Hepatitis B, hepatitis C and HIV transfusion-transmitted infections in the 21st century.

In the past, transfusion-transmitted virus (TTV) infections were not uncommon. In recent years with advanced technologies and improved donor screening, the risk of viral transfusion transmission has been markedly reduced. Hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) have all shown marked reduction in transmission rates. However, the newer technologies, including nucleic acid technology (NAT) testing, have affected the residual rates differently for these virally transmitted diseases. Zero risk, which has been the goal, has yet to be achieved. False negatives still persist, and transmissions of these viruses still occur, although rarely. It is known that HBV serological testing misses some infected units; likewise, HBV NAT-negative units have also been known to transmit the virus. Similarly, HIV minipool NAT-negative units have transmitted HIV, as recently as 2007; likely, these transmissions would have been prevented with single-unit NAT testing. Newer technologies, such as pathogen inactivation (PI), will (ideally) eliminate these falsely test negative components, regardless of the original testing method used for detecting the viruses.

TRALI is due to pulmonary venule damage from leucocytes with cholesterol crystal formation.

BACKGROUND: There are two presumed mechanisms for the pulmonary oedema in transfusion-related acute lung injury (TRALI). One is antibodies to leucocytes while the other is biologically active lipids. We evaluated the vascular injury due to the former. METHODS: The pulmonary vasculature was studied by light microscopy (LM) and scanning electron microscopy (SEM) in three fatal cases of TRALI and compared with that of two autopsied control patients. Lung tissue from two of the TRALI cases and both controls was studied by gas chromatography-mass spectroscopy (GC-MS) to identify crystals present in the former. RESULTS: All three TRALI cases exhibited massive pulmonary oedema by weight and light microscopy and extensive defects by SEM in the endothelium of venules of the lungs. Such endothelial defects were absent in controls. Thrombi, composed of crystals, were present in venules and small veins diffusely throughout the lungs in Case 1. Similar crystals were identified in Case 2. The crystals in the lung vessels were identified morphologically as cholesterol and were proximate to the cytoplasmic defects of the endothelial surfaces. By GC-MS, there were markedly elevated levels of cholesterol and fatty acids in the two TRALI lungs tested compared with the lungs of the two controls. CONCLUSIONS: Pulmonary damage in TRALI is related to formation of cholesterol crystals that appear to pierce endothelial membranes of venules. The endothelial defects lead to plasma extravasation into the alveoli causing TRALI.

Does donating blood for the first time during a national emergency create a better commitment to donating again?

BACKGROUND AND OBJECTIVES: Emergency situations often elicit a generous response from the public. This occurred after attacks on the US on September 11, 2001 when many new blood donors lined up to donate. This study was performed to compare return rates for first time donors (FTD) after September 11th, 2001 to FTD during a comparable period in 2000. MATERIALS AND METHODS: A total of 3315 allogeneic whole blood donations from FTD at a regional blood centre were collected between September 11th and 30th, 2001. Subsequent donations by the FTD before March 31, 2002 were reviewed. This (test) group was compared to 1279 FTD (control group) donating during the same time period in September 2000 and to their return rate in the subsequent 6 months. RESULTS: Following September 11, 2001, 1087/3315 (32.8%) FTD returned by March 31, 2002. This return rate was similar to the control group [427/1279 (33.4%)]. The deferral rate during the donor screening process for the control group was significantly higher than the deferral rate for the September 11-30, 2001 group (P < 0.01). The odds of an individual FTD returning increased with age, and the chance of a female donor returning was 1.13 times higher than a male (P = 0.06). There was a carryover effect after September 11, 2001 too. CONCLUSION: A national emergency, September 11, 2001, inspired people to donate blood for the first time. However, the proportion of return donations amongst them was not increased. Females and males in certain age groups were more likely to become repeat donors due to the residual effect of September 11, 2001. Additional efforts are needed to retain eligible FTD in donor pools.

Transfusion-associated graft-versus-host disease.

