Path. Res. Pract. 188,817-823 (1992)

Aplastic Crisis in Haemolytic Anaemia due to Infection Parvovirus B 19 As Expert Quiz published in Vol. 185,533-535 (1989)

H. E. Schaefer Pathologisches Institut der Universität Freiburg, FRG

This Quiz refers to an iliac crest biopsy performed in a 32-year-old man suffering from haemolytic anaemia. Deficiency of glucose 6-phosphate dehydrogenase was known in this patient for many years. Due to an aeute haemolytic crisis accompanied by pancytopenia he had to be hospitalised. The clinical examination revealed a haemoglobin concentration drastically decreased to 5 g/dl, whereas the reticulocyte count was reported to be slightly subnormal with an actual value of 1.2%. The blood concentration for lactate. dehydrogenase was significantly increased up to 500 U/m!. Leucocytes (2.3 X 1091) and platelets (87 x 10 91) were both decreased in number.

The trephine biopsy taken from the iliac crest consisted of a cylinder of cancellous bone measuring 30 mm in length. The specimen was fixed in a mixture of glutaraldehyde (0.5°/', viv), formaldehyde (1.2% viv) and calcium acetate (1.85% w/v), subsequently decalcified in neutral EDTA and embedded in paraffin. Sections were stained with Giemsa, Gomori and PAS according to routine procedures, and for chloroacetate esterase and tartrate resistant acid phosphatase l . The cancellous bone did not show any signs of an enhanced turnover. By means of the tartrate resistant acid phosphatase reaction only very few inactive osteroclasts

Fig. 1. The slightly hyperplastic bone marrow is mainly composed of granulopoietic cells exhibiting a positive chloroacetate ester ase reaction (dark staincd cytoplasm) while the large blasts (arrows) remain unstained, x 1070. © J 992 by Gustav Fiscber Verlag, Stuttgart

0344-0338/92/0188-0817$3.50/0

818 . H. E. Schader

Fig. 2. Thc blasts (arrows) are composed of a smooth basophilic cytoplasm surrounding a large nucleus with an extremely delicate chromatin and a prominent nucleolus. Erythroblastic islands are virtually missing. Giemsa, differential-interference contrast, xl070.

Fig. 3. Bone marrow macrophages contain phagocytosed erythrocytes and shown an enhanced iron content. Large blasts (arrows) are diffusely distributed. Perl's reaction, x650.

Aplastic Crisis in Haemolytic Anaemia duc to Infection Parvovirus B19 . 819

were demonstrable. - Whereas fat cells, slightly reduced in number, showed a regular distribution and the reticular fibres revealed a typical architecture, the cellularity of the haematopoietic bone marrow was slightly increased. Granulocytopoiesis and megakaryocytes where moderately increased and left-shifted. An almost complete absence of erythroid islands was rcmarkable. The siderin content of the bone marrow macrophages was significantly increased. These cells frequently contained phagocytoscd erythrocytes (Fig.3). There was no increase in plasma cells. Large, diffusely disseminated blastic cells formed a salient feature in the histologie picture of this case (Fig. 1-3). These cells contained large vesicular nuclei with an extremely loosely distributed chromatin. Prominent, inclusion body-like nucleoli appeared in a more or less marginated position. Few of these cells showcd mitotic activity. Their nuclei were surrounded by a basophilic cytoplasm which, in contrast to granulopoietic cells, reacted negatively for chloroacetate esterase. Duc to size and shape of their nuclei, some of these blasts reminded of Hodgkin cells. Neither multinuclear Sternbcrg-Reed cells nor a granulomatous reaction were observcd. The final question arises as to whether this suddenly developing severe anaemia may be explained by the histological findings presented. Which role do the peculiar blasts play? Are these cells to be considered of neoplastic or of reactive nature?

Solution Summary Presented as a Quiz reporting on a suddenly developing severe anaemia in a 32-year-old man with known glucose-6-phosphatc dehydrogenase deficiency, the characteristics of bone marrow morphology leading to the dia gnosis of transient erythroid aplasia are outlined. As po in ted out in a short overview, this type of acute febrile erythroblastophthisis is due to the cytopathic effect of an infection by parvovirus B19. Due to its marked erythroid tropism this viral agent blocks the growth of erythroblasts at an early stage of maturation. The ensuing erythropoietic aplasia recovers within few days with the advent of humoral immunity. In subjects with a constitutionally reduced life span of crythrocytes, the inhibited recruitment of red blood cells may lead to crises of severe anaemia. Apart from this complicated occurrcnce, it emerges from the recent literature that previous B19 infections rank amongst the most frequent viral diseases prcsenting as crythcma infectiosum or a mild febrile illness.

