Functional Features of Hematopoiesis in Patients with Aplastic Anemia During High-Altitude Climate Therapy

A.R. Raimjanov MD, S.M. Mamatov MD

Kyrgyz Scientific Center of Hematology, Bishkek, Kyrgyzstan

Key Words · Aplastic anemia · climate therapy.

Introduction

Aplastic anemia (AA) is one of the most severe hematological diseases. Although the clinical picture of AA was first described more than 100 years ago (Erlich, 1888), it still remains a problem today. Prolonged anemia, progressing hemorrhagic syndrome, and severe infectious complications are the main causes of death among AA patients, especially in severe AA (SAA). In spite of intensive efforts to find an effective treatment for AA, the mortality rate of this disease is still at a high level.^1,2 Allosensitization resulting from repeated transfusions of erythro- and thrombocytic elements , significantly decreases the efficacy of hemotransfusion, hampers the subsequent substitutive therapy and increases hemorrhagic complications. At the same time, the long-term administration of glucocorticoids and anabolic hormones may cause many side effects. Splenectomy has proved to be ineffective in severe AA.^3,4,5 Progress achieved in the treatment of AA during recent years, is mainly linked to the introduction of bone marrow transplantation (BMT).^6,7 However, BMT has limited application due to problems inherent in the selection of a histocompatible donors and subsequent immunological problems. Immunosuppressive therapy comprising cyclosporin A and antilymphocyte globulin/antimonocyte globulin (ALG/AMG) though initially used in pre-transplantation preparation programs has proved to be a potent therapeutic modality in the majority of patients with AA even as an independent treatment.^1,5,6,8-11

Along the classic approaches, investigations by M.M. Mirrakhimov and A.R. Raimjanov^12,13 have revealed the efficacy of yet another therapeutic modality namely high-altitude climatotherapy in these patients. Since 1967, 202 AA patients received mountain climatotherapy at the High-Altitude Station of the National Center for Cardiology and Internal Medicine, the Ministry of Health of the Kyrgyz Republic (Tuya-Ashu Pass, 3200 m).

Material and Methods

Two hundred and two patients aged 15 to 50 years entered this study. According to the criteria of Cammitta et al.¹⁴ 54 subjects were diagnosed as severe AA (SAA) and 148 (83 males and 65 females) as non-severe AA (reticulocyte count less than 2% after correction, platelet count less than 50.0´ 10⁹/1, and neutrophil count less than 1.0x10⁹/l).

Determination of relevant parameters of peripheral blood, bone marrow, serum iron, as well as 17-oxyketosteroids, were conducted as described elsewhere [G.V. Docie, S.M. Lewis, 1995]. Investigations included analysis of erythrocyte functions by interferometry for measurement of the dry weight of individual erythrocytes. Evaluation of the erythrocyte dry weight was carried out using an interference microscope "MBIN-4" LOMO on peripheral blood smears fixed by methanol. The dry weight of 50-100 erythrocytes from each subject was measured. To verify diagnosis in these patients, bone marrow aspiration was performed from the iliac crest in 48.3% of cases to monitor the dynamics of therapy as recommended by Docie G.V., and Lewis S.M.,(1995).

Erythroid cells were studied using autoradiography with H³ - thymidine. Smears were dried and stained by azure-eosine for 15 minutes through a photoemulsion layer. Cells were counted on the entire smear areas. Labeled cells incorporated 5 or more granules of reduced silver and the total labelling index (TLI) was evaluated in all nucleated erythroid elements (250-500 cells), and all stages of erythronormoblast maturation. Investigation of polysaccharides in marrow erythroid cells (PAS reaction) was carried out. Bone marrow smears were fixed in formalin and then incubated in iodide solution for 5 minutes. To enhance nuclear contrast, 2% solution of methyl-green was used. Thus, mucoproteids, neutrophilic mucopolyssacharides, glucoproteids, glycogen were colored purple-red.

Marrow fibroblast culturing was conducted in sterile flasks on the home medium 199 with supplementation of 15% of human serum albumin. Results were evaluated in a number of colony-forming units (FCFU). Fibroblast colony-forming efficacy (FCFE) was measured by a value of FCFU per 10⁵ marrow cells [G.V. Docie, S.M. Lewis, 1995].

