Ann Hematol (1992) 65:247-252
Annals of
Hematology 9 Springer-Verlag 1992
Original article Hypoplastic acute leukemia: description of eight cases and search for hematopoietic inhibiting activity R. de Bock, M. de Jonge, M. Korthout, E. Wouters, D. van Bockstaele, M. van der Planken, and M. Peetermans Department of Hematology,Universityof Antwerp (UZA), Wilrijkstraat 10, B-2650 Edegem, Belgium Received January 15, 1992/Accepted September 30, 1992
Summary. Hypoplastic acute leukemia (HAL) is characterized by pancytopenia and by hypocellularity of the bone marrow. The marrow contains equal to or more than 30~ myeloblasts. Absence of tissue infiltrates and/or tumor masses is mandatory. Eight patients are described here. They do not fit into the FAB classification for either acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS), except for one patient who subsequently proved to have a chronic myelomonocytic leukemia (CMML). The median age is 65 years. Two patients, including the CMML patient, are alive, 22 and 6 months from diagnosis. Six patients have died. The median survival is 8 months. Normal bone marrow cells cultured either with HAL sera or with HAL peripheral blood mononuclear cells as feeders and exogenous GMCSF yielded subnormal CFU-GM counts. This might indicate inhibitory activity of HAL serum and defective stimulatory activity of HAL peripheral blood mononuclear cells. Key words: Leukemia - Aplasia - Myelosuppression
Introduction The French-American-British (FAB) classification for acute leukemias and myelodysplastic syndromes is now accepted worldwide. Only a minority of acute leukemias, including hypoplastic acute leukemia (HAL), are difficult to classify in this system [3,4]. The incidence of HAL is 5% - 7 % of all acute leukemias [2,15]. HAL is characterized by a hypocellular bone marrow (less than 50% cellularity on core biopsy) and more than 30% marrow blasts. The differential diagnosis between HAL and hypoplastic Correspondence to: R. de Bock, University Hospital Antwerp,
Department of Haematology, Wilrijkstraat 10, B-2650 Edegem, Belgium
myelodysplastic syndrome [22,24] is easily made based on the number of marrow blasts and the lack of a preceding history of dysplasia. In the majority of cases blasts are of myeloid lineage According to some reports, hypocellularity correlates with a good prognosis [20]. Patients present with cytopenias and only very few peripheral blasts. The disease mainly afflicts the elderly [2, 5,15]. Median survival without therapy reported in the literature is about 6 - 9 months [2, 5, 15]. Most patients die of infections. Aggressive remission induction treatment can contribute to prolonged survival in patients younger than 65 years [5]. Complete remissions with aggressive chemotherapy have been reported [2,13]. The number of blasts increases very slowly in most patients, and peripheral blood cell counts usually remain fairly constant. Thus, one is confronted with the dilemma of supportive treatment versus aggressive remission induction chemotherapy in HAL patients. This problem remains unsolved [21]. The use of hematopoietic growth factors could be of benefit [12]. The hypocellularity in HAL remains a riddle: Does the leukemic clone produce an inhibitory factor for normal hematopoiesis [1,6,11, 19]? Do defective accessory cells have a diminished hematopoiesis stimulatory activity, or is there an intrinsic defect of progenitor ceils rendering them less responsive to regulatory mechanisms? Is the primum movens marrow hypoplasia, and is the "leukemia" a secondary clonal evolution [7, 8]? In genuine acute leukemia the deficient normal hematopoiesis is explained by "crowding out" due to overgrowth by the leukemic clone. This obviously cannot be the case in HAL, were only a few blast clusters are present and no myelophthisis is seen. We set out to test the hypothesis that the marrow hypoplasia is caused by inhibitory factors produced by the leukemic clone. The possible lack of stimulatory activity of accessory cells needed further investigation. We studied the effects on short-term progenitor cultures of normal BM cells in sera of seven HAL patients and of their peripheral blood mononuclear cells.