The condition presented in this Quiz 18 in principle has been known for ninety years. In 1900 Minkowsk y l2 for the first time reported on a 42-year-old man suffering from recurrent jaundice, slowly increasing splenomegaly, enhanced urobilinuria and pigmented cholelithiasis. Other members of his family were affected by the same syndrome in three generations. This patient detiorated suddenly with influenza-like symptoms and severs malaise. He died of a finallobar pneumonia.

This early description of a suddcnly ending congenital acholuric jaundice was later on followed by repeated casuistic reports on congenital haemolytic diseases, mostly cases of familial spherocytosis, developing severc acute anaemia combined with fever, anorexia, nausea, vomiting, headache, abdominal pain and chills 4, 6, 9,10,14. In instances there occurred epistaxis, delirium and syncope 4 . These severe symptoms were in part attributable to a pronounced anaemia in general attaining its nadir within a few days with haemoglobin concentrations of about 5 g/dl, sometimes necessitating blood transfusions. For all, most patients recovered with ten days from their severe illness. Initially this transient severe anaemia complicating diverse conditions of congenital haemolytic anaemia was designated a "haemolytic crisis" on account of an alleged enhancement of haemolysis 6 or blood cell sequestration in the spleen9 • However, in 1949 Owren 14 deduced from his metaculous analysis of six cases that the crises are due to a sudden cessation in the formation of red cells because of a transient aeute aplasia of the erythroblasts. During the crises he noticed a disappearance of reticulocytes from the blood and a decrease of jaundice, serum bilirubin and urobilinuria to normal values. These findings, clearly disproving an enhanccd haemolysis, had escaped attention in the previous studies which had regarded mostly the phases of recovery from the crises where rebound erythroblastic hyperplasia and a more or less pronounced increase of the blood reticulocyte count ensue. A later study conducted by Gasser 8 fully confirmed the pathogenetic concept of Owren 14. Moreover, for the first time this author 8 succeeded in demonstrating that the same febrile infection, obviously triggering conditions of chronic haemolytic disorders to develop crises of severe anaemia, does provoke aeute erythroblastic aplasia lasting for a few days even in normal subjects. This finding was recently confirmed with infection studies in volunteers 16 . While the erythropoietic aplasia of a 5 -lO-day duration does not produce any significant anaemia in healthy persons, whose erythrocytes have a life span of about 120 days, in a11 instances of a shortened erythrocytic survival time (e.g.: 15 days in familial spherocytosis1 4 ) this occurrence regularly produced a decompensation of erythropoietic system with severe anaemia. Indeed, apart from familial spherocytosis aeute aplastic crises have been reported to occur in sickle cell anaemia I, acquired immune haemolytic anaemia, hereditary elliptocytosis, thalassaemia, glucose-6-phosphate dehydrogenase deficiency 2, pyruvate kinase deficiency 3 etc. 4 . On account of the febrile symptoms and repeated observation of aplastic crises developing almost simultaneously in members of one family, a causative agent of infective nature had been postulated beginning with the first observation of Minkowski 12 . Finally, this enigma was solved in 1981, when Pattison et al.1 5 and Serjeant et a1. 20 identified parvovirus B19 as the infective cause of epidemical outbreaks of aplastic crises in sickle cell anaemia 17. Later on the same agent was found to provoke aplastic crises also in hereditary spherocytosis3 , 7, 11, 17, haemoglobin-SC disease l7 , sickte beta-thalassaemia 17 and in pyruvate kinase deficiency 3.

820 . H. E. Schaefer

Parvovirus B19 was discovered in 1975 by Cossart et a1. 5 . In a relatively short period this virus has been established as a wide-spread infective agent causative in childhood for erythema infectiosum (fifth disease of slapped cheeck syndromeJ7,21, polyarthralgia syndrome in adults or hydrops fetalis and hepatic iron deposition in the still-born ensuing from intra uterine infection 23 . The human parvovirus B19 is related to several animal parvoviruses, as for instance to agents causing canine and feline panleucopenia, catastrophic epidemias amongst domestic swine and geese, or the immune complex mediated aleutian mink disease. Parvovirus B19 is a rather small virus measuring 15-28 nm in diameter and is composed of single stranded DNA, two or three capsid proteins and at least one noncapsid protein. The virus shows icosahedral symmetry and lacks an envelope 23 . Parvovirus B19 seems to be transmitted via airways. In volunteers intranasal inoculation proved to be effective l6 • Viraemia II follows after 8-9 days, attaining virus concentrations of up to 10 12 viral particles/ml acute phase serum l7 . In this period, the virus is shed from the upper repiratory tract and into the urine. With the advent of specific IgM antibodies 17 the aeute illness resolves. The prevalence of IgG antibodies in 30-61 % in healthy adults 13 (blood donors)3 points to the fact that parvovirus B19 infections rank among the most widespread viral diseases. One of the salinet features accounting for the phenomenon of acute aplastic crises is the marked erythroblastic

