Scand J Haematol(l975) 15,261-271
Congenital Dyserythropoietic Anaemia with Peculiar Nuclear Abnormality
s.WEISS, M.D., u. GAFTER,M.D., E. VAN DER LYN &
M. DJALDETTI, M.D.
Department of Internal Medicine ‘B’, Hematology Clinic and Electron Microscope Unit, (Chief, M . Djaldetti), Hasharon Hospital, Petah - Tiqva and Tel-Aviv University Medical School, Israel
A patient with clinical and haematological findings compatible with congenital dyserythropoietic anaemia (CDA) most probably type I1 is described. In contradistinction to previous observations, electron microscope examination of the erythroid cells in the bone marrow showed the concomitant appearance of nuclei at different maturation stages in a single cell. In addition, a marked tendency toward multinuclearity of the megakaryocytes was found. The mature erythroblasts showed an increased uptake of 3H-uridine, while the proerythroblasts revealed an uptake of 3H-thymidine less than is normally found in these cells. Thes findings suggest an impairment in RNA and DNA synthesis. Key words: congenital dyserythropoietic anaemia - nuclear abnormality uridine and thymidine uptake
Accepted for publication August 30, 1975 Correspondence to: Prof. Meir Djaldetti, Department of Medicine ‘B’, Hasharon Hospital, Petah-Tiqva, Israel
In recent years there has been an increased number of reports concerning the congenital dyserythropoietic anaemias (CDA). Several types of this disorder have been described: type I (Heimpel et a1 1971, Queisser et a1 1971, Lewis et a1 1972, Meuret et a1 1972, Maldonado & Taswell 1974, Bethlenfalvay 1974), type I1 (HEMPAS) (Valentine et a1 1972, Hug et a1 1972, Wong et a1 1972, Verwilghen et a1 1973, Mouriquand et a1 1973), type I11 (Goudsmit et a1 1972) and combined types (Morgenstern et a1 1973, Dvilansky et a1 1974). The various types are distinguished by specific haematological and ultrastructural features.
In the present report, we describe functional studies and the ultrastructure of the erythroblasts in a patient with CDA, most probably type 11; in addition to the findings typical for this disorder, two nuclei were foand to appear at different maturation stages in a single cell. The megakaryocytes also showed a striking tendency to multinuclearity. CASE REPORT A 42-year-old patient of Iraqi origin, married, without children, was admitted to another hospital in 1970 because of fever and weakness. Physical examination at that time showed pallor, palpable
262
S. WEISS, U. GAFTER, E. VAN DER LYN ’& M. DJALDETTI
liver 2-3 cm below the costal margin and mild splenomegaly. The pertinent laboratory findings Radioautography were: Hb 9-10 g per 100 ml; haematocrit, 27 %; Bone marrow cells were resuspended in autologous reticulocytes, 0.1-0.2 %. Aspiration bone marrow plasma and counted. To 1 ml of cell suspension biopsy revealed hyperplasia of the red cell series containing 17 X lo6 cells was added 50 pCi of with the presence of multinucleated normoblasts. 3HH-uridine(spec. act. 2.9 mCi/m Mol) or 50 pCi The patient was diagnosed as suffering from Di- 3H-thymidine (spec. act. 5 Ci/m Mol), both obGuglielmo disease and was discharged without tained from the Radiochemical Centre Amersham, treatment. During the next few years he felt well, England. The cell suspensions were incubated for although somewhat weak and pale, but without 60 and 30 min respectively, at 37” C in a moist jaundice. atmosphere containing 5 % CO,. The cells were On June 1974 he was admitted to our Depart- then washed three times in autologous plasma, ment because of weakness and pallor. There was smeared on glass slides and prepared for radiono family history of jaundice or anaemia. On phy- autography (Djaldetti et a1 1970). The results are sical examination the patient was found to be in expressed as mean grain count per cell. At least good general condition, pale, without jaundice. The 