PROGNOSTIC VALUE OF ERYTHROCYTES STRUCTURAL AND ULTRASTRUCTURAL CHANGES IN THE COURSE OF METASTATIC ADENOCARCINOMA OF THE PROSTATE

Reviewable on-line scientific magazine “Caucasus Medical Herald”, N28 april, 2011

PROGNOSTIC VALUE OF ERYTHROCYTES STRUCTURAL AND ULTRASTRUCTURAL CHANGES IN THE COURSE OF METASTATIC ADENOCARCINOMA OF THE PROSTATE

Veshapidze N., Alibegashvili M., Kotrikadze N., Gabunia N., Chigogidze T.

Departments of  Exact and Natural Sciences and Medicine, Iv. Javakhishvili Tbilisi State University

It’s known that erythrocytes defense function, that is absorption, transportation and participation in metabolic processes is an important part of organizm`s regulative mechanism [6,5] but its alteration plays pathogenetic role in pathogenesis of cancer diseases [8,2,3]. Considering the above mentioned hypothesis it is obvious that the changes of erythrocytes defense function to the structural and ultrastructural levels may have a diagnostic importance, it`s reflect the complicative course of the disease and  therefore can be an objective criterion of  treatment effectiveness.

Insofar our studies stipulated for revealing alterations of the defense function of the erythrocytes in the men with metastatic adenocarcinoma of  the prostate, before the castration and thereafter, a morphological pattern of the erythrocytes was studied with an aid of the light and electron microscopy. It is considered that changes of the erythrocytes defense function are reflected primarily in the altered morphology of the erythrocytes.

MATERIAL AND METHODS

The erythrocytes of 15 men with the metastatic adenocarcinoma of  the prostate, before castration, and of the 15 men ~ 6 months after castration, served as experimental material. The erythrocytes of the 15 apparently healthy men was used as the control group material. The patients age was ranged between 60-75 years.

The method of the light microscopy [7]

The blood was sampled from a finger, on an empty stomach. The smear was fixed, dried and stained according to the Anders’ method (I azur – II eosin). The material was observed in the light microscope Photo-Microscope (Opton, Germany). Each specific parameter was calculated per 100 cells.

Experimental data were processed by means of standard variation statistics method  MINITAB (Basic statistic). P<0,05 was taken as statistically significant indice.

The method of the electron microscopy [1]

The ultrastructural observation of the erythrocytes was made by means of the electron microscope BS-500 (“Tesla”, Czech Republic), an acceleration voltage of  microscope  was    about  80 kVT. Blood sample was fixed (double fixing) in the 1% osmium dioxide solution in  buffer. Fixed material was sliced with the ultra-microtome OmU2 (Reichert, Austria), the slices width was 1200-1500 Ǻ. Contrasting of the slices was performed  by the  Reinold’s method.

RESULTS

The light microscopic study of erythrocytes has shown: that in the blood of control group men were fixed: round and long shaped erythrocytes, erythrocytes with central unstained part (CUP) (big, medium and small diameter) and erythrocytes wich did not have central unstained parts (Table. I), (Table. II) (Fig. 1,a.).

Concerning the light microscopic examination of the erythrocytes in the men with metastatic adenocarcinoma of  the prostate (pCa), investigations performed prior to castration have shown that in the blood of the patients are present all those structural properties of the erythrocytes, which are characteristic of the blood in the control group. However quantitative  changes of the structural indices were manifested quite conspicuously (Tables I and II; Fig. 1b.). In addition, unlike the blood of the control group in the blood of the metastatic prostate cancer patients, prior to castration, the pathological erythrocytes (the erythrocytes with protuberances and folds) and adhesion of the degenerated thrombocytes on the erythrocytes’ surface have been revealed (Table II). These results certify for decrease function of erythropoiesis, accompanied with sharp increase of percentage of the acanthocytes – (senescent erythrocytes [11]),  (Table III).