tropism of this virus. In vitro inoculation provokes in erythroblastic burst cells identical cytopathic changes as known to occur in the bone marrow in vivo 22 • Prolifera tion of erythroblasts stops at an early developmental stage. During the aeute phase of the infection, or on in vitro inoculation, the cytopathic property of the virus induces progressive loss of erythroblasts lasting for several days. In the second half or so of the aplastic phase there appear in the bone marrow beside pyknotic regressive forms abnormally large proerysthroblasts - "gigantoproerythroblasts"3 (Fig. 1,2 and 3). Between the fifth and the tenth day after onset of the disease, erythropoiesis quickly re covers (Fig.4) due to humoral immunity while the cultured erythroblasts finally die. Beside the severe erythroblastic aplasia, only rarely combined with a moderate depression of granulocytopoiesis and thrombocytopoiesis 14 , the appearance of a few scattered gigantoproerythroblasts is diagnostic for erythroblastic aplasia of the aeute transient type. As with other virogenic cytopathic lesions this hallmark allows the diagnosis of active parvovirus B19 infection. The erythroblastic nature of these unusual blasts can be proved by the immunocytochemical demonstration of haemoglobin (Fig. 1). The large spherical nuclei of the gigantoproerythroblasts contain prominent, in part band-like nucleoli surrounded by a homogeneously distributed delicate chromatin while the periphery of the nuclei is made up by a narrow rim of condensed heterochromatin. This zonal distribution of chromatin becomes apparent only in thin

Fig. 1. The cyroplasm ob solirary gigantoproeryrhroblasr (arrows) found in acute eryrhroid aplasia srains posirively for haemoglobin. A srrong positiv reaction is also seen in reticular macrophages scavenging red cells. Modified ABC immunostainingl 8 , paraffin section of decalcified iliac crest biopsy obtained from the case reported on in the Quiz. 600 x

Aplastic Crisis in Haemolytic Anaemia du e to Infection Parvovirus B19 . 821

..

•

Fig. 2. Cytological aspect of acute erythroid aplasia. In complete absence of normoblasts there are at least two gigantoproerythroblasts (a rrows), the middle one undergoing cytolytic decay. Smear from sternal puncrure obtaine from a 38-year-old woman suffering from congenital spherocytic anae mia, now experiencing acute febrile illness with severe decompensated anaemia. May-Grünwald-Giemsa stail1, 670 x

Fig. 3. Gigantoproerythroblast with a prominet nucleolus, loosely distributed chromatin and its deeply basophilic cytoplasm. Same ca se as reported on in Fig. 2. May-Grüntwald-Giemsa stain, 1,700 x

822 . H. E. Schaefer

Fig. 4. Recovery from acute erythroid aplasia with normonoblastic rebound hyperplasia. Gigantoproerythroblasts have disappeared. Bone marrow smear obtained onJy fcw days after recovery. Same case as presented in Fig. 2-3. At the time when this sequential bone marrow puncture was done, the reticulocyte count had raised from 1.6 to 5.0%. May-Grünwald-Giemsa stein, 670 x

sections. For technical reasons, in smear prepartions the nuclear structure appears more or less diffusely leptochromie. The cytoplasm stains deeply basophilic (Fig. 2 and 3). Virions have been demonstrated by dect1'on microscopy and immunohistochemistry to be present in the cytoplasm and in the outer rim of the densely marginated nuclear chromatin. As has been demonstrated in vitro, the viruses are replicated in these abnormal erythroblast 22 . Within few days gigantoproerythroblasts completely disappear from the bone marrow and are followed by regenerating normoblasts (Fig. 4). While this pathognomonic type of anomalous erythroblasts is better known from the cytologicalliterature, to the best of our knowledge, histological documents obtained from biopsies have been lacking up to this Quiz report. Thus, the pattern recognition strategy usually applied by pathologists to read a slide did not work, and the Quiz remained unsolved.