In addition, an electron microscopic study of erythroid cells of bone marrow was also carried out.

Climatic and geographical characteristics of the studies' zones:

Bishkek city is situated in the Chui valley at the northern foot of the Kyrgyz range of Ala-Too (760 m). The Chui valley forms part of the foothills rising at an altitude of 250-500 to 1000-2000 m. The climate is continental, the spring is short but warm and the average summer temperature varies from 20 to 25° C. The region receives about 400 mm of precipitation a year.

The high-altitude station of the National Center for Cardiology and Internal Medicine is situated on the northern portal of Tuya-Ashu Pass at an altitude of 3200 m (129 km from Bishkek). The pass crosses the Kyrgyz mountain range at an altitude of 3600 m. The climate is very harsh and due to hard frosts and permafrost there are no forests around the Pass and the ground is covered with grass in summer only. The summer barometric pressure ranges from 527 to 529 mm Hg. The average summer temperature reaches +17-22° C. The region of Tuya-Ashu Pass receives about 753 mm of annual precipitation, mainly in the warm seasons (638 mm). Air humidity varies within a wide range (48-95%), 71% on average.

The method of mountain climatotherapy (MCT):

Mountain climatotherapy was conducted according to the method of M.M. Mirrakhimov and A.R. Raimjanov.^12,13 After a comprehensive examination performed in Bishkek, patients accompanied by medical personnel, were transported along the Tuya-Ashu Pass. First, a semi-ambulatory state was prescribed for all patients. From the 7-10th days of their stay, short walks were recommended, then the ambulation protocol was gradually extended to include respiratory exercise, physical therapy, graded walking and sports. The therapy duration spanned over 40 days at high altitude. Repeated examination was carried out on the 40th day of adaptation and 3 months after MCT.

Results and Discussion

An increase in reticulocyte count was noted from the first day after ascent and achieved the greatest values on the 20th day (45% increase, p<0.05) (Table 1). During subsequent adaptation and after descent, reticulocytosis decreased but still exceeded the initial level. Quantitative changes induced by high altitude were also characterized by a gradual rise of granulocyte and platelet counts that rose to a maximum on the 40th day after ascent (by 1.4 and 1.5 - fold respectively, p<0.05). Three months after therapy the blood parameters showed a decreasing tendency, though they still exceeded initial values significantly.

The effect of high-altitude hypoxia on the values of acidified serum lysis test from AA patients has not yet been described in literature. There are some differences between the acidified serum lysis test of patients and healthy subjects. Thus, a peak of erythrocyte maximal hemolysis in the patients corresponded to the 4th minute vs 3.5th minute in normal individuals. The hemolysis interval was extended to 11 minutes vs 8 minutes in healthy subjects. Significant differences were observed in the qualitative characteristics: low-resistant erythrocytes made up 12% which was less than two-fold compared to healthy subjects (p<0.05). Simultaneously, a decrease of midresistant erythrocyte count (37% vs 50.8% in controls, p<0.05) and an increase of high-resistant erythrocyte count (1.8-fold, as compared to healthy individuals p<0.05) were found. By the end of the month, the sloping curve of acidified serum lysis test remained unchanged. The peak of erythrocyte maximal destruction was registered on the 5th minute (vs 4 minute in health), and hemolysis duration made up 13.5 minutes, exceeding values of the control group by 4 minutes. Thus, high-altitude adaptation stimulated the formation of young ultra-resistant red cells enriched with fetal hemoglobin due to shunt erythropoiesis.

The dry weight of a number of erythrocytes rose from 10-19 pg to 15.7±0.3 pg (Table 2). These data were evidence that during altitude climatotherapy, the number of erythrocytes with a low level of hemoglobin diminished while their hemoglobinization improved. By the end of high-altitude adaptation, the myelokaryocyte count exceeded the initial values 1.88-fold (p<0.05), pronormoblast count -1.6-fold (p<0.05), polychromatophilic normocytes -1.2-fold (p<0.05), oxyphilic -1.4-fold (p<0.05), and the number of basophilic normocytes decreased 1.4-fold (p<0.05) and lymphocytes to 19.5±0.85% (by 28%, p<0.05).

