Human immunodeficiency virus 1 dual-target nucleic acid technology improves blood safety: 5 years of experience of the German Red Cross blood donor service Baden-Württemberg-Hessen.

BACKGROUND: RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015. STUDY DESIGN AND METHODS: Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays. RESULTS: Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays. CONCLUSION: The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.

Evaluation of algorithms for the diagnostic assessment and the reentry of blood donors who tested reactive for antibodies against hepatitis B core antigen.

BACKGROUND: Screening of blood donations for antibodies against hepatitis B core antigen (anti-HBc) is an accepted method to prevent some transfusion-transmitted hepatitis B virus (HBV) infections. However, anti-HBc testing may result in donor loss due to unspecific results in the currently available anti-HBc tests. Algorithms to distinguish true-positive from false-positive results and for reentry of those donors who tested false anti-HBc positive were evaluated retrospectively. STUDY DESIGN AND METHODS: Samples that tested reactive for anti-HBc by chemiluminescent microparticle immunoassay (CMIA) were investigated for anti-HBc by microparticle immunoassay, for anti-HBs and hepatitis B surface antigen (HBsAg) by CMIA, and for HBV DNA by individual-donor nucleic acid testing. Results were classified true positive, indeterminate, and false positive for anti-HBc. Donors who tested indeterminate and false positive were admitted for reentry if follow-up testing for anti-HBc became negative and no further evidence for an HBV infection was apparent. RESULTS: A total of 554 of 148,000 samples, taken from 30,000 individuals within 3 years tested reactive for anti-HBc by CMIA. Of those, 553 could be further classified: 142 (26%) true positive, 76 (14%) indeterminate, and 335 (60%) false positive. A total of 214 of 411 (52%) samples termed indeterminate or false positive were admitted for reentry and able to provide further donations. In one donor, anti-HBc-positive/HBsAg- and HBV DNA-negative HBV DNA was detectable during follow-up. CONCLUSION: According to our proposed algorithm, 26% of anti-HBc-reactive results tested by CMIA were true positive. Many donors tested indeterminate or false positive can provide future donations if our proposed algorithm for reentry is applied. One donor at risk for transmitting HBV was identified solely by anti-HBc testing.

Prolactin triggers pro-inflammatory immune responses in peripheral immune cells.

The peptide hormone prolactin (PRL) is produced by specialized cells in the anterior pituitary gland and in a number of sites outside the pituitary. Its biological actions consist of various roles in reproduction, lactation, and of a number of homeostatic biological activities that also include immune functions. Elevated serum PRL concentrations often correlate with abnormalities in immune responses. To determine the influence of PRL on human immune cells, human whole blood cultures were stimulated with lipopolysaccharide (LPS), supplemented with various concentrations of human recombinant PRL. We found that PRL, at concentrations achievable during pregnancy, anesthesia and medication, significantly amplified interleukin (IL)-12 and tumor necrosis factor-alpha (TNF-alpha) synthesis in LPS-stimulated cultures, in a dose-dependent manner. Conversely, synthesis of the anti-inflammatory cytokine IL-10 only increased significantly at very high concentrations of supplemented PRL. PRL alone was not able to induce any measurable secretion of TNF-alpha, IL-10, or IL-12 in non-stimulated, whole blood cultures. However, we demonstrated that PRL, by itself or in combination with LPS, causes an increase in the binding activity of the transcription factors nuclear factor-kappaB (NFkappaB) and interferon regulatory factor-1 (IRF-1), which are known to promote TNF-alpha and IL-12 secretion. These data suggest that PRL promotes pro-inflammatory immune responses via NFkappaB and IRF-1, which may affect pathophysiological processes in physiological hyperprolactinemic states.

Early alterations in the number of circulating lymphocyte subpopulations and enhanced proinflammatory immune response during opioid-based general anesthesia.

The balance between proinflammatory and anti-inflammatory processes is of key importance in the reaction of the body to infection, injury, and surgical trauma. Drugs commonly used in anesthesia and intensive care may modulate immunological reactions by influencing intercellular communication through modification of cytokine response and fluctuation of peripheral immune cells such as natural killer (NK) cells, B cells, and T lymphocyte subpopulations (CD4+ and CD8+ cells). To examine the effects of general anesthesia with the hypnotic agent propofol and the opioid fentanyl, 30 patients undergoing minor elective orthopedic surgery were studied before and 20 min after application of the anesthetic drugs, but before the start of surgery. We found a significant enhancement of TNF-alpha and IL-1beta release in lipopolysaccharide (LPS)-stimulated whole blood cultures after induction of anesthesia. Similar results were observed with interferon-gamma (IFN-gamma) in cultures stimulated with phytohemagglutinin (PHA). Conversely, synthesis of the anti-inflammatory cytokine interleukin 10 (IL-10) decreased significantly in LPS-stimulated cultures. During general anesthesia, we found a decrease of circulating lymphocytes, characterized by a significant increase in the percentage of T lymphocytes in favor of CD4+ cells, increased B lymphocytes, and a significant decrease of NK cells. These data suggest that anesthesia with propofol and fentanyl promotes proinflammatory immune responses and influences peripheral lymphocyte composition in patients, which may subsequently affect pathophysiological processes during opioid-based anesthesia.

Probability of anti-D development in D- patients receiving D+ RBCs.