References 1 Chernoff Al, ]osepi}son AM (195 1) Acute erythroblastopenia in sickle cel! anemia and infecrious mOllollucleosis. AMA Am] Dis Child 82: 310-322

2 Cloutier MD, Burgerr EO (1966) Congenital nonspheroeytie hemolytic disease secondary to glueose-6-phosphate dehydrogen ase deficiency: report of three cases. Mayo Clin Proc 41: 316-325 .J Cohcn B], Mortimer PP, Pereira MS (1983) Diagnosrie assays with monoclonal antibodies for the human serum parvovirus-like virus (SPLV). J Hyg (Lond) 91 : 113-130 4 Conklin GT, George JN, Sears DA (1974) Transient erythroid aplasia in hemolytie anemia: A review of the literature with two case reports. Texas Rep Biol Med 32: 391-411 5 Cossart YE, Cant B, Field AM, Widdows D (1975) Parvovirus-like particles in human sera. Lancet I: 72-73 6 Dameshek W, Bloom ML (1948) The events in the hemolytie erisis of hereditary spheroeytosis, with particular reference to the reticulocytopenia, pancytopenia and an abnormal splenie meehanism. Blood 3: 1381-1410 7 Davidsoll RK, Brown T, Wiseman D (1984) Human parvovirus infcction and aplastic crisis in hereditary spheroeytosis. ] Infeer 9: 298-300 8 Gasser C (1950) Erythroblastopenie aigue dans les anemies hemolytiques. Sang 21: 237-245 9 Heilmeyer L (1 950) Die hämolytisehen Allämien. Sang 21: 105-141 10 Horne ]L, Kirkpatrick HJR, Lederer H, Leys DG (1945) Fqmilial crises in congenital haemolyric disease. Lancet TI: 33 -36 11 KelleherJF, Luban NLC; Mortimcr PP, Kamimura T (1983) Human serum "parvovirus": A specific cause of aplastic crisis in children with hereditary spherocyrosis. J Pediatr 102: 720-722

Aplastic Crisis in Hacmolytic Anaemia due to Infection Parvovirus B19 . 823 12 Minkowski 0 (1900) Über eine hereditäre, unter dem Bilde eines chronischen Icterus mit Urobilinurie, Splenomegalie und Nierensiderosis verlaufende Affection. Verh Congr Innere Med 18:316-319 13 Mortimer PP, Humphries RK, Moore JG, PureeIl RH, Young NS (1983) A human parvovirus-like virus inhibits haematopoietic colony formation in vitro. Nature 302: 426-429 14 Owren PA (1948) Congenital hemolytic jaundiee. The pathogenesis of the "hemolytic crisis" Blood 3: 231-248 15 Pattisan ]R, ]ones SE, Hodgson ], Davis LR, White JM, Stround CE, Murtaza L (1981) Parvovirus infections and hypoplastic crisis in sickle-cell anaemia. Laneet 1: 664-665 16 Potter CG, Potter AC, Hatton CSR, Chapel HM, Anderson Mj, Pattison ]R, Tyrell DA], Higgins PG, Will man JS, Parry HF, Cotes PM (1987) "Variation of erythroid and myeloid precursors in the marrow and peripheral blood of volunteer subjects infected with human parvovirus (B19).] Clin Invest 79: 1486-1492 17 Saarinen UM, Chorba TL, Tattersall P, Young NS, Anderson L], Palmer E, Coccia PF (1986) Human parvovirus ß19-

induced epidemie aeute red cell aplasia in patients with hereditary hemolytic anemia. Blood 67: 1411-1417 18 Schaefer HE (1989) Unusual blasts in bone marrow. Path Res Pract 185: 533-535 19 Schaefer HE (1984) Methoden zur histologischen, zytologischen und zytochemischen Diagnostik von Blut und Knochenmark. In: Pathologie. Remmele W (Ed), Springer-Verlag. Voll; 435-452. Berlin 20 Serjeant GR, Mason K, Topley JM, Serjeant BE (1981) Outbreak of aplastic crises in sickle eell anaemia associated with parvovirus-like agent. Laneet 11: 595-597 2\ Shneerson JM, Mortimer PP, van der Velde EM (1980) Febrile illness duc to a parvovirus. Br Med ] 280: 1580 22 Young N, Harrison M, Moore J, Mortimer Ph, Humphries RK (1984) Direct demonstration of the human parvovirus in erythroid progenitor cells infected in vitro. J Clin luvest 74: 2024-2032 23 Young N (1988) Hcmatologic and hematopoietic consequences of B19 parvovirus infection. Semin Hematol 25: 159-172

Author's address: Prof. Dr. Hans-Eckart Schaefer, Pathologisches Institut der Universität Freiburg, Albertstr. 9, D-W-7800 Freiburg, FRG

Remark of the Editor: Unfortunately we did not receive any satisfactory diagnoses and answers to the three qucstiol1S.