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The work deals with issue of workers motivation on projects managed by method of Theory of constraints (TOC) which improves efficiency of projects. But this management method requires completely different manner of project workers consideration which can be unnatural for many people. This can cause same problems that in extreme cases may involve unsuccessful implementation of TOC. This is the reason why importance of motivation rapidly increases in connection with deployment and sustainment of Critical Chain Project Management. Main goals of this work are to describe most important differences between TOC and original methods of project management, to indentify impact of TOC on project workers, and (in relationship with previous findings) to evaluate existing theories of motivation and make a proposal of some measures for people motivation which are based on these theories.

The work is concerned with stakeholder analysis in information system project. This theme is a part of project management. The main goal is to describe process of stakeholder analysis, which is solved very poorly in technical literature. Other goals are: describe the influence on the project and possibility of prevention and solution of the most common stakeholders in detail, and briefly search the level of solution of this issue in technical literature. The work mainly draw from personal ideas, party from experiences contained in mentioned literature. The process of analysis, which is the main theme of this work, is processed almost completely on basis of personal creative activity. The document is divided into six chapters. The first one is introducing the theme, second chapter deals with basic terms, third chapter describes the stakeholder analysis, fourth chapter presents the influence of most common stakeholders and fifth chapter mentions the level of solution in source literature. The last chapter summarize all gained knowledge.

The author describes a peculiar case of a paternity suite where the accused died and his parents were also dead. Possible paternity was assessed by examination of HLA antigen of his ex-wife and daughter from his former marriage and in his sisters.

The author described an uncommon case when neither of two men (former husband and another man) who were considered possible fathers of the child, could be ruled out as potential fathers based on examination of signs of 19 erythrocyte, serum and enzyme systems and 59 HLA-A, B, C antigens and their subtypes. The eliminating capacity of the examination was 99.86%; paternity was, based on probability calculation, practically proved (p = 99.9925% and 99.90%). According to statements of parties in the dispute the other man is the child's father. The author discusses perspectives of DNA examinations.

The authors describe another subtype of antigen HLA-B27, described as HLA-B27P3. It was assessed, using 5 anti-HLA-B27 sera. It is identical with the weak antigen HLA-B27(HLA-B27w) described by the author in 1976.

In 79 leukaemic subjects (42 with acute leukaemia, 37 with chronic myelosis) the authors assessed HLA-DR antigens 1-5.7 and their frequency. As compared with a group of 130 healthy subjects, no significant difference was found. The results do not support a possible relationship of HLA-DR antigens and leukaemia.

In 1974-1992 in the HLA reference laboratory of the Institute of Haematology and Blood Transfusion in Prague 15,731 sera sent by transfusion departments were examined; 4368 sera met the standard required for HLA typing, detecting 9 HLA-A, 18 HLA-B and 3 HLA-C antigens. Against these antigens with the exception of HLA-B41 and public antigens HLA-Bw4 and HLA-Bw6 monospecific sera were found: against 15 HLA antigens in a large number and against 12 HLA antigens--2 to 21 for the given antigen. Sera against subtypes of HLA antigens were sought only in rare instances, as in common practice it is sufficient (and sometimes essential) to type only basic HLA antigens.

Lymphocytes treated with a 6% and 4% AET solution after 60 or 120 minutes at 20 degrees or 37 degrees C display polyreactivity. Polyreactivity does not develop after short-term treatment (1-10 mins.). After treatment of lymphocytes with a 3% or 2% AET solution for a period of 20 or 30 mins. at 20 degrees or 37 degrees C the specific expresivity of HLA--A, B antigens rises; they are manifested, contrary to untreated lymphocytes, also by weak HLA sera. Addition of AET to the mixture of lymphocytes--HLA serum causes in the first stage of the microlymphocytotoxic test negativity of the test due to a block of the haemolytic activity of rabbit complement.

For evaluation of the results of HLA examinations in paternity disputes the objective finding that the man has HLA antigens which the child inherited from the father, is decisive. The controversy as regards heredity ensuing from assessment of HLA haplotypes of the man by family studies is caused by recombination. The author presents a case of this type.