Following the castration, light microscopic investigation of the erythrocytes also revealed all the structural features characteristic of the control group, although in a post-castration material, quantitative alterations of the structural indices have been observed: macrocytosis, microcytosis, acanthocytosis, increase of the pathologic and elongated erythrocytes number, and decrease of the normocytes (Tables II and III; Fig. 1c.). The data obtained point at existence of the post-surgery course of the disease. All the changes in the above structural properties of the erythrocytes, in their turn, indicate decrease of the erythropoesis. It should be considered, also that after the castration some structural indices of the erythrocytes normalized relatively and approached somewhat the indices of the control group (Table I) – number of the erythrocytes without the CUP decreased, number of the normocytes with the large diameter CUP-s increased, and the number of the erythrocytes with the small diameter CUP-s equaled the control group indices. This, presumably, should be considered as strengthening of the erythrocytes’ absorption capacity and improvement of respective defense ability. Validity of this suggestion is supported by the fact that after the castration complete adhesion of thrombocytes on the erythrocytes surface was not found (Fig. 1c.) . Contrariwise, in the metastatic adenocarcinoma of  the prostate (pCa), before the castrations, adhesion of the degenerated thrombocytes on the erythrocytes surface was obvious (Fig. 1b.). Adhesion of the thrombocytes onto the surface of the pathological erythrocytes plays role of filter for disabled erythrocytes with lowered functional activity. Considering the above-mentioned, the fact that following the castration there is no adhesion of the thrombocytes on the erythrocytes surface, once more certifies for recovered defense response of the erythrocytes against the tumor pathology. As to appearance of the giant and degenerated thrombocytes in the blood after castration (on the background of decreased amount of the mature thrombocytes), this must point at weakening of functional activity of the thrombocytes. Thus, notwithstanding the partial recovery of the erythrocytes defense function in the post-surgery period, functional activity of the thrombocytes remained deteriorated.

At the next stage of the study, morphological pattern of the erythrocytes was studied by the electron microscopy. The studies have shown that in the blood of the control group men, mostly the biconcave disk-shaped cells were presented (normocytes) (Fig. 2,a.) The borders of such cells were sharply contoured, while there were almost no changes in the staining distribution zones. In the center of the cells there were usually located the CUP-s of various diameters (large, medium, and small). Just to this region is connected the defense function of the erythrocytes, which implies absorption and metabolism of the toxic compounds circulating in the blood [6,2,10].

The electron microscopic investigation of the erythrocytes in patients with metastatic adenocarcinoma of  the prostate (pCa) (before- and after the castration) has shown the weakly contoured, misshaped cells – the pathologic erythrocytes (Fig. 2 d,e.). On the surface of such cells there were almost no alterations in the staining zones’ distribution, although the some regions with relatively weak staining were manifested in the blood of both prior and following the castration. In the blood of the men with the metastatic pCa, the elongated erythrocytes (Fig. 2b,c.) were shown, which frequently built the sludges, although, it should be noted that the borders of these cells were isolated from each other; there was no adhesion between them and respectively, there was no contact between such cells.

Revealing of the elongated erythrocytes in the peripherial blood and increase of their number, on the one hand, is an index of the erythropoesis process deterioration [12], which was certified also by our light microscope studies (Tab. II). On the other hand, this must point at involvement of the central nervous system (CNS) [9]. Functional deterioration of the latter, in its turn, reflects an alteration of the general homeostasis of the organism [ 13].

Besides the above-mentioned, investigation of the erythrocytes with the electron microscopy method has shown that prior to castration and thereafter, buildup of the sludge between the erythrocytes did occur (Fig. 2 b.), while after the castration the sludge were noted not only between the erythrocytes (Fig. 2c), but between the erythrocytes and thrombocytes as well (Fig. 2 d, e.). As it is evident in the morphological pictures of the erythrocytes, the erythrocytes and thrombocytes are presented in the “inter-squeezed” mode, although the full adhesion between them did not occur. Respectively, there should be no direct contact between the cells. The latter is evidenced by the area of the sharply contoured surfaces of the thrombocytes. It is suggested that only small area of the thrombocytes must contact with the erythrocytes, because in these sites deterioration of the thrombocytes’ edges does occur (Fig. 2 d,e). Putatively, typical mechanical squeezing of the thrombocytes between the erythrocytes, which occurs following the castration, determines alteration of the electric potential and superficial electric charge on the erythrocyte surface [4]. Probably, just such interrelation between the erythrocytes and thrombocytes promotes transformation of the altered thrombocytes into the targets for the immunocompetent cells, which either immediately destruct them, or facilitate further phagocytosis of the pathological erythrocytes by the macrophages. It is suggested that incomplete adhesion of the thrombocytes on the erythrocytes surface promotes production of the dwarf erythrocytes (microcytes) and their release into the circulation (Fig. 2 d,e).

Therefore, microscopic investigation of the erythrocytes’ morphological pattern in the genesis of the prostate cancer, has a prognostic value, because investigations in the above direction provide respective information concerned with the changes in the organism subjected to the tumorous damage.

DISCUSSION

  • The alterations occurring in the erythrocytes before the castration have been determined at the structural and ultrastructural levels. This fact have shown that decrease of the defense functions of the erythrocytes and weakening of an organism’s controlling systems’ responses are the indices of the responses against the tumor pathology.
  • It was found that following ~6 months after castration, some structural features of the erythrocytes underwent relative normalization. This fact is an index of improvement of the erythrocytes’ absorption ability and, respectively, defense capacity.
  • Following the castration,  the changes of the erythrocytes at the structural and ultrastructural levels have been found, which indicate the post-surgery course of the disease.