248
Materials and Methods Peripheral counts and bone marrow cytology and histology were performed according to standard methods. For bone marrow biopsies Vim-Silverman and Islam needles were used. Supportive treatment consisted of transfusions of leukocyte-poor packed cells and platelet concentrates. Infection prophylaxis with oral quinolones and antimycotics was given, and febrile episodes were treated with empiric i.v. antibiotics. Aggressive chemotherapy was not given. Bone marrow and peripheral blood progenitor cultures
Bone marrow and peripheral blood were collected in sterile tubes containing 2 rnl Iscove's Modified Dulbecco's medium (IMDM) (Gibco-BRL) with 200 IU preservative-free heparin. The cell suspensions were separated over lymphocyte separation medium (LSM), density 1.077 g/ml (Boehringer Mannheim). The lowdensity cell fraction was harvested and washed once with RPMI 1640 (Flow Laboratories). For diagnostic purposes cultures were performed in a standard way with 5637 conditioned medium as a stimulator. An excess of 200 U GM-CSF (Sandoz) was added in repeat cultures. Influence of HAL sera on normal bone marrow
Myeloid progenitor cells (CFU-GM) were cultured in a monolayer agar system. One milliliter of culture consisted of 1• 105 normal bone marrow mononuclear cells (BM MNC), 200 U GM-CSF, 0.307o agarin IMDM, and 20070fetal calf serum (FCS) as control 1 (CI), 10070 FCS plus 1007o pooled human serum derived from blood donors as control 2 (C2), or 10% FCS plus 10% HL 60-conditioned medium as control 3 (Ca). Triplicates of 2 ml were plated into 35-ram Petri dishes. Influence of H A L peripheral blood mononuclear cells on normal B M cells
In this experiment, CFU-GM were established in a double-layer system with peripheral blood mononuclear cells (PBL MNC) from HAL patients. Feeder layers were prepared with 1.166 HAL PBL MNC, 10% FCS, and 0.5% agar in IMDM. Overlayers were then prepared with 1• 105 normal BM MNC in 1 ml of 10% FCS and 0.3070 agar in IMDM. Controls were: C~ - GM-CSF 200 U/ml without feeder ceils; C2 - normal peripheral blood mononuclear cells; and C3 - HL 60 cells. Triplicates of 2 ml were plated into 35-ram Petri dishes. Influence of HAL PBL MNC on normal BM cells stimulated by 200 U o f GM-CSF
CFU-GM were established as above, but 200 U GM-CSF/ml were added to the normal BM MNC. Cultures were incubated for 14 days in 5070 CO2 at 37~ After 14 days the culture dishes were examined under an inverted microscope for colony (> 40 cells) growth. Statistics
Minimum significant differences at the 5070experiment-wise significance level between pairs of means with heterogeneous variances were calculated (according to the method of Games and Howell [91). Flow-cytometric immunophenotyping
The mononuclear cells from peripheral blood and bone marrow samples were obtained through a Ficoll-Paque density separation procedure. Cell pellets consisting of 106 cells were incubated with directly conjugated [chromophoric group either fiuorescein isothiocyanate (FITC) or phycperythrin (PE)] monoclonal (aCD2-FITC, aDC4-PE, aDC8-FITC, aCD20-FITC, aHLA-DR-PE, aCD38-PE, aCD 11b-PE), or polyclonal antibodies (a-human total Ig-FITC),
obtained from either Becton Dickinson (Erembodegem, Belgium, monoclonal ab) or Kallestad (Austin, TX, polyclonal ab). After two PBS washes, cells were resuspended in PBS and immediately measured on a Facscan (Becton Dickinson, Erembodegem, Belgium) analytical flow cytometer. Isotype negative controls were included to account for background fluorescence and proper threshold settings. Normal bone marrow was obtained with informed consent from cardiac surgery patients.
