BritishJoumal OfHaematology, 1977,35,613.
Granulocytic Progenitor Cells in Aplastic Anaemia P. KERN,H. HEIMPEL, W. HEIT AND B. KUBANEK
Department of Internal Medicine and Pediatrics, Division of Haematology, University of Ultn, UIm/Dotiau, Gerttrany (Received 23 August 1976; acceptedfir publication 13 September 1976) SUMMARY. The characteristics and the concentration of granulopoietic colony forming cells (CFC) were examined in 22 patients with aplastic anaemia at different stages of their disease. Additionally the ability of the patients’ peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (CSA) was studied. The ability of the patients’ cells to generate CSA was shown to be unaffected. However, the incidence of CFC within the marrow and peripheral blood suspensions was significantly reduced in all patients. The results suggest a reduced compartment size of CFC even in those patients who have recovered fiom aplastic anaemia. This may indicate that the disturbances in the preceding compartments of the haemopoietic cell renewal system still persist after recovery from the acute bone marrow failure. Aplastic anaemia is defined as an acute or chronic bone marrow failure with fatty replacement of the haemopoietic tissue and peripheral pancytopenia. This condition may be regarded as the nonspecific result of disturbances in the preceding Compartments of the haemopoietic cell renewal system. The pathophysiology of these disturbances is largely unknown. Hypothetical mechanisms involve ‘primary’ defects of maturation and proliferation of the haemopoietic stem cells, changes in their stromal and cellular microenvironment and aberrations of their humoral regulation (Heimpel & Kubanek, 197s). The chain of events leading to histological and functional aplasia may be caused or triggered off by external agents, such as chloramphenicol, phenylbutazone or the hepatitis virus. Some insight into the function of the haemopoietic stem cell compartments may be gained by the application of iti vitro methods for the assessment of progenitor cells committed to granulo-monocytopoiesis (Pike & Robinson, 1970). In this study, the characteristics and concentration of the granulopoietic progenitor cells assessed as colony forming cells (CFC) were examined in patients with aplastic anaemia at different stages of their disease as well as the ability of the patients’ peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (colony stimulating activity, CSA). MATERIAL AND METHODS
Patients The diagnosis of aplastic anaemia in 22 patients (17 female, five male) was based on the observation of a hypo- or aplastic marrow in more than one biopsy and a pancytopenia at Correspondence:Dr P. Kern, Clinical Department. Bemhard-Nocht Institute for Nautical and Tropical Diseases, Bernhard Nocht Str. 74. D-2000 Hamburg 4, West Germany.
P. Kern et al
614
some time during the course of the disease. Table I shows the period of observation and the haematological data at the time of in-vitro culture. The age of the patients varied between 12 and 67 years. Four patients were studied twice and one patient three times during followup. Thus, in all, the findings from 28 observations are described in this paper. TABLE I. Haeniatological data of patients with aplastic anaemia at the time of in-vitro culture of bone marrow and peripheral blood Peripheral blood characteristics Follow-up from Case diagnosis Haemoglobin Reticulocytes Platelets Lmcocytes Nos. per pl x 103 per rl x 103 (g/dl) (months) per pl I 2
3 4 5 6 7 8
9 I0 I1 1a
I I
a
4 4 15
9.6 6.8 9.7
9 1 I4 a73
6.6
10.7 13.9
w Ia
5
I8 I8 aa
10.1
0
69
5.2
I8
I3
5.5
1s
10.1 1a.r
5 6
I4
37 40 44 46 54
15
58
16 I7 18
60 78
12.2
24 56 27 39 47 66
6.6
I0
0
6
9.9 12.7
84 170
98 I9 57
I 5 I
8.3 7.1 9.6
a1
I1
0
7 8 4 47
I3
I9 ao
6 a1
I1
8.5
115
I0
13.0
6
9.8 10.6 12.4 12.7
37 n.d. 63
24
aa
15.6 14.1 14.0
71 85
Bone marrow cellularity
I1
38 24 n.d. 27 54 45 I4
8.3
Neutrophils per pl
21
198
69 126 I0
I18
54
327 53
35
81
* Iahomogeneous distribution of the haemopoietic cells within the bone marrow. As aetiological agent chloraniphenicol was suspected in four cases (cases 7, 8, 13 and Is), phenylbutazone in three cases (cases I, 2 and 4) and in one patient (case 3) the aplasia developed during the course of acute viral hepatitis. None of the patients had an enlarged spleen. None of the patients had received chemotherapy for malignant tumours. At the time of culture 18 cases were under treatment for aplastic anaemia, e.g. blood transfixions, anti-
CFC in Aplastic Anaemia 615 biotics, anabolic steroids, or corticosteroids. In 10 cases investigations were made when the patients were recovering or had recovered from aplastic anaemia. As controls we studied the peripheral blood of healthy blood donors, and the marrow specimens of healthy volunteers, donors for bone marrow transplantation and patients undergoing bone marrow examination in the out-patient clinic as part of their diagnostic study. Those who were found to have a morphologically normal bone marrow, normal peripheral cell counts and no evidence of infection were included in this group. The ages of 17 control subjects (12 female, five male) varied from 16 to 63 years. Cell Suspensions and Morphological Examinations Bone marrow was aspirated from the posterior iliac crest. One part of the aspirate was processed for routine morphological examination. The nucleated cell count per ml of the remaining suspension was determined. Red cells were sedimented either by mixing three parts of the cell suspension with one part of 3% Dextran (Pharmacia, Uppsala) in saline or by the method of Hulliger & Blascovec (1967). The supernatant containing the nucleated cells was washed three times in saline, resuspended in McCoys 5A Medium (Gibco) containing 10% fetal calf serum (Biocult). Smears were obtained by means of a cytocentrifuge and a differential count of zoo cells was made on each marrow suspension. At the same time a needle biopsy was performed and the bone marrow cylinder was prepared for histological examination. All available bone marrow smears and histological sections were evaluated by tivo independent observers in order to confirm the diagnosis of aplastic anaemia. The cellularity was judged as aplastic (dd) when the marrow spaces contained less than 10% marrow cells, hypoplastic (d)when the cellularity ranged between 10 and 30%. A normal cellularity was described within the range 3 ~ 5 0 % (n), and hypercellularity when the marrow spaces contained more than 50% of cells. In a number of cases the cellular distribution was found to be inhomogeneous, i.e. hypercellular foci between empty marrow spaces. Heparinized peripheral blood samples were separated using Isopaque-Ficoll-Gradient (Lymphoprep, Nygaard) (Boyum, 1968). Mononuclear cells at the top of the Lymphoprep solution were washed twice in buffered saline and resuspended in McCoy’s gA medium containing 10% fetal calf serum. The polymorphonuclear cells were prepared from the bottom layer of the gradient. A differential count of the peripheral blood smears was performed; on each slide at least 50 neutrophils were counted in order to maintain a constant statistical error.