Transfusion-associated graft-versus-host disease (TA-GvHD) is a rare complication of transfusion of cellular blood components producing a graft-versus-host clinical picture with concomitant bone marrow aplasia. The disease is fulminant and rapidly fatal in the majority of patients. TA-GvHD is caused by transfused blood-derived, alloreactive T lymphocytes that attack host tissue, including bone marrow with resultant bone marrow failure. Human leucocyte antigen similarity between the transfused lymphocytes and the host, often in conjunction with host immunosuppression, allows tolerance of the grafted lymphocytes to survive the host immunological response. Any blood component containing viable T lymphocytes can cause TA-GvHD, with fresher components more likely to have intact cells and, thus, able to cause disease. Treatment is generally not helpful, while prevention, usually via irradiation of blood components given to susceptible recipients, is the key to obviating TA-GvHD. Newer methods, such as pathogen inactivation, may play an important role in the future.

Hepatitis B virus (HBV) DNA screening of blood donations in minipools with the COBAS AmpliScreen HBV test.

BACKGROUND: The risk of hepatitis B virus (HBV) transmission by blood transfusion (estimated at 1 in 63,000-1 in 205,000 units in the United States) exceeds that of hepatitis C virus (HCV) or human immunodeficiency virus (HIV). Reduction of window-period HBV transmissions through detection of HBV DNA-positive units by minipool nucleic acid testing (MP NAT) would be expected to decrease this risk. STUDY DESIGN AND METHODS: A large multicenter study of the COBAS AmpliScreen HBV test (Roche Molecular Systems) was conducted on minipools of 24 blood donation specimens. The yield of HBV DNA-positive, hepatitis B surface antigen (HBsAg)-negative window-period donations was determined relative to current and newly licensed HBsAg assays. Donors with selected HBV DNA, HBsAg, and anti-hepatitis B core antigen (HBc) results were further evaluated. RESULTS: The detection rate of window-period units was 1 in 352,451 (95% confidence interval, 1 in 2,941,176-1 in 97,561). Assay specificity was high (99.9964%). HBV DNA was detected in 84 percent of HBsAg-positive, anti-HBc-positive donations by MP NAT and in 94 percent when individual-donation (ID) NAT was added. HBV DNA was detected in 0.03 percent of HBsAg-negative, anti-HBc-positive donations by MP NAT and in 0.41 percent when ID NAT was added. CONCLUSIONS: Implementation of HBV MP NAT will provide an increment in safety relative to HBV serologic screening, similar to that for HCV and in excess of that for HIV. Our data indicate that the implementation of HBV MP NAT would likely interdict 39 HBV window-period units and prevent 56 cases of transfusion-transmitted HBV infection annually. The current data indicate that HBV MP NAT should not lead to discontinuation of anti-HBc testing but that discontinuation of HBsAg testing with retention of anti-HBc testing may be possible.

Visual assessment of hemolysis in red blood cell units and segments can be deceptive.

BACKGROUND: Blood components that appear hemolyzed are discarded. However, visual inspection is subjective and criteria for excessive hemolysis are poorly defined. STUDY DESIGN AND METHODS: Packed RBCs (10 CPDA-1, 10 Adsol) were collected. Half of each unit was leukoreduced. Plasma Hb was measured and compared in segments and units by three methods: 1) a HemoCue Plasma/Low Hb Photometer system; 2) a tetramethyl-benzidine (TMB) chemical method, and 3) a free Hb visual comparator. RESULTS: Visual assessment tended to overestimate hemolysis. Chemical methods were comparable (r(2)= 0.894; HemoCue = 0.043 +[0.770]x TMB; n = 400; range, 0.01-0.5 g/dL), although the mean plasma Hb (g/dL) for the HemoCue method was higher than that of the TMB method (0.12 vs. 0.10 g/dL, respectively; p < 0.001). No units would have been discarded based on a hemolysis level of at least 0.6 g/dL (approx. 1%) if measured by a chemical method. However, 50 percent of CPDA-1 and 10 percent of Adsol units would have been discarded if only visual criteria were used. Leukoreduction did not increase plasma Hb levels. Discrepancies in plasma Hb levels were noted between units and their corresponding segments. CONCLUSION: Visual assessment of hemolysis can result in unnecessary wastage of blood components. HemoCue offers an alternative, objective method to assess plasma Hb in the setting of blood collection and processing facilities for routine quality control and process validation, and may aid in the development of objective criteria for excessive hemolysis in blood components.

Platelet function abnormalities in qualified whole-blood donors: effects of medication and recent food intake.