100 cells from each maturation stage were counted. liver was palpated 3 cm and the spleen 6 cm below the costal margin. Laboratory examinations showed: the patient’s Electron microscopy blood type was 0, Rh positive, Hb 9.3 g per 100 Bone marrow was immediately fixed in cold, 1 % ml; haematocrit, 27 %; reticulocytes, 3.1 %; WBC, glutaraldehyde in phosphate buffer, pH 7.4, post6,400 per pl with a normal differential count; fixed in osmium tetroxide, dehydrated in graded platelet count, 170,000 per pl. The peripheral blood alcohols and embedded in Epon 812. Thin sections smear showed hypochromia, anisocytosis and poiki- were cut with an LKB ultratome 111 and examined locytosis. Aspiration biopsy of the bone marrow with a Philips 300 electron microscope. revealed hyperplasia of the red blood series; 75 % of the erythroblasts possessed one nucleus, 22 %, two and 3 %, three or more nuclei. In some of the RESULTS multinucleated cells, the presence of nuclei at different maturation stages was observed. (Figures 1 Incorporation of 3H-uridine and 3H-thymiand 2). The osmotic and mechanical fragilities of dine in the patient’s and control erythroblasts the red blood cells were normal; serum iron was is compared in Table I. Uptake of 3H-uridine 67 pg per 100 ml and TIB 206 ,ug per 100 ml. was similar in the case of the proerythroTotal serum bilirubin 1.0 mg per 100 ml; vitamin O.l), but a higher uptake was B,, 542 p g per ml, red cells folate content, 320 ng/ blasts (P found in the patient’s basophilic, poly- and ml of packed cells, plasma Hb 2.3 mg per 100 ml. Haemoglobin electrophoresis gave normal findings; orthochromatic erythroblasts (P 0.001). 24 hs stercobilinogen excretion ranged from 368The patient’s proerythroblasts showed a low1347 mg. (Norman range 140-180 mg). The red er uptake of 3H-thymidine than in the corcell half life examined by labelling with W r was responding control (p O.Ol), while the up29 days. Direct and indirect Coombs tests gave take in the basophilic, and the poly- and negative results. The Ham test gave negative findings with the patient’s own serum but was positive orthochromatic erythroblasts was similar in in 5 out of 10 cases using normal acidified sera. both samples (P 0.1 and P 0.9 respecPatient’s red cells were lysed by anti-i serum with tively). The labelling index of patient’s a titer of 1:80.
>
erythroblasts was higher in the patient’s sample than in the control. In Table 11, uptake of 3H-uridine and 3Hthymidine in the nuclei of mono- and binucleated erythroblasts is presented. The re-
CONGENITAL ANAEMIA WlTH NUCLEAR ABNORMALITY
263
TABLE I Uptake of 3H-uridine and 3H-thymidine in patient's (P) and control's (C) erythroblasts' and their labelling index -n-uiiuine u p i a ~ e
Type of cells
l
-n-mymlume
c
Proerythroblasts
60.26 f 25.10
56.06 f9.23
64.40k 25.10
78.04 f 30.10
92
80
Basophilic erythroblasts
25.08 f 10.90
16.61 k 5.56
42.96f 20.30
48.32f 19.10
71
65
Poly- and orthochromatic erythroblasts
11.46f 7.99
6.89 f 5.56
25.68 k 17.90
26.12 k 12.80
19
9
*
Average grains per cell.
sults are expressed as average grain number per nucleus. For both radioactive precursors, there was no difference in uptake by the basophilic erythroblasts (P > 0.9), while for the more mature erythroblasts, the nuclei of binucleated cells displayed a lower uptake than the nuclei of mononuclear cells at identical maturation stages (P > 0.01). Electron microscopy
nuclearity appeared first at the stage of basophilic erythroblasts, when most of the cells were binucleated (Figure 3), the nucleus still showing normal structure. Occasionally, basophilic erythroblasts with 4 nuclei were seen (Figure 4). With advance in maturation, the tendency to multinuclearity increased and polychromatophilic erythroblasts with 4 nuclei (Figure 5 ) or orthochromatic erythroblasts with more than 10 nuclei (Figure 6 ) were found. The nuclei of