Agar Cultures The double layer agar culture system was used (Pike & Robinson, 1970). An appropriate concentration of the cells was suspended in modified McCoy’s 5A medium, supplemented with 15% fetal calf serum and 5% horse serum (Biocult) and purified agar (Difco) resulting in a final agar concentration of 0.3% for the upper layers and 0.55% for the feeder layers. Aliquots of I ml were plated in 3 5 nim petri dishes (Greiner). Colony stimulating activity was provided by feeder layers of a defined leucocyte composition (Heit et al, 1974), in order to obtain optimal colony stimulation, When evaluating the ability of the patients’ leucocytes to stimulate normal CFC, normal marrow suspensions were used as target cell suspensions.
616
P. Kern et a1
Two to three cell concentrations of the bone marrow and the peripheral blood specimens were plated in the upper layers. Duplicate or triplicate cultures of each cell concentration were incubated in sterile air-tight glass containersat 37.s°C, 5% carbon dioxide and high humidity. After 10 d all aggregates of more than five cells were counted using an Olympus dissecting microscope. Colonies were defined as having more than 50 cells, clusters less than 50 cells. The concentration of CFC was calculated from the dose response results under optimal stimulation and expressed as CFC per 2 x 105 nucleated bone marrow cells or as CFC per I x 106peripheral mononuclear cells. The statistical variation was thought to follow a Poisson distribution and accordingly the standard error was calculated (Blackett, 1974). Bone morrow/ 2x0' nucleated ECIIS
Peripheral blood /
1x10' mononuclear WIIS
A
- -
4 -s-
t
FIGI. Mean colony and duster incidencewithin the bone marrow and the peripheral blood of patients with aphtic anaemia. The shaded areas represent the standard deviations of the controls.
RESULTS The colony and cluster incidence within the bone marrow and the peripheral blood suspensions of all 28 observations in aplastic anaemia are summarized in Fig I. In the control group we found a mean colony incidence of 75 per 2 x 10' marrow nucleated cells, in the peripheral blood a mean of 28 per I x 106 mononuclear cells. From this it is evident that in all cases of aplastic anaemia the incidence of CFCs in the bone marrow and in the peripheral blood is markedly reduced, whereas the colony to cluster ratio fills within the normal
CFC in Aplastic Anaemia
617
limits. The individual results of each bone marrow examination are listed in Table I1 and summarized and compared with the results of the control marrow suspensions. The mean nucleated cell count of the aspirated marrow suspensions was 11x 106/ml TABLE 11. Bone marrow results of patients with aplastic anaemia and normal controls Di$erential count (percentages) Case Nos. I 2
3 4 5
6 7 8 9 I0 I1 I2 13
14 IS
16 17 I8
19 20
21
22
All patients (mean) Controls (n = 17) Mean (range)
Nucleated cells per ml aspirate x I06
Cells of GMP*
Colonyforming cells Total granupn 2 x 105nucleated locytic cells Lymphocytes c e h (rage)
3
9
2
I0
4 8 9 84
6 3
I1
I8
22
I0
34 I8
69 37 49
30 33
6
3
I0
I3
0
2
I
0
5 8 39 4 6
0 20
I
22 I
30 4 45 47 41 49 45 28
4 8
I8 2
28
20
I8 I2
I0 I
43 17 64 78 36 I4 8 40 60 4 65 8 31 46 30 I0
61 I8 8
6
32 27 I8
40
0
24
70
2
I
IS
2
50
14
2 I2
79 26 6
IS
17
29 42
20
I0
50
15
8 20
I4 9
32 57
20
11.6
10.7
33.0
34.6
29.7
18.8
48.2
13.5
(12-54
(8-29)
(3347)
5
7
(5-22)
GMP:cells of the granulocytic mitotic pool, i.e. myeloblasts, promyeiocytcs, myelocytes.
compared with a mean of 30 x Io6/ml in the controls. This difference suggests that the low colony incidence in the marrow suspension could be due to the contamination with peripheral blood. Because this could not be excluded the results have been grouped together firstly
P. Kern et a1
618
according to the patients' marrow cellularity and secondly according to the number of nucleated cells counted in the aspirate (Fig 2). It can be seen that there was a definite reduction of CFCs in all classes when compared with the controls. It is highly significant even in that class of patients whose marrow aspirates had a nucleated cell count of more than 15 x Io6/ml (k 0.001).This cell count falls within the range of that of normal marrow suspensions. 75
-
3.
n
ha
50-
GI? 3
br
25rcl
n
rcI
A
A
A
A
x $50
l
A & A
k AA A
A A
A
a
0
20
30
Rtcentopc of qonulocytic precursor cells
FIG 3. Number of CFC plotted against the percentage of cells of the granulocytic mitotic pool (i.e. myeloblasts, promyelocytes and myelocytes) within the marrow suspension. ,Individual observations of the patients; A, controls.
As can be seen from Table 11, the percentage of cells of the granulocytic mitotic pool (GMP),i.e. myeloblasts, promyelocytes and myelocytes, was considerably reduced in the patients, whereas the proportion of marrow lymphocytes was markedly increased. This finding was observed particularly in cases with severe hypoplasia and pancytopenia. However, in a considerablenumber of patients the relative proportion of granulocytic cells within the marrow suspension fell within the normal range. The relation between the relative concentration of cells of GMP and CFC is illustrated in Fig 3 and compared with the controls.
CFC in Aplastic Anaemia
419
The clear-cut reduction of CFC even in suspensions showing a normal proportion of cells of GMP becomes obvious. This indicates that even in cases in which the compartments of proliferating and differentiating granulocytic cells have regained a nornlal size, the relative concentration of their progenitor cells (CFC) is reduced. Patients who were observed for more than 2 years after diagnosis of aplastic anaemia (see Tables I and 11), continued to show a low colony incidence in marrow suspensions. Furthermore, marrow suspensions prepared at an interval of several months were found to have a
/\'
Months
FIG4. Follow-up of one patient during recovery from aplastic anaemia (case 20). Colony incidence in three marrow suspensions is shown in the upper graph, the peripheral cell counts (polymorphonuclear cells, reticulocytes and platelets) in the lower sections.
