been determined to be about threefold that in the West.European studies have confirmed and quantified medi-cal drugs as risks for the development of marrow failure(Table 1); surprisingly, drug use accounts for only a smallfraction of the disease in Thailand, almost all of which isidiopathic.

PathophysiologyThe hallmark of aplastic anemia is the empty bone mar-row, and by all measures hematopoiesis is markedly re-duced.4 Not only are the distinctive hematopoietic pre-cursor cells, the young forms of the erythroid and my-eloid lineages and megakaryocytes, absent by visual ex-amination of the aspirate and biopsy morphology, butimaging of the vertebrae shows uniform replacement of

Table 1. A classification of acquired aplastic anemia.

Idiopathic

Secondary

Radiation

Drugs and chemicals

Regular effects:

Cytotoxic agents

Benzene

Idiosyncratic reactions:

Chloramphenicol

Nonsteroidal anti-inflammatory drugs

Sulfas

Antithyroid drugs

Antiepileptics and psychotropics

Cardiovascular drugs

Gold, penicillamine, allopurinol

Viruses

Epstein-Barr virus (infectious mononucleosis)

Non-A, non-B, non-C, non-G hepatitis virus

Human immunodeficiency virus(acquired immunodeficiency syndrome)

Immune diseases

Eosinophilic fasciitis

Hypoimmunoglobulinemia

Thymoma and thymic carcinoma

Graft-versus-host disease in immunodeficiency

Paroxysmal nocturnal hemoglobinuria

Pregnancy


marrow with fat. CD34+ cells measured by flow cyto-metry are decreased in blood and marrow. Functionally,cells capable of forming erythroid, myeloid, and mega-karyocytic colonies in semisolid media are much reduced,and in vitro assays of very primitive, quiescent hemato-poietic cells, closely related to if not identical with truestem cells, show a similar consistent and severe deficit.5

Estimating from these assays, it is likely that patients withaplastic anemia present with pancytopenia when theirstem cell and progenitors have fallen to -1% or less ofnormal numbers. While some blood cells are produced,evidence of the enormous compensatory capacity of themarrow, such a profound deficiency has important quali-tative consequences, perhaps reflected in the shortenedtelomere length measured in granulocytes of patients withaplastic anemia,6 compatible with extremely stressed he-matopoiesis.

The viability and differentiation of blood cell pro-genitors depend on specific hematopoietic growth fac-tors, mainly produced by the marrow stroma. However,laboratory studies have generally shown normal functionof aplastic anemia patients’ stroma, and the circulatingblood levels or in vitro production of almost all cyto-kines is normal and indeed elevated in the great majorityof patients.7 Stromal dysfunction also appears unlikelyfrom clinical observations; after bone marrow transplan-tation, many stromal elements remain of host origin, andgrowth factor administration is usually ineffective in pa-tients with severe disease.

Certainly the commonest form of hematopoietic fail-ure is iatrogenic—the transient aplasia that follows cyto-toxic chemotherapy or radiation treatment. Benzene alsohas been thought to directly affect the marrow. However,patients with community-acquired aplastic anemia sel-dom have a history of such obvious exposures. The me-tabolism of common drugs can lead to the formation oftoxic intermediate forms that can bind to protein, DNA,or RNA and lead to cellular injury; under certain circum-stances, these metabolites have been assumed to accu-mulate specifically and harmfully in the marrow. Such amechanism requires a potency for extremely low concen-trations of metabolites equal to massive quantities of can-cer chemotherapeutic agents specifically designed as cel-lular toxins and which in themselves do not usually lead topermanent marrow damage.

Clinical observations first suggested an alternativepathophysiology for marrow failure when Mathé reportedunexpected blood count improvement in transplant pa-tients who had rejected their grafts. He inferred that theconditioning regimen, which included an antilymphocyteserum to suppress the host’s rejection response, had for-tuitously treated an underlying autoimmune process.8 Theefficiency of immune system destruction of hematopoie-sis is also obvious in animal runt disease and in human

transfusion-associated graft-versus-host-disease(GVHD); in these syndromes, small numbers of allore-active T cells uniformly lead to fatal aplastic anemia.9 Alarge amount of laboratory data supports the hypothesisthat, in most patients with acquired aplastic anemia, lym-phocytes are responsible for the destruction of the he-matopoietic cell compartment.10 Early experimentsshowed a suppressive effect of patients’ lymphocytes onhematopoietic colony formation of normal persons andon the patients’ own bone marrow. These cells produceda soluble inhibitory factor that ultimately was identifiedas �-interferon, and indeed activation of a TH1-type Tcell response was inferred from excessive production ofinterferon, tumor necrosis factor, and interleukin-2. Cy-totoxic lymphocyte activation, and most recently the in-tracellular presence of type 1 cytokines,11 can be mea-sured by flow cytometric methods in patients’ blood andmarrow. The result of this immune process is destruc-tive, with Fas-mediated CD34+ cell death and activationof other intracellular pathways leading to cell cycle ar-rest and the release of nitric oxide. Immunity is local andhas been modeled in cell culture systems in which lowconcentrations of �-interferon are secreted into the mar-row microenvironment. In an animal model of aplasticanemia, bone marrow failure is produced by injection ofalloreactive lymphocytes, but pancytopenia can be pre-vented by treatment with a monoclonal antibody to �-interferon.12 Acquired aplastic anemia appears to sharean autoimmune pathophysiology with other human dis-eases in which TH1/TC1 cells effect organ-specific de-struction, like multiple sclerosis, ulcerative colitis, uvei-tis, and type I diabetes.

EtiologiesAlthough most often aplastic anemia occurs without asuggestive prior history and is labeled idiopathic, in somecases a clear inciting event can be identified (Table 2).In contrast to sometimes nebulous histories of drug orchemical exposure, hepatitis has objective markers, andthe post-hepatitis aplastic anemia syndrome has been wellcharacterized clinically.13 Seronegativity for the usualviruses implicates a novel infectious agent (likely alsoresponsible for fulminant hepatitis of childhood). Bothlaboratory and clinical studies suggest an immune patho-physiology, and the liver inflammation as well as mar-row aplasia respond to immunosuppressive therapy.

It is more difficult to confidently implicate a medi-cal drug as a causative agent in an individual case. Aplasticanemia follows as a rare idiosyncratic reaction in per-haps 1/100,000 to 1/200,000 individuals exposed to adrug. Patients whose marrow failure has a presumed drugetiology are demographically identical and respond totreatment similarly to those with idiopathic aplastic ane-mia. The mechanism of drug-induced aplastic anemia is

Hematology 2000 21

unknown and may involve specific metabolic pathwaysas well as aberrant immune responses.

Host factors have been suggested by higher specifichistocompatibility antigen frequencies among patients.HLA-DR2 is about twice as frequent as in the normalpopulation,14 and in Japanese patients a specific class IIhaplotype (DRB*1501) has been strongly associated withcyclosporine-responsive and -dependent disease.15 In oneparticularly well studied case, altered drug metabolismwas incriminated as responsible for aplastic anemia inan epileptic man.16 However, why the immune responseis set on a devastating pathologic course only in rare in-dividuals exposed to a frequently used medication, or toa probably ubiquitous virus, is a central unresolved ques-tion.

TherapyThe underlying pathology of aplastic anemia can be ad-dressed by replacing the marrow through stem cell trans-plantation or by quelling lymphocyte attack through im-munosuppressive therapies.17,18 Bone marrow or, morerecently, peripheral blood stem cell transplantation froma histocompatible sibling usually cures the underlyingbone marrow failure. Survival rates have been reported

Table 2. Drugs associated with aplastic anemia in theInternational Aplastic Anemia Agranulocytosis Study.*

MultivariateStratified Relative

Drug Risk Estimate# Risk Estimate#

Nonsteroidal analgesicsbutazones 3.7 (1.9-7.2)# 5.1 (2.1-12)#indomethacin 7.1 (3.4-15) 8.2 (3.3-20)piroxicam 9.8 (3.3-29) 7.4 (2.1-26)diclofenac 4.6 (2.0-11) 4.2 (1.6-11)

Antibioticssulfonamides** 2.8 (1.1-7.3) 2.2 (0.6-7.4)

Antithyroid drugs 16 (4.8-54) 11 (2.0-56)

Cardiovascular drugsfurosemide 3.3 (1.6-7.0) 3.1 (1.2-8.0)

Psychotropic drugsphenothiazines 3.0 (1.1-8.2) 1.6 (0.4-7.4)

Corticosteroids 5.0 (2.8-8.9) 3.5 (1.6-7.7)

Penicillamine �

Allopurinol 7.3 (3.0-17) 5.9 (1.8-19)

Gold 29 (9.7-89)

# 95% confidence interval* Extracted from1.**other than trimethoprim/sulfonamide combination

as high as 90% from a single experienced institutions19

and at 75–80% for registry data, which reflect more gen-eral experience20,21 (Figure 1A, B). Death rates for thefirst 100 days post transplant have fallen, probably dueto lower rates of graft rejection and better control of in-fections. GVHD, the frequency and severity of whichcorrelate with patient age, continues to limit the successof transplantation, largely accounting for the lower sur-vival of adults compared to children in most analyses(Figure 1C).22 In the most recently published Seattle data,41% of 212 patients who had survived more than 2 yearsafter transplant suffered chronic GVHD, and their mor-tality rate was three times higher than for patients with-out this complication23 (and of course GVHD contrib-uted to earlier deaths as well). Efforts to address GVHDin aplastic anemia by T cell depletion of the marrow in-oculum were disastrous, resulting in high rates of pri-mary graft failure.

Allogeneic transplantation is available only to a mi-nority of patients, as about 70% will lack a suitablymatched sibling donor. Phenotypically identical alterna-tive family donors are acceptable but are found for onlyan occasional patient. Many more donors are availableoutside the family and can now be located through largeregistries in the United States and Europe. Relatively goodresults have been achieved at Children’s Hospital in Mil-waukee, where T cell depletion of the graft is combinedwith cytosine arabinoside, cyclophosphamide, and totalbody irradiation; survival at a median follow-up of aboutthree years in 28 children was 54%, despite the heavytransfusion burden and previous treatment, and GVHDwas infrequent.24,25 Results elsewhere have been more dis-appointing, especially among adults, owing to high ratesof graft rejection, GVHD, and infection caused by de-layed immune system reconstitution; in general, survivalhas been about half that observed with standard trans-plants, 29%26 to 34%.27 The rigorous conditioning regi-mens required for engraftment are poorly tolerated byolder patients and, even among children, seem likely toexact a delayed toll in late malignant disease. In aplasticanemia, malignant tumors occur at a higher than expectedrate among patients undergoing standard conditioning;28,29

intensive chemo- and radiotherapies used in unrelateddonor regimens would be predicted to eventually resultin higher rates.30

Immunosuppression is employed in patients who arenot candidates for stem cell transplantation due either toage or the lack of a donor. Horse antithymocyte globulin(ATG) and rabbit antilymphocyte globulin (ALG) are nowboth licensed for use in the United States. Hematologicresponses, which are usually defined as sufficiently im-proved blood counts such that the patient no longer re-quires transfusions of red blood cells or platelets and isnot susceptible to infection, occur in 40–50% of those


treated with either ATG or ALG alone.17,31 Forpatients with severe aplastic anemia, the ad-dition of cyclosporine to ATG or ALG hasimproved response and survival rates. In Eu-ropean32 and American33 studies, responserates have been 70–80%, and survival at 5years among responders is about 90%. Com-bined treatment with cyclosporine and ATGhas been particularly beneficial for childrenand patients with absolute neutropenia, com-pared with results for ATG alone. However,cyclosporine as a single agent of immuno-suppression is inferior to ATG or ALG.34

ATG and ALG have distinctive toxicities.As foreign proteins, they can elicit anaphy-laxis in the host; we routinely skin test forevidence of sensitivity and desensitize pa-tients who have a positive reaction. ATG isnot specific for lymphocytes and can reducealready low platelet and neutrophil levels andcause a positive direct antiglobulin test. An-tibodies produced by the patient to horse pro-teins can lead to immune complex formationand serum sickness, usually about 11 daysafter initiation of treatment.35 Cyclosporineis nephrotoxic, and both serum creatinine anddrug levels should be monitored to avoid ir-reversible renal damage; hypertension, gin-gival hyperplasia, and gastrointestinal andneurologic symptoms are other common sideeffects, and because of the risk ofPneumocystis carinii, we administer prophy-laxis with monthly pentamidine inhalations.