Forty-five specimens of lymphocytes from HLA-B27 positive subjects were examined by the micro-lymphocytotoxic test with 48 anti-HLA-B27 sera and the results were evaluated statistically by calculation of the correlation coefficient r. Based on the results of the statistical evaluation two groups of sera were determined which determined two groups of lymphocytes, carriers of different HLA-B27 subtypes. Six sera determined subtype B27P1, detected in four lymphocyte samples, another six sera subtype B27P2 found in three lymphocytes. The results of the statistical evaluation correspond to the serological reactions. The results indicate that HLA antigens can be reliably differentiated by simple methods.

Analysis of the results of check-up examinations of HLA-A,B,C antigens in 62 cases of paternity demonstrated complete agreement of the first and second expert in 58.06% cases. Differences in 26 cases (= 41.94%) consisted in 10 cases in the detection of different HLA antigens from the first and second expert (owing in 6 cases to the different conclusion of the experts' account) and in 16 cases in the evidence of some HLA antigen only from one expert (usually by the revision). The most frequent errors pertained to the evidence of HLA antigens A28, A29, A30/31, A33, B16, B17, B21, B18, Bw22 and of the antigens of HLA-C locus; however, the well demonstrable HLA antigens A9, A10, B8, B13 were also not proved. The causes of the mistakes were analysed. It is necessary to avoid the errors of the administrative character and by the evaluation of the results in doubtful cases.

The author analyzed possibilities as regards the solution of paternity suits where paternity depends whether on the HLA 1 locus he is a homozygote or not. The authors presents a case of solution of a suit where the result depended on whether the man is a HLA-A2 homozygote or whether he has on the locus along with HLA-A2 also HLA-28 antigen.

The author investigated the effect of 20%, 15%, 10%, 5%, 2.5%, 1.25% chloroquine on HLA-A,B antigens on lymphocytes and on the reaction HLA-A,B antigen-antibody. HLA-A,B antigens are not released by chloroquine from the lymphocyte surface and their expressivity does not change, as was revealed by examination of the micro-lymphocytotoxic and absorption test. Higher concentrations of chloroquine cause poly-reactivity of lymphocytes. A 20%, 15% and sometimes also 10% and 5% of chloroquine solution block the reaction HLA-A, B antigen--HLA antibodies. The reason for this is the destructive action of chloroquine on rabbit complement.

Anti-idiotype antibodies against HLA specificities anti-HLA-A1, -A2, -A9, -A10, -B7, -B8, -B12 were detected in sera of 13 patients after transplantation of bone marrow and in five patients who had no transplantation but many blood transfusions. They had a multispecific character, inhibited concurrently 4-5 of the mentioned HLA antibodies. HLA antibodies were not present in sera of these patients and did not develop even after treatment with dithiothreitol. It seems therefore that anti-idiotypic antibodies have also a IgG structure. The bond of anti-idiotype antibodies with HLA specificities was firm and maximal: they were not released after 24 hours incubation at room temperature and did not form bonds with other HLA antibodies with the same specificity. The authors discuss the importance of anti-idiotype antibodies against HLA specificities for the outcome of bone marrow transplantation and therapeutically successful thrombocyte transfusion.

HLA-DR antigens were examined in peripheral lymphocytes by means of a one-colour fluorescent microcytotoxic test using ethidium-bromide. Evidence of HLA-DR antigens was equally reliable as the two-colour fluorescent test. Contrary to the latter, however, faint non-specific positive results were obtained in sera not containing HLA-DR antibodies. This is caused probably by the fact that ethidium-bromide penetrates into cells with a very slightly damaged membrane. This reduces substantially the possibility of introduction of the method into common practice.

As part of research of juvenile hypertension the authors assessed the types of HLA antigens loci A and B in 242 children, adolescents and young adults with asymptomatic primary hypertension. The control group was formed by 300 normotonic blood donors. In the group of hypertonic subjects a significantly higher frequency of HLA-B5 antigen was found and a lower frequency of HLA-B8 and B35. Classification of the group of patients by the type of raised BP revealed the following: in subjects with a variable BP, as compared with the control group, antigens HLA-A1, B8, B35 and B40 were less frequent, in subjects with a permanently elevated blood pressure antigen HLA-B5 was more frequent and conversely antigen HLA-B35 was less frequent than in healthy subjects. From the investigation ensues a probable relationship between the presence of antigen HLA-B5 and the greater risk of the subsequent manifestation of the disease and conversely between the presence of antigen HLA-B35 in subjects at risk and probably a favourable prognosis of blood pressure readings. Contemporary findings cannot yet be considered unequivocal and it will be necessary to test their validity by extending the number of examined subjects.