Patients During the period 1988-1991 we saw eight patients with hypoplastic acute leukemia in the Department of Hematology of the University Hospital in Antwerp (Table 1). They represented 9.6% of all patients with acute leukemia (n = 83) seen in the same period. Median age was 65 years (range 5 6 - 7 4 years). Sex ratio was 6/2 (M/F). None of the patients had a known history of exposure to myelotoxic or leukemogenic agents. The diagnosis was preceded by symptomatic anemia in six patients, leukopenia in two, and thrombocytopenia in four patients during periods of 1 - 6 months (Table 2). All but one patient had pancytopenia at diagnosis. The median Hb level was 7.8 g/di (range 4 . 9 - 9 . 9 ) , the median leukocyte count was 2.1• (range 0.8-8.4• the median granulocyte count was 0.13• (range 0.04-0.19• There were very few or no peripheral blasts at diagnosis, but five patients subsequently had more than 5% blasts, with a maximum of 20%. We did not observe an excess of CD 8-positive cells in peripheral blood or bone marrow (Table 3). Bone marrows were hypoceUular in all cases, with small islands o f blasts (Table 4). Median marrow blast percentage at diagnosis was 39% (Range 28% to 5507o). No Auer rods were seen. A preleukemic phase (11%, 14%, and 18% blasts in the marrow) was observed in three patients lasting from 3 weeks to 1 month before the diagnosis of H A L could be made. A > 15% increase in marrow blast occurred in only two patients. All blasts were myeloblasts. None of the patients fitted the FAB classification for A N L L or MDS. Karyotypes were normal, but because o f low cell numbers only 6 - 1 0 (mean 8.3) metaphases were examined for each patient. Supportive treatment consisted of transfusions of packed cells and platelet concentrates. Transfusion needs were 2 units o f packed cells per 1 - 8 weeks and 8 units of platelet concentrates per 1 - 5 days. Intravenous antibiotics were given to seven of the eight patients. Six patients have since died of infection 621, 561, 480, 311, 296, and 264 days from diagnosis, and two patients are alive: one is the C M M L patient, 817 days from diagnosis, and the other is an H A L patient recently observed and now 93 days from diagnosis (see Table 1).
Results At diagnosis all patients showed an abnormal growth pattern o f bone marrow in progenitor cell culture, with near total absence o f growth (Table 5). The addition o f GMCSF yielded no increase of growth, except in one patient (no. 3) who later proved to have CMML.
249 Table 1. Patierrt characteristics
Table 2. Peripheral blood counts at diagnosis
Patient no.
1
2
3
4
5
6
7
8
Age (years)
65
56
74
66
59
64
65
62
Sex
M
M
F
M
M
M
M
F
Survival in days from diagnosis
264
296
817+ alive
621
561
311
480
93+ alive
1
2
3
4
5
6
7
8
5.3 115
6 102
8.1 118
4.9 123
8.6 108
9.9 112
7.8 99
8.5 100.9
8.4 1.5 47 50 1.5 265
1.1 11 86 1.5 0 37
1.7 11 84 3.3 0 21
1 10 86 0.5 0 25
0.8 5 81 4 0 49
16 12 86 0 0 47
Patient no. Hb MCV
(g/dl) (fl)
WBC PMN LY Mono Blasts PLT
(109/1) (%) (%) (%) (%) (109/1)
1.2 14 83 1 0 12
1.7 10 89 1 0 35
Table 3. Immunophenotyping of PB and BM cells by flow cytometry at diagnosis Patient no.
CD2 CD4 CD 8 CD20 CDlla CD38 HLADR
1
2
3
4
5
6
7
8
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
80 60 17 2 2.7 19 6
54 87 17 -3.8 28 24
86 63 19 4 3 15 5
70 17 --4.5 46 30
--------
--------
88 38 46 9.5 26 18 21
66 38 19 -5.6 7 41
96 70 24 2.8 4.5 9.2 2.7
--------
81 59 18 3 2 19 7
55 29 24 -18 7 41
87 38 39 4 25 43 15
76 31 41 --12 43
91 60 23 5 14 11 11
57 34 17 41 48 11.4 25.1
6
7
8
hypo 34 68 1.6
hypo 40 20 0.3
PB, Peripheral blood; BM, bone marrow.
Table 4. Bone marrow at diagnosis Patient no.