CONCLUSIONS

The above changes may be used as one of the additional prognostic tests, in order to determine the level of the disease progression following the castration. However, it should be considered that this surgical method of treatment is a palliative one and does not imply a total cure of a patient.

References:

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Pjysiol. 1995; 22:1-25.

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Table I

Variation of morphological index (percentage) in erythrocytes
(100 cells for each parameter) 

Erythrocytes Control group PCa  

(before castration)

PCa 

(after castration)

Erythrocytes without CUP 38  ± 1 %  

62 ± 5 %

 

50 ± 3 %

Erythrocytes with CUP 62  ± 1.5 %  

38 ± 3 %

 

50 ± 2.2 %

Big diameter erythrocytes with CUP 15 ± 1,6 %  

20 ± 2 %

 

35 ± 3 %

Medium  diameter erythrocytes with CUP 68 ± 3,4 %  

40 ± 4 %

 

41 ± 2 %

Small diameter erythrocytes with CUP 17 ± 2,2 %  

45 ± 1 %

 

24 ± 5 %

N=15 (number of patients in each group), the patients and controls  age was 60-75 years.

p<0,0001

Table II

Variation of patholological erythrocytes concentration

(100 cells for each parameter)

Erythrocytes Control group PCa  

(before castration)

PCa 

(after castration)

Erythrocytes with protuberances and  fold biological  net __ 28 ± 3 % 0,2 ± 0,01
Long erythrocytes 2 ± 0,2 % 15 ± 0,3 % 

p<0,0001

12 ± 0,3 % 

p<0,0001

Pathological erythrocytes __ 22 ± 1 % 12 ± 1 %

N=15 (number of patients in each group), the patients and controls  age was 60-75 years.


Table III

Variation of structural index (percentage) in  erythrocytes (100cells for each parametr) 

Control group PCa  

(before castration))

PCa 

(after castration)

Normocytes (7-8 MKM) 90,8±± 4% 64 ±±5% 

65±± 4% 

Macrocytes (>8 MKM) 2,2±± 0,4% ± 0,4% 

15 ±±0,3% 

Microcytes (<6 MKM) 1,8±± 0,2% 12±± 0,1% 

9 ±±0,1% 

Acanthocytes 0,2±± 0,05% 15±± 0,2% 

11 ±±0,1% 

Ghost-cells ± 0,05% ± 0,1% 

± 0,3% 

N=15 (number of patients in each group), the patients and controls  age was 60-75 years.

p<0,0001

Figure  1. Erythrocytes  morphological picture (light microscope) (10x2x100)

a) Control group; b) Metastatic PCa, before castration c) Metastatic PCa, after castration:

1. Erythrocytes with CUP; 2.Erythrocytes without  CUP.; 3.Big diameter erythrocytes with  CUP; 4.Medium diameter erythrocytes with CUP; 5.Small diameter erythrocytes with CUP; 6.Adhesion of degenerative thrombocytes on erythrocytes’ surface;

Figure  2. Erythrocytes  morphological picture (electron  microscope). (8000×2);

a) Control group b) Metastatic PCa, before castration;  c,d,e) Metastatic PCa after castration:

1. Pathological erythrocytes; 2.Long erythrocytes; 3. Sludges between erythrocytes; 4. Sludges between erythrocytes and thrombocytes; 5. Typical mechanical squeezing of the thrombocytes between the erythrocytes; 6. Dwarf erythrocytes.

Resume:

Objectives: In order to determine prognostic value of erythrocytes structural and ultrastructural changes  in the course of prostate malignant tumors, alterations of the defense function of the erythrocytes were investigated in the apparently healthy men (control group) and the men with metastatic adenocarcinoma of  the prostate (pCa), before the castration and thereafter.

Materials and Methods: The erythrocytes of 15 men with metastatic adenocarcinoma of  the prostate (pCa), before castration, and of the 15 men  ~ 6 months after castration, served as experimental material. The erythrocytes of the 15 apparently healthy men was used as the control group material. The patients age was ranged between 60-75 years.

Results: The alterations occurring in the erythrocytes before and after the castration have been determined by light and electron microscopy at the structural and ultrastructural levels.The experiments have shown that decrease of the defense function of the erythrocytes and weakening of an organism’s controlling systems’ responses are the indices of the responses against the tumor pathology before the castration. It has been established, that following ~ 6 months after castration, some structural features of the erythrocytes underwent relative normalization. This fact is an index of improvement of the erythrocytes’ absorption ability and, respectively, defense capacity.