The proerythroblasts did not show ultrastructural alterations. The tendency to multi-
Figure 1. Light micrograph of an erythroblast. Nuclei are visible at different maturation stages. t x 1,000)
Figure 2. Light micrograph of an erythroblast possessing four nuclei of which two pairs are at different maturation stages. (x 1,000)
S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
264
TABLE I1 Uptake of JH-uridine and 3H-thymidine in patient’s mono- and binucleated erythroblasts (Average grains per cell)
3H-uridine uptake Type of cells
~
one nucleus
I
3H-thymidine uptake
binucleated
one nucleus
I
binucleated
~
Basophilic erythroblasts
25.08 k 10.90
24.34 k 14.59
42.96 k20.30
47.04 k 25.04
Poly- and orthochromatic erythroblasts
11.46k 7.99
7.98k 5.60
25.68 f 17.90
17.14f13.60
the latter type of cells showed marked irregularity and a ‘spongy’ appearance, with wide nuclear pores (Figures 7 and 8). Myeline formations were detected in the nucleus (Figure 9), as well as in the cytoplasm. In some cases, there were cells containing nuclei at different maturation stages, such as nuclei typical for baso- and polychromatophilic erythroblasts or poly- and orthochromatic erythroblasts (Figures 10 and 11). Marked deposition of iron was seen both in the mitochondria and the cytoplasm (Figure 7). Centrioles located in pairs were found (Figure 12). Occasionally, membranous formations crisscrossing the nucleus and the
Figure 3. Electron micrograph of basophilic erythroblast with two nuclei. (X 7,700)
cytoplasm were seen. In some of the erythroblasts, numerous tubular formations were detected (Figure 13). The megakaryocytes showed a marked tendency to multinuclearity, the nuclear lobes displaying striking irregularity (Figure 14). DISCUSSION
The patient possessed the clinical and haematological features characteristic of CDA type I1 (Valentine et a1 1972, Bright et a1 1972, Hug et a1 1972, Wong et a1 1972, Enquist et a1 1972, Venvilghen et a1 1973). The anaemia was moderated and macro-
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
Figure 4. Basophilic erythroblast possessing four nuclei. (x 5,130)
Figure 5. Polychromatophilic erythroblasts displaying four nuclei. Note the wide nuclear pores and the iron deposits in the cytoplasm. (x 9,540)
265
266
S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
cytic, there was mild splenomegaly, slightly elevated indirect bilirubin and low haptoglobin. The high serum iron level and increased stercobilinogen excretion suggest ineffective erythropoiesis. The shortened life span of the red blood cells could be compatible with an accompanying haemolytic pro-
Figure 6. Excessive polynuclearity in a polychromatophilic erythroblast. ( x 7,730)
Figure 7. Polychromatophilic erythroblast with marked irregularity and ‘spongy’ appearance of the nucleus. Wide nuclear pores permit ‘invasion’ of the cytoplasm into the nucleus, which shows a ‘spongy’ appearance. (x 9,370)
cess. It has been shown that in CDA type 11, the anti-i and anti-I tests as well as the Ham test using acidified serum from normal donors (Bright et a1 1972, Lewis & Venvilghen 1972, Lewis et a1 1972) are positive. The patient’s serum was positive for anti-i and anti-I antigens, although the score of the
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
Figure 8. Orthochromatic erythroblasts with irregular nucleus. The cytoplasm shows a vacuole containing most probably degenerated haemoglobin. ( x 13,800)
Figure 9. Myeline figures and membranous formations in the nucleus of a polychromatophilic erythroblast. The nucleus displays wide nuclear pores. ( x 51,120)
267
268
S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
reaction was not convincingly high as is found in CDA type I1 (HEMPAS). Slightly increased agglutinability by anti-i was found in diverse forms of dyshaemopoietic anaemias (Venvilghen et a1 1973).