consistently decreased number of CFC (cascs I 8-22). Thc hacmatological recovcry of one patient is shown in Fig 4 (case 20). Within 9 months thc granulocytes aiid reticulocytes returned to low/normal levels and the platelets counts showed a clinically relevant recovery. However, no increase in the number of CFC was found. Morphologically the cells within the colonies consisted mainly of differentiating granulocytic cells but macrophage colonies were often seen to develop from peripheral blood suspensions. In three cases examined the size of the colonies and the histogram of the aggregates
620
P. Kern et a1
did not differ from that of two controls. In order to evaluate the growth characteristics of the bone marrow cells in agar an aggregate count was performed on each second day during the incubation period, A rapid rise of the numbers of aggregates was observed on days 3-5 in a control marrow suspension as well as in three marrow suspensions from patients with aplastic anaemia (cases 18, 20 and 22). Thereafter the number of aggregates during the remaining incubation time declined. When the aggregate count on day 10 was compared with that on days 3-5 a two- to three-fold higher incidence was seen in the early incubation period. This indicates that the pattern of cell proliferation in agar was not altered in marrow suspensions of the patients. The ability of the patients’ peripheral leucocytes to stimulate normal bone marrow CFC was studied in I I cases. No significant difference between the stimulation by control feeder layers and the stimulation by the patients’ cells was found as computed by the Wilcoxon rank test. DISCUSSION
The observed reduction of granulopoietic progenitor cells within the bone marrow and the peripheral blood of our patients with aplastic anaemia agrees with findings reported in the literature although the diagnosis of aplastic anaemia and the stage of the disease is not always as strictly defined as in our patients (Kurnick et a/, 1971 ; Greenberg & Schrier, 1973; Dicke & Lowenberg, 1974; Howell et a!, 197s). Using the same method all these authors found a significantly lower number of CFC in the bone marrows of 28 patients than in the corresponding control group with the exception of three out of 16 cases examined by Dicke & Lijwenberg (1974). There are several possible interpretations of the low CFC numbers in aplastic anaemia. First, they could be due simply to the dilution of the marrow suspensions with peripheral blood leucocytes. This interpretation may be relevant to those marrow suspensions which were found to contain considerably less nucleated cells than those of the controls. However, there was still a significantly reduced concentration of CFC in those of our patients who reached a remission as judged from the cellularity and the differential count of their marrow. Furthermore, in all degrees of hypoplasia the reduction becomes more pronounced when the colony formation is related to the number of the cells within the GMP appearing only in the bone marrow but not in the peripheral blood of the patients, as can be calculated from Table 11. In the controls a mean of 439 CFC/2 x 10’ cells of GMP (range 222-693) was obtained compared with a mean of 73 CFC/2x 10’ (range 0-400) in patients of aplastic anaemia. Greenberg & Schrier (1973) analysed their bone marrow findings on patients with neutropenic disorders using a similar calculation. A significant reduction of CFC was only found in those five patients who had, in addition to the neutropenia, a hypoplastic bone marrow and were thus comparable to our patients although a higher percentage of cells of GMP was observed in their hypoplastic group than in ours. Secondly, it could be argued that the low colony incidence as related to mononuclear marrow cells may be due to a dilution by an increase of marrow lymphocytes within the suspension. However, there is an inverse relationship between the degree of marrow cellularity and the percentage of marrow lymphocytes (Frisch & Lewis, 1974). This suggests that
CFC in Aplastic Anaemia
62 I
the loss of recognizable proliferating and differentiating haemopoietic cells leads to an increased proportion of lymphocytes. However, the ratio of CFC to cells of the GMP indicates that the increase of marrow lymphocytes contributes only little to the dilution of CFC. Thirdly, one must reject the argument that the low colony incidence is due to poor culture conditions or to colony inhibition. The latter niay occur when the colony stimulating activity is influenced by cellular interactions within the feeder layers (Heit et al, 1977). These factors were eliminated as far as possible by using at least three different qualities of CSA and by plating a control marrow in order to evaluate the colony stimulating activity derived by the feeder layers with a defined cellular composition. Additionally the aggregate formation during the culture period reflected a pattern which is not different from that found in cultures from normal human bone marrow. This finding indicates that in aplastic anaemia the growth pattern of marrow cells differs only quantitatively from normals without representing a qualitative variation in culture growth, as has been shown for instance in most cases of acute leukaemia (Moore et al, 1974). On the basis of these considerations it seems most likely that the low colony incidence would reflect a real reduction of the number of granulocytic progenitor cells, i.e. cells which are not identifiableby means of morphology, but which differentiate into the morphologically recognizable cells of the GMP. Of particular interest is the observed reduction of granulocytic progenitor cells in those cases which have recovered from aplastic anaemia for several months to years and which show a near normal to normal granulocyte production. These observations indicate that in aplastic anaemia clinical remissions which are characterized by a sufficient production of functional cells can occur, although the state of the preceding progenitor compartment is still abnormal. Due to the lack of a suitable assay system the concentration of the pluripotent stem cells in humans remains uncertain. Indirect evidence for the involvement of pluripotent stem cells can be derived from the clinical observation of the course of the disease and in particular from the recovery which takes place over a long period of time. Furthermore, comparable observations have been made in animal models in which a hypoplastic bone marrow failure developed after long-termed treatment with busulfan, a cytostatic drug known to act especially on resting cells of the pluripotent stem cell compartment (Morley & Blake, 1974; Dunn, 1974). The compartment of the pluripotent stem cells as well as the compartment of the granulopoietic progenitor cells were found to be markedly diminished, whereas the proliferating and differentiating pool of recognizable haemopoietic cells as well as the peripheral cell counts remained between low normal to normal numbers, until a frank marrow failure developed and the animals died of aplasia. It seems that the influx of cells into the more differentiated and specialized compartment of precursor cells is sufficient to restore the recognizable compartments, whereas the ability of the progenitor cells to sustain themselves is defective. A similar situation has been observed in our patients with aplastic anaemia who have recovered. Since our measurements detect oiily a static value, i.e. pool size of CFC, turnover studies of the CFC compartment would be helpful to discriminate the different possibilities which lead to a reduced compartment size. Attempts to measure the turnover rate by the tritiated thymidine suicide technique were unsuccessful due to the low incidence of CFC in the uiiseparated marrow suspensions. Our data can therefore not definitely exclude the possibility of a near normal to normal influx,
622
P. Kern et a1
in terms of fractional differentiationrate, of the CFC into the GMP in those cases showing a compensated production of functional cells. However, if one assumes that there is a greater turnover within the granulocytic progenitor cell compartment one would expect an earlier recovery to a normal compartment size than was observed. Thus, the most likely explanation for the long-lasting reduction of the CFC-compartment would be a defect within the pluripotent stem cell compartment. The nature of the hypothetical stem cell defect and the preceding events of such a longlasting failure of stem cell recovery are not understood. Exhaustion of pluripotent stem cells call only occur as a consequence of an impairment of their ‘unlimited’ growth potential. A decreasing potential of stem cells for self-renewal is suggested by the decline of the repopulation efficiency of haemopoietic tissue when serially transplanted (Siminovitch et al, 1964; Cudkowicz et al, 1964) or following chronic irradiation (Blackett, 1967). Due to the hypothetical injury (drugs, viruses) of the haemopoietic stem cells followed by an extensive demand for proliferation a senescent population of stem cells may be selected. These cells would have a poorer proliferative potential to sustain their numbers but their ability to differentiate seems to be affected less. Additionally, cellular immune reactions have to be taken into consideration. In the course of an immunological response lymphocytic suppressor cells may interact directly or by short-range factors with the stem cells. The cells may be blocked in their ability to proliferate, as has recently been suggested by AscensZo (1976). When considering other pathogenetic mechanisms of aplastic anaemia, no data on the role of the bone marrow matrix have been obtained in this work. Successhl bone marrow transplantation in patients with aplastic anaemia seems to indicate that the homing of haeniopoietic stem cells is not impaired (Storb & Thomas, 1975). This is supported by our own observation on the CFC incidence in the bone marrow suspensions of three patients with aplastic anaemia who have been followed up in the post transplantation period. A considerable increase of CFC was seen in three cases 10-20 d after transplantation (p. Kern and W. Heit, unpublished observations). In addition, environmental conditions do not influence the growth and differentiationof transplanted stem cells in the above-mentioned animal model of hypoplastic bone marrow fiilure (Morley et al, 1975). No evidence for abnormal regulation could be obtained by our experiments. The ability of the patients’ leucocytes to elaborate factors for colony stimulation of normal marrow suspensions was unaffected. However, in the series of pancytopenic patients reported by Dicke & Liiwenberg (1974) peripheral leucocytes of five patients showed a reduced ability to produce CSA. In the series of Senn et a1 (1974) describing neutropenic patients, two out of six suspensions of adherent mononuclear cells of the peripheral blood did not lead to an appropriate colony stimulation. This could indicate a deficient factor production by a defect or the lack of those cells which are responsible for CSA production. However, one has to interpret such findings with care, since it seems most unlikely that the fiilure of all three haemopoietic cell renewal systems (erythropoiesis, granulopoiesis and thrombopoiesis) is caused by a defect of several unrelated regulation factors. Furthermore the fict that erythropoietin levels in most cases of aplastic anaemia are found to be raised according to the degree of anaemia (Kubanek et al, 1975) makes it less likely that abnormal regulation plays an important role in the pathogenesis of the disease.