BACKGROUND AND OBJECTIVES: Platelet function abnormalities have been reported in blood donors who have not consumed aspirin. Our objective was to identify factors other than aspirin that may contribute to impaired platelet function in qualified volunteer blood donors. MATERIALS AND METHODS: Blood samples were obtained from 24 donors following routine blood donation. Donors completed a study questionnaire that included questions about recent food consumption, medication and medical history. Platelet activation was measured using monoclonal antibodies and flow cytometry. CD62P expression and PAC-1 binding on platelets were used as indicators of platelet activation. Platelet function was measured on a platelet function analyser (PFA-100) using both collagen/epinephrine (cEPI) and collagen/ADP (cADP) cartridges. RESULTS: Fifty-four per cent of donors (13 of 24) had normal platelet function. Thirty-eight per cent (nine of 24) had prolonged cEPI closure times, of whom four (17%) had no cEPI closure (> 300 seconds). No closure was associated with aspirin use (two donors) or chocolate consumption (two donors) before donation. Two donors (8%) had either a shortened cEPI or cADP closure time. CONCLUSIONS: Platelet dysfunction in qualified blood donors is underestimated. Platelet function screening can identify donors with diet-related platelet dysfunction or with poor recollection of aspirin use.

Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components.

BACKGROUND: Universal leukoreduction of blood components is becoming the standard of care. Flow cytometry methods are being used for quality control of the leukoreduction process. METHODS: We provide an atlas of atypical flow cytograms generated by a commercial LeucoCOUNT assay that was used to enumerate residual leukocytes in leukoreduced red blood cell components. Numeric results are derived from a flow cytogram generated by the assay. RESULTS: Three types of atypical flow cytogram patterns were observed during process validation or routine quality control of leukoreduced red blood cell components. (a) Fixation artifact: Fixation of control or test samples can alter the staining intensity compared with fresh cells. (b) "Rain" pattern: Flow cytometry methods count slightly damaged leukocytes not removed during leukoreduction. Slightly damaged leukocytes appear on a flow cytogram like "rain" falling from a well-defined "cloud" of intact residual leukocytes. Discrepancies between automated flow cytometry results and subjective manual counting methods can occur. (c) Autofluorescence-debris pattern: Cell debris and age-related changes in the sample can cause shifts in the fluorescence staining pattern, resulting in erroneous test results. CONCLUSION: Review of flow cytograms is essential for accurate reporting of flow cytometry-based methods for enumerating residual leukocytes in leukoreduced blood components.

Fluctuation of serum HCV-RNA levels in untreated blood donors with chronic hepatitis C virus infection.

The hepatitis C virus (HCV)-RNA levels were measured in 281 serum samples from 32 untreated volunteer blood donors prospectively collected over a period of 14-73 months. The HCV-RNA levels were tested by the branched DNA signal amplification assay. The mean HCV-RNA levels of each donor ranged from 4.92 log10-6.36 log10 gene equivalents/mL (25%, median, 75% percentile; 5.51, 5.79, 6.12 log10 gene equivalents/mL). The fluctuations of HCV-RNA levels in individuals, represented by the ratio of the maximum value divided by the minimum value, ranged from a 1.7- to a 141-fold change. Fluctuations with more than a 10-fold change were observed in five subjects: 11-, 15-, 17-, 96- and 141-fold changes. Eleven subjects were followed for at least 5 years; all subjects had fluctuations of HCV-RNA levels greater than 3-fold during the observation period. No blood donor was observed whose HCV levels changed from a high-level phase to a low-level phase or from low to high. No subjects cleared HCV during follow-up, although two had undetectable HCV-RNA levels transiently. These findings reveal that changes in HCV-RNA levels occur which are unrelated to treatment with interferon and ribavirin.

The disappearance of transfusion-transmitted hepatitis C virus infections in the United States.

Because of anti-HCV testing, rates of transfusion-transmitted HCV infections have dropped from a high level (approximately 1 per 200 units, even using volunteer, repeat donors) to an extremely low one (approximately 1 per 125,000 units). Moreover, preliminary data indicate that pooled- (and perhaps, eventually, single-) specimen NAT for HCV-RNA or EIA for HCV core antigen may reduce this risk even further. It is anticipated that implementation of one or more of these methods, coupled with one or more pathogen-inactivation steps, may functionally eliminate the risk of transmitting HCV by transfusions.

HIV transmissions from a window-period platelet donation.