Many (perhaps most) patients with aplas-tic anemia are not adequately treated by asingle 4-day course of ATG followed by 6 to12 months of cyclosporine. Slowly decliningblood counts signal a need to reinstitute treat-ment and usually respond to either an increasein the dose or the reinstitution of cyclosporine.Some patients appear to depend on contin-ued administration of cyclosporine, often atrelatively low doses. Frank pancytopenia canrecur and prompt a second course of ATG.However, long-term prognosis does not ap-pear to be affected by relapse. Patients whorespond to immunosuppression often con-tinue to have blood counts that, while ad-equate for full activities, remain below nor-mal. Incomplete responses, frequent relapses,and cyclosporine-dependence are probablyevidence of chronic immune system activityon a hematologically compensated bone mar-row. A further problem is the development of

Figure 1. (A) Survival after allogeneic bone marrow transplant: data fromindividual hospital series in peer-reviewed publications, 1991–1997 (1–15,with 95% confidence intervals); shaded area represents 5-year survival(with the same confidence intervals) of patients reported to theInternational Bone Marrow Transplant Registry during the same timeperiod (reprinted from 21, which provides details).

(B) The continuing influence of age on survival as reflected in IBMTRdata (reprinted from 21).

(C) Comparative probability of survival after immunosuppression (IS)and bone marrow transplant (BMT) for patients treated in the 1980s and1990s, from the Working Party on Severe Aplastic Anemia of the EuropeanBlood and Marrow Transplant Group (reprinted from 51).

Hematology 2000 23

late clonal diseases (see below). Small numbers of patients at Johns Hopkins were

treated with cyclophosphamide at high doses equivalentto those employed in transplant conditioning regimensbut without stem cell rescue, administered during inter-vals in the 1980s when ATG was temporarily not avail-able; of the seven responding patients, six had normalblood counts, never had relapsed, and were without clini-cal evidence of clonal disease.36 Based on this report, weinitiated a randomized trial to compare ATG to high dose-cyclophosphamide; patients in both arms received cyclo-sporine. However, this study was terminated prematurelydue to excess toxicity, mainly severe fungal infections,and deaths in the group that received cyclophosphamide.37

High-dose cyclophosphamide is a much more aggressiveform of immunosuppression than antilymphocyte sera andresults in profound and sustained suppression of bonemarrow function as well; profound, virtually absoluteneutropenia can persist for many weeks, necessitating pro-longed courses of antibiotics and even granulocyte trans-fusions, and several of our patients were iatrogenicallyconverted from severe to super-severe disease, with itsassociated poorer immediate prognosis. Whethercyclophosphamide’s putative advantages are worth suchacute risks seems doubtful, especially when other thera-peutic approaches are promising.

One such strategy is based on induction of tolerance.ATG and ALG reduce lymphocyte numbers, but tran-siently and modestly compared with cytotoxic chemo-therapy. Part of their beneficial activity may be to inducetolerance, perhaps by specific deletion of activated lym-phocytes; indeed, the concurrent use of cyclosporine,which blocks T cell activation, may blunt ATG’s ef-fects.38,39 In our new NIH protocol for severe aplasticanemia, we have delayed the introduction of cyclosporineand added a novel immunosuppressive drug, myco-phenolate mofetil; mycophenolate, by inhibiting inosinemonophosphate, is cytotoxic for cycling T cells. Acti-vated lymphocytes should be subject to elimination bytheir characteristic cell surface antigens (recognized byATG) and their mitotic activity. To date, 21 patients haveentered this protocol; mycophenolate’s activity has beendramatically apparent in the early and substantial in-creases in neutrophil counts in patients with extremelysevere disease. Mycophenolate lacks nephrotoxicity, andits use may also allow decreased doses and earlier termi-nation of cyclosporine as well as prevent relapse. Othermilder but more specific forms of immunosuppressionmight also be effective. For example, ATG contains anti-body specificities for the interleukin-2 receptor, presenton activated lymphocytes; we are testing daclizumab(Zenapax®), a commercially available monoclonal anti-body to the receptor in patients with moderate aplasticanemia. Other monoclonal antibodies, recombinant

soluble cytokine receptors, and new immunosuppressivedrugs like rapamycin deserve examination in immune-mediated marrow failure syndromes.

There are no clear guidelines for the treatment ofrefractory aplastic anemia. Multiple courses of immuno-suppression are commonly administered at European cen-ters: the majority of patients who received rabbit ALGafter failing horse ALG became transfusion-independentin a recent Italian study.40 Androgens in various formula-tions have been employed in aplastic anemia for manydecades and they are occasionally effective, particularlyif hematopoietic failure is not complete;41,42 their mecha-nism of action in aplastic anemia is not well understoodbut may relate to immunomodulation rather than to ef-fects on erythropoietin production or on hematopoieticcells. There is little justification for either a therapeutictrial of corticosteroids as primary treatment or theirchronic use to prevent bleeding; aplastic anemia patientsappear particularly susceptible to aseptic necrosis as acomplication of steroid use.43 Therapeutic trials of he-matopoietic growth factors are not appropriate as first-line treatment of severe aplastic anemia (G-CSF and GM-CSF can increase granulocyte counts in aplastic anemia,but this effect is almost always transient and rarely oc-curs in patients with very severe neutropenia).44 G-CSFalso does not improve the response rate or survival inpatients undergoing standard immunosuppressive treat-ment.45 However, growth factors can be useful in refrac-tory aplastic anemia, with clinically meaningful improve-ment in blood counts after prolonged administration ofG-CSF, erythropoietin, and stem cell factor, usually insome combination.46-48 While in Japan chronic G-CSFtherapy has been linked to the development of the dire cy-togenetic abnormality monosomy 749,50 (see below), thisdangerous relationship has not been reported elsewhere.

Only a few patients face an actual choice betweenallogeneic transplantation and immunosuppressivetherapy. Analyses of large databases have not shown majordifferences in outcomes between these two therapeuticapproaches.51 Nevertheless, transplant is probably pref-erable for certain defined subgroups, especially youngpatients and those with very severe neutropenia. Patientswho failed immunosuppressive therapy have later under-gone successful transplantation from matched siblingsor from unrelated donors.

Late EvolutionAplastic anemia is closely related to other bone marrowfailure syndromes. Myelodysplasia, paroxysmal noctur-nal hemoglobinuria (PNH), and aplastic anemia are eas-ily confused in the clinical classification of a patient withpancytopenia and a hypocellular bone marrow, and theymay indeed share common pathophysiologic features. Aspatient survival has improved, so too has the opportunity


to observe the chronic course of marrow failure. Evolu-tion of aplastic anemia has been reported in a substantialminority of patients undergoing immunosuppressivetherapy: in a large European series of more than 200 pa-tients, the actuarial risk of developing PNH was 13% at7 years, and 15% for myelodysplasia and leukemia.52 Inour series of more than 100 patients followed at the NIHClinical Center, the actuarial risk for all late clonal evo-lution was 16% at 7 years. Immunosuppression itself isnot etiologic, as similar data have been acquired in pa-tients treated with androgens.42 Myelodysplastic marrowchanges and cytogenetic abnormalities tended to appearlate, sometimes years after initiation of therapy, and of-ten (but not always) in the setting of a poor response totreatment. In contrast, PNH was usually diagnosed withinthe first or second year in patients who had shown im-provement in blood counts, and often was purely a labo-ratory finding.

Indeed, whether “evolution” accurately describes therelationship among these diseases is questionable. Forthe diagnosis of PNH, highly sensitive flow cytometrictests, which detect the absence of those proteins anchoredto the cell surface by glycosylphosphoinositol anchors,has replaced the classic Ham test. By flow cytometry, alarge proportion of bone marrow failure patients at thetime of presentation show deficient granulocytes. In ourseries, 15% of aplastic anemia patients and 23% of my-elodysplasia patients showed PNH cells early in theirdisease and before any treatment.53 Clones also disap-pear over time, especially those present at low levels ini-tially. Of interest will be comparable analyses utilizingmore sensitive assays to detect chromosomal abnormali-ties typical of myelodysplasia, such as fluorescent in situhybridization. Patients with myelodysplasia, especiallyof the refractory anemia subtype, can show blood countimprovement with the same immunosuppressive treat-ments employed in aplastic anemia.54 One tenable unify-ing hypothesis is that both PNH and myelodysplasia rep-resent clonal stem cell escape from immune system at-tack.

Supportive CareAnemia and thrombocytopenia can be corrected by trans-fusion. Limited numbers of blood transfusions probablydo not affect the outcome of stem cell transplantation.Red blood cells should be infused to achieve hemoglo-bin levels compatible with full activity, usually above 70gr/L (90 gr/L in patients with cardiopulmonary compro-mise). Platelet collection by cytopheresis and leucocytereduction by ultraviolet light or filtration are measuresthat reduce alloimmunization from transfusions.55 Thelong-term benefit of a prophylactic platelet transfusionprogram is unclear; when patients require periodic plate-let administration to control hemorrhage, maintenance

of levels above 10 x 109/L is adequate. Neutropenic fe-vers must be treated aggressively with parenteral, broad-spectrum antibiotics, and antifungal therapy should beadded for persistent fever. Attention to details of oralhygiene and hand washing and avoidance of minor inju-ries or casual exposure to infectious agents can reducethe risk of serious complications. Good medical practicealso requires a relationship between physician and pa-tient that provides for honest discussion of therapeuticoptions, prompt response to crises, and psychologicalsustenance. Frightening symptoms, delayed or absentimprovement in blood counts, relapses, and complica-tions of therapy must be dealt with realistically but alsoreassuringly. Even refractory patients occasionally makeunexpected and sustained recoveries.