CFC in Aplastic Anaemia
623
ACKNOWLEDGMENTS
The authors are indebted to Mrs G. Neu, Mrs S. Miehe and Miss U. Schmidt for technical assistance throughout this work and to Dr Steinbach for his statistical advice. The authors gratefully acknowledge the financial support given by Deutsche Forschungsgenieinschaft
(SFB
112).
REFERENCES ASCENS~O, J., PAHWA,R., KAGAN,W., HANSEN, J., MOORE, M.A.S. & GOOD, R. (1976) Aplastic anaemia : evidence for an immunological mechanism. Lancet, i, 669. BLACKETT, N.M. (1976) Erythropoiesis in the rat under continued y-irradiation of 46 rads/day. British Journal ofhlaernatology, 13, 91 5 . BLACKETT, N.M. (1974) Statistical accuracy to be expected from cell colony assays; with special reference to the spleen colony assay. Cell and Tissue Kinetics, 7, 407. B~YUM A.. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 21, Suppl. 97, 77. G.. UPTON, A.C., SHEARBR, G.M. & CUDKOWICZ, HUGHES,W.L. (1964) Lymphocyte content and proliferative capacity of serially transplanted mouse bone marrow. Nature, 201, 165. DICKE,K.A. & LOWENBERG, B. (1974) In vitro analysis of pancytopenia: its possible relevance to the clinical course and the preleucaemic state in the aplastics. Proceedings of the 8th Leukocyte Culture Conference (ed. by K. Lindahl-Kiessling and D. Osoba). Academic Press, London. DUNN,C.D.R. (1974) The chemical and biological properties of Busulfan. Experimental Hematology, 2. 101.
FRISCH, B. & LEWIS,S.M. (1974) The bone marrow in aplastic anaemia : diagnostic and prognostic features. Journal of Clinical Pathology, 27, 2 3 I. GREENBERG, P.L. & SCHRIER,S.L. (1973) Granulopoiesis in neutropenic disorders. Blood, 41,753. HEWEL, H. & KUBANEK, B. (1975) Pathophysiology of aplastic anaemia. British Journal qf Haematology, 31 (sUPP1.h 57. B. & HEIMPEL,H. HEIT, W., KERN,P., KUBANEK, (1974) Some factors influencing granulocytic colony formation in vitro by human white blood cells. Blood, 44, 5 I I. HEIT, W., KERN,P., HEIMPEL, H. & KUBANEK. B. (1977) The role of granulocytes in colony stimulation by human white blood cells in agar cultures. Scandinavian Journal of Huematology (in press). HOWELL,A., ANDREWS, T.M. & WATTS,R.W.E. (1975) Bone-marrow cells resistant to chlorampheni-
col in chloramphenicol-induced aplastic anaemia. Lancet, i, 65. HULLIGER, L. & BLASCOVEC, A.A. (1967) A simple and efficient method of separating peripheral bloodleukocytes for in-vitro studies. Lancet, i, 1304. KUBANEK, B., HEIT, W. & BOCK,E. (1975) Regelmechanismen der Hamopoese bei der Knochenniarksinsuffizienz. Hdmatologie und Bluttransjusion, Bd. 16, Knochenmarksinn?Bixieni (ed. by W. Stich and G. Rubenstroh-Bauer), p 76. Lehmann, Miinchen. KURNICK, J.E., ROBINSON, W.A. & DICKEY, C.A. (1971) In vitro granulocytic colony-forming potential of bone marrow from patients with granulocytopenia and aplastic anemia. Proceedings qf the Society for Experimental Biology and Medicine, 137, 917. MOORE,M.A.S., SPITZER,G., WILLIAMS, N., METJ. (1974) Agar culture studies CALP,D. & BUCKLEY, in 127 cases of untreated acute leukemia. The prognostic value of reclassification of leukemia according to in vitro growth characteristics. Blood,
44, I. MORLEY,A. & BLAKE,J. (1974) Haemopoietic p r e cursor cells in experimental hypoplastic marrow failure. Australian Journal of Evperimental Biology and Medical Sciences, 52, 909. MORLEY,A., TRAINOR,K. & BLAKE,J. (1975) A primary cell lesion in experimental chronic hypoplastic marrow failure. Blood, 45, 681. PIKE,P.L. & ROBINSON, W.A. (1970) Human bone marrow colony growth in agar-gel. Journal o j Cellular Physiology, 76, 77. SENN,J.S., MESSNER, H.A. & STANLEY, E.R. (1974) Analysis of interacting cell populations in cultures of marrow from patients with neutropenia. Blood, 44.33.
SIMINOVITCH, L., TILL,J.E. & MCCULLOCH,E.A. (1964) Decline in colony-forming ability of marrow cells subjected to serial transplantation into irradiated mice. journal of Cellular and Comparative Physiology, 64, 23. STORB, R. &THOMAS, E.D. (197s) Bone marrow transplantation for aplastic anaemia. British Journal of Haematology, 31 (Suppl.), 83.
