A polymerase chain reaction-sequence-specific oligonucleotide procedure for HLA class II typing using biotin- and digoxigenin-labeled probes simultaneously in hybridization.

To simplify PCR-SSO HLA-DRB generic typing, we labeled eight of 15 oligonucleotide probes with DIG and the others with biotin, and hybridized each dot blot with both a biotin-labeled probe and a DIG-labeled probe simultaneously. We chose oligonucleotide pairs which require the same hybridization and stringent washing conditions and do not compete with each other during hybridization. After incubation with a mixture of anti-DIG Fab fragment-alkaline phosphatase and streptavidin-peroxidase conjugates, specific binding of the DIG-labeled probe was revealed by a chemiluminescent substrate (CSPD) and specific binding of the biotin-labeled probe was subsequently visualized by a chromogenic substrate (TMB). The sensitivity of both probes was similar and gave comparable hybridization signals. Using this simplified procedure, the number of hybridizations or dot blots can be reduced to half the usual amount and the labor involved in PCR-SSO typing significantly reduced.

Simplified PCR-SSO procedure for DQA and DQB typing. Simultaneous hybridization of two probes to one membrane.

To simplify DQA and DQB oligotyping, we applied our improved PCR-SSO procedure for DR typing. We used 12 oligonucleotide probes for DQA typing and 18 for DQB typing. Oligonucleotide probes that require the same hybridization and stringent washing conditions were selected as pairs for simultaneous hybridization to a dot-blot membrane containing various DNA samples. One probe of each pair was labeled with digoxigenin and the other with biotin. After hybridization, the dot-blot membranes were incubated with a mixture of conjugates. Specific binding of the corresponding DNA probes was visualized on an X-ray film using a chemiluminescent substrate (CSPD) and by staining using a chromogenic substrate (TMB). This approach, previously employed for DR typing, is also suitable for DQA and DQB oligotyping and significantly reduces the labor inherent in PCR-SSO typing.

Multidrug resistance in heart transplant patients: a preliminary communication on a possible mechanism of therapy-resistant rejection.

Multidrug resistance refers to a complex cellular phenotype, the hallmark of which is cross-resistance to multiple drugs, for example, chemotherapeutic agents, that are unrelated to the selecting agent in structure, cellular target, and mode of action. The expression of this multidrug resistance is connected with the overexpression of P-glycoprotein. By applying the method of immunocytochemical assay, we have demonstrated the appearance of the multidrug-resistant phenotype (P-glycoprotein+ cells, multidrug-resistant cells) in mononuclear cells of the peripheral blood from 32/49 patients receiving triple-drug (azathioprine, steroids, cyclosporine) immunosuppressive therapy after heart transplantation. In the group of patients showing not only the presence of cells with multidrug-resistant phenotype in the peripheral blood, but also a significant increase in the number of these cells during the interval of observation (0 to 767 days)-16/32/49 cases--a significantly increased incidence of acute rejection episodes could be demonstrated. This supports the hypothesis of a possible existence of a therapy-resistant form of acute rejection, with an involvement of mechanisms of multidrug-resistance playing a role in its causal development.

[10 years transplantation of bone marrow and hematopoietic stem cells in adults at the Hannover Medical School]. / Zehn Jahre Transplantation von Knochenmark und hämatopoetischen Stammzellen bei Erwachsenen an der Medizinischen Hochschule Hannover.