Figure 10. Erythroblast containing nuclei typical of the basophilic (lower) and polychromatophilic (upper) stage. (x 14,000)
Figure 11. Binucleated erythroblast with nuclei at different maturation stages. (x 9,450)
The fact that the Ham test showed positive results with 5 out of 10 acidified normal sera, suggests the possibility that the anaemia is type 11. The electron microscopic findings in the
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
erythroblasts are more characteristic for CDA type I1 (Hug et a1 1972, Wong et a1 1972, Mouriquand et a1 1973) and allow the differentiation to be made from type I (Heimpel et a1 1971, Meuret et a1 1972,
269
Maldonado & Taswell 1974). They include the multinucleartity and the multiple nuclear protrusions merging with the haemoglobin in the cytoplasm. On the other hand, the cells did not show the perimembranous cysternae
Figure 12. Two pairs of centrioles in a polychromatophilic erythroblast. (x 17,440)
Figure 13. Multiple tubular formations in an erythroblast. (x 5,200)
270
S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
which are characteristic of CDA type 11. The unusual observations in this case are the appearance of multinuclearity in the immature erythroblasts and the finding of nuclei at various maturation stages in a single cell. These findings most probably indicate a disturbed coordination of DNA synthesis and of the cell cycle, which results also in the appearance of hypertetraploid and octoploid cells (Queisser et a1 1971, Meuret et a1 1972). Heimpel et a1 (1971) reported a case of CDA type I in which double nucleated cells with nuclei of different size, chromatin structure and staining ability were demonstrated by light microscopy. Another peculiar feature in the present case, concerning the ultrastructural findings, was the tendency to multinuclearity of the megakaryocytes. This suggests a common defect in DNA synthesis in cells different from the red blood series. The 3H-uridine incorporation experiments showed statistically significant differences in the uptake of radioactive material in the haemoglobin producing erythroblasts of the patient as compared to the corresponding
Figure 14. Early megakaryocyte with a tendency to multinuclearity. (x 7,270)
cells. Since haemoglobin and RNA-values in maturing erythroblasts are negatively correlated (Thorell 1967, Djaldetti et a1 1970) it may be concluded that there is impaired RNA synthesis in the patient’s maturing erythroblasts, with subsequent delay in haemoglobin synthesis. This observation differs from the findings of Meuret et a1 (1972) in patients with CDA type I, in whom a high proportion of the erythroblasts showed only traces of RNA, or no RNA at all. The uptake of 3H-thymidine was lower in the patient’s proerythroblasts than in the corresponding control cells, whereas in the mature forms there was no difference, although the labelling index was higher in cells at all maturation stages. These findings are consistent with the observations of Queisser et a1 (1971) and Meuret et a1 (1972) who showed that, in CDA type 11, only erythroblasts with minimal haemoglobin content incorporated thymidine in vitro. On the other hand, these authors found an increased number of diploid unlabelled polychromatophilic cells and a reduction in the labelling index, which was not observed in our case.
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
The lower incorporation of both 3H-uridine and 3H-thymidine in the nuclei of binucleated cells as compared with their uptake by the nuclei of mononucleated cells is an additional evidence for impaired RNA and DNA synthesis in these cells.
ACKNOWLEDGEMENTS Our thanks are given to Prof. S. Levine of the Central Laboratories, Ministry of Health, Jerusalem for the performance of the serological studies. The technical assistance of Mrs. Fishman and the photographic work of Mr. Sadovnik are greatly appreciated. This work was supported by a grant from the Chief Scientist’s Bureau, Ministry of Health.
REFERENCES Bethlenfalvay N C (1974) Letter to the editor. Blood, 43, 155. Bright M, Cobb J, Evans B & Parry T E (1972) Congenital dyserythropoietic anaemia with erythroblastic multinuclearity. J Clin Pathol 25, 561-69. Djaldetti M, Chui D, Marks P A & Rifkind R A (1970) Erythroid cell development in fetal mice. Stabilization of the haemoglobin synthetic capacity. J. Mol Biol 50, 345-58. Dvilansky A, Sukenik S, Stern J & Djaldetti M (1974) Congenital dyserythropoietic anaemia with ultrastructure findings compatible with both types I and 11. Acta Haematol (Basel) 52, 161-68. Enquist R W, Gockerman J P, Jenis E H, Warkel R L I& Dillon D E (1972) Type I1 congenital dyserythropoietic anaemia. A n n Znt Med 77, 371-76. Goudsmit R, Beckers D, de Bruijne J I, Engelfriet C P, James J, Morselt A F W & Reynierse E (1972) Congenital dyserythropoietic anaemia, type 111. Br J Haematol 23, 97-105. Heimpel H, Forteza-Vila J, Queisser W & Spiertz E (1971) Electron and light microscopic study of the erythroblasts of patients with congenital dyserythropoietic anaemia. Blood 37, 299-310.