Granulocytic Progenitor Cells in Aplastic Anaemia P. KERN,H. HEIMPEL, W. HEIT AND B. KUBANEK
Department of Internal Medicine and Pediatrics, Division of Haematology, University of Ultn, UIm/Dotiau, Gerttrany (Received 23 August 1976; acceptedfir publication 13 September 1976) SUMMARY. The characteristics and the concentration of granulopoietic colony forming cells (CFC) were examined in 22 patients with aplastic anaemia at different stages of their disease. Additionally the ability of the patients’ peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (CSA) was studied. The ability of the patients’ cells to generate CSA was shown to be unaffected. However, the incidence of CFC within the marrow and peripheral blood suspensions was significantly reduced in all patients. The results suggest a reduced compartment size of CFC even in those patients who have recovered fiom aplastic anaemia. This may indicate that the disturbances in the preceding compartments of the haemopoietic cell renewal system still persist after recovery from the acute bone marrow failure. Aplastic anaemia is defined as an acute or chronic bone marrow failure with fatty replacement of the haemopoietic tissue and peripheral pancytopenia. This condition may be regarded as the nonspecific result of disturbances in the preceding Compartments of the haemopoietic cell renewal system. The pathophysiology of these disturbances is largely unknown. Hypothetical mechanisms involve ‘primary’ defects of maturation and proliferation of the haemopoietic stem cells, changes in their stromal and cellular microenvironment and aberrations of their humoral regulation (Heimpel & Kubanek, 197s). The chain of events leading to histological and functional aplasia may be caused or triggered off by external agents, such as chloramphenicol, phenylbutazone or the hepatitis virus. Some insight into the function of the haemopoietic stem cell compartments may be gained by the application of iti vitro methods for the assessment of progenitor cells committed to granulo-monocytopoiesis (Pike & Robinson, 1970). In this study, the characteristics and concentration of the granulopoietic progenitor cells assessed as colony forming cells (CFC) were examined in patients with aplastic anaemia at different stages of their disease as well as the ability of the patients’ peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (colony stimulating activity, CSA). MATERIAL AND METHODS
Patients The diagnosis of aplastic anaemia in 22 patients (17 female, five male) was based on the observation of a hypo- or aplastic marrow in more than one biopsy and a pancytopenia at Correspondence:Dr P. Kern, Clinical Department. Bemhard-Nocht Institute for Nautical and Tropical Diseases, Bernhard Nocht Str. 74. D-2000 Hamburg 4, West Germany.
P. Kern et al
614
some time during the course of the disease. Table I shows the period of observation and the haematological data at the time of in-vitro culture. The age of the patients varied between 12 and 67 years. Four patients were studied twice and one patient three times during followup. Thus, in all, the findings from 28 observations are described in this paper. TABLE I. Haeniatological data of patients with aplastic anaemia at the time of in-vitro culture of bone marrow and peripheral blood Peripheral blood characteristics Follow-up from Case diagnosis Haemoglobin Reticulocytes Platelets Lmcocytes Nos. per pl x 103 per rl x 103 (g/dl) (months) per pl I 2
3 4 5 6 7 8
9 I0 I1 1a
I I
a
4 4 15
9.6 6.8 9.7
9 1 I4 a73
6.6
10.7 13.9
w Ia
5
I8 I8 aa
10.1
0
69
5.2
I8
I3
5.5
1s
10.1 1a.r
5 6
I4
37 40 44 46 54
15
58
16 I7 18
60 78
12.2
24 56 27 39 47 66
6.6
I0
0
6
9.9 12.7
84 170
98 I9 57
I 5 I
8.3 7.1 9.6
a1
I1
0
7 8 4 47
I3
I9 ao
6 a1
I1
8.5
115
I0
13.0
6
9.8 10.6 12.4 12.7
37 n.d. 63
24
aa
15.6 14.1 14.0
71 85
Bone marrow cellularity
I1
38 24 n.d. 27 54 45 I4
8.3
Neutrophils per pl
21
198
69 126 I0
I18
54
327 53
35
81
* Iahomogeneous distribution of the haemopoietic cells within the bone marrow. As aetiological agent chloraniphenicol was suspected in four cases (cases 7, 8, 13 and Is), phenylbutazone in three cases (cases I, 2 and 4) and in one patient (case 3) the aplasia developed during the course of acute viral hepatitis. None of the patients had an enlarged spleen. None of the patients had received chemotherapy for malignant tumours. At the time of culture 18 cases were under treatment for aplastic anaemia, e.g. blood transfixions, anti-
CFC in Aplastic Anaemia 615 biotics, anabolic steroids, or corticosteroids. In 10 cases investigations were made when the patients were recovering or had recovered from aplastic anaemia. As controls we studied the peripheral blood of healthy blood donors, and the marrow specimens of healthy volunteers, donors for bone marrow transplantation and patients undergoing bone marrow examination in the out-patient clinic as part of their diagnostic study. Those who were found to have a morphologically normal bone marrow, normal peripheral cell counts and no evidence of infection were included in this group. The ages of 17 control subjects (12 female, five male) varied from 16 to 63 years. Cell Suspensions and Morphological Examinations Bone marrow was aspirated from the posterior iliac crest. One part of the aspirate was processed for routine morphological examination. The nucleated cell count per ml of the remaining suspension was determined. Red cells were sedimented either by mixing three parts of the cell suspension with one part of 3% Dextran (Pharmacia, Uppsala) in saline or by the method of Hulliger & Blascovec (1967). The supernatant containing the nucleated cells was washed three times in saline, resuspended in McCoys 5A Medium (Gibco) containing 10% fetal calf serum (Biocult). Smears were obtained by means of a cytocentrifuge and a differential count of zoo cells was made on each marrow suspension. At the same time a needle biopsy was performed and the bone marrow cylinder was prepared for histological examination. All available bone marrow smears and histological sections were evaluated by tivo independent observers in order to confirm the diagnosis of aplastic anaemia. The cellularity was judged as aplastic (dd) when the marrow spaces contained less than 10% marrow cells, hypoplastic (d)when the cellularity ranged between 10 and 30%. A normal cellularity was described within the range 3 ~ 5 0 % (n), and hypercellularity when the marrow spaces contained more than 50% of cells. In a number of cases the cellular distribution was found to be inhomogeneous, i.e. hypercellular foci between empty marrow spaces. Heparinized peripheral blood samples were separated using Isopaque-Ficoll-Gradient (Lymphoprep, Nygaard) (Boyum, 1968). Mononuclear cells at the top of the Lymphoprep solution were washed twice in buffered saline and resuspended in McCoy’s gA medium containing 10% fetal calf serum. The polymorphonuclear cells were prepared from the bottom layer of the gradient. A differential count of the peripheral blood smears was performed; on each slide at least 50 neutrophils were counted in order to maintain a constant statistical error.