1
2
3
4
hypo 36 27 1.3
hypo 28 52 1.2
hypo 53 25 4.2
hypo 55 28 3.6
15
20
5
Cytology Cellularity Blasts (%) Lymphocytes (%) M E ratio
hypo h y p o 30 . 36 55 18 i.2 1.7
Histology Cellularity
(%)
Significant inhibitory activity was demonstrated in t h r e e o f five H A L s e r a as c o m p a r e d w i t h n o r m a l s e r a (Fig. 1). T h i s i n h i b i t o r y a c t i v i t y w a s less p r o n o u n c e d t h a n t h a t o f H L 6 0 - c o n d i t i o n e d m e d i u m (C3). Stimulatory activity of peripheral blood mononuclear cells w a s l a c k i n g i n t h r e e o f f o u r H A L p a t i e n t s ( F i g . 2). Samples from patient 2 were not obtained. HL 60 feeder
20
< 10
25
20
Annals of
Hematology 9 Springer-Verlag 1992
Original article Hypoplastic acute leukemia: description of eight cases and search for hematopoietic inhibiting activity R. de Bock, M. de Jonge, M. Korthout, E. Wouters, D. van Bockstaele, M. van der Planken, and M. Peetermans Department of Hematology,Universityof Antwerp (UZA), Wilrijkstraat 10, B-2650 Edegem, Belgium Received January 15, 1992/Accepted September 30, 1992
Summary. Hypoplastic acute leukemia (HAL) is characterized by pancytopenia and by hypocellularity of the bone marrow. The marrow contains equal to or more than 30~ myeloblasts. Absence of tissue infiltrates and/or tumor masses is mandatory. Eight patients are described here. They do not fit into the FAB classification for either acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS), except for one patient who subsequently proved to have a chronic myelomonocytic leukemia (CMML). The median age is 65 years. Two patients, including the CMML patient, are alive, 22 and 6 months from diagnosis. Six patients have died. The median survival is 8 months. Normal bone marrow cells cultured either with HAL sera or with HAL peripheral blood mononuclear cells as feeders and exogenous GMCSF yielded subnormal CFU-GM counts. This might indicate inhibitory activity of HAL serum and defective stimulatory activity of HAL peripheral blood mononuclear cells. Key words: Leukemia - Aplasia - Myelosuppression
Introduction The French-American-British (FAB) classification for acute leukemias and myelodysplastic syndromes is now accepted worldwide. Only a minority of acute leukemias, including hypoplastic acute leukemia (HAL), are difficult to classify in this system [3,4]. The incidence of HAL is 5% - 7 % of all acute leukemias [2,15]. HAL is characterized by a hypocellular bone marrow (less than 50% cellularity on core biopsy) and more than 30% marrow blasts. The differential diagnosis between HAL and hypoplastic Correspondence to: R. de Bock, University Hospital Antwerp,
Department of Haematology, Wilrijkstraat 10, B-2650 Edegem, Belgium
myelodysplastic syndrome [22,24] is easily made based on the number of marrow blasts and the lack of a preceding history of dysplasia. In the majority of cases blasts are of myeloid lineage According to some reports, hypocellularity correlates with a good prognosis [20]. Patients present with cytopenias and only very few peripheral blasts. The disease mainly afflicts the elderly [2, 5,15]. Median survival without therapy reported in the literature is about 6 - 9 months [2, 5, 15]. Most patients die of infections. Aggressive remission induction treatment can contribute to prolonged survival in patients younger than 65 years [5]. Complete remissions with aggressive chemotherapy have been reported [2,13]. The number of blasts increases very slowly in most patients, and peripheral blood cell counts usually remain fairly constant. Thus, one is confronted with the dilemma of supportive treatment versus aggressive remission induction chemotherapy in HAL patients. This problem remains unsolved [21]. The use of hematopoietic growth factors could be of benefit [12]. The hypocellularity in HAL remains a riddle: Does the leukemic clone produce an inhibitory factor for normal hematopoiesis [1,6,11, 19]? Do defective accessory cells have a diminished hematopoiesis stimulatory activity, or is there an intrinsic defect of progenitor ceils rendering them less responsive to regulatory mechanisms? Is the primum movens marrow hypoplasia, and is the "leukemia" a secondary clonal evolution [7, 8]? In genuine acute leukemia the deficient normal hematopoiesis is explained by "crowding out" due to overgrowth by the leukemic clone. This obviously cannot be the case in HAL, were only a few blast clusters are present and no myelophthisis is seen. We set out to test the hypothesis that the marrow hypoplasia is caused by inhibitory factors produced by the leukemic clone. The possible lack of stimulatory activity of accessory cells needed further investigation. We studied the effects on short-term progenitor cultures of normal BM cells in sera of seven HAL patients and of their peripheral blood mononuclear cells.