PATIENTS AND METHODS: From January 1986 until August 1995 230 adult patients received an allogeneic or autologous transplantation of bone marrow or hematopoietic blood stem cells. The conditioning and myeloablative treatment regimens were chosen according to the underlying disease and type of transplant. RESULTS: The observation period comprises 1 to 115 months after transplantation. After allogeneic transplantation from HLA-identical family donors, the probabilities of disease-free survival were for acute myeloid leukemia in first complete remission (CR) (n = 35) 77%, for acute lymphoid leukemia in 1st CR (n = 7) 72% and in 2nd CR (n = 10) 40%, in first chronic phase of chronic myeloid leukemia (n = 34) 50% and in severe aplastic anemia (n = 7) 100%. Following myeloablative therapy and autologous transplantation the probabilities of disease-free survival were 47% in relapsed Hodgkin's disease (n = 22) and 42% for relapsed high-grade non-Hodgkin's lymphoma (n = 12). Eight of 10 patients with acute myeloid and 7 of 8 with acute lymphoid leukemia suffered a leukemic relapse after autologous bone marrow transplantation. Three of 8 patients with relapsed testicular cancer survived relapse-free. Treatment failures were due to more advanced acute graft versus host disease after allogeneic transplantation and caused by relapse after autologous transplantation. Current protocols evaluate the allogeneic transplantation of enriched CD34+ blood stem cells. In chronic myeloid leukemia the autologous transplantation of blood stem cells after myeloablative therapy is being studied.

[Experiences with a Token Ring Network in blood bank administration of the Hannover medical university]. / Erfahrungen mit einem Token-Ring-Netzwerk in dem Blutbankverwaltungssystem der Medizinischen Hochschule Hannover.

The Token Ring Network is a Local Area Network of IBM. It is a very helpful tool for modulating working processes by personal computers. The Token Ring is a network of the third generation and constructed like a star net. The blood bank of the Medical University of Hannover (MHH) is using the Token Ring for the administration of blood storage and donors. Medical data of foreign laboratories are transmitted into the blood bank computer. During the last year, we had no difficulties working with this software tool in response time and data security and it seemed to us a very cheap opportunity for data integration and data collection on decentralized systems.

[Experience with a microcomputer-controlled blood bank administration system in the blood bank of the Hannover Medical University]. / Erfahrungen mit einem PC-gestützten Blutbank-verwaltungssystem in der Blutbank der Medizinischen Hochschule Hannover.

During the last three years two components of a modular administration system were implemented in the blood bank of the Medical University of Hannover (MHH). Considering the integration of the blood bank system to the central hospital information system of the clinic the aspect of independence and self-controlling by the blood bank was the most important point. This goal was reached by a couple of PC's which are compounded by a local area network (LAN). This LAN can communicate with the host of the computer center of the MHH. By this way the short staff capacity was not stressed more than absolute necessary by taking over all parameters of the central data base. On the other hand all functions for administrating the blood bank were in self-responsibility of the department. This concept of different modules has the possibility of using single parts like donor- or storage-system in other blood banks without implementing the whole system.

[Familial investigations of carriers of Rh 33]. / Familienuntersuchung einer Trägerin von Rh 33.

The erythrocytes of a blood donor (P) showed an unusual pattern with anti-D sera: no agglutination with incomplete sera, and a strong agglutination with 2 out of 5 complete, monoclonal sera. These findings suggested the presence of an Rh 33, which was then confirmed in two external laboratories as the genotyp R0Har. Using anti-e sera, the titer scores of the cells of P were comparable with those of Ee cells but were only half as high as the scores of ee cells. This is consistent with a weak e in R0Har. The red cells of the donor's daughter (T) gave a positive reaction with all anti-D sera, but we observed no or only weak agglutination with several anti-e sera. It is very probable that T is also carrier of R0Har.

[Effect of temperature on the donor and the thrombocyte function in thrombopheresis with a cell separator (Aminco)]. / Temperatureinfluss auf Spender und Thrombozytenfunktion bei der Thrombopherese am Zellseparator (Aminco.

In the continuous cell separation (Aminco) the temperature of the extracorporal blood is diminished by 9.3 degrees C. The calculated cooling of the intracardiac blood of 0.1 degrees C does not lead to an irregular cardiac rhythm. The donor only needs 0.339 kcal/min to rewarm the blood. It is necessary to rewarm the blood before retransfusion only if you have proved evidence of coldness antibodies with an extremely wide amplitude of temperature.