Agar Cultures The double layer agar culture system was used (Pike & Robinson, 1970). An appropriate concentration of the cells was suspended in modified McCoy’s 5A medium, supplemented with 15% fetal calf serum and 5% horse serum (Biocult) and purified agar (Difco) resulting in a final agar concentration of 0.3% for the upper layers and 0.55% for the feeder layers. Aliquots of I ml were plated in 3 5 nim petri dishes (Greiner). Colony stimulating activity was provided by feeder layers of a defined leucocyte composition (Heit et al, 1974), in order to obtain optimal colony stimulation, When evaluating the ability of the patients’ leucocytes to stimulate normal CFC, normal marrow suspensions were used as target cell suspensions.
616
P. Kern et a1
Two to three cell concentrations of the bone marrow and the peripheral blood specimens were plated in the upper layers. Duplicate or triplicate cultures of each cell concentration were incubated in sterile air-tight glass containersat 37.s°C, 5% carbon dioxide and high humidity. After 10 d all aggregates of more than five cells were counted using an Olympus dissecting microscope. Colonies were defined as having more than 50 cells, clusters less than 50 cells. The concentration of CFC was calculated from the dose response results under optimal stimulation and expressed as CFC per 2 x 105 nucleated bone marrow cells or as CFC per I x 106peripheral mononuclear cells. The statistical variation was thought to follow a Poisson distribution and accordingly the standard error was calculated (Blackett, 1974). Bone morrow/ 2x0' nucleated ECIIS
Peripheral blood /
1x10' mononuclear WIIS
A
- -
4 -s-
t
FIGI. Mean colony and duster incidencewithin the bone marrow and the peripheral blood of patients with aphtic anaemia. The shaded areas represent the standard deviations of the controls.
RESULTS The colony and cluster incidence within the bone marrow and the peripheral blood suspensions of all 28 observations in aplastic anaemia are summarized in Fig I. In the control group we found a mean colony incidence of 75 per 2 x 10' marrow nucleated cells, in the peripheral blood a mean of 28 per I x 106 mononuclear cells. From this it is evident that in all cases of aplastic anaemia the incidence of CFCs in the bone marrow and in the peripheral blood is markedly reduced, whereas the colony to cluster ratio fills within the normal
CFC in Aplastic Anaemia
617
limits. The individual results of each bone marrow examination are listed in Table I1 and summarized and compared with the results of the control marrow suspensions. The mean nucleated cell count of the aspirated marrow suspensions was 11x 106/ml TABLE 11. Bone marrow results of patients with aplastic anaemia and normal controls Di$erential count (percentages) Case Nos. I 2
3 4 5
6 7 8 9 I0 I1 I2 13
14 IS
16 17 I8
19 20
21
22
All patients (mean) Controls (n = 17) Mean (range)
Nucleated cells per ml aspirate x I06
Cells of GMP*
Colonyforming cells Total granupn 2 x 105nucleated locytic cells Lymphocytes c e h (rage)
3
9
2
I0
4 8 9 84
6 3
I1
I8
22
I0
34 I8
69 37 49
30 33
6
3
I0
I3
0
2
I
0
5 8 39 4 6
0 20
I
22 I
30 4 45 47 41 49 45 28
4 8
I8 2
28
20
I8 I2
I0 I
43 17 64 78 36 I4 8 40 60 4 65 8 31 46 30 I0
61 I8 8
6
32 27 I8
40
0
24
70
2
I
IS
2
50
14
2 I2
79 26 6
IS
17
29 42
20
I0
50
15
8 20
I4 9
32 57
20
11.6
10.7
33.0
34.6
29.7
18.8
48.2
13.5
(12-54
(8-29)
(3347)
5
7
(5-22)
GMP:cells of the granulocytic mitotic pool, i.e. myeloblasts, promyeiocytcs, myelocytes.
compared with a mean of 30 x Io6/ml in the controls. This difference suggests that the low colony incidence in the marrow suspension could be due to the contamination with peripheral blood. Because this could not be excluded the results have been grouped together firstly
P. Kern et a1
618
according to the patients' marrow cellularity and secondly according to the number of nucleated cells counted in the aspirate (Fig 2). It can be seen that there was a definite reduction of CFCs in all classes when compared with the controls. It is highly significant even in that class of patients whose marrow aspirates had a nucleated cell count of more than 15 x Io6/ml (k 0.001).This cell count falls within the range of that of normal marrow suspensions. 75
-
3.
n
ha
50-
GI? 3
br
25rcl
n
rcI
A
A
A
A
x $50
l
A & A
k AA A
A A
A
a
0
20
30
Rtcentopc of qonulocytic precursor cells
FIG 3. Number of CFC plotted against the percentage of cells of the granulocytic mitotic pool (i.e. myeloblasts, promyelocytes and myelocytes) within the marrow suspension. ,Individual observations of the patients; A, controls.
As can be seen from Table 11, the percentage of cells of the granulocytic mitotic pool (GMP),i.e. myeloblasts, promyelocytes and myelocytes, was considerably reduced in the patients, whereas the proportion of marrow lymphocytes was markedly increased. This finding was observed particularly in cases with severe hypoplasia and pancytopenia. However, in a considerablenumber of patients the relative proportion of granulocytic cells within the marrow suspension fell within the normal range. The relation between the relative concentration of cells of GMP and CFC is illustrated in Fig 3 and compared with the controls.
CFC in Aplastic Anaemia
419
The clear-cut reduction of CFC even in suspensions showing a normal proportion of cells of GMP becomes obvious. This indicates that even in cases in which the compartments of proliferating and differentiating granulocytic cells have regained a nornlal size, the relative concentration of their progenitor cells (CFC) is reduced. Patients who were observed for more than 2 years after diagnosis of aplastic anaemia (see Tables I and 11), continued to show a low colony incidence in marrow suspensions. Furthermore, marrow suspensions prepared at an interval of several months were found to have a
/\'
Months
FIG4. Follow-up of one patient during recovery from aplastic anaemia (case 20). Colony incidence in three marrow suspensions is shown in the upper graph, the peripheral cell counts (polymorphonuclear cells, reticulocytes and platelets) in the lower sections.