248
Materials and Methods Peripheral counts and bone marrow cytology and histology were performed according to standard methods. For bone marrow biopsies Vim-Silverman and Islam needles were used. Supportive treatment consisted of transfusions of leukocyte-poor packed cells and platelet concentrates. Infection prophylaxis with oral quinolones and antimycotics was given, and febrile episodes were treated with empiric i.v. antibiotics. Aggressive chemotherapy was not given. Bone marrow and peripheral blood progenitor cultures
Bone marrow and peripheral blood were collected in sterile tubes containing 2 rnl Iscove's Modified Dulbecco's medium (IMDM) (Gibco-BRL) with 200 IU preservative-free heparin. The cell suspensions were separated over lymphocyte separation medium (LSM), density 1.077 g/ml (Boehringer Mannheim). The lowdensity cell fraction was harvested and washed once with RPMI 1640 (Flow Laboratories). For diagnostic purposes cultures were performed in a standard way with 5637 conditioned medium as a stimulator. An excess of 200 U GM-CSF (Sandoz) was added in repeat cultures. Influence of HAL sera on normal bone marrow
Myeloid progenitor cells (CFU-GM) were cultured in a monolayer agar system. One milliliter of culture consisted of 1• 105 normal bone marrow mononuclear cells (BM MNC), 200 U GM-CSF, 0.307o agarin IMDM, and 20070fetal calf serum (FCS) as control 1 (CI), 10070 FCS plus 1007o pooled human serum derived from blood donors as control 2 (C2), or 10% FCS plus 10% HL 60-conditioned medium as control 3 (Ca). Triplicates of 2 ml were plated into 35-ram Petri dishes. Influence of H A L peripheral blood mononuclear cells on normal B M cells
In this experiment, CFU-GM were established in a double-layer system with peripheral blood mononuclear cells (PBL MNC) from HAL patients. Feeder layers were prepared with 1.166 HAL PBL MNC, 10% FCS, and 0.5% agar in IMDM. Overlayers were then prepared with 1• 105 normal BM MNC in 1 ml of 10% FCS and 0.3070 agar in IMDM. Controls were: C~ - GM-CSF 200 U/ml without feeder ceils; C2 - normal peripheral blood mononuclear cells; and C3 - HL 60 cells. Triplicates of 2 ml were plated into 35-ram Petri dishes. Influence of HAL PBL MNC on normal BM cells stimulated by 200 U o f GM-CSF
CFU-GM were established as above, but 200 U GM-CSF/ml were added to the normal BM MNC. Cultures were incubated for 14 days in 5070 CO2 at 37~ After 14 days the culture dishes were examined under an inverted microscope for colony (> 40 cells) growth. Statistics
Minimum significant differences at the 5070experiment-wise significance level between pairs of means with heterogeneous variances were calculated (according to the method of Games and Howell [91). Flow-cytometric immunophenotyping
The mononuclear cells from peripheral blood and bone marrow samples were obtained through a Ficoll-Paque density separation procedure. Cell pellets consisting of 106 cells were incubated with directly conjugated [chromophoric group either fiuorescein isothiocyanate (FITC) or phycperythrin (PE)] monoclonal (aCD2-FITC, aDC4-PE, aDC8-FITC, aCD20-FITC, aHLA-DR-PE, aCD38-PE, aCD 11b-PE), or polyclonal antibodies (a-human total Ig-FITC),
obtained from either Becton Dickinson (Erembodegem, Belgium, monoclonal ab) or Kallestad (Austin, TX, polyclonal ab). After two PBS washes, cells were resuspended in PBS and immediately measured on a Facscan (Becton Dickinson, Erembodegem, Belgium) analytical flow cytometer. Isotype negative controls were included to account for background fluorescence and proper threshold settings. Normal bone marrow was obtained with informed consent from cardiac surgery patients.