Recently, blood centers began investigational testing for HIV RNA by pooled nucleic acid testing (NAT). A 35-year-old frequent platelet donor tested HIV p24 antigen positive, antibody negative before implementation of NAT. He made 2 platelet donations (day -4 and -11) immediately before testing positive for HIV. The donor's HIV seroconversion was monitored, and stored samples were tested retrospectively for HIV RNA. Platelet recipients were tested for HIV infection. The day -4 sample tested positive for HIV RNA by pooled and individual sample NAT. The day -11 sample tested negative for HIV RNA by both NAT tests. The 2 recipients of the day -4 platelets tested HIV RNA and p24 antigen positive. The recipient of the day -11 platelets could not be tested because he had died. HIV NAT would have prevented transmission of HIV had it been available at the time of this donor's HIV seroconversion.

HLA class II antibodies in transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a serious, sometimes fatal, complication of transfusion. Granulocyte and HLA class I antibodies present in blood donors have been associated with TRALI. HLA class II antibodies have recently been described in a few cases of TRALI. STUDY DESIGN AND METHODS: Donors involved in TRALI reactions reported to a blood center over an 18-month period were tested for HLA class I and II antibodies as well as granulocyte antibodies, if HLA antibodies were not identified. RESULTS: HLA class II antibodies were identified, in at least one donor, in 7 (64%) of 11 cases of TRALI. HLA class I antibodies were identified in combination with HLA class II antibodies in 5 of these 7 cases. HLA class I antibodies were exclusively identified in 2 cases. Granulocyte antibodies were identified in 1 case, and no antibodies were identified in another. CONCLUSION: In addition to HLA class I antibodies, HLA class II antibodies are associated with TRALI. Testing of donors for HLA class II antibodies as well as HLA class I and granulocyte antibodies is recommended as part of the investigation of suspected cases of TRALI.

Mechanisms of severe transfusion reactions.

Serious adverse effects of transfusion may be immunologically or non-immunologically mediated. Currently, bacterial contamination of blood products, particularly platelets, is one of the most significant causes of transfusion-related morbidity and mortality. Septic transfusion reactions can present with clinical symptoms similar to immune-mediated hemolytic transfusion reactions and transfusion-related acute lung injury. Extremely high fever and/or gastrointestinal symptoms, in a transfusion recipient, may be indicative of sepsis. The diagnosis is based upon culturing the same organism from both the patient and the transfused blood component. Numerous organisms have been implicated as the cause of septic transfusion reactions. Due to different storage conditions, gram negative organisms are more often isolated from red blood cell components; gram positive organisms are more often isolated from platelets. Prevention of septic transfusion reactions is primarily dependent on an adequate donor history and meticulous preparation of the donor phlebotomy site. Visual inspection of blood components prior to transfusion is also vital to preventing these reactions. Several methods of detection of bacterial contamination and inactivation of pathogens are currently under active investigation.

Leukocyte-reduced red blood cell transfusions in patients with anemia and human immunodeficiency virus infection: the Viral Activation Transfusion Study: a randomized controlled trial.

CONTEXT: Allogeneic blood transfusions have immunomodulatory effects and have been associated with activation of human immunodeficiency virus (HIV) and cytomegalovirus (CMV) in vitro and of HIV in small pilot studies. Retrospective studies suggest that transfusions adversely affect the clinical course of HIV. Data in selected non-HIV-infected patients requiring blood transfusion have suggested clinical benefit with leukocyte-reduced red blood cells (RBCs). OBJECTIVE: To compare the effects of leukoreduced and unmodified RBC transfusions on survival, complications of acquired immunodeficiency syndrome, and relevant laboratory markers in HIV-infected patients. DESIGN AND SETTING: Double-blind randomized controlled trial conducted in 11 US academic medical centers from July 1995 through June 1999, with a median follow-up of 12 months (24 months in survivors). PATIENTS: A total of 531 persons infected with HIV and CMV, aged 14 years or older, who required transfusions for anemia; 259 received leukoreduced transfusions and 262 received unmodified transfusions (10 did not receive the planned transfusion). MAIN OUTCOME MEASURES: Survival and change in plasma HIV RNA level 7 days after transfusion, compared by type of transfusion. RESULTS: At entry, the groups were similar in demographic, clinical, and relevant laboratory characteristics. A total of 3864 RBC units were transfused. Two hundred eighty-nine deaths occurred (151 with leukoreduced transfusion; 138 with unmodified transfusion); median survival was 13.0 and 20.5 months, respectively (relative hazard [RH], 1.20; 95% confidence interval [CI], 0.95-1.51; log-rank P =.12). Analyses adjusted for prognostic factors suggested possible worse survival with leukoreduction (RH, 1.35; 95% CI, 1.06-1.72). There was no difference in time to new opportunistic event/death or frequency of transfusion reactions. No changes in plasma HIV RNA level were seen in either group at days 7, 14, 21, or 28, even in patients not taking antiretroviral drugs. There were no differences in trends between groups in CMV DNA, CD4 cell counts, activated (CD38% or human leukocyte antigen-DR) CD8 cell counts, or plasma cytokine levels. CONCLUSIONS: We found no evidence of HIV, CMV, or cytokine activation following blood transfusion in patients with advanced HIV infection. Leukoreduction provided no clinical benefit in these patients. These data demonstrate the importance of conducting controlled studies of effects of leukoreduction in different patient populations, since smaller studies in other patient populations have suggested leukoreduction may be beneficial.