BACKGROUND: In some situations, the administration of D+ RBCs to D- patients is necessary. The probability of a subsequent anti-D formation is assumed to be around 80 percent, a figure based primarily on studies in healthy volunteers. It was hypothesized that patients requiring blood transfusion have a much lower probability of developing antibodies. STUDY DESIGN AND METHODS: A retrospective analysis was performed whereby 78 D- patients were evaluated for the development of RBC antibodies after administration of D+ RBCs. For the analysis of the cross-sectional observations, parametric models were used for interval-censored data. RESULTS: Anti-D was detected in 16 of 78 patients. Considering the individual patient's inspection times, the calculated probability of developing antibody following D+ RBC supply was shown to be below 41.7 percent (upper 95% confidence bound) and estimated as 30.4 percent. The data hinted toward an inverse correlation between the number of transfused units and the probability of antibody formation. Interestingly, 6 of these 16 patients developed additional IgG autoantibody. In 3 of those cases, evidence for prolonged hemolysis was found. CONCLUSION: The actual frequency of antibody formation in our patients is much lower than assumed. On the other hand, prolonged hemolysis probably induced by additional autoreactive antibodies might occur. This possible complication has not yet been addressed. Further studies might reveal whether a less restricted transfusion policy with respect to D matching is justified in selected patients.

Prolactin induces enhanced interferon gamma release in peripheral whole blood after stimulation with either PHA or LPS.

A number of recent studies have demonstrated the importance of prolactin as a key mediator in immune-neuroendocrine communication. Using a whole blood assay and various concentrations of prolactin, we stimulated cell cultures with either the plant lectin PHA or the endotoxin LPS, a widespread agent in common infectious diseases. Studying 15 healthy blood donors we found that human recombinant prolactin, at concentrations from 5 ng/ml to 100 ng/ml, significantly amplified IFN-gamma yields after stimulation with either PHA or LPS. PHA-stimulated cultures revealed a significant dose-dependent enhancement of IFN-gamma release. Our results indicate that prolactin can upregulate IFN-gamma secretion from immune cells in whole blood cell cultures in response to both PHA or LPS. Since IFN-gamma is suspected to play a key role in the cytokine cascade, amplifying the toxic effect of other pro-inflammatory cytokines and ultimately leading to augmented inflammatory tissue damage, our findings point to a modulatory role of prolactin in infection. Special interest should therefore be directed towards any naturally occurring hyperprolactinemia, caused for instance by stress, a number of drugs, and some chronic diseases.

Anti-myeloma activity of natural killer lymphocytes.

Natural killer (NK) cells are assumed to contribute to a graft-versus-leukaemia effect. In vitro experiments have shown that many leukaemic cells are NK-cell sensitive. Nevertheless, no data concerning the influence of purified NK cells on malignant myeloma (MM) cells exist. We co-incubated NK cells with three different MM cell lines and fresh bone marrow samples of nine MM patients. The proportion of vital MM cells was determined before and after co-cultivation by a flow-cytometry-based assay. All MM cells tested, with the exception of one cell line (NCI H929), were susceptible to a NK-cell attack even without exogenous interleukin 2 (IL-2). The mean killing of the native MM samples was 23.1 +/- 5.4% and 34.5 +/- 6.5% at 10:1 and 20:1 effector:target ratio respectively, This corresponded to about 2/3 of those values obtained with the highly sensitive line K562. In contrast, CD34-positive haematopoietic stem cells as well as peripheral mononuclear cells were completely resistant under similar experimental conditions (1.3% killing). To elucidate the underlying triggering mechanisms, we measured human leucocyte antigen (HLA)-class I expression of the MM cells. No evidence for HLA loss, which could have explained the NK-cell recognition if it occurred, was demonstrated. These findings may contribute to the understanding of in vivo NK-cell activation and encourage clinical applications of NK cells for MM patients.

Frequency and load of hepatitis B virus DNA in first-time blood donors with antibodies to hepatitis B core antigen.

The objective of this study was to determine the frequency and load of hepatitis B virus (HBV) DNA in anti-HBc-positive first-time blood donors; it was designed to contribute to determining whether anti-HBc screening of blood donations might reduce the residual risk of posttransfusion HBV infection. A total of 14 251 first-time blood donors were tested for anti-HBc using a microparticle enzyme immunoassay; positive results were confirmed by a second enzyme-linked immunosorbent assay (ELISA). For the detection of HBV DNA from plasma samples, we developed a novel and highly sensitive real-time polymerase chain reaction (PCR) assay. The 95% detection limit of the method amounted to 27.8 IU/mL, consistent with the World Health Organization (WHO) international standard for HBV DNA. A total of 216 blood donors (1.52%) tested anti-HBc-positive in both tests, and 205 of them (16 HBsAg(+), 189 HBsAg(-)) were tested for HBV DNA. In 14 (87.5%) of the HBsAg-positive blood donors, HBV DNA was repeatedly detected, and in 3 (1.59%) of the HBsAg-negative donors, HBV DNA was also found repeatedly. In the 3 HBV DNA-positive, HBsAg-negative cases, anti-HBe and anti-HBs (> 100 IU/L) were also detectable. HBV DNA in HBsAg-negative as well as HBsAg-positive samples was seen at a low level. Thus, HBV DNA is sometimes found in HBsAg-negative, anti-HBc-positive, and anti-HBs-positive donors. Retrospective studies on regular blood donors and recipients are necessary to determine the infection rate due to those donations. Routine anti-HBc screening of blood donations could probably prevent some transfusion-transmitted HBV infections.

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells.

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.

Large-scale generation of natural killer lymphocytes for clinical application.

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.