In all patients marrow aspiration from the iliac bone was performed before climatotherapy. We obtained high quality smears from 118 (58%) patients. Complete myeloid aplasia was observed in 38 (32%) patients and hypoplasia (in the interbeam spaces filled with fat there were few sites with cellular elements)- in 80 subjects (68%). In the final stages of high-altitude adaptation, bone marrow biopsy of good quality were obtained from 62 patients. In 34 (54%) subjects with hypoplasia of bone marrow the interbeam spaces were filled with red bone marrow, in 2 - the ratio of red and yellow bone marrow was equalized and in the others (16 patients with hypoplasia of bone marrow and 10 with aplasia) the picture remained invariable but individual megakaryocytes were still observed.

To study erythropoiesis in 20 AA patients in the Hematological Department (Bishkek), the ratio of nucleated erythroid cells were counted at different stages of maturation. Analysis of pronormoblast revealed a statistically significant increase in the number of basophilic erythroblasts up to 9.9±0.57% vs 1.8±0.35% (p<0.05) in healthy donors which indicates intramyeloid destruction of erythroid cells. Measurement of the ineffective erythropoiesis value was conducted by polysaccharide detection in erythroid cells of bone marrow (PAS reaction). In this study, an increase of polysaccharide concentration in erythroblasts of bone marrow was observed in diffuse form (18.4 vs 4.6% in norm, p<0.001) (Fig. 1). Basophilic erythroblast made up the major part of PAS-positive cells. During high-altitude adaptation, a growth in nucleated erythroid cell count and an improvement in their maturation was noted. The content of polychromatophilic erythroblasts (on average 11.9±0.58%) differed insignificantly from initial values. At the same time, an increase in the pronormoblast count and a considerable decrease in the number of basophilic erythroblast was observed, suggestive of diminution of ineffective erythropoiesis under high-altitude conditions. It was also corroborated by a considerable decrease of PAS positive erythroid cell count of 5.27 vs 18.4% before therapy. Three months after climatotherapy their number made up 4.8%.

The proliferative activity of erythroid cells of bone marrow was determined by using DNA progenitor H³ - thymidine. In AA patients it was initially diminished as compared with controls (Fig. 2). Thus, the labelling index of pronormoblasts decreased 6.7-fold, basophilic erythroblast 4.5-fold (p<0.001), polychromatophilic erythroblasts 5.6-fold (p<0.001). According to the autoradiography data mountain climatotherapy, induced statistically significant growth of proliferative activity of nucleated myeloid cells (Table 3). Thus, after therapy, the pronormoblast labelling index increased 4-fold (p<0.01), basophilic erythroblasts 2.8-fold (p<0.05), polychromatophilic cells 3.1-fold (p<0.01), total labelling index 2.1-fold (p<0.05). Three months after descent, an insignificant decrease of the mentioned parameters was found though they remained statistically greater compared to initial values.

Studies of iron metabolism revealed that during the 40 days sojourn at high altitude, a significant diminution of serum iron level (34.2% from the initial level, p<0.05), as well as sideroblast and siderocyte counts by 51% and 60%, respectively (p<0.05) were observed.

Experimental investigations demonstrated that in bone marrow, besides hematopoietic stem cells, there were a line of self-maintaining stromal cells. It is known that stromal cells of hematopoietic organs play an important role in the prolonged maintenance of normal hematopoiesis, and they participate in the formation of a hematopoietic microenvironment. For this reason, the study of the ability of human normal and pathological bone marrow for fibroblast colony-forming efficacy (FCFE) is of undoubted interest. In 18 patients, fibroblast colonies from the bone marrow was cultured and FCFE changes examined. Before climatotherapy, FCFC of bone marrow in AA patients was extremely low, and in 10, the fibroblast did not proliferate (Table 4). In the other, from 0.1 to 0.5 colonies were formed. By the end of adaptation a growth of bone marrow fibroblasts in the culture was significantly activated in 9 subjects, 5 especially. Thus, studies of fibroblast growth in the monolayer culture showed that sojourn at high-altitude could cause the growth activation and 2-4-fold ECFE augmentation in 50% of the patients. In 4 patients with iron-deficiency anemia (control) the effectiveness of fibroblast growth was similar to the lower limits of the norm both before and after a stay in the mountains.