[Studies with radiolabeled autologous and heterologous thrombocytes to determine life-span and destruction sites]. / Untersuchungen mit radioaktiv markierten Eigen- und Fremdthrombozyten zur Ermittlung von Uberlebensdauer und Abbauort

The kinetic of 51Cr-labelled autologeous and heterologeous thrombocytes was studied in 12 healthy volunteers. The mean recovery value was 63% (SX = +/- 15%). The survival times (t50% and t10%) were determined as 94 hrs. (SX = +/- 11 hrs.) and as 202 hrs. (SX = +/- 13 hrs.) respectively. The "excess counts" over spleen and liver were measured and the spleen to liver ratio calculated. In every parameter investigated no significant difference between autologeous and heterologeous labelled thrombocytes could be found. The "excess counts" over spleen and liver were calculated on the basis of the radioactivity of blood and of the heart surface area. The values obtained from the blood radioactivity calculations showed significant lower variation coefficients than the corresponding values derived from the heart measurements.

[Status of data of HLA-typed donors for unrelated bone marrow transplantation in Hannover: organization and results]. / Stand der Datei HLA-typisierter Spender für die nichtverwandte Knochenmarktransplantation in Hannover: Organisation und Ergebnisse.

The pool of HLA-A, B and C-typed blood donors in Hannover for cell-donation increased from 2162 in 1988 to 2406 in 1989. 1032 blood donors declared their readiness to donate thrombocytes; 667 would like to donate bone marrow, 453 of them have undergone complete HLA-A, B, C and DR-typing. Until August 31, 1989 we looked in our data bank for an HLA-identical bone marrow donor for 84 patients from other transplantation centers and for 19 patients who are being looked after in Hannover and in national and international data banks.

[Analysis of donor self exclusion in repeat blood donors]. / Analyse des Spenderselbstausschlusses (SSA) bei Dauerblutspendern.

A confidential self-exclusion (self-exclusion means that the donor doesn't want his blood to be used for transfusion purposes) questionnaire was given during 12 months to 19,108 periodical blood donors in the College of Medicine in Hannover. 237 donors stated that their blood was not to be used for transfusion purposes. 70% of them were males, 30% females. 53% of these self-exclusion donors were between 21 and 30 years old. Among these self-exclusion donor, all the HIV-antibody-test showed negative results. The blood units from these donors were destroyed. The financial loss was DM 48,000. From among donors who thrice wished self-exclusion, one third declared promiscuity as the motive for self-exclusion, one third did not understand the questionnaire and one third could not or did not want to explain the self-exclusion. The question about the efficiency of the confidential donor self-exclusion process may be answered positively compared to the results of Nusbacher's study, which evaluated five times as many blood donations.

Transfusion results of cytapheresis platelet concentrates stored in two different polyolefin containers for five days.

OBJECTIVE: Two different types of polyolefin storage containers were compared in order to estimate their ability to preserve apheresis platelet concentrates for 5 days. MATERIAL AND METHODS: Ten pairs each consisting of one patient with thrombocytopenia following chemotherapy and one healthy platelet apheresis donor were examined. The platelet concentrates stored in the LE-2 bag were collected with a Fresenius AS 104 cell separator and those stored in the PL 732 with a Fenwal CS 3000. RESULTS: The separation efficiency of both cell separators was similar; the mean yields were 3.37 +/- 0.83 x 10(11) platelets in 274 +/- 26 ml for the AS 104 and 3.87 +/- 1.31 x 10(11) platelets in 318 +/- 22 ml for the CS 3000 (mean +/- SD). Storage for 5 days did not influence the platelet count significantly. The platelet loss due to filtration was 16 and 13%, respectively. The mean platelet volume obtained with both systems was reduced from a mean of 8.4 fl immediately after harvesting to 7.6 fl after storage (p < 0.0001) and to 7.3 fl after filtration (p = 0.001). The established corrected count increments (CCI) and the pre- and posttransfusion platelet counts were satisfactory and comparable for the 2 systems tested. The mean CCI of the AS 104-LE-2 system was 14.5 1 h and 7.4 x 10(9) platelets 24 h after transfusion, the mean CCI of the CS 3000-PL 732 system was 12.5 and 5.2 x 10(9) platelets, respectively. CONCLUSIONS: Single-donor apheresis concentrates with a large platelet content may be stored in the new LE-2 polyolefin container for up to 5 days and used in clinical transfusion.