ConclusionsIn the last century, both our understanding of the originof aplastic anemia and the definitive and supportive treat-ment of the individual patient have improved enormously.Aplastic anemia is usually immunologically mediated,sharing pathophysiologic mechanisms with other autoim-mune diseases. Unfortunately, stem cell destruction maybe quite advanced by the time the patient presents withpancytopenia. Replacement of hematopoietic tissue byeither stem cell transplantation or suppression of thepathologic immune response to allow recovery of thepatient’s own marrow are both effective. Improvementsin methods for monitoring hematopoiesis and immunesystem activity and especially in the application ofimmunomodulatory drugs should be of clinical benefit.Major research questions remain as to the nature of in-citing antigens and the determinant of the aberrant im-mune response, as well as the fundamental pathophysi-ologic relationship among aplasia, dysplasia, and parox-ysmal nocturnal hemoglobinuria.

II. ACQUIRED PURE RED CELL APLASIA:PHYSIOLOGY AND THERAPY

Janis L. Abkowitz, M.D.*

Acquired pure red cell aplasia (PRCA) is characterizedby severe anemia, reticulocytopenia, and the absence ofhemoglobin-containing cells in an otherwise normalmarrow aspirate. It can occur independently or in asso-ciation with systemic disorders, including lymphoma andrheumatologic disease. Sometimes, proerythroblasts per-sist, while in other circumstances the block in erythroiddifferentiation occurs earlier. With cell culture studies,the level of differentiation that is impaired can be de-fined as prior to burst-forming unit erythroid (BFU-E)

* Hematology Division, University of Washington, Box 357710,Seattle WA 98195-7710

Hematology 2000 25

(23% of the 35 cases in one study1), BFU-E to colony-forming unit erythroid (CFU-E) (29% of cases), CFU-Eto proerythroblast (31% of cases), and after proerythro-blast (17% of cases). As PRCA involves a single (eryth-roid) lineage in a stage-specific fashion, studies of thisdisorder provide insights into the physiology of differen-tiation, and specifically those events that are importantfor erythropoiesis.

There are three etiologies of PRCA: 1) immunologic,2) viral (human parvovirus B19 [B19]), and 3) myelod-ysplasia. Understanding the etiology of PRCA is impor-tant for clinical decision making and implementation ofcorrect therapies.

Immunologic PRCAMost cases of PRCA can be attributed to immunologicinteractions. PRCA has been associated with thymoma,drugs such as azathioprine and procainamide, rheuma-toid arthritis, systemic lupus erythematosis (SLE), hepa-titis, mononucleosis, lymphoma, B cell chronic lympho-cytic leukemia (CLL), T cell CLL, large granular lym-phocytic (LGL) leukemia, and angioimmunoblastic lym-phadenopathy.1-4 Although these are a diverse group ofdiseases, there are common pathophysiologies linkingthem and red cell failure. Antibodies have been describedin patients’ sera that are selectively cytotoxic for marrowerythroid cells or are directed against erythropoietin.2,5,6

T cells from patients with PRCA associated with thy-moma,2,7 chronic Epstein-Barr viral infection,8 lym-phoma,9 CLL,10-12 and LGL leukemia3,4,13,14 have beenshown to suppress erythropoiesis in vitro. Theanemia in patients with red cell aplasia asso-ciated with these disorders generally respondsto immunosuppressive therapies, includingcorticosteroids, cyclosporine, and low-dosecyclophosphamide.1,3,15

A particularly instructive case was re-cently described by Rupert Handgretinger andcolleagues.4 This 56-year-old man developedPRCA in the setting of LGL leukemia. Thelarge granular lymphocytes were defined as�� T-cells by flow cytometry (the rearrangedreceptor variable-region genes were V�4 andV�1). They were CD2, CD3, CD5, and CD7positive and failed to express antigens char-acteristic of natural killer (NK) cells (CD16and CD56). BFU-E and CFU-E were ob-served in marrow culture studies, butproerythroblasts were absent on the marrowaspirate, suggesting that the block in erythro-poiesis occurred as CFU-E matured toproerythroblasts. In subsequent studies, theseinvestigators demonstrated that the �� T-cellslysed target cells in an MHC unrestricted

manner. However, as these cells also expressed killer-cell inhibitory receptors (KIR), target cells that displayHLA class I proteins were spared. Although all normalearly hematopoietic progenitor cells express HLA class Ideterminants, surface class I antigen expression isdownregulated as CFU-E mature to proerythroblasts. Theauthors demonstrated that at this stage of differentiation,erythroid cells became sensitive to lysis by the expanded(clonal) population of �� T-cells, resulting in the pheno-type of pure red cell aplasia (Figure 2). It is possible thatcomparable mechanisms will be implicated in the patho-physiology of other autoimmune disorders.

There are many therapeutic options for immunologicPRCA. Patients generally respond to steroids,cyclosporine, cyclophosphamide, or antithymocyte globu-lin (ATG).1,2,15,16 Responses to androgens, plasmapheresis,and splenectomy have been described.15,16 Treatment ofan underlying lymphoproliferative disorder with combi-nation chemotherapies or fludarabine16 can lead to a re-mission of PRCA, but this is not often required. An inde-pendent clinical decision should be made about the ap-propriateness of treating the underlying disorder, and thetreatment of PRCA should be approached as one wouldapproach the treatment of idiopathic thrombocythemiapurpura (ITP) or hemolytic anemia complicating stage 0CLL. We generally begin with a therapeutic trial of pred-nisone (1 mg/kg/d po x 4–6 weeks) and follow the trans-fusion interval, reticulocyte count, and hematocrit as theindication of response. If this is unsuccessful (only 27–37% of patients appear to respond1,15), we begin a sec-

Figure 2. The patient’s �� T cells express KIR (killer inhibitory receptors).When KIR bind HLA class I antigens on the surface of a target cell, the ability ofthe effector cell to lyse its target is inhibited. Normal BFU-E express class Ideterminants, but this expression is down-regulated as CFU-E matureproerythroblasts. This patient had a clonal expansion (LGL leukemia) involving�� T cells, resulting in the “autoimmune” lysis of proerythroblasts and PRCA.


ond-line agent, either cyclophosphamide (75–100 mg poqd in adults, decreased if needed to maintain the granu-locyte count > 1.5–2.0 x 103/ml) or cyclosporine (stan-dard dosing). For each drug, we allow a 8–10 week trial.Because red cell transfusion is of low morbidity, we havechosen this approach rather than one with more aggres-sive additional therapies. In patients with underlying sys-temic lupus erythematosus (SLE) or rheumatologic dis-orders, we generally use cyclophosphamide as the sec-ond agent. In children and in individuals with B cell CLLcyclosporine is used.6,18 We continue with sequential treat-ment trials, including ATG,19 until we find that agent towhich the patient responds. Responding individuals aretreated for 3–6 months. Most do not relapse with discon-tinuation of therapies. In fact, of 24 patients that achievedcomplete response in the studies of reference 1, 20 (83%)had a normal hematocrit (without continued therapy) atthe time (median 5 years) of follow-up observations. Inpatients with LGL leukemia of an NK cell phenotype,low-dose methotrexate (i.e., 15 mg po q week) is oftenefficacious.1

Viral PRCAA second physiology of PRCA is viral-induced disease.Human parvovirus B19 (B19) (reviewed in 20) is a com-mon viral disorder, usually acquired early in life. By age15, 50% of children have detectable IgG to B19, con-firming prior exposure, and infection is prevalent, so that90% of the elderly are seropositive.21 B19 is the cause offifth disease in young children and of an arthropathy syn-drome in adults. B19 specifically infects and lyses eryth-roid precursor cells.20 The cellular receptor is the bloodgroup P antigen, globoside, which is expressed on someCFU-E and all proerythroblasts and subsequent eryth-roid cells.22 B19 infection can produce characteristic gi-ant proerythroblasts with eosinophilic nuclear inclusionbodies in marrow aspirates.20 This morphologic feature,however, can also be seen in patients with HIV-1 in theabsence of B19 infection.23

In a patient with a chronic hemolytic anemia (e.g.,hereditary spherocytosis, sickle cell anemia), B19 infec-

tion results in profound anemia (an acute PRCA), alsotermed aplastic crisis. It is likely that all individuals whoacquire B19 will develop marrow manifestations consis-tent with PRCA. However, an immunologically normalhost clears this viral infection and recovers normal he-matopoiesis within a few weeks. As the lifespan of a redcell is 120 days, anemia never develops. Because of theshort circulatory red cell lifespan in patients with chronichemolysis, severe anemia quickly occurs. Symptomaticanemia can be treated with red cell transfusions. B19 in-fection will (because of the intact immune status of thehost) spontaneously resolve and the patient’s hematocritreturn to its baseline value. Patients with chronic immu-nologic abnormalities, including HIV-1 infection, maybe unable to resolve B19 infection.23 In this circumstance,an erythroid marrow failure, clinically indistinguishablefrom idiopathic or other secondary forms of PRCA, de-velops (Table 3).

Chronic B19 infection should be considered as anetiology for PRCA in all individuals with HIV-1 infec-tion (independent of the stage of disease),23-25 as well asin individuals with other immunologic impairments (con-genital immunodeficiency or immunodeficiency second-ary to cytotoxic or immunosuppressive drugs).26 PRCAdue to persistent B19 has been reported in patients with-out clinically evident immunodeficiency.26 It is impor-tant to diagnose PRCA secondary to B19 infection, be-cause patients are uniformly responsive to immunoglo-bulin therapy.20,23-26 Because of the high prevalence of B19in the general population, commercial sources of IgGcontain high titers of antibodies to the virus. Therapy withIgG (dose options equivalent to ITP therapy) results in areticulocytosis within 3–5 days and the subsequent reso-lution of the anemia. B19 titers decrease from 1012 B19DNA copies/ml serum to 106 (i.e., detectable only byPCR) in HIV-1-positive patients, but the infection likelynever totally resolves.23 Therefore, these individualssometime recur with PRCA 6–9 months later and respondto the readministration of IgG.23-26

To diagnose B19 as the cause of PRCA, the appro-priate study is a dot blot analysis of serum (see discus-

sion in reference 25). PCRstudies may be too sensi-tive as treated (and re-sponding) HIV-1-positivepatients have persistentPCR positivity, and normalindividuals may remainpositive for B19 by PCRfor many months afterclearing clinical infec-tion.20,21 Thus, resultscould be falsely positive ifobtained at a time of a

Table 3. Human parvovirus B19 infection and anemia.

Time Required toRBC Immune Mount an Effective

Patient Lifespan Status Immune Response Outcome

Normal individual 120 d Normal 14–21 d Normal Hct is maintained

Chronic hemolysis 5–10 d Normal 14–21 d Acute (transient) PRCA

HIV 120 d Abnormal Unable to clear B19 PRCA

Erythropoiesis is suppressed in all patients infected with B19. Recovery occurs at 14–21 days (as B19is cleared) in those individuals with normal immune function. Patients with chronic hemolysis develop asevere (transient) anemia, termed “aplastic crisis,” because their RBC lifespan is short. Patients withHIV may be unable to clear B19 and thus can develop anemia after 2–4 months.

Hematology 2000 27

clinical epidemic. Currently, DNA analyses are not com-mercially available. Therefore, we often use the standardPCR assay (available in many reference laboratories) asa screening test. If this is negative, the patient does nothave B19 as the etiology of PRCA. If it is positive, oneneeds to consider the cost/benefit of a IgG treatment trialversus a confirmatory assay by DNA hybridization (avail-able in the research setting).