271
Hug G, Wong K Y & Lampkin B C (1972) Congenital dyserythropoietic anaemia type 11. Ultrastructure of erythroid cells and hepatocytes. Lab Invest 28, 11-21. Lewis S M, Nelson D A & Pitcher C S (1972) Clinical and ultrastructural aspects of congenital dyserythropoietic anaemia type J. Br J Haematol 23, 113-19. Lewis S M & Verwilghen R L (1972) Dyserythropoiesis and dyserythropoietic anaemia. Br J Haematol 23, 1-3. Maldonado J E I& Taswell H F (1974) Type I Dyserythropoietic anemia in an elderly patient. Blood 44, 495-500. Meuret G, Tschan P, Schliiter G, Keyserlingk D G & Boll I (1972) DNA - , histone - , RNA - , hemoglobin - content and DNA - synthesis in erythroblasts in a case of congenital dyserythropoietic anemia type I. Blut 24, 32-41. Morgenstern E, Schatanek W, Meiser J R & Hufnag1 D (1973) Ultrastructural studies in a particular case of congenital dyserythropoietic anemia (CDA). Blut 27, 307-21. Mouriquand C, Bachelot C, Lovis J, Beaudoing A & Berthier R (1973) Dyserythropoitse congknitale de type I1 avec anBmie hCmolytique nBonatale chez deux soeurs: Btude hBmatologique et ultrastructurale. Nouv Rev Fr Hematol 13, 85772. Queisser W, Spiertz E, Jost E i& Heimpel H (1971) Proliferation disturbances of erythroblasts in congenital dyserythropoietic anemia type I and 11. Acta Haematol (Basel) 45, 65-76. Thorell B (1947) Studies on the formation of cellular substances during blood cell production Kimpton. London. Valentine W N, Crookston J H, Paglia D E & Konrad P N (1972) Erythrocyte enzymatic abnormalities in HEMPAS (hereditary erythroblastic multinuclearity with a positive acidified - serum test) Br J Haernatol 23, 107-12. Verwilghen R L, Lewis S M, Dacie J V, Crookston J H & Crookston M C (1973) HEMPAS: Congenital dyserythropoietic anaemia (type 11). Quart J M e d 62, 257-78. Wong K Y,Hug G Lk Lampkin B C (1972) Congenital dyserythropoietic anemia type 11; ultrastructural and radioautographic studies of blood and bone marrow. Blood 39, 23-30.
Congenital Dyserythropoietic Anaemia with Peculiar Nuclear Abnormality
s.WEISS, M.D., u. GAFTER,M.D., E. VAN DER LYN &
M. DJALDETTI, M.D.
Department of Internal Medicine ‘B’, Hematology Clinic and Electron Microscope Unit, (Chief, M . Djaldetti), Hasharon Hospital, Petah - Tiqva and Tel-Aviv University Medical School, Israel
A patient with clinical and haematological findings compatible with congenital dyserythropoietic anaemia (CDA) most probably type I1 is described. In contradistinction to previous observations, electron microscope examination of the erythroid cells in the bone marrow showed the concomitant appearance of nuclei at different maturation stages in a single cell. In addition, a marked tendency toward multinuclearity of the megakaryocytes was found. The mature erythroblasts showed an increased uptake of 3H-uridine, while the proerythroblasts revealed an uptake of 3H-thymidine less than is normally found in these cells. Thes findings suggest an impairment in RNA and DNA synthesis. Key words: congenital dyserythropoietic anaemia - nuclear abnormality uridine and thymidine uptake
Accepted for publication August 30, 1975 Correspondence to: Prof. Meir Djaldetti, Department of Medicine ‘B’, Hasharon Hospital, Petah-Tiqva, Israel
In recent years there has been an increased number of reports concerning the congenital dyserythropoietic anaemias (CDA). Several types of this disorder have been described: type I (Heimpel et a1 1971, Queisser et a1 1971, Lewis et a1 1972, Meuret et a1 1972, Maldonado & Taswell 1974, Bethlenfalvay 1974), type I1 (HEMPAS) (Valentine et a1 1972, Hug et a1 1972, Wong et a1 1972, Verwilghen et a1 1973, Mouriquand et a1 1973), type I11 (Goudsmit et a1 1972) and combined types (Morgenstern et a1 1973, Dvilansky et a1 1974). The various types are distinguished by specific haematological and ultrastructural features.