consistently decreased number of CFC (cascs I 8-22). Thc hacmatological recovcry of one patient is shown in Fig 4 (case 20). Within 9 months thc granulocytes aiid reticulocytes returned to low/normal levels and the platelets counts showed a clinically relevant recovery. However, no increase in the number of CFC was found. Morphologically the cells within the colonies consisted mainly of differentiating granulocytic cells but macrophage colonies were often seen to develop from peripheral blood suspensions. In three cases examined the size of the colonies and the histogram of the aggregates
620
P. Kern et a1
did not differ from that of two controls. In order to evaluate the growth characteristics of the bone marrow cells in agar an aggregate count was performed on each second day during the incubation period, A rapid rise of the numbers of aggregates was observed on days 3-5 in a control marrow suspension as well as in three marrow suspensions from patients with aplastic anaemia (cases 18, 20 and 22). Thereafter the number of aggregates during the remaining incubation time declined. When the aggregate count on day 10 was compared with that on days 3-5 a two- to three-fold higher incidence was seen in the early incubation period. This indicates that the pattern of cell proliferation in agar was not altered in marrow suspensions of the patients. The ability of the patients’ peripheral leucocytes to stimulate normal bone marrow CFC was studied in I I cases. No significant difference between the stimulation by control feeder layers and the stimulation by the patients’ cells was found as computed by the Wilcoxon rank test. DISCUSSION
The observed reduction of granulopoietic progenitor cells within the bone marrow and the peripheral blood of our patients with aplastic anaemia agrees with findings reported in the literature although the diagnosis of aplastic anaemia and the stage of the disease is not always as strictly defined as in our patients (Kurnick et a/, 1971 ; Greenberg & Schrier, 1973; Dicke & Lowenberg, 1974; Howell et a!, 197s). Using the same method all these authors found a significantly lower number of CFC in the bone marrows of 28 patients than in the corresponding control group with the exception of three out of 16 cases examined by Dicke & Lijwenberg (1974). There are several possible interpretations of the low CFC numbers in aplastic anaemia. First, they could be due simply to the dilution of the marrow suspensions with peripheral blood leucocytes. This interpretation may be relevant to those marrow suspensions which were found to contain considerably less nucleated cells than those of the controls. However, there was still a significantly reduced concentration of CFC in those of our patients who reached a remission as judged from the cellularity and the differential count of their marrow. Furthermore, in all degrees of hypoplasia the reduction becomes more pronounced when the colony formation is related to the number of the cells within the GMP appearing only in the bone marrow but not in the peripheral blood of the patients, as can be calculated from Table 11. In the controls a mean of 439 CFC/2 x 10’ cells of GMP (range 222-693) was obtained compared with a mean of 73 CFC/2x 10’ (range 0-400) in patients of aplastic anaemia. Greenberg & Schrier (1973) analysed their bone marrow findings on patients with neutropenic disorders using a similar calculation. A significant reduction of CFC was only found in those five patients who had, in addition to the neutropenia, a hypoplastic bone marrow and were thus comparable to our patients although a higher percentage of cells of GMP was observed in their hypoplastic group than in ours. Secondly, it could be argued that the low colony incidence as related to mononuclear marrow cells may be due to a dilution by an increase of marrow lymphocytes within the suspension. However, there is an inverse relationship between the degree of marrow cellularity and the percentage of marrow lymphocytes (Frisch & Lewis, 1974). This suggests that
CFC in Aplastic Anaemia
62 I
the loss of recognizable proliferating and differentiating haemopoietic cells leads to an increased proportion of lymphocytes. However, the ratio of CFC to cells of the GMP indicates that the increase of marrow lymphocytes contributes only little to the dilution of CFC. Thirdly, one must reject the argument that the low colony incidence is due to poor culture conditions or to colony inhibition. The latter niay occur when the colony stimulating activity is influenced by cellular interactions within the feeder layers (Heit et al, 1977). These factors were eliminated as far as possible by using at least three different qualities of CSA and by plating a control marrow in order to evaluate the colony stimulating activity derived by the feeder layers with a defined cellular composition. Additionally the aggregate formation during the culture period reflected a pattern which is not different from that found in cultures from normal human bone marrow. This finding indicates that in aplastic anaemia the growth pattern of marrow cells differs only quantitatively from normals without representing a qualitative variation in culture growth, as has been shown for instance in most cases of acute leukaemia (Moore et al, 1974). On the basis of these considerations it seems most likely that the low colony incidence would reflect a real reduction of the number of granulocytic progenitor cells, i.e. cells which are not identifiableby means of morphology, but which differentiate into the morphologically recognizable cells of the GMP. Of particular interest is the observed reduction of granulocytic progenitor cells in those cases which have recovered from aplastic anaemia for several months to years and which show a near normal to normal granulocyte production. These observations indicate that in aplastic anaemia clinical remissions which are characterized by a sufficient production of functional cells can occur, although the state of the preceding progenitor compartment is still abnormal. Due to the lack of a suitable assay system the concentration of the pluripotent stem cells in humans remains uncertain. Indirect evidence for the involvement of pluripotent stem cells can be derived from the clinical observation of the course of the disease and in particular from the recovery which takes place over a long period of time. Furthermore, comparable observations have been made in animal models in which a hypoplastic bone marrow failure developed after long-termed treatment with busulfan, a cytostatic drug known to act especially on resting cells of the pluripotent stem cell compartment (Morley & Blake, 1974; Dunn, 1974). The compartment of the pluripotent stem cells as well as the compartment of the granulopoietic progenitor cells were found to be markedly diminished, whereas the proliferating and differentiating pool of recognizable haemopoietic cells as well as the peripheral cell counts remained between low normal to normal numbers, until a frank marrow failure developed and the animals died of aplasia. It seems that the influx of cells into the more differentiated and specialized compartment of precursor cells is sufficient to restore the recognizable compartments, whereas the ability of the progenitor cells to sustain themselves is defective. A similar situation has been observed in our patients with aplastic anaemia who have recovered. Since our measurements detect oiily a static value, i.e. pool size of CFC, turnover studies of the CFC compartment would be helpful to discriminate the different possibilities which lead to a reduced compartment size. Attempts to measure the turnover rate by the tritiated thymidine suicide technique were unsuccessful due to the low incidence of CFC in the uiiseparated marrow suspensions. Our data can therefore not definitely exclude the possibility of a near normal to normal influx,
622
P. Kern et a1
in terms of fractional differentiationrate, of the CFC into the GMP in those cases showing a compensated production of functional cells. However, if one assumes that there is a greater turnover within the granulocytic progenitor cell compartment one would expect an earlier recovery to a normal compartment size than was observed. Thus, the most likely explanation for the long-lasting reduction of the CFC-compartment would be a defect within the pluripotent stem cell compartment. The nature of the hypothetical stem cell defect and the preceding events of such a longlasting failure of stem cell recovery are not understood. Exhaustion of pluripotent stem cells call only occur as a consequence of an impairment of their ‘unlimited’ growth potential. A decreasing potential of stem cells for self-renewal is suggested by the decline of the repopulation efficiency of haemopoietic tissue when serially transplanted (Siminovitch et al, 1964; Cudkowicz et al, 1964) or following chronic irradiation (Blackett, 1967). Due to the hypothetical injury (drugs, viruses) of the haemopoietic stem cells followed by an extensive demand for proliferation a senescent population of stem cells may be selected. These cells would have a poorer proliferative potential to sustain their numbers but their ability to differentiate seems to be affected less. Additionally, cellular immune reactions have to be taken into consideration. In the course of an immunological response lymphocytic suppressor cells may interact directly or by short-range factors with the stem cells. The cells may be blocked in their ability to proliferate, as has recently been suggested by AscensZo (1976). When considering other pathogenetic mechanisms of aplastic anaemia, no data on the role of the bone marrow matrix have been obtained in this work. Successhl bone marrow transplantation in patients with aplastic anaemia seems to indicate that the homing of haeniopoietic stem cells is not impaired (Storb & Thomas, 1975). This is supported by our own observation on the CFC incidence in the bone marrow suspensions of three patients with aplastic anaemia who have been followed up in the post transplantation period. A considerable increase of CFC was seen in three cases 10-20 d after transplantation (p. Kern and W. Heit, unpublished observations). In addition, environmental conditions do not influence the growth and differentiationof transplanted stem cells in the above-mentioned animal model of hypoplastic bone marrow fiilure (Morley et al, 1975). No evidence for abnormal regulation could be obtained by our experiments. The ability of the patients’ leucocytes to elaborate factors for colony stimulation of normal marrow suspensions was unaffected. However, in the series of pancytopenic patients reported by Dicke & Liiwenberg (1974) peripheral leucocytes of five patients showed a reduced ability to produce CSA. In the series of Senn et a1 (1974) describing neutropenic patients, two out of six suspensions of adherent mononuclear cells of the peripheral blood did not lead to an appropriate colony stimulation. This could indicate a deficient factor production by a defect or the lack of those cells which are responsible for CSA production. However, one has to interpret such findings with care, since it seems most unlikely that the fiilure of all three haemopoietic cell renewal systems (erythropoiesis, granulopoiesis and thrombopoiesis) is caused by a defect of several unrelated regulation factors. Furthermore the fict that erythropoietin levels in most cases of aplastic anaemia are found to be raised according to the degree of anaemia (Kubanek et al, 1975) makes it less likely that abnormal regulation plays an important role in the pathogenesis of the disease.