Although B19 is the only described viral cause ofhuman PRCA, feline leukemia virus subgroup C/Sarma(FeLV-C) has been identified as the virologic agent re-sponsible for PRCA in cats. This animal model of PRCA,is of interest from a physiologic standpoint. FeLV-C ap-pears to block BFU-E to CFU-E differentiation by im-pairing the cell surface expression or function of theretroviral receptor. Quigley et al27 and Tailor et al28 haverecently identified the feline and human FeLV-C recep-tors (FLVCR), respectively. The receptor cDNA is pre-dicted to encode a 560 amino acid protein with 12 mem-brane-spanning domains that shares homology with re-ceptor for D-glucarate (an anionic sugar) in C. elegansand bacteria. It is likely that this molecule will have aspecific role in erythroid differentiation, and that under-standing its action will provide insights into events thatare critical to the normal maturation of erythroid cellsfrom BFU-E to CFU-E.

PRCA as the Initial Presentation of MyelodysplasiaMyelodysplasia is a clonal disorder originating in hemato-poietic stem or early progenitor cells. Generally, all threelineages (red cells, white cells, platelets) are affected andthe marrow is hyperproliferative and dysmorphic. A vari-ant of myelodysplasia has been described in which theneoplastic progenitor cell has a limited differentiationcapacity, leading to a clinical phenotype that is indistin-guishable from aplastic anemia.29 Similarly, in rare cir-cumstances, PRCA can be the only or initial manifesta-

tion of myelodysplasia.1,3,30 The anemia is often unrespon-sive to immunosuppressive agents.1,3,30 In marrow culturestudies, BFU-E are not detected, suggesting that the ori-gin of this disease is in a multipotent stem/precursor cellthat cannot undertake the erythroid differentiation path-way.1,31 Sometimes there are subtle dysmorphic featuresof granulocytes (e.g., a Pelger-Huet abnormality), thepresence of excess basophils, or unexplained marrow fi-brosis that raise the suspicion of myelodysplasia. How-ever, the marrow may be indistinguishable from that oftypical acquired PRCA.1 Perhaps the simplest diagnosticstudy is cytogenetics. An abnormal marrow karyotype(in the absence of leukemia/lymphoma involving themarrow) establishes this diagnosis.1,3,30

A Clinical Approach to Patients with Acquired PRCAIn patients with profound anemia and reticulocytopenia,yet with normal platelet and white blood cell counts, weobtain a marrow aspirate and biopsy to establish a diag-nosis of PRCA. If lymphoid cells are increased in num-ber in the blood or marrow or demonstrate aberrant mor-phology (i.e., LGL), immunophenotyping and studies ofclonality by T cell or immunoglobulin gene rearrange-ment (when appropriate) are obtained. We do a carefulphysical examination and history and obtain blood stud-ies if needed to consider the diagnosis of associated dis-orders such as SLE or rheumatoid arthritis. Although thy-moma is uncommonly associated with PRCA, we gener-ally check a chest x-ray or computerized tomogram (CT).A history of thymoma, concurrent thymoma, or the sub-sequent development of thymoma is reported in 8% and5% of PRCA patients in two large series.1,15 Earlier fre-quency estimates (30–50%; derived from the analysis ofcase presentations) reflect significant reporting bias. Asmost patients respond to prednisone or a second line agent(e.g., cyclophosphamide, cyclosporine), we generallyinitiate treatment trials prior to any further investigationssuch as marrow culture studies or cytogenetics. In re-fractory patients with normal cytogenetics and whereserum does not contain B19 DNA, culture studies can beinformative (Figure 3). If erythroid bursts are present innormal numbers, it implies that BFU-E are present in thepatient and, when removed from serum antibody and thepatient’s T (or NK) cells, can mature into hemoglobinizedcolonies. This finding justifies sequential therapies withadditional immunologic agents. In general, PRCA is avery treatable disorder (80% response rate1), as well asone that provides unique insights into the physiology oferythroid differentiation.Figure 3. The clinical value of in vitro culture.

The relationship of normal BFU-E growth (30 bursts/105 marrowmononuclear cells) and remission (CR and PR) is shown. BFU-Ematuration in vitro was a superb predictor of clinical response. Itssensitivity was 96%, its specificity was 78% and its predictivevalue was 93% (p = .0001 with 2-tailed chi-square analysis).Complete data are in reference 1.

BFU-E Maturation

Remissionobtained

yes no

yes 27 1

no 2 7


III. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Lucio Luzzatto, M.D.*

The phrase paroxysmal nocturnal hemoglobinuria reflectsthe most dramatic manifestation of a blood disorder thatwe must now regard as quite complex. Indeed, hemoglo-binuria follows hemoglobinemia, which in turn resultsfrom intravascular hemolysis; therefore, PNH has beentraditionally and correctly classified as a hemolytic ane-mia. However, often the anemia is associated also with adecrease in neutrophils, or platelets, or both, hinting to abroader pathology of the hematopoietic system. In addi-tion, patients with PNH are liable to the potentially dev-astating consequence of venous thrombosis, particularlyin the veins of the porto-hepatic system. The triad ofhemolytic anemia, pancytopenia and thrombosis makesPNH a truly unique clinical syndrome (see Table 4).

EpidemiologyPNH is encountered in all populations and can affectpeople of all ages and socio-economic groups. However,PNH has never been reported as a congenital disease,and there is no report of family clustering. Thus, PNH isan acquired disease. There is little information on theincidence of PNH, but the rate is estimated to be 5–10times less than that of aplastic anemia; thus, PNH is arare disease. It has been suggested that, like aplastic ane-mia, PNH may be more frequent in South East Asia andin the Far East.1

Clinical FeaturesIn most cases2 the patient presents as a problem in thedifferential diagnosis of anemia, whether symptomaticor discovered incidentally. The diagnosis is immediatelymade much easier if the patient reports having passeddark urine, which is due to hemoglobinuria, the land-mark of intravascular hemolysis. In some patients PNHmay present with typical signs and symptoms of throm-

bosis in a deep vein in one of the limbs; in others withrecurrent attacks of severe abdominal pain, which mayprove to be due to intra-abdominal thrombosis. The clas-sic site is in the hepatic veins, causing the Budd-Chiarisyndrome, but the mesenteric vessels, the splenic, or theportal vein may be also involved. Not infrequently, theanemia is associated with other cytopenias, suggestingsome degree of bone marrow failure (BMF). Indeed,sometimes a PNH patient may become less hemolyticand more pancytopenic, converting in fact to aplasticanemia. Conversely, PNH can emerge in patients with aprevious diagnosis of aplastic anemia; indeed, in somerecent series small PNH clones have been detected in upto 50% of cases of aplastic anemia.3

The natural history of PNH is that of a very chronicdisorder, which may afflict the patient continuously fordecades. Without treatment the median survival is esti-mated to be about 8 years; in the past the most commoncauses of death have been thrombosis or hemorrhage as-sociated with severe thrombocytopenia. Rarely, PNH mayterminate in acute myeloid leukemia. On the other hand,full spontaneous recovery from PNH also has been welldocumented.4

HemolysisHemolysis in PNH is due to an intrinsic abnormality ofthe red cell. This defect was first characterized serologi-cally through the finding that, unlike in auto-immune orallo-immune hemolytic anemia, there was no specificantibody involved. Rather, the red cells hemolyze when-ever they are in the presence of activated complement(C), whether it is activated by an antibody (as with anti-I, present at low titer in most normal people), or throughthe alternative pathway (by lowering pH or ionic strength).Indeed, acidification of serum was used to develop a di-agnostic test that is still in use in laboratories,5,6 and lowionic strength has been used for a screening test (sucrosehemolysis), which should be regarded as obsolete. It tookhalf a century before the biochemical basis for this pecu-liar hypersusceptibility to C was clarified. We now knowthat several membrane proteins protect cells—includingred cells—against damage from the membrane attackcomplex of C (C5-C9). One of these proteins, CD59,

Table 4. PNH: clinical heterogeneity and proposed terminology (from 40).

Predominant Size ofClinical Features Blood Findings PNH Clone Designation

Hemolysis ± thrombosis Anemia; little or no other cytopenia Large Florid PNH

Hemolysis ± thrombosis Anemia; mild to moderate other cytopenia(s) Large PNH, hypoplastic

Purpura and/or infection Moderate to severe pancytopenia Large AA/PNH

Purpura and/or infection Severe pancytopenia Small AA with PNH clone

Thrombosis Normal or moderate cytopenia(s) Small Mini-PNH

* Department of Human Genetics, Memorial Sloan Kettering Can-cer Center, 1275 York Avenue, New York, NY 10021, USA; andIstituto Nazionale Ricerca sul Cancro, IST, Largo Rosanna Benzi10, Genova, Italy.

Hematology 2000 29

specifically hinders the insertion into the membrane ofC9 polymers.7,8 CD59 is severely deficient or completelyabsent from the membrane of PNH red cells.9 This ex-plains why there is chronic hemolysis in PNH, why thehemolysis is largely intravascular, and why it can be dra-matically exacerbated in the course of a viral or bacterialinfection, when antigen-antibody reactions associated withthe infection will cause bursts of C activation.

Biochemical Abnormalities and Somatic CellMosaicism

Serological studies had suggested long ago that certainnormal antigens were poorly expressed on the surface ofPNH red cells. With the introduction and increasing useof flow cytometry, it was discovered that a bewilderingmultitude of membrane proteins is deficient in the ab-normal population of blood cells of different lineages inpatients with PNH.10 In most cases there is no obvioussimilarity in the function of these proteins; rather, theyhave a common structural element, namely a phospho-lipid moiety: a glycosyl phosphatidyl inositol (GPI) thatincludes an ethanolamine which can form a peptide linkwith a C-terminal amino acid of certain proteins. BecauseGPI is embedded in the lipid bilayer of the membrane,and it serves to retain the protein attached to the mem-brane, it is referred to as a GPI anchor. The fact that allGPI-linked proteins are deficient on the membrane ofPNH cells suggested that the underlying defect might bein the complex biochemical pathway through which theGPI is synthesized in mammalian cells.