In the present report, we describe functional studies and the ultrastructure of the erythroblasts in a patient with CDA, most probably type 11; in addition to the findings typical for this disorder, two nuclei were foand to appear at different maturation stages in a single cell. The megakaryocytes also showed a striking tendency to multinuclearity. CASE REPORT A 42-year-old patient of Iraqi origin, married, without children, was admitted to another hospital in 1970 because of fever and weakness. Physical examination at that time showed pallor, palpable
262
S. WEISS, U. GAFTER, E. VAN DER LYN ’& M. DJALDETTI
liver 2-3 cm below the costal margin and mild splenomegaly. The pertinent laboratory findings Radioautography were: Hb 9-10 g per 100 ml; haematocrit, 27 %; Bone marrow cells were resuspended in autologous reticulocytes, 0.1-0.2 %. Aspiration bone marrow plasma and counted. To 1 ml of cell suspension biopsy revealed hyperplasia of the red cell series containing 17 X lo6 cells was added 50 pCi of with the presence of multinucleated normoblasts. 3HH-uridine(spec. act. 2.9 mCi/m Mol) or 50 pCi The patient was diagnosed as suffering from Di- 3H-thymidine (spec. act. 5 Ci/m Mol), both obGuglielmo disease and was discharged without tained from the Radiochemical Centre Amersham, treatment. During the next few years he felt well, England. The cell suspensions were incubated for although somewhat weak and pale, but without 60 and 30 min respectively, at 37” C in a moist jaundice. atmosphere containing 5 % CO,. The cells were On June 1974 he was admitted to our Depart- then washed three times in autologous plasma, ment because of weakness and pallor. There was smeared on glass slides and prepared for radiono family history of jaundice or anaemia. On phy- autography (Djaldetti et a1 1970). The results are sical examination the patient was found to be in expressed as mean grain count per cell. At least good general condition, pale, without jaundice. The 100 cells from each maturation stage were counted. liver was palpated 3 cm and the spleen 6 cm below the costal margin. Laboratory examinations showed: the patient’s Electron microscopy blood type was 0, Rh positive, Hb 9.3 g per 100 Bone marrow was immediately fixed in cold, 1 % ml; haematocrit, 27 %; reticulocytes, 3.1 %; WBC, glutaraldehyde in phosphate buffer, pH 7.4, post6,400 per pl with a normal differential count; fixed in osmium tetroxide, dehydrated in graded platelet count, 170,000 per pl. The peripheral blood alcohols and embedded in Epon 812. Thin sections smear showed hypochromia, anisocytosis and poiki- were cut with an LKB ultratome 111 and examined locytosis. Aspiration biopsy of the bone marrow with a Philips 300 electron microscope. revealed hyperplasia of the red blood series; 75 % of the erythroblasts possessed one nucleus, 22 %, two and 3 %, three or more nuclei. In some of the RESULTS multinucleated cells, the presence of nuclei at different maturation stages was observed. (Figures 1 Incorporation of 3H-uridine and 3H-thymiand 2). The osmotic and mechanical fragilities of dine in the patient’s and control erythroblasts the red blood cells were normal; serum iron was is compared in Table I. Uptake of 3H-uridine 67 pg per 100 ml and TIB 206 ,ug per 100 ml. was similar in the case of the proerythroTotal serum bilirubin 1.0 mg per 100 ml; vitamin O.l), but a higher uptake was B,, 542 p g per ml, red cells folate content, 320 ng/ blasts (P found in the patient’s basophilic, poly- and ml of packed cells, plasma Hb 2.3 mg per 100 ml. Haemoglobin electrophoresis gave normal findings; orthochromatic erythroblasts (P 0.001). 24 hs stercobilinogen excretion ranged from 368The patient’s proerythroblasts showed a low1347 mg. (Norman range 140-180 mg). The red er uptake of 3H-thymidine than in the corcell half life examined by labelling with W r was responding control (p O.Ol), while the up29 days. Direct and indirect Coombs tests gave take in the basophilic, and the poly- and negative results. The Ham test gave negative findings with the patient’s own serum but was positive orthochromatic erythroblasts was similar in in 5 out of 10 cases using normal acidified sera. both samples (P 0.1 and P 0.9 respecPatient’s red cells were lysed by anti-i serum with tively). The labelling index of patient’s a titer of 1:80.