HLA sera were absorbed with erythrocytes with proved HLA antigens and blood cells with HLA-A, B antigens absorbed in vitro from serum. Both types of red cells were 5x to 15x washed in saline and exposed for two hours to the action of 15% chloroquine. The degree of absorption of HLA antibodies and the decline of the score were equal or slightly different after absorption with erythrocytes with own or quinked HLA antigens. Washing in saline or processing of blood cells with chloroquine did not affect the resulting absorption. From the results of the experiment ensues that the link of HLA-A, B antigens is equally firm blood cells isolated from the peripheral blood stream and blood cells where the antigens were linked to them in vitro from serum. This suggests that the view is justified that the majority of HLA-A, B antigens detected in blood cells is secondarily linked from plasma.

Ethanol in a concentration of 0.001% to 2% increases the expressivity of HLA-A, B antigens on lymphocytes. In 26.13% of the examinations HLA antigens were proved only on lymphocytes processed with ethanol. The expressivity is maximally enhanced already by short-term incubation of lymphocytes with ethanol; if prolonged to 120 mins at 20 degrees C ethanol does not produce an unspecific positivity of results. There is no difference between processing of lymphocytes at 20 degrees C and 37 degrees C. 1% and 2% ethanol facilitates detection of HLA-A, B antigens after addition to serum and lymphocytes in the first stage of the microlymphocytotoxic test.

Typing of 38-43 HLA antigens ruled out paternity in 33.75% of 726 men suspected, during 198 revisions in 71.72%. In 63.57% the man was ruled out concurrently in loci HLA-A and B incl. in 79.33% where the man and child had all HLA-A, B antigens different. Locus HLA-A alone was ruled out in 8.01%, locus HLA-B in 28.17%; in a single case the man was ruled out in locus HLA-C only. By family investigations the man was ruled out in three of 11 cases. In two cases of paternity of twins the man was ruled out from paternity only of one child. In one case superfecundation was involved, confirmed in systems Duffy, Inv, Hp, AP, in the second case the man was ruled out from paternity of the second child in the Hp system. Male carriers of antigens of haplotypes frequent in the population were not ruled out from paternity in high percentages: with HLA antigens A1, B8 in 37.19%, A2, B7 in 19.54%, A2, B12 in 24.72%, A3, B7 in 37.14%.

The author describes his own modification of a two-colour fluorescent technique for typing of HLA-DR antigens. Concurrent examination of HLA-DR antigens in 34 subjects by this method and the monocytotoxic or B-lymphocytotoxic test revealed a greater sensitivity of the fluorescent technique; there was however also a tendency of non-specific positivity of results. More reliable results were obtained when the fluorescent technique was performed on Hammat plates, as compared with examinations using Terasaki plates. To eliminate erroneous results, the author recommends to use for HLA-DR typing two methods concurrently.

After saturation of the anti-A monoclonal antibody USOL by A1 erythrocytes sensitized with various human anti-A sera and or by several specimens of A1 blood cells sensitized with the same anti-A serum the authors detected a different decline of the titre and score of antibody. Similar results were obtained also in attempts to saturate various human anti-A sera with A1 erythrocytes sensitized with monoclonal anti-A. This means that A epitopes on A1 blood cells of different subjects are not identical as regards the content of A determinants, and that different human anti-A sera an d anti-A monoclonal antibodies differ as to the structure of anti-A specificities.

In 10 patient-sibling pairs (sibling perspective bone marrow donor) the author typed HLA-A, B, DR and specific monocyte antigens MoP-1 to 6. Concurrent HLA-A, B and MoP identity was revealed in 80% of the cases, concurrent HLA-A, B, DR and MoP agreement in 60%. The author discusses the importance of this observation for the solution of the relationship between HLA and specific monocyte antigens. He recommends typing of specific monocyte antigens before transplantation of bone marrow as another way of donor selection.

On the model of the Soviet monoclonal anti-A and anti-B antibody--erythrocytes of the groups A1, A2, A1B and A2B the conditions for the elaboration of the branch standard for anti-A and anti-B monoclonal antibodies were worked out. In comparison with the standard for human sera determining the ABO groups, the requirement for avidity can be increased, furthermore it seems necessary to determine the percentage of non-A blood cells in the A2B group, which should not exceed 30%, and the percentage of non-B blood cells in A1B erythrocytes, which should not exceed 10%.