A remarkable feature of patients with PNH, whencompared with those who have hemolytic anemia due tosome other intracorpuscular cause, is that not all theirred cells participate in the hemolytic process and thatsome erythrocytes are qualitatively normal. This obser-vation led to the idea that PNH is a clonal disorder due toa somatic mutation.11 Direct supporting experimental evi-dence was obtained three decades ago in PNH patientswho were heterozygous for the X-linked gene encodingglucose 6-phosphate dehydrogenase12 and in whom allPNH cells expressed the same G6PD allele. Thus, wehave true somatic cell mosaicism in patients with PNH,complicating efforts to identify the biochemical abnor-mality of PNH, since patient material always consists ofa mixture of both types of cells. However, a feasible ap-proach was to study cloned lymphoblastoid cell lines(LCL) that displayed the PNH phenotype from PNH pa-tients and to use as controls LCLs with normal pheno-type obtained from the same patients. Comparative analy-sis of these two sets of cell lines revealed normal synthe-sis of phosphatidylinositol, but failure to incorporatemannose or NAcGlcN, thus pinpointing a block at thelevel of the NAcGlcN transfer.13

Molecular GeneticsThe PIG-A gene (so called for Phosphatidyl Inositol Gly-can complementation group A) was isolated by expres-sion cloning,14 i.e. through its ability to restore the ex-pression of GPI-anchored proteins on the surface of cellslacking those proteins, including those from PNH pa-tients.15 PIG-A maps to the short arm of the X chromo-some on Xp22.1, within a region almost completely cov-ered by physical contigs.16 It quickly became clear thatacquired mutations of the PIG-A gene are responsiblefor PNH; to date, a total of 174 somatic mutations in thePIG-A gene (see Figure 4) have been identified by dif-ferent investigators in more than 28 reports in 146 pa-tients.17 Of these, (a) 135 are such that (large deletions,frameshifts, nonsense) we can predict complete functionalinactivation of the PIG-A gene product (PIG-Ao); (b) 35are missense mutations and 4 are small in frame dele-tions. These two groups presumably account for the ex-istence in PNH patients of blood cells with complete de-ficiency of GPI-anchored proteins (PNH III) or partialdeficiency of GPI-anchored proteins (PNH II). The twotypes not infrequently co-exist in the same patient, indi-cating that two different clones are present. PNH III redcells are naturally even more susceptible to C than PNHII red cells, and therefore the absolute and relative amountsof these and of the residual normal red cell population af-fect the rate of hemolysis considerably.

The predicted protein product of PIG-A consists of484 amino acids. A hydrophobic region near the carboxylterminus may be a transmembrane domain (amino acids415-442). The hydrophilic carboxyl terminal region of42 residues corresponds to the lumenal domain of PIG-A. Watanabe et al18 have shown that PIG-A, together withthree other proteins (PIG-H, PIG-C and GPI1), consti-tutes a complex that mediates the first reaction step ofthe GPI anchor biosynthesis, the transfer of GlcNAc tophosphatidylinositol consistent with the biochemical stud-ies mentioned above. The fact that PIG-A is part of anenzyme is also in keeping with the fact that PNH muta-tions are recessive.

PIG-A mutations can be demonstrated in the bloodcells of a large majority of patients with PNH. Failure tofind a mutation in the coding region of PIG-A in anyindividual patient is most likely due to technical reasons;however, it has not been formally excluded that in rarecases the PNH phenotype may arise through mutation ofboth alleles of an autosomal gene encoding any of theproteins required for the GPI biosynthetic pathway. Thevast predominance of PIG-A mutations (over hypotheti-cal mutations of such other genes) must be due to thefact that, because PIG-A is X-linked, an inactivatingmutation (one-hit) will cause the PNH phenotype directly.


Fig

ure

4. S

tru

ctu

re a

nd

mu

tati

on

s o

f th

e P

IG-A

gen

e.

The

cod

ing

regi

on is

sho

wn

in th

e fo

rm o

f box

es; i

ntro

ns a

re s

how

n in

the

form

of d

ashe

d lin

es (

not d

raw

n to

sca

le).

Nuc

leot

ide

num

bers

are

sho

wn

abov

e th

e ex

ons.

Nul

l mut

atio

ns (

fram

eshi

ft,no

nsen

se a

nd s

plic

ing)

are

indi

cate

d ab

ove

the

exon

s. M

isse

nse

mut

atio

ns a

nd in

fram

e de

letio

ns a

re in

dica

ted

belo

w th

e ex

ons.

Eac

h sy

mbo

l rep

rese

nts

one

indi

vidu

al m

utat

ion.

All

mut

atio

nsar

e so

mat

ic, e

xcep

t for

the

two

show

n in

gre

en. F

rom

ref

eren

ce 1

7 .

Hematology 2000 31

ThrombosisThrombosis is one of the most immediately life-threat-ening complications of PNH and yet one of the least un-derstood in its pathogenesis. In principle, we can envis-age three sorts of mechanisms: (a) Impaired fibrinolysis.The urokinase plasminogen activator receptor (uPAR) isa GPI-linked protein and is deficient in leukocytes be-longing to the PNH clone.19 If leukocytes play a role inendogenous fibrinolysis, PNH leukocytes might be lessefficient in this function due to their inability to bindurokinase. In addition, the levels of soluble u-PAR (whichmay normally help to regulate urokinase activity) are sig-nificantly increased in PNH patients.20 This could resultin reduced endogenous fibrinolysis. However, tissue plas-minogen activator is believed to be the main effector ofthe conversion of plasminogen to plasmin. (b) Hyperco-agulability. The coagulation cascade, and therefore ulti-mately thrombin generation, may become activated inPNH. For instance, the C5b-9 complex has been shownto induce release of platelet micro-particles that expressreceptors for factor Va and exhibit prothrombinase21 and“tenase” (factor X cleavage) activity.22 Intravascularhemolysis also may release from red cells substances thathave thromboplastin activity. However, in a series of 11PNH patients, no abnormalities were seen in levels ofthrombin/anti-thrombin complexes or in thrombin acti-vation fragment F1.2.23 Thrombosis can occur in PNHpatients even when they are fully anticoagulated withwarfarin or hirudin, both of which would be expected toeffectively prevent hypercoagulability through their ef-fects on the common coagulation pathway. Therefore,activated coagulation itself is not likely to be the mainculprit for thrombosis in PNH. (c) Hyperactivity of plate-lets. After treatment with C5b-9 PNH platelets undergoincreased vesiculation and thrombin generation,24 andelevated levels of platelet derived micro-particles havebeen demonstrated in some PNH patients.25 The expres-sion of activation markers is increased on the surface ofplatelets from PNH patients,23 probably because of ab-normal C regulation, which may result in platelet activa-tion.26,27 The lack of CD59 on the PNH platelet couldlead to abnormal insertion of the C5b-9 complex in theplatelet membrane, as is the case with the red cell.

Though these three factors may play a role in pro-ducing a thrombophilic state in PNH, it seems very likelythat the primary cause lies in the PNH platelets, whichare abnormal precisely because they belong to the PNHclone. This notion is supported by the occurrence of cere-bral infarction in a patient who did not have PNH, but whohad congenital CD59 deficiency and chronic hemolysis.28

Bone Marrow FailureThe close association between PNH and aplastic anemia(mentioned above) may reflect a shared pathogenetic

basis.29 Aplastic anemia is essentially an organ-specificautoimmune disease. In the peripheral blood and bonemarrow of patients with aplastic anemia it is possible tofind increased numbers of ‘activated’ CD8+ T-lympho-cytes, which are able to inhibit the growth in vitro ofboth autologous and HLA-identical hematopoietic colo-nies.30 T-lymphocytes might be implicated in causingaplastic anemia in two ways. On one hand, by secretinginterferon-� and tumor necrosis factor-� they induce Fasexpression on CD34+ cells. On the other hand, cytotoxicT-lymphocytes (CTLs) may ultimately cause apoptosisthrough Fas-FasL interaction (although this sequence ofevents has not yet been conclusively demonstrated), lead-ing to depletion of HSC. The putative auto-antigens thatincites and serves as a target of the autoreactive CTLs isnot known. The strongest evidence for the immune basisof aplastic anemia is that the majority of patients respondto immunosuppressive treatment,31 Recently, skewing ofthe T cell repertoire has been demonstrated in a subset ofpatients with aplastic anemia by immunoscope analysis,32

and a detailed study conducted by the same approach hasshown that one or several expanded T cells clones are presentin PNH patients three times more frequently than in con-trol subjects.33

Although PNH has elements of BMF, it is obviouslydifferent from aplastic anemia because of its other promi-nent clinical features (hemolysis and thrombosis). Onepossible way to look at the pathophysiology of PNH isthat it results precisely from the co-existence of BMFwith a large PIG-A mutant clone.34 In order to explainthe co-existence of these two components, we can sur-mise in principle three possibilities: (i) The PNH cloneand BMF co-exist by a sheer coincidence. (ii) The PNHclone causes BMF. (iii) BMF favors the development orthe expansion of the PNH clone. Because AA and PNHare both rare diseases, the first possibility can be dis-carded on statistical grounds as being too improbable.The second possibility is unlikely, since PNH often de-velops in patients originally suffering from aplastic ane-mia, who therefore already had established BMF at a timewhen no PNH clone could be demonstrated. Thus, thethird possibility seems the most likely, and it has recentlyobtained support through experimental findings in mice,as well as observations in humans.

Consequences of PIG-A Inactivation in MiceWhen the PIG-A gene is targeted by homologous recom-bination in mouse embryonic stem (ES) cells, and theseare then injected into blastocysts, viable chimeric ani-mals can be obtained only if both the contribution to theembryo by the PIG-A null cells and the numbers of GPI-negative cells in their blood are low. Recently, by usingthe technique of conditional ‘knock-out’ based on whatis known in jargon as the cre-lox system, two groups have


succeeded in producing mice that can be regarded as truemodels for human PNH.35,36 Indeed, the mice have twodiscrete populations of red cells, granulocytes, monocytes,and lymphocytes: in first approximation, the flowcytometry patterns are remarkably similar to those seenin patients with PNH. In addition, although the mice arenot anemic, they do have evidence of hemolysis and theirred cells have increased susceptibility to complement(data on platelets and thrombosis are not yet available).Interestingly, the clinical course of the mice is very be-nign, and the proportion of PNH cells is remarkablystable, in spite of the fact that these mice are born with alarge number of pre-formed PNH cells. In humans, bycontrast, before development of the clinical picture ofPNH, the PNH cell population must have undergone sub-stantial expansion, since it arises from a single mutantstem cell.37 It is tempting to surmise that the PNH cells inmice are very similar to PNH cells in human patients;but the mice do not have the human disease, PNH, be-cause they do not have BMF. The fact that this is lackingsupports the notion that the causation of BMF is indepen-dent of the PNH cell population itself.

PNH Clones and Microclones in HumansWe have already mentioned that PNH III and PNH II canco-exist in the same patient; indeed, by mutation analy-sis it is not infrequent to find two or more clones in thesame patient. These findings are strongly suggestive ofthe hypothesis that these clones expand, simultaneouslyor in sequence, in response to a certain selective agentpresent in the patient’s bone marrow environment.

On the other hand, is there evidence for PNH clonesin normal people, and what is the fate of such clones? Ithas been shown recently that very small populations ofPNH granulocytes and PNH erythrocytes are present innormal people, probably generated simply by the ‘back-ground’ level of somatic mutations in the PIG-A gene.Exactly as in patients with PNH, missense, frameshifts,and nonsense mutation have been identified. Two of thesemutations had been found previously in patients with clas-sical PNH, thus showing formally and conclusively thatPIG-A gene mutations are not sufficient for the develop-ment of PNH.38 The fact that these very tiny PNH cellpopulations—microclones—do not expand clearly meansthat they do not have any intrinsic growth advantage and,conversely, that in PNH patients clones have expandedin a favorable environment.