>
erythroblasts was higher in the patient’s sample than in the control. In Table 11, uptake of 3H-uridine and 3Hthymidine in the nuclei of mono- and binucleated erythroblasts is presented. The re-
CONGENITAL ANAEMIA WlTH NUCLEAR ABNORMALITY
263
TABLE I Uptake of 3H-uridine and 3H-thymidine in patient's (P) and control's (C) erythroblasts' and their labelling index -n-uiiuine u p i a ~ e
Type of cells
l
-n-mymlume
c
Proerythroblasts
60.26 f 25.10
56.06 f9.23
64.40k 25.10
78.04 f 30.10
92
80
Basophilic erythroblasts
25.08 f 10.90
16.61 k 5.56
42.96f 20.30
48.32f 19.10
71
65
Poly- and orthochromatic erythroblasts
11.46f 7.99
6.89 f 5.56
25.68 k 17.90
26.12 k 12.80
19
9
*
Average grains per cell.
sults are expressed as average grain number per nucleus. For both radioactive precursors, there was no difference in uptake by the basophilic erythroblasts (P > 0.9), while for the more mature erythroblasts, the nuclei of binucleated cells displayed a lower uptake than the nuclei of mononuclear cells at identical maturation stages (P > 0.01). Electron microscopy
nuclearity appeared first at the stage of basophilic erythroblasts, when most of the cells were binucleated (Figure 3), the nucleus still showing normal structure. Occasionally, basophilic erythroblasts with 4 nuclei were seen (Figure 4). With advance in maturation, the tendency to multinuclearity increased and polychromatophilic erythroblasts with 4 nuclei (Figure 5 ) or orthochromatic erythroblasts with more than 10 nuclei (Figure 6 ) were found. The nuclei of
The proerythroblasts did not show ultrastructural alterations. The tendency to multi-
Figure 1. Light micrograph of an erythroblast. Nuclei are visible at different maturation stages. t x 1,000)
Figure 2. Light micrograph of an erythroblast possessing four nuclei of which two pairs are at different maturation stages. (x 1,000)
S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
264
TABLE I1 Uptake of JH-uridine and 3H-thymidine in patient’s mono- and binucleated erythroblasts (Average grains per cell)
3H-uridine uptake Type of cells
~
one nucleus
I
3H-thymidine uptake
binucleated
one nucleus
I
binucleated
~
Basophilic erythroblasts
25.08 k 10.90
24.34 k 14.59
42.96 k20.30
47.04 k 25.04
Poly- and orthochromatic erythroblasts
11.46k 7.99
7.98k 5.60
25.68 f 17.90
17.14f13.60
the latter type of cells showed marked irregularity and a ‘spongy’ appearance, with wide nuclear pores (Figures 7 and 8). Myeline formations were detected in the nucleus (Figure 9), as well as in the cytoplasm. In some cases, there were cells containing nuclei at different maturation stages, such as nuclei typical for baso- and polychromatophilic erythroblasts or poly- and orthochromatic erythroblasts (Figures 10 and 11). Marked deposition of iron was seen both in the mitochondria and the cytoplasm (Figure 7). Centrioles located in pairs were found (Figure 12). Occasionally, membranous formations crisscrossing the nucleus and the
Figure 3. Electron micrograph of basophilic erythroblast with two nuclei. (X 7,700)
cytoplasm were seen. In some of the erythroblasts, numerous tubular formations were detected (Figure 13). The megakaryocytes showed a marked tendency to multinuclearity, the nuclear lobes displaying striking irregularity (Figure 14). DISCUSSION
The patient possessed the clinical and haematological features characteristic of CDA type I1 (Valentine et a1 1972, Bright et a1 1972, Hug et a1 1972, Wong et a1 1972, Enquist et a1 1972, Venvilghen et a1 1973). The anaemia was moderated and macro-
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
Figure 4. Basophilic erythroblast possessing four nuclei. (x 5,130)
Figure 5. Polychromatophilic erythroblasts displaying four nuclei. Note the wide nuclear pores and the iron deposits in the cytoplasm. (x 9,540)
265
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S. WEISS, U. GAFTER, E. VAN DER LYN & M. DJALDETTI
cytic, there was mild splenomegaly, slightly elevated indirect bilirubin and low haptoglobin. The high serum iron level and increased stercobilinogen excretion suggest ineffective erythropoiesis. The shortened life span of the red blood cells could be compatible with an accompanying haemolytic pro-
Figure 6. Excessive polynuclearity in a polychromatophilic erythroblast. ( x 7,730)
Figure 7. Polychromatophilic erythroblast with marked irregularity and ‘spongy’ appearance of the nucleus. Wide nuclear pores permit ‘invasion’ of the cytoplasm into the nucleus, which shows a ‘spongy’ appearance. (x 9,370)
cess. It has been shown that in CDA type 11, the anti-i and anti-I tests as well as the Ham test using acidified serum from normal donors (Bright et a1 1972, Lewis & Venvilghen 1972, Lewis et a1 1972) are positive. The patient’s serum was positive for anti-i and anti-I antigens, although the score of the
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
Figure 8. Orthochromatic erythroblasts with irregular nucleus. The cytoplasm shows a vacuole containing most probably degenerated haemoglobin. ( x 13,800)
Figure 9. Myeline figures and membranous formations in the nucleus of a polychromatophilic erythroblast. The nucleus displays wide nuclear pores. ( x 51,120)
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reaction was not convincingly high as is found in CDA type I1 (HEMPAS). Slightly increased agglutinability by anti-i was found in diverse forms of dyshaemopoietic anaemias (Venvilghen et a1 1973).