HLA antigens on erythrocytes were assessed concurrently by three tests--the indirect antiglobulin, two-stage bromelin and absorption test--using polyspecific and monospecific sera against HLA antigens A1, A2, B7, B8 and B12. Evidence obtained by the bromelin test was successful only in rare instances. The positivity of the absorption test increased with the number of saturations: after triple saturation of the HLA serum with erythrocytes of the same subject readings were obtained twice to three times more frequently than after single absorption. As compared with the indirect antiglobulin test, the positive erythrocyte reactions in the absorption test were less frequent: with polyspecific HLA sera in 36.97% as compared with 78.15%, with monospecific HLA sera in 63.64% as compared with 100%. The absorption test is a suitable method for detection of HLA antigens on erythrocytes, supplementing the indirect antiglobulin test.

Weak accessory HLA antibodies in HLA sera were assessed by three test--the microlymphocytotoxic, antiglobulin cytotoxic and fluorescent cytotoxic test. Most frequently they were detected by the antiglobulin cytotoxic test (69.64%), followed by the fluorescent cytotoxic test (55.73%) and least frequently by the microlymphocytotoxic test (45.77%). From the results ensues that the antiglobulin cytotoxic test is a suitable method of crossmatch before thrombocyte transfusions to patients who developed reactions after transfusions or where therapy failed although the crossmatch was performed by the microlymphocytotoxic test between the donor's lymphocytes and the patient's serum and the test was negative.

The authors describe a rare paternity suit where paternity was ruled out in the HLA system only in locus HLA-C. The absence of HLA-Cw2 antigen on the lymphocytes was proved by the negative results confirmed by the same result on examination of the erythrocytes Ss system.

The authors describe four cases of paternity suits where the paternity was not ruled out according to HLA antigens and was ruled out in signs of erythrocyte, serum and enzyme systems, although the capacity of the HLA system was high, in two instances above 98%. From the results ensues that it is useful to supplement the examination by erythrocyte, serum and enzyme systems in those instances where HLA typing was performed first and the man was not ruled out.

HLA-A, B antigens and the ABO group were examined in 184 patients with aplasia of bone marrow and leukaemia and in 373 of their relatives, mostly siblings. A HLA-A, B donor, identical or compatible, was found for 35.87% patients, a HLA-DR identical for 84.21% of 38 patients who had a HLA-A, B identical relative. Bone marrow was transplanted to 16 patients (10 with bone marrow aplasia, 6 with acute or chronic leukaemia), with one exception bone marrow from a sibling. The bone marrow was accepted in all patients but two where the transplantation was made despite MLC positivity. From the results ensues that it is essential for successful transplantation of bone marrow to ensure maximal identity between donor and recipient as regards the ABO group, HLA antigens and negativity of the MLC reaction. The negativity of the MLC reaction is more important than HLA-DR identity; when assessing only one HLA-DR antigen in a donor identical with the patient, it cannot be ruled out that on the lymphocytes of the donor there exists another one which was not detected. The authors discuss the causes of different results of the MLC reaction and HLA-A, B, DR typing.

Twelve monocytotoxic sera were examined with 15 samples of monocytes, using the micromonocytotoxic and micromonoagglutination test. Weak agglutinins [+/-, +] were detected concurrently with cytotoxins only in 3 of 180 examinations at 37 degrees C. Monocytotoxins were found in 23.33% of reactions. From the results ensues that the existence of monocytic agglutinins is dubious.

In a group of 69 insulin dependent diabetics aged 19-59 years (mean 25.5 years) with a duration of diabetes of 2 to 34 years (mean 12.5) the authors assessed the incidence of diabetic retinopathy, nephropathy and neuropathy in relation to the duration of diabetes, to its long-term compensation and HLA antigens. In 45 the diabetes was manifested before the age of 15 years. The authors found a rising trend of retinopathy (12-14-25-75-86%) and neuropathy (0-50-60-85-83%) in five groups with a duration of diabetes up to 5, 10, 15, 20 and above 20 years. 15% of the patients with a duration of diabetes of more than 15 years had positive microalbuminuria or permanent proteinuria and hypertension. In diabetic patients with long-term satisfactory compensation there was a lower incidence of these complications than in patients with poorer compensation. The presence of HLA B8 antigen was associated with a prolonged favourable course of diabetes, with a lower incidence and later manifestation of complications.