Clinical Implications

Classification of PNHHow well do these concepts fit the clinical reality of pa-tients with PNH? This can be tested by considering, ineach patient, the relative roles of the PNH clone(s) and

of BMF in determining the clinical picture (Table 4). Atone end of the spectrum we may find a patient with aPNH clone so large that it masks BMF (almost) com-pletely. The patient will have signs and symptoms of briskhemolysis and may have serious thrombotic complica-tions, but the peripheral blood count is normal or nearnormal; the patient can be said to have “florid PNH.” Atthe other end of the spectrum is a patient who meets allcriteria for severe aplastic anemia and who is found (bysensitive analytical techniques) to have a very small pro-portion of GPI-negative cells in the blood. In such a pa-tient the presence of the PNH clone is not likely to sig-nificantly affect the clinical course, and therefore we pre-fer to designate the patient as having “aplastic anemiawith a PNH clone,” because it is the BMF rather than thePNH that must dictate therapeutic decisions. Intermedi-ate situations are not uncommon. Most important, sincethere is a dynamic relationship between PNH clone(s)and BMF, transitions from one form to another may takeplace.

TherapyIt is clear from the above that in a patient with PNH weare dealing with two problems: the PNH clone and BMF(see Figure 5). The management of patients we havecalled “aplastic anemia with a PNH clone” does not dif-fer from that of patients with straightforward aplastic ane-mia. By contrast, patients with “florid PNH” may presentunique clinical problems, mainly massive hemolytic at-tacks and serious thrombotic complications. For thesepatients the two extreme choices are radical treatment bybone marrow transplantation (BMT) or supportive treat-ment only (of which red cell transfusion is the major com-ponent). When an HLA-identical sibling is available, al-logeneic BMT is probably the treatment of choice foryounger patients with moderate to severe cytopenias and,in view of the risk of life-threatening complications, mustbe regarded as an option to be offered to any young pa-tient with PNH. By contrast, the past record of BMT fromunrelated donors in PNH is poor, and it should still beregarded as experimental in the treatment of this condi-tion. For patients who do not have an appropriate donor(and perhaps also for those who do), immunosuppres-sive treatment with ALG or ATG and cyclosporine A maybe a good alternative. This type of treatment cannot beexpected to eradicate the PNH clone because it aims in-stead to relieve the immune-mediated inhibition of nor-mal hematopoiesis. However, in doing so it will limit ifnot eliminate the abnormal marrow environment in whichthe PNH clone thrives. Thus, for treating the BMF com-ponent of PNH we can capitalize on what has been learnedfrom aplastic anemia. Confronting the consequences ofhaving a large PNH clone (hemolysis and thrombosis) isa difficult challenge at the moment. Thrombosis, particu-

Hematology 2000 33

larly in the abdomen, poses an immediate threat to thepatient. Unfortunately even well-managed anticoagulanttreatment does not always prevent thrombosis, althoughin some cases thrombolytic therapy can resolve it.39 Asfor intravascular hemolysis, despite the impressive ad-vances of complement research we still do not have aclinically applicable method to inhibit the effector path-way—there is a dire need to develop new ideas in thisarea.

Conclusion: A Coherent Model for thePathogenesis of PNH

In terms of the nosologic classification of human dis-eases, PNH is rather special and perhaps unique for atleast two reasons: (a) Although it results from somaticmutations, it is not a malignant disease. (b) It is probablythe only acquired hemolytic anemia that is due to an in-trinsic red cell abnormality.

On the basis of current knowledge, we can formu-late a model for the pathogenesis of PNH (Figure 6)which explains at least most of its clinical and hemato-logical features, as follows:

• PNH always co-exists with BMF.• BMF is clinically obvious in patients who first mani-

fest aplastic anemia and then develop PNH. In pa-tients who initially present with PNH, BMF may notbe obvious, because at diagnosis the PNH clone hasexpanded to the point where it provides a substantialproportion of the patient’s hematopoiesis.

• The PNH clone has a long but probably finite lifespan. If, by the time the PNH clone is exhausted,BMF has not improved, the patient evolves clinicallyfrom PNH to aplastic anemia. If, by the time the PNHclone is exhausted, BMF has improved, the patientwill be ‘cured’ of PNH.

• A PNH clone arises through a PIG-A mutation in aHSC. Since there is only one active X-chromosomein each HSC, the mutated stem cell and its progenywill acquire the PNH phenotype, and this can hap-pen in any normal person. Cells with the PNH phe-notype have no intrinsic growth advantage, and there-fore PNH clones will not normally expand. As longas there is no BMF, clinical PNH will not develop.

• The existence of a florid PNH clone while the rest of

Figure 5. Guidelines for the management of PNH.

This algorithm40 is based on the consideration that patients with this condition vary considerably (a) in terms of clinical severity, and (b) interms of the contributions of the PNH clone and of bone marrow failure (BMF) respectively to determining the overall clinical picture.Some patients have been cured by bone marrow transplantation (BMT); at the other end of the spectrum, some of the patients who for along time have been ‘living with PNH’—by choice or otherwise—have eventually experienced spontaneous recovery.4

Diagnosis of PNH

Determine Clinical Form

Florid PNH PNH, hypoplastic AA.PNH AA with PNH clone

Evaluate: Treat as per AA3 months follow-up

Severe Less Severe

HLA-identical No HLA-identical Living with PNHsibling available sibling available

BMT ATG Progressive disease Stable

Living with PNH

➤

➤➤

➤➤

➤➤

➤

➤

➤

➤

➤

➤

➤

➤

➤

➤➤➤➤


hematopoiesis is depressed suggests that the PNHclone can be spared selectively from the injury af-fecting the rest of the bone marrow.

• In order to explain the previous statement, we maysurmise specifically that the damage to stem cellscausing BMF is mediated through a GPI-linked sur-face molecule: in this case, the PNH cells lackingthese molecules will survive.

• Thus, the very defect of the PNH clone may endowit with a relative survival or growth advantage in apatient with BMF. If the patient has such a clone, heor she will present with PNH; otherwise he or she

would present with overt aplastic anemia. Thus, thedevelopment of PNH is conditional on a backgroundof BMF.

(8) A large PNH clone carries with it intravascularhemolysis and thrombosis, the ‘classical’ manifesta-tions of PNH.

Acknowledgements: I thank all my colleagues with whomI have been fortunate to work on PNH over the years,and I am very grateful to D. Araten, A. Karadimitris, R.Notaro and D. Nafa for help with this paper.

Figure 6. The role of somatic mutation and auto-immune mediated bone marrow failure in the pathophysiology of PNH.

The top panel is a cartoon of normal hematopoietic stem cells (HSC): the arrow indicates a somatic mutation in the PIG-A gene in one ofthe HSC. As a result, the cell and its progeny lose surface GPI-anchored proteins. As time goes on, micro clones arising from such mutantcells may become exhausted, and new ones may arise: however, there is no clonal expansion. The middle panel illustrates the presencein the bone marrow of auto reactive immune cells, which may be cytotoxic T cells: these attack the HSC, which gradually decrease innumbers, eventually resulting in the picture of aplastic anemia (AA). The bottom panel illustrates the consequences of the co-existence ofa PIG-A somatic mutation and autoreactive immune cells in the bone marrow. If we make the specific hypothesis that the target of theautoimmune attack is a GPI-anchored protein, the mutant clone will expand as a result of negative selection against the normal HSC. Asa result, the majority of hematopoiesis will consist of GPI-anchored protein deficient cells. The large numbers of c-susceptible red cellswill cause hemolytic anemia; the large numbers of abnormal platelets will cause a high risk of thrombotic complications.

Hematology 2000 35

REFERENCES

I. Acquired Aplastic Anemia1. Kaufman DW, Kelly JP, Levy M, Shapiro S: The Drug Etiology

of Agranulocytosis and Aplastic Anemia. New York: Oxford;1991.

2. Issaragrisil S, Leaverton PE, Chansung K, Thamprasit T,Porapakham Y, Young NS, The Aplastic Anemia Study Group:The incidence of aplastic anaemia in Thailand. Am J Hematol.1999;61:164-168.

3. Chongli Y, Xiaobo Z. Incidence survey of aplastic anemia inChina. Chin Med Sci J. 1991;6:203-207.

4. Young NS. Hematopoietic cell destruction by immune mecha-nisms in aquired aplastic anemia. Semin Hematol. 2000;37:3-14.

5. Maciejewski JP, Selleri C, Sato T, Anderson SA, Young NS. Asevere and consistent deficit in marrow and circulating primitivehematopoietic cells (long term culture-initiating cells) inacquired aplastic anemia. Blood. 1996;88:1983-1991.

6. Ball SE, Gibson FM, Rizzo S, Tooze JA, Marsh JCW, Gordon-Smith EC. Progressive telomere shortening in aplastic anemia.Blood. 1998;91:3582-3592.

7. Marsh JC. Hematopoietic growth factors in the pathogenesis andfor the treatment of aplastic anemia. Semin Hematol.2000;37:81-90.

8. Mathé G, Amiel JL, Schwarzenberg L, et al. Bone marrow graftin man after conditioning by antilymphocytic serum. Br Med J.1970;2:131-136.

9. Anderson KC, Weinstein HJ. Transfusion-associated graftversus-host disease. N Engl J Med. 1990;323:315-321.

10. Young NS, Maciejewski J. The pathophysiology of acquiredaplastic anemia. N Engl J Med. 1997;336:1365-1372.

11. Sloand E, Kim S, Maciejewski JP, Chaudhuri A, Kirby M,Young NS. Presence of intracellular interferon-gamma (IFN-gamma) in circulating lymphocytes and response to immunosup-pressive therapy in patients with aplastic anemia. Blood.1998;10 (Suppl 1):158a. (abstr)

12. Wolk A, Simon-Stoos K, Nami I, et al. A mouse model ofimmune-mediated aplastic anemia. Blood. 1998;10 (Suppl.1):158a-159a. (abstr)

13. Brown KE, Tisdale J, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia. N Engl J Med. 1997;336:1059-1064.

14. Nimer SD, Ireland P, Meshkinpour A, Frane M. An increasedHLA DR2 frequency is seen in aplastic anemia patients. Blood.1994;84:923-927.

15. Nakao S, Takamatsu H, Chuhjo T, et al. Identification of aspecific HLA class II haplotype strongly associated withsusceptibility to cyclosporine-dependent aplastic anemia. Blood.1994;84:4257-4261.

16. Gerson WT, Fine DG, Spielberg SP, Sensenbrenner LL.Anticonvulsant-induced aplastic anemia: increased susceptibilityto toxic drug metabolites in vitro. Blood. 1983;61:889-893.

17. Young NS, Barrett AJ. The treatment of severe acquired aplasticanemia. Blood. 1995;85:3367-3377.

18. Tsai TW, Freytes CO. Allogeneic bone marrow transplantationfor leukemias and aplastic anemia. Adv Int Med.1997;42:423-451.

19. Storb R, Etzioni R, Anasetti C, et al. Cyclophosphamidecombined with antithymocyte globulin in preparation forallogeneic marrow transplants in patients with aplastic anemia.Blood. 1994;84:941-949.

20. Bacigalupo A, Brand R, Oneto R. Treatment of acquired severeaplastic anemia: bone marrow transplantation compared withimmunosuppressive therapy—The European Group for Bloodand Bone Marrow Transplantation experience. Sem Hematol.2000;37:69-80.