CFC in Aplastic Anaemia
623
ACKNOWLEDGMENTS
The authors are indebted to Mrs G. Neu, Mrs S. Miehe and Miss U. Schmidt for technical assistance throughout this work and to Dr Steinbach for his statistical advice. The authors gratefully acknowledge the financial support given by Deutsche Forschungsgenieinschaft
(SFB
112).
REFERENCES ASCENS~O, J., PAHWA,R., KAGAN,W., HANSEN, J., MOORE, M.A.S. & GOOD, R. (1976) Aplastic anaemia : evidence for an immunological mechanism. Lancet, i, 669. BLACKETT, N.M. (1976) Erythropoiesis in the rat under continued y-irradiation of 46 rads/day. British Journal ofhlaernatology, 13, 91 5 . BLACKETT, N.M. (1974) Statistical accuracy to be expected from cell colony assays; with special reference to the spleen colony assay. Cell and Tissue Kinetics, 7, 407. B~YUM A.. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 21, Suppl. 97, 77. G.. UPTON, A.C., SHEARBR, G.M. & CUDKOWICZ, HUGHES,W.L. (1964) Lymphocyte content and proliferative capacity of serially transplanted mouse bone marrow. Nature, 201, 165. DICKE,K.A. & LOWENBERG, B. (1974) In vitro analysis of pancytopenia: its possible relevance to the clinical course and the preleucaemic state in the aplastics. Proceedings of the 8th Leukocyte Culture Conference (ed. by K. Lindahl-Kiessling and D. Osoba). Academic Press, London. DUNN,C.D.R. (1974) The chemical and biological properties of Busulfan. Experimental Hematology, 2. 101.
FRISCH, B. & LEWIS,S.M. (1974) The bone marrow in aplastic anaemia : diagnostic and prognostic features. Journal of Clinical Pathology, 27, 2 3 I. GREENBERG, P.L. & SCHRIER,S.L. (1973) Granulopoiesis in neutropenic disorders. Blood, 41,753. HEWEL, H. & KUBANEK, B. (1975) Pathophysiology of aplastic anaemia. British Journal qf Haematology, 31 (sUPP1.h 57. B. & HEIMPEL,H. HEIT, W., KERN,P., KUBANEK, (1974) Some factors influencing granulocytic colony formation in vitro by human white blood cells. Blood, 44, 5 I I. HEIT, W., KERN,P., HEIMPEL, H. & KUBANEK. B. (1977) The role of granulocytes in colony stimulation by human white blood cells in agar cultures. Scandinavian Journal of Huematology (in press). HOWELL,A., ANDREWS, T.M. & WATTS,R.W.E. (1975) Bone-marrow cells resistant to chlorampheni-
col in chloramphenicol-induced aplastic anaemia. Lancet, i, 65. HULLIGER, L. & BLASCOVEC, A.A. (1967) A simple and efficient method of separating peripheral bloodleukocytes for in-vitro studies. Lancet, i, 1304. KUBANEK, B., HEIT, W. & BOCK,E. (1975) Regelmechanismen der Hamopoese bei der Knochenniarksinsuffizienz. Hdmatologie und Bluttransjusion, Bd. 16, Knochenmarksinn?Bixieni (ed. by W. Stich and G. Rubenstroh-Bauer), p 76. Lehmann, Miinchen. KURNICK, J.E., ROBINSON, W.A. & DICKEY, C.A. (1971) In vitro granulocytic colony-forming potential of bone marrow from patients with granulocytopenia and aplastic anemia. Proceedings qf the Society for Experimental Biology and Medicine, 137, 917. MOORE,M.A.S., SPITZER,G., WILLIAMS, N., METJ. (1974) Agar culture studies CALP,D. & BUCKLEY, in 127 cases of untreated acute leukemia. The prognostic value of reclassification of leukemia according to in vitro growth characteristics. Blood,
44, I. MORLEY,A. & BLAKE,J. (1974) Haemopoietic p r e cursor cells in experimental hypoplastic marrow failure. Australian Journal of Evperimental Biology and Medical Sciences, 52, 909. MORLEY,A., TRAINOR,K. & BLAKE,J. (1975) A primary cell lesion in experimental chronic hypoplastic marrow failure. Blood, 45, 681. PIKE,P.L. & ROBINSON, W.A. (1970) Human bone marrow colony growth in agar-gel. Journal o j Cellular Physiology, 76, 77. SENN,J.S., MESSNER, H.A. & STANLEY, E.R. (1974) Analysis of interacting cell populations in cultures of marrow from patients with neutropenia. Blood, 44.33.
SIMINOVITCH, L., TILL,J.E. & MCCULLOCH,E.A. (1964) Decline in colony-forming ability of marrow cells subjected to serial transplantation into irradiated mice. journal of Cellular and Comparative Physiology, 64, 23. STORB, R. &THOMAS, E.D. (197s) Bone marrow transplantation for aplastic anaemia. British Journal of Haematology, 31 (Suppl.), 83.