Severe hemolysis caused by antibodies against the mushroom Paxillus involutus and its therapy by plasma exchange.

It has been shown that fatal "poisoning" with the mushroom species Paxillus involutus is caused by antibodies against the fungus in sensitized patients. Because circulating immune complexes play an important role, therapeutic procedures which can eliminate those complexes could stop immune hemolysis. A 37-year-old patient became severely ill after repeated ingestion of sufficiently cooked Paxillus involutus. As a result of hemolysis with reversible shock symptoms, acute renal failure developed. Plasma exchange with 3,000 ml albumin 5% was carried out daily during the first 3 days after admission. Each plasma exchange lowered free hemoglobin and immune complex levels by 60%-75%. Acute renal failure was successfully treated with hemodialysis. Specific IgG-antibodies against membrane particles of Paxillus involutus were detected by hemagglutination tests in the serum of the patient. The sequence of reactions resulting from the testing procedures strongly suggests the formation of immune complexes. These complexes are likely to bind to erythrocytes acting as innocent bystanders. Activation of the complement system finally results in hemolysis and shock. In addition to adequate shock treatment elimination of these immune complexes by plasma separation seems to be the therapy of choice.

Stress, cancer and immunity. New developments in biopsychosocial and psychoneuroimmunologic research.

Research in biobehavioral oncology has been focused on stress as one dispositional factor in the multifactorial origin and in the clinical progression of malignant disease. New insights into the transduction of environmental influences to the immune system and to other body systems by the brain and neurotransmitters have increased the salience of this approach. Behavioral medicine in the area of cardiovascular disease has been successful due to the introduction of a "Type A" or coronary prone behavior pattern in large epidemiologic studies. This pattern is marked by both psychologic and physiologic hyperresponsiveness. Type A persons appear to be hostile, easily angered, competitive and hard-driving. More recently, behavioral oncologists have similarly attempted at conceptualizing a "Type C" or biopsychosocial cancer risk pattern, as they have noted the denial and suppression of emotions, in particular anger. Other features of this pattern are "pathological niceness", avoidance of conflicts, exaggerated social desirability, harmonizing behavior, over-compliance, over-patience, as well as high rationality and a rigid control of emotional expression ("anti-emotionality"). This pattern, usually concealed behind a facade of pleasantness, appears to be effective as long as environmental and psychological homeostasis is maintained, but collapses in the course of time under the impact of accumulated strains and stressors, especially those evoking feelings of depression and reactions of helplessness and hopelessness. As a prominent feature of this particular coping style, excessive denial, avoidance, suppression and repression of emotions and own basic needs appears to weaken the organism's natural resistance to carcinogenic influences. This may mean that the excessive use of denial and suppression/repression has important psychophysiologic effects linked to tumor biology and host-defense. Recent studies reveal that psychosocial stressors which are met by inadequate and repressive coping styles are associated with changes in immunocompetence, including both humoral and cell-mediated immunity. Relationships between different immune parameters (natural killer cell activity, lymphocytes, serotonin uptake, mean platelet volume) and mood states, psychological coping styles and personality variables are outlined. Recent findings indicate also that in certain malignancies (eg. breast cancer) the clinical course of the disease is influenced by psychosocial factors and coping style, as well as that the risk of cancer recurrence and metastasis is influenced by the type and duration of a given stressor. Individuals with a more favorable outcome have higher fighting spirit, a greater potential for aggression and lesser suppressive tendencies. Psychological intervention in cancer patients in its different forms and within the frame of the over-all treatment has now become a matter of scientific discussion and research.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of maximal bactericidal activity in human granulocytes.