21. Horowitz MM. Current status of allogeneic bone marrow

transplantation in acquired aplastic anemia. Semin Hematol.2000;37:30-42

22. Bacigalupo A. Treatment of severe aplastic anaemia. In Gordon-Smith EC, ed. Aplastic Anaemia. Clin Haematol. v. 2. London:Bailliere Tindall; 1989:19-36.

23. Deeg HJ, Leisenring W, Storb R, et al. Long-term outcome aftermarrow transplantation for severe aplastic anemia. Blood.1998;91:3637-3645.

24. Margolis D, Camitta B, Pietryga D, Keever-Taylor C. Unrelateddonor bone marrow transplantation to treat severe aplasticanaemia in children and young adults. Br J Haematol.1996;94:65-72.

25. Margolis DA, Casper JT. Alternative-donor hematopoietic stem-cell transplantation for severe aplastic anemia. Semin Hematol.2000;37:43-55.

26. Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462transplantations from unrelated donors facilitated by theNational Marrow Donor Program. N Engl J Med. 1993;328:593-602.

27. Bacigalupo A. Severe Aplastic Anaemia Working Party, EBMTWorking Parties Reports. Harrogate, European Group for BoneMarrow Transplantation, 1994:49-62

28. Witherspoon RP, Fisher LD, Schock G, et al. Secondary cancersafter bone marrow transplantation for leukemia or aplasticanemia. N Engl J Med. 1989;321:784-789.

29. Socié G, Henry-Amar M, Cosset JM, Devergie A, Girinsky T,Gluckman E. Increased incidence of solid malignant tumorsafter bone marrow transplantation for severe aplastic anemia.Blood. 1991;78:277-279.

30. Curtis ER, Rowlings PA, Deeg J, et al. Solid cancers after bonemarrow transplantation. N Engl J Med. 1997;336:897-904.

31. Frickhofen N, Rosenfeld SJ. Immunosuppressive treatment ofaplastic anemia with antithymocyte globuilin and cyclosporine.Semin Hematol. 2000;37:56-68.

32. Bacigalupo A, Broccia G, Corda G, et al. Antilymphocyteglobulin, cyclosporin, and granulocyte colony-stimulating factorin patients with acquired severe aplastic anemia (SAA): a pilotstudy of the EBMT SAA working party. Blood. 1995;85:1348-1353.

33. Rosenfeld SJ, Kimball J., Vining D, Young NS: Intensiveimmunosuppresion with antithymocyte globulin andcyclosporine as treatment for severe acquired aplastic anemia.Blood. 1995;85:3058-3065.

34. Raghavachar A, Kolbe K, Höffken K, et al. A randomized trialof standard immunosuppression versus cyclosporine andfilgastrim in severe aplastic anemia. Blood. 1997;90 (Suppl1):439a. (abstr)

35. Bielory L, Lawley T, Gascon P, Yancey K, Young N, Frank MM.Human serum sickness after equine antithymocyte globulin inpatients with bone marrow failure. Medicine. 1988;67:40-57.

36. Brodsky RA, Sensenbrenner LL, Jones RJ. Complete remissionin severe aplastic anemia after high-dose cyclophosphamidewithout bone marrow transplantation. Blood. 1996;87:491-494.

37. Tisdale JF, Dunn DE, Geller NL, et al. Excessive toxicity ofhigh dose cyclophosphamide in severe aplastic anemia: resultsof a randomized trial. Lancet. In press.

38. Genestier L, Fournel S, Flacher M, Assossou O, Revillard J-P,Bonnefoy-Berard N. Induction of Fas (Apo-, CD95)-mediatedapoptosis of activated lymphocytes by polyclonal antithymocyteglobulin. Blood. 1998;91:2360-2368.

39. Merion RM, Howell T, Bromberg JS: Partial T-cell activationand anergy induction by polyclonal antithymocyte globulin.Transplantation. 1998;65:1481-1489.

40. Di Bona E, Rodeghiero F, Bruno B, et al. Rabbit antithymocyteglobulin (r-ATG) plus cyclosporine and granulocyte colonystimulating factor is an effective treatment for aplastic anaemiapatients unresponsive to a first course of intensive immunosup-


pressive therapy. Br J Haematol. 1999;107:330-334.41. Gardner FH, Juneja HS: Androstane therapy to treat aplastic

anaemia in adults: an uncontrolled pilot study. Br J Haematol.1987;65:295-300.

42. Najean Y, Haguenauer O. Long-term (5 to 20 years) evolution ofnongrafted aplastic anemia. Blood. 1990;76:2222-2228.

43. Marsh JCW, Zomas A, Hows JM, Chapple M, Gordon-SmithEC. Avascular necrosis after treatment of aplastic anaemia withantilymphocyte globulin and high-dose methylprednisolone. BrJ Haematol. 1993;84:731-735.

44. Marsh JCW, Socie G, Schrezenmeier H, et al. Haemopoieticgrowth factors in aplastic anaemia: a cautionary note. Lancet.1994;344:172-173.

45. Gluckman E, Rokicka-Milewska R, Gordon-Smith EC, et al.Results of a randomized study of glycosylated rHuG-CSFLenogastrim in severe aplastic anemia. Blood. 1998;92 (Suppl1):376a. (abstr)

46. Kojima S, Matsuyama T. Stimulation of granulopoiesis by high-dose recombinant human granulocyte colony-stimulating factorin children with aplastic anemia and very severe neutropenia.Blood. 1994;83:1474-1478.

47. Bessho M, Hirashima K, Asano S, Ikeda Y. Treatment of theanemia of aplastic anemia patients with recombinant humanerythropoietin in combination with granulocyte colony-stimulating factor: a multicenter randomized controlled studyMulticenter study group. Eur J Haematol. 1997;58:265-272.

48. Kurzrock R, Paquette R, Gratwohl A, et al. Use of stem cellfactor (Stemgen, SCF) and filgastrimm (GCSF) in aplasticanemia (AA) patients who have failed ATG/ALG therapy.Blood. 1997;90(Suppl 1):173a. (abstr)

49. Imashuku S, Hibi S, Mitsui T, et al. A review of 125 cases todetermine the risk of myelodysplasia and leukemia in pediatricneutropenic patients after treatment with recombinant humangranulocyte colony-stimulating factor. Blood. 1996;84:2380-2381.

50. Kaito K, Kobayashi M, Katayama T, et al. Long-termadmiistration of G-CSF for aplastic anaemia is closely related tothe early evolution of monosomy 7 MDS in adults. Br JHaematol. 1998;103:297-303.

51. Bacigalupo A, Brand R, Oneto R, et al. Treatment of acquiredsevere aplastic anemia: bone marrow transplantation comparedwith immunosuppressive therapy—The European Group forBlood and Marrow Transplantation experience. Semin Hematol.2000;37:69-80.

52. Socié G, Henry-Amar M, Bacigalupo A, et al. Malignant tumorsoccurring after treatment of aplastic anemia. N Engl J Med.1993;329:1152-1157.

53. Dunn DE, Tanawattanacharoen P, Boccuni P, et al. Paroxysmalnocturnal hemoglobinuria populations in patients with bonemarrow failure: prevalence, progression, and relationship toimmunosuppressive therapy. Blood. 1998;92 (Suppl 1):15b.(abstr)

54. Molldrem J, Caples M, Mavroudis D, Plante M, Young NS,Barrett AJ. Antithymocyte globulin (ATG) abrogates cytopeniasin patients with myelodysplastic syndrome. Br J Haematol.1997;99: 699-705.

55. Killick SB, Win N, Marsh JCW, et al. Pilot study of HLAalloimmunization after transfusion with pre-storageleucodepleted blood products in aplastic anemia. Br J Haematol.1997;97:677-684.

56. Kaufman DW, Kelly JP, Levy M, Shapiro S. The Drug Etiologyof Agranulocytosis and Aplastic Anemia. New York: OxfordPress; 1991.

II. Acquired Pure Red Cell Aplasia1. Charles RJ, Sabo KM, Kidd PG, Abkowitz JL. The pathophysi-

ology of pure red cell aplasia: implications for therapy. Blood.1996;87:4831-4838.

2. Dessypris EN. The biology of pure red cell aplasia. SeminHematol. 1991;28:275-284.

3. Lacy MQ, Kurtin PJ, Tefferi A. Pure red cell aplasia: associationwith large granular lymphocyte leukemia and the prognosticvalue of cytogenetic abnormalities. Blood. 1996;87:3000-3006.

4. Handgretinger R, Geiselhart A, Moris A, et al. Pure red-cellaplasia associated with clonal expansion of granular lympho-cytes expressing killer-cell inhibitory receptors. N Engl J Med.1999;340:278-284.

5. Peschle C, Marmont AM, Marone G, Genovese A, Sasso GF,Condorelli M. Pure red cell aplasia: studies on an IgG seruminhibitor neutralizing erythropoietin. Br J Haematol.1975;30:411-417.

6. Tsujimura H, Sakai C, Takagi T. Pure red cell aplasia compli-cated by angioimmunoblastic T-cell lymphoma: humoral factorplays a main role in the inhibition of erythropoiesis fromCD34(+) progenitor cells. Am J Hematol. 1999;62:259-260.

7. Mangan KF, Volkin R, Winkelstein A. Autoreactive erythroidprogenitor-T suppressor cells in pure red cell aplasia associatedwith thymoma and panhypogammaglobulinemia. Am J Hematol.1986;23:167-173.

8. Socinski MA, Ershler WB, Tosato G, Blaese RM. Pure redblood cell aplasia associated with chronic Epstein-Barr virusinfection: evidence for T cell-mediated suppression of erythroidcolony forming units. J Lab Clin Med. 1984;104:995-1006.

9. Reid TJ III, Mullancy M, Burrell LM, Redmond J III, ManganKF. Pure red cell aplasia after chemotherapy for Hodgkin’slymphoma: in vitro evidence for T cell mediated suppression oferythropoiesis and response to sequential cyclosporin anderythropoietin. Am J Hematol. 1994;46:48-53.

10. Chikkappa G, Zarrabi MH, Tsan M-F. Pure red-cell aplasia inpatients with chronic lymphocytic leukemia. Medicine.1986;65:339-351.

11. Akard LP, Brandt J, Lu L, Jansen J, Hoffman R. Chronic T celllymphoproliferative disorder and pure red cell aplasia. Am JMed. 1987;83:1069-1074.

12. Hoffman R, Kopel S, Hsu SD, Dainiak N, Zanjani ED. T cellchronic lymphocytic leukemia: presence in bone marrow andperipheral blood of cells that suppress erythropoiesis in vitro.Blood. 1978;52:255-260.

13. Abkowitz JL, Kadin ME, Powell JS, Adamson JW. Pure red cellaplasia: lymphocyte inhibition of erythropoiesis. Br J Haematol.1986;63:59-67.

14. Levitt LJ, Reyes GR, Moonka DK, Bensch K, Miller RA,Engleman EG. Human T cell leukemia virus-I-associated T-suppressor cell inhibition of erythropoiesis in a patient with purered cell aplasia and chronic Tg-lymphoproliferative disease. JClin Invest. 1988;81:538-548.

15. Clark DA, Dessypris EN, Krantz SB. Studies on pure red cellaplasia. XI. results of immunosuppressive treatment of 37patients. Blood. 1984;63:277-286.