Figure 10. Erythroblast containing nuclei typical of the basophilic (lower) and polychromatophilic (upper) stage. (x 14,000)
Figure 11. Binucleated erythroblast with nuclei at different maturation stages. (x 9,450)
The fact that the Ham test showed positive results with 5 out of 10 acidified normal sera, suggests the possibility that the anaemia is type 11. The electron microscopic findings in the
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
erythroblasts are more characteristic for CDA type I1 (Hug et a1 1972, Wong et a1 1972, Mouriquand et a1 1973) and allow the differentiation to be made from type I (Heimpel et a1 1971, Meuret et a1 1972,
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Maldonado & Taswell 1974). They include the multinucleartity and the multiple nuclear protrusions merging with the haemoglobin in the cytoplasm. On the other hand, the cells did not show the perimembranous cysternae
Figure 12. Two pairs of centrioles in a polychromatophilic erythroblast. (x 17,440)
Figure 13. Multiple tubular formations in an erythroblast. (x 5,200)
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which are characteristic of CDA type 11. The unusual observations in this case are the appearance of multinuclearity in the immature erythroblasts and the finding of nuclei at various maturation stages in a single cell. These findings most probably indicate a disturbed coordination of DNA synthesis and of the cell cycle, which results also in the appearance of hypertetraploid and octoploid cells (Queisser et a1 1971, Meuret et a1 1972). Heimpel et a1 (1971) reported a case of CDA type I in which double nucleated cells with nuclei of different size, chromatin structure and staining ability were demonstrated by light microscopy. Another peculiar feature in the present case, concerning the ultrastructural findings, was the tendency to multinuclearity of the megakaryocytes. This suggests a common defect in DNA synthesis in cells different from the red blood series. The 3H-uridine incorporation experiments showed statistically significant differences in the uptake of radioactive material in the haemoglobin producing erythroblasts of the patient as compared to the corresponding
Figure 14. Early megakaryocyte with a tendency to multinuclearity. (x 7,270)
cells. Since haemoglobin and RNA-values in maturing erythroblasts are negatively correlated (Thorell 1967, Djaldetti et a1 1970) it may be concluded that there is impaired RNA synthesis in the patient’s maturing erythroblasts, with subsequent delay in haemoglobin synthesis. This observation differs from the findings of Meuret et a1 (1972) in patients with CDA type I, in whom a high proportion of the erythroblasts showed only traces of RNA, or no RNA at all. The uptake of 3H-thymidine was lower in the patient’s proerythroblasts than in the corresponding control cells, whereas in the mature forms there was no difference, although the labelling index was higher in cells at all maturation stages. These findings are consistent with the observations of Queisser et a1 (1971) and Meuret et a1 (1972) who showed that, in CDA type 11, only erythroblasts with minimal haemoglobin content incorporated thymidine in vitro. On the other hand, these authors found an increased number of diploid unlabelled polychromatophilic cells and a reduction in the labelling index, which was not observed in our case.
CONGENITAL ANAEMIA WITH NUCLEAR ABNORMALITY
The lower incorporation of both 3H-uridine and 3H-thymidine in the nuclei of binucleated cells as compared with their uptake by the nuclei of mononucleated cells is an additional evidence for impaired RNA and DNA synthesis in these cells.
ACKNOWLEDGEMENTS Our thanks are given to Prof. S. Levine of the Central Laboratories, Ministry of Health, Jerusalem for the performance of the serological studies. The technical assistance of Mrs. Fishman and the photographic work of Mr. Sadovnik are greatly appreciated. This work was supported by a grant from the Chief Scientist’s Bureau, Ministry of Health.
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