Growth of bacteria in inoculated platelets: implications for bacteria detection and the extension of platelet storage.

BACKGROUND: Recent reports from Europe have advocated the use of bacterial culturing of platelets on Day 2 or 3 of storage to extend the shelf life of platelets to 7 days, thereby reducing the outdating of platelets and preserving a limited medical resource. To assess the optimal timing, the necessary sensitivity, and the possible efficacy of bacterial detection, the bacterial growth characteristics were reviewed in 165 platelet units, each inoculated on the day of collection with one of the following organisms: Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermidis from four previously published studies. STUDY DESIGN AND METHODS: Quantitative culture data from inoculated platelet concentrates from five sites and four studies were combined into one database and analyzed for bacterial concentration thresholds (> or =10(1), > or =10(2), > or =10(3), > or =10(4), > or =10(5) CFU/mL) by day of storage. RESULTS: All examples of B. cereus, P. aeruginosa, K. pneumoniae, S. marcescens, and S. aureus had concentrations > or =10(2) CFU per mL by Day 3 after inoculation. By Day 4, all units with these organisms contained > or =10(5) CFU per mL. Units contaminated with S. epidermidis showed slower and more varied growth. By Day 3 after inoculation, 81.3 percent had 10(2) CFU per mL. By Day 4 after inoculation, 46 (95.8%) of 48 units had concentrations > or =10(2) CFU per mL. CONCLUSION: These experiments suggest that an assay capable of detecting 10(2) CFU per mL on Day 3 of storage would detect the vast majority of bacterially contaminated platelet units, prevent many cases of platelet-associated bacterial sepsis, and provide a scientific basis for the extension of the current platelet storage time. It would be expected that a rare, slow-growing organism could escape such a detection scheme.

Universal leukocyte reduction.

Leukocyte reduction of blood components, in the United States, is generally reserved for conditions in which a clinical indication has been documented. There is no evidence that either Creutzfeldt-Jakob disease or variant Creutzfeldt-Jakob disease are transmitted by transfusion in humans or that leukocyte reduction of blood components could reduce their transmission. A number of adverse outcomes following transfusion are alleged to be the result of white blood cells. At this point in time, there are insufficient clinical data to justify the universal leukocyte reduction of blood components.

Old and new tests: where will it end?

Testing has improved the safety of the blood supply. We have excellent serologic tests in place now and are implementing nucleic acid based tests to identify asymptomatic carriers of viruses during the infectious part of the pre-seroconversion (window) period. However, the blood supply was already quite safe after a variety of other mechanisms had been put into place besides testing to screen out individuals at risk of carrying the most important transfusion transmissible agents. An important safety factor is the use of volunteer, unpaid (unremunerated) blood donors. The best alternative to implementing yet more tests to reduce, but not eliminate, the minute residual risks of transfusion transmission of such agents as HIV, HBV and HCV is the application of microbial inactivation technology to blood and blood components. Such microbially inactivated, cellular blood components should not have the risk of transmitting infectious agents, but may have other, different risks, since nothing has yet been shown to be one hundred percent safe (i.e., risk free). The use of a test to detect carriers of spongiform encephalopathies to prevent their theoretical transmission by transfusion may cause harm to donors and might increase risk for recipients by decreasing the available blood supply.