16. Raghavachar A. Pure red cell aplasia: review of treatment andproposal for a treatment strategy. Blut. 1990;61:47-51.

17. Serra S, Real E, Pastor E, Grau E. Refractory pure red-cellaplasia associated with B chronic lymphocytic leukemiasuccessfully treated by fludarabine. Haematologica.1999;84:1154-1155.

18. Chikkappa G, Pasquale D, Zarrabi MH, Weiler RJ, Divakara M,Tsan MF. Cyclosporine and prednisone therapy for pure red cellaplasia in patients with chronic lymphocytic leukemia. Am JHematol. 1992;41:5-12.

19. Abkowitz JL, Powell JS, Nakamura JM, Kadin ME, AdamsonJW. Pure red cell aplasia: response to therapy with anti-

Hematology 2000 37

thymocyte globulin. Am J Hematol. 1986;23:363-371.20. Brown KE, Young NS. Parvoviruses and bone marrow failure.

Stem Cells. 1996;14:151-163.21. Cohen BJ, Buckley MM. The prevalence of antibody to human

parvovirus B19 in England and Wales. J Med Microbiol.1988;25:151-153.

22. Brown KE, Anderson SM, Young NS. Erythrocyte P antigen:cellular receptor for B19 parvovirus. Science. 1993;262:114-116.

23. Frickhofen N, Abkowitz JL, Safford M, et al. Persistent B19parvovirus infection in patients infected with human immunode-ficiency virus Type 1 (HIV-1): a treatable cause of anemia inAIDS. Annals Intern Med. 1990;113:926-933.

24. Gottlieb F, Deutsch J. Red cell aplasia responsive to immunoglo-bulin therapy as initial manifestation of human immunodefi-ciency virus infection. Am J Med. 1992;92:331-333

25. Abkowitz JL, Brown KE, Wood RW, Kovach NL, Green SW,Young NS. Clinical relevance of parvovirus B19 as a cause ofanemia in patients with human immunodeficiency virusinfection. J Infect Dis. 1997;176:269-273.

26. Frickhofen N, Chen ZJ, Young NS, Cohen BJ, Heimpel H,Abkowitz JL. Parvovirus B19 as a cause of acquired chronicpure red cell aplasia. Br J Haematol. 1994;87:818-824.

27. Quigley JG, Burns CC, Anderson MM, et al. Cloning of thecellular receptor for feline leukemia virus subgroup C inducesred cell aplasia. Blood. 2000;95:1093-1099.

28. Tailor CS, Willett BJ, Kabat D. A putative cell surface foranemia-inducing feline leukemia virus subgroup C is a memberof a transporter superfamily. J Virol. 1999;73:6500-6505.

29. Appelbaum FR, Barrall J, Storb R, et al. Clonal cytogeneticabnormalities in patients with otherwise typical aplastic anemia.Exp Hematol. 1987;15:1134-1139.

30. Dessypris EN. Fogo A, Russell M, Engel E, Krantz SB. Studieson pure red cell aplasia. X. association with acute leukemia andsignificance of bone marrow karyotype abnormalities. Blood.1980;56:421-426.

31. Lacombe C, Casadevall N, Muller O, Varet B. Erythroidprogenitors in adult chronic pure red cell aplasia: relationship ofin vitro erythroid colonies to therapeutic response. Blood.1984;64:71-77.

III. Paroxysmal Nocturnal Hemoglobinuria1. Issaragrisil S. Epidemiology of aplastic anemia in Thailand.

Thai Aplastic Anemia Study Group. Int J Hematol.1999;70:137-40.

2. Rosse W, Paroxysmal nocturnal hemoglobinuria. In Handin LS,Stossel TP, eds. Blood—Principles and Practice of Hematology.Lippincott: Philadelphia; 1995:367-376.

3. Schrezenmeier H, et al. A pathogenetic link between aplasticanemia and paroxysmal nocturnal hemoglobinuria is suggestedby a high frequency of aplastic anemia patients with a deficiencyof phosphatidylinositol glycan anchored proteins. Exp Hematol.1995;23:81-87.

4. Hillmen P, et al. Natural history of paroxysmal nocturnalhemoglobinuria. N Engl J Med. 1995;333:1253-1258.

5. Ham TH. Chronic hemolytic anemia with paroxysmal nocturnalhemoglobinuria. A study of the mechanism of hemolysis inrelation to acid-base equilibrium. N Engl J Med. 1937;217: 915-918.

6. Dacie JV, Israels MCG, Wilkinson JF. Paroxysmal nocturnalhaemoglobinuria of the Marchiafava type. Lancet 1938;i:479-482.

7. Davies A, et al. CD59, an Ly-6-like protein expressed in humanlymphoid cells, regulates the action of the complementmembrane attack complex on homologous cells. J Exper Med.1989. 170: p. 637-654.

8. Rosse WF. The control of complement activation by the blood

cells in paroxysmal nocturnal haemoglobinuria. Blood.1986;67:268-269.

9. Marchiafava E, Nazari A. Nuovo contributo all studio degli ittericronici emolitici. Policlinico (Sez. Med.), 1911;18:241.

10. Giffen HZ. Hemoglobinuria in hemolytic jaundice. Arch InternMed. 1922;31:573-578.

11. Dacie JV. Paroxysmal nocturnal haemoglobinuria. Proc RoyalSoc Med. 1963;56:587-596.

12. Oni S.B, Osunkoya BO, Luzzatto L. Paroxysmal nocturnalhemoglobinuria: evidence for monoclonal origin of abnormalred cells. Blood. 1970;36:145-152.

13. Hillmen P, et al. Specific defect in N-acetylglucosamineincorporation in the biosynthesis of theglycosylphosphatidylinositol anchor in cloned cell lines frompatients with paroxysmal nocturnal hemoglobinuria. Proc NatlAcad Sci USA. 1993;90:5272-5276.

14. Miyata T, et al. The cloning of PIG-A, a component in the earlystep of GPI-anchor biosynthesis. Science. 1993;259:1318-1320.

15. Bessler M, et al. Paroxysmal nocturnal haemoglobinuria (PNH)is caused by somatic mutations in the PIG-A gene. EMBOJournal. 1994;13:110-117.

16. Ferrero, G.B., et al. An integrated physical and genetic map of a35 Mb region on chromosome Xp22.3-Xp21.3. Hum MolGenet, 1995. 4(10): p. 1821-7.

17. Luzzatto L, Nafa K. Genetics of PNH. In Young NS, Moss J(Eds). Paroxysmal Nocturnal Hemoglobinuria and the GPI-Linked Proteins. Academic Press: New York; 2000:21-47.

18. Watanabe R, et al. The first step of glycosylphosphatidylinositolbiosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-Cand GPI1. Embo J. 1998;17(4):877-85.

19. Plough M, et al. The receptor for urokinase-type plasminogenactivator is deficient on peripheral blood leukocytes in patientswith paroxysmal nocturnal hemoglobinuria. Blood.1992;79:1447-1455.

20. Ronne E, et al. The receptor for urokinase plaminogen activatoris present in plasma from healthy donors and elevated in patientswith paroxysmal nocturnal haemoglobinuria. Br J Haematol.1995;89:576-581.

21. Sims PJ, et al. Complement proteins C5b-9 cause releast ofmembrane vesicles from the platelet surface that are enriched inthe membrane receptor for coagulation factor Va and expressprothrombinase activity. J Biol Chem. 1988;263:18205-18212.

22. Gilbert GE, et al. Platelet-derived microparticles express highaffinity receptors for factor VIII. J Biol Chem. 1991;266:17261-17268.

23. Granlick HR, et al. Activated platelets in paroxysmal nocturnalhaemoglobinuria. Br J Haematol. 1995;91:697-702.

24. Wiedmer T, et al. Complement-induced vesiculation andexposure of membrane prothrombinase sites in platelets ofparoxysmal nocturnal hemoglobinuria. Blood. 1993;82:1192-1196.

25. Hugel B, et al. Elevated levels of circulating procoagulantmicroparticles in patients with paroxysmal nocturnal hemoglobi-nuria. Blood. 1999;93:3451-3456.

26. Polley MJ, Nachman RL. Human complement in thrombin-mediated platelet function. J Exp Med. 1979;150:633-645.

27. Zimmerman TS, Kolb WP. Human platelet-initiated formationand uptake of the C5-9 complex of human complement. J ClinInvest. 1976;57:203-211.

28. Shichishima T, et al. Complement sensitivity of erythrocytes in apatient with inherited complete deficiency of CD59 or with theInab phenotype. Br J Haematol. 1999;104:303-6.

29. Rotoli B, Luzzatto L. Paroxysmal nocturnal hemoglobinuria.Semin Hematol. 1989;26:201-207.

30. Zoumbos N.C, et al. Circulating activated suppressor Tlymphocytes in aplastic anemia. N Engl J Med. 1985;312:257-265.


31. Bacigalupo A, et al. Antilymphocyte globulin, cyclosporin, andgranulocyte colony-stimulating factor in patients with acquiredsevere aplastic anemia (SAA): a pilot study of the EBMT SAAWorking Party. Blood. 1995;85:1348-53.

32. Zeng W, et al. Characterization of T-cell repertoire of the bonemarrow in immune-mediated aplastic anemia: evidence for theinvolvement of antigen-driven T-cell response in cyclosporine-dependent aplastic anemia. Blood. 1999;93:3008-16.

33. Karadimitris A, Thaler HT, Notaro R, et al. Abnormal T-cellrepertoire is consistent with immune process underlying thepathogenesis of PNH. Blood. 2000;96:2617-2620.

34. Luzzatto L, Bessler M. The dual pathogenesis of paroxysmalnocturnal hemoglobinuria. Curr Opin Hematol. 1996;3:101-110.

35. Tremml G, Dominguez C, Rosti V, Zhang Z, Pandolfi PP, KellerP, Bessler M. Increased sensitivity to complement and adecreased red cell life span in mice mosaic for a non-functionalPiga gene. Blood. 1999;93:2945-54.

36. Murakami Y, Kinoshita T, Maeda Y, Nakano T, Kosaka H,Takeda J. Different roles of glycosylphosphatidylinositol invarious hematopoietic cells as revealed by model mice ofparoxysmal nocturnal hemoglobinuria. Blood. 1999;93:2963-70.

37. Luzzatto L. Paroxysmal murine hemoglobinuria (?): A modelfor human PNH. Blood. 1999;94:2941-2944.

38. Araten D, Nafa K, Pakdeesuwan K, Luzzatto L. Clonalpopulations of hematopoietic cells with paroxysmal nocturnalhemoglobinuria genotype and phenotype are present in normalindividuals. Proc Natl Acad Sci USA. 1999;96:5209-5214.

39. McMullin MF, et al. Tissue plasminogen activator for hepaticvein thrombosis in paroxysmal nocturnal haemoglobinuria. J IntMed. 1994;235:85-89.

40. Tremml G, Karadimitris A, Luzzatto L. Paroxysmal nocturnalhemoglobinuria: learning about PNH cells from patients andfrom mice. Haematology. 1998;1:12-20.

new insights into the pathophysiology of acquired...

Documents