A conventional in vitro test assay was used to determine maximal bactericidal capabilities of human granulocytes. By means of a mathematical model the maximal phagocytosis and killing activity could be calculated for S. aureus and P. aeruginosa serving as test organisms. The evaluation allowed moreover the determination of the optimal bacterial load and also of critical bacterial concentrations leading to a complete depression of observable granulocyte killing functions. In contrast to other studies frozen suspensions of bacteria were used allowing the employment of identical microorganisms within a complete series of experiments. On average one granulocyte was found to ingest a maximum of 17 CFU of S. aureus with 9 CFU killed under optimal ratios of bacteria per granulocyte. For P. aeruginosa the granulocyte function reached peak values of 96 CFU ingested and 62 CFU killed per one granulocyte. The new assay might provide a highly reproducible method for clinical assessment of granulocyte dysfunctions in various diseases.

Polycythemia vera: electron microscopy of the bone marrow in 10 non-treated patients. A thin section and freeze-fracture study.

Electron microscopy (thin sections and freeze-fracture replicas) was performed on the bone marrow of ten patients with Polycythemia vera prior to any treatment. In addition to a hyperplasia of all three cell lineages and the sinuses, atypias were observed in the maturing erythroblasts. These aberrations of normal development consisted mainly of deep invaginations of the nuclear envelope in proerythroblasts and conspicuous nuclear clefts in erythro- and normoblasts. In comparison with similar changes in dyserythropoietic and aplastic anemia as well as leukemia these alterations are discussed in connection with disturbances of DNA synthesis. Further atypias involved megakaryopoiesis which displayed microforms probably as an evidence for maturation arrest. These ultrastructural abnormalities and their morphological features of a neoplastic proliferation of all three cell lineages in Polycythemia vera are in good agreement with the new concept of a transformation of a pluripotent stem cell with clonal character.

[Thrombophoresis I. Influence of centrifuge speed on thrombocyte yield, distribution of thrombocytes among centrifuge zones, and thrombocyte spreading (author's transl)]. / Thrombophorese I. Einfluss der Zentrifugendrehzahl auf Thrombozytenausbeute, Thrombozytenverteilung im Zellseparator und Ausbreitungsfähigkeit der Thrombozyten.

Thrombocyte yields up to 1,4 X 10(11) per litre of separated whole blood were obtained using centrifuge speed up to 2000 RPM. With increasing speed, higher platelet concentrations in the thrombocyte zone were accompanied by a progressive decline of thrombocyte counts in the plasma zone, whereas the thrombocyte contamination in the erythrocyte zone remained unchanged. Taking into account both high extraction efficiency and good preservation of platelet function as measured by Breddin's spreading test, optimal conditions for thrombophoresis with the Aminco Celltrifuge have been obtained at 1600 RPM. Since repeated separation of the donor's blood resulted in a rising number of platelets with impaired spreading capacity, the total blood flow should be limited to the blood volume of the donor.

[Fatal immunohaemolytic anaemia after eating the mushroom Paxillus involutus (author's transl)]. / Tödlich verlaufene immunhämolytische Anämie nach Genuss des Kahlen Kremplings (Paxillus involutus).

A 49-year-old previously healthy man fell gravely ill after repeatedly eating the mushroom Paxillus involutus. Haemolysis and circulatory shock caused acute renal failure, acute respiratory failure and disseminated intravascular coagulation. The patient died three-and-a-half days after the mushroom meal in protracted shock. In addition to the signs of haemolysis, post-mortem examination revealed signs of intravascular coagulopathy in lungs, kidneys, adrenals, myocardium, liver and spleen. There were also extensive fat emboli to both lungs. An IgG antibody against paxillus involutus extract was demonstrated in the patient's serum. The immunological reaction pattern suggests primarily the formation of an immune complex, which secondarily attaches itself to the erythrocyte surface and causes intravascular haemolysis by activation of the complement chain. In the first instance the diagnosis is made from anamnestic data. In addition to adequate treatment of circulatory shock, immediate plasma separation could provide a chance of effective treatment.