Patients During the period 1988-1991 we saw eight patients with hypoplastic acute leukemia in the Department of Hematology of the University Hospital in Antwerp (Table 1). They represented 9.6% of all patients with acute leukemia (n = 83) seen in the same period. Median age was 65 years (range 5 6 - 7 4 years). Sex ratio was 6/2 (M/F). None of the patients had a known history of exposure to myelotoxic or leukemogenic agents. The diagnosis was preceded by symptomatic anemia in six patients, leukopenia in two, and thrombocytopenia in four patients during periods of 1 - 6 months (Table 2). All but one patient had pancytopenia at diagnosis. The median Hb level was 7.8 g/di (range 4 . 9 - 9 . 9 ) , the median leukocyte count was 2.1• (range 0.8-8.4• the median granulocyte count was 0.13• (range 0.04-0.19• There were very few or no peripheral blasts at diagnosis, but five patients subsequently had more than 5% blasts, with a maximum of 20%. We did not observe an excess of CD 8-positive cells in peripheral blood or bone marrow (Table 3). Bone marrows were hypoceUular in all cases, with small islands o f blasts (Table 4). Median marrow blast percentage at diagnosis was 39% (Range 28% to 5507o). No Auer rods were seen. A preleukemic phase (11%, 14%, and 18% blasts in the marrow) was observed in three patients lasting from 3 weeks to 1 month before the diagnosis of H A L could be made. A > 15% increase in marrow blast occurred in only two patients. All blasts were myeloblasts. None of the patients fitted the FAB classification for A N L L or MDS. Karyotypes were normal, but because o f low cell numbers only 6 - 1 0 (mean 8.3) metaphases were examined for each patient. Supportive treatment consisted of transfusions of packed cells and platelet concentrates. Transfusion needs were 2 units o f packed cells per 1 - 8 weeks and 8 units of platelet concentrates per 1 - 5 days. Intravenous antibiotics were given to seven of the eight patients. Six patients have since died of infection 621, 561, 480, 311, 296, and 264 days from diagnosis, and two patients are alive: one is the C M M L patient, 817 days from diagnosis, and the other is an H A L patient recently observed and now 93 days from diagnosis (see Table 1).
Results At diagnosis all patients showed an abnormal growth pattern o f bone marrow in progenitor cell culture, with near total absence o f growth (Table 5). The addition o f GMCSF yielded no increase of growth, except in one patient (no. 3) who later proved to have CMML.
249 Table 1. Patierrt characteristics
Table 2. Peripheral blood counts at diagnosis
Patient no.
1
2
3
4
5
6
7
8
Age (years)
65
56
74
66
59
64
65
62
Sex
M
M
F
M
M
M
M
F
Survival in days from diagnosis
264
296
817+ alive
621
561
311
480
93+ alive
1
2
3
4
5
6
7
8
5.3 115
6 102
8.1 118
4.9 123
8.6 108
9.9 112
7.8 99
8.5 100.9
8.4 1.5 47 50 1.5 265
1.1 11 86 1.5 0 37
1.7 11 84 3.3 0 21
1 10 86 0.5 0 25
0.8 5 81 4 0 49
16 12 86 0 0 47
Patient no. Hb MCV
(g/dl) (fl)
WBC PMN LY Mono Blasts PLT
(109/1) (%) (%) (%) (%) (109/1)
1.2 14 83 1 0 12
1.7 10 89 1 0 35
Table 3. Immunophenotyping of PB and BM cells by flow cytometry at diagnosis Patient no.
CD2 CD4 CD 8 CD20 CDlla CD38 HLADR
1
2
3
4
5
6
7
8
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
PB
BM
80 60 17 2 2.7 19 6
54 87 17 -3.8 28 24
86 63 19 4 3 15 5
70 17 --4.5 46 30
--------
--------
88 38 46 9.5 26 18 21
66 38 19 -5.6 7 41
96 70 24 2.8 4.5 9.2 2.7
--------
81 59 18 3 2 19 7
55 29 24 -18 7 41
87 38 39 4 25 43 15
76 31 41 --12 43
91 60 23 5 14 11 11
57 34 17 41 48 11.4 25.1
6
7
8
hypo 34 68 1.6
hypo 40 20 0.3
PB, Peripheral blood; BM, bone marrow.
Table 4. Bone marrow at diagnosis Patient no.
1
2
3
4
hypo 36 27 1.3
hypo 28 52 1.2
hypo 53 25 4.2
hypo 55 28 3.6
15
20
5
Cytology Cellularity Blasts (%) Lymphocytes (%) M E ratio
hypo h y p o 30 . 36 55 18 i.2 1.7
Histology Cellularity
(%)
Significant inhibitory activity was demonstrated in t h r e e o f five H A L s e r a as c o m p a r e d w i t h n o r m a l s e r a (Fig. 1). T h i s i n h i b i t o r y a c t i v i t y w a s less p r o n o u n c e d t h a n t h a t o f H L 6 0 - c o n d i t i o n e d m e d i u m (C3). Stimulatory activity of peripheral blood mononuclear cells w a s l a c k i n g i n t h r e e o f f o u r H A L p a t i e n t s ( F i g . 2). Samples from patient 2 were not obtained. HL 60 feeder
20
< 10
25
20