It is known that adrenal hormones possess the ability to improve erythropoiesis, as they are useful in the immune genesis of AA, and high-altitude hypoxia stimulates the activity of adrenal glands in healthy subjects and intact animals. So in 43 AA patients the 17-OCS in blood plasma and in 28 AA subjects the urinary excretion of total, free and combined fractions of ketosteroids were studied. Simultaneously, in 33 patients 17-OCS and 17-KS urinary metabolites were investigated and in 32 subjects the cortisol concentrations were measured. Activation of adrenal cortical function in AA patients under the influence of high altitude manifested by an increase of 17-OCS content in blood plasma (by 26%, p<0.05) and of all the urinary metabolites of 17-OCS and 17-KS (2-3-fold, p<0.05). Changes of androgen function of the adrenal glands determined by altitude hypoxia promoted the normalization of the menstrual cycle in 6 females with AA, as well as the occurrence of pregnancy and normal delivery in some patients after repeated (2-3) courses of mountain therapy.

To understand the pathogenesis of the disease and to determine whether changes of hematopoiesis have been superficial or if they had affected a bone marrow ultrastructure, we conducted comprehensive studies of the intimate hematopoietic mechanisms, in particular, erythropoiesis. As a result, electron microscopic examination of the bone marrow revealed that during mountain climatotherapy the stage sequence of erythroid cell maturation did not change. Erythroblast cellular surface became even with insignificant invaginations, while in the foothill conditions erythroblasts had variegated shapes. Activation of a lamellar complex occurred, manifested by the appearance of a great number of follicles and vacuoles of different sizes, especially on the border with endoplasmic reticulum. Such an electron microscopic picture was characteristic in the patients who received the course of climatotherapy. In addition, a decrease of ferritin accumulation in cytoplasm and the extent of destructive changes in mitochondria was also observed, i.e. swelling and enlightenment of the mitochondrial matrix diminished. The greatest response to the high-altitude hypoxia was found in the nuclear structure. Expansion of the perinuclear space filled with pellucid for electron microscopic contents significantly decreased; nuclear chromatin was condensed; there were nuclear pores especially where light areas of chromatin came into contact with nuclear membrane. Therefore, the effect of high-altitude hypoxia on erythroid bone marrow cells in patients with AA causing observed destructive changes have a reversible character.

The survival rate of AA patients treated at a high altitude showed that six months after climatotherapy, the survival rate was 73.8%, after a year the rate was 61.5%. Fifty-eight and four-tenth percent of the patients survived up to 3 years and 52.1% up to 5 years. The 7-year survival figure amounted to 38.5%, and 22% of the patients survived for 10 years (Fig. 3).

Analysis of the findings reveals that high-altitude climatotherapy can be an important supplementation to the available methods of treatment for AA. In the case of insufficient efficacy of a single course of climatotherapy, repeated courses are recommended, especially for patients suffering from severe AA.

Conclusion

The influence of a high-altitude climate on erythropoiesis in patients with AA is manifested by reticulocytosis, appearance of ultra-resistant erythrocytes enriched with fetal hemoglobin, improving erythrocyte hemoglobinization, decreasing the signs of ineffective erythropoiesis, amplifying the processes of differentiation and proliferation in erythroid cells of the bone marrow, an increase of bone marrow cellularity and a considerable growth of fibroblast colony-forming efficacy.

* High altitude exerts a stimulating effect not only on erythro-, leuko- and thrombopoiesis but also on corticosteroid and androgen functions of adrenal cortex, and according to the data of electron microscopy, destructive changes in erythroid cells of the bone marrow can be reversed.

Single and especially repeated (3-4) courses of climatotherapy produce a significant decrease in the signs of anemic and hemorrhagic syndromes, improving the course of the disease and extending the period of remission.

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