Analytical performance of RNA isolated from BD SurePath™ cervical cytology specimens by the PreTect™ HPV-Proofer assay.

Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.

Validation of a microbial detection system for use with ACD-A platelets with PAS III platelet additive solution.

BACKGROUND: The BacT/ALERT microbial detection system (BTA) is used for testing leukoreduced apheresis platelets (LR-AP) in plasma. Platelet additive solutions (PASs) such as InterSol (PAS III) may be used to reduce the amount of plasma transfused in LR-AP. This study evaluated the performance of the two-bottle BTA testing scheme in the recovery of seeded microorganisms from LR-AP in InterSol-plasma compared to a reference plate culture method. STUDY DESIGN AND METHODS: Hyperconcentrated, double LR-AP were collected from healthy donors; InterSol was added (65% Intersol:35% plasma), equally divided into two containers, and then inoculated with an isolate of 1 of 10 clinically relevant index organisms at two levels. Aerobic (BPA) and anaerobic (BPN) BTA bottles were inoculated with 4 mL each of the inoculated LR-AP, and blood agar plates (BAPs) for aerobic and anaerobic culture (0.5 mL each). RESULTS: Zero false-positives from 103 bottle pairs were observed. All 400 two-bottle BTA tests were positive within 24 hours, except for Propionibacterium acnes (maximum time-to-detection of 86.4 hr) and 13 of 20 pairs of Streptococcus viridans (maximum time-to-detection of 31.7 hr). Thirteen of 400 BAP two-plate tests were negative for starting bacterial concentrations of 10 colony-forming units (CFUs)/mL or less. At 40 CFUs/mL or less, BTA was 100% positive while BAP was 94% positive. CONCLUSION: Seeded organism recovery was superior in the two-bottle BTA test system compared to the two-plate BAP system using InterSol platelets (PLTs). This performance is comparable to previously published results for PLTs in plasma. The use of InterSol does not appear to have a detrimental effect on the performance of the two-bottle BTA system.

Thrombotic thrombocytopenic purpura and urinary tract infections: is there a connection?

Most cases of thrombotic thrombocytopenic purpura (TTP) are considered idiopathic without an identifiable etiologic agent. It has been previously reported that a number of patients with TTP had a urinary tract infection (UTI). Apheresis records were searched for patients with TTP from 1999 through 2007. Records were examined for evidence of UTI, and the patients were divided into 4 groups: 1, laboratory evidence of UTI on admission; 2, UTI just before admission; 3, UTI that developed during hospitalization; and 4, weak laboratory evidence of a UTI. The study included 90 TTP "visits." (A visit was defined as all admissions for TTP for a specific patient within a 1-month period.) Of the TTP visits, 21 (23%) were associated with UTIs. Group 1 included 7 patients; group 2, 10 patients; group 3, 3 patients; and group 4, 1 patient. This suggests that UTIs might serve as a TTP stimulus. Owing to the relatively strong association of UTIs with TTP, all patients with TTP should be screened for UTI and treated accordingly.

ABO blood type and longevity.

To assess the observation that blood type B might be a marker for longevity, we reviewed the records and determined the ABO blood types of all patients who died in our hospital in 2004. Age was stratified by decade of death, and linear regressions were calculated by ABO percentage. ABO survival curves were compared. In 2004, 906 patients died; 35 were excluded (stillborn infants). Of the remaining 871 patients, ABO types were available for 772 (88.6%). The percentage of patients with group B blood declined with age (P < .01). None of the other blood type percentages had a statistically significant increase or decrease. The group B survival curve was statistically worse than non-B groups (P ≤ .01); there were no differences in survival among groups A, O, and AB (P =.47). In our patient population, the percentage of patients with group B blood declines with age. The survival curve in group B was worse than that in groups A, O, and AB. These findings suggest that in our patient population, blood group B is not a marker for longevity but may be a marker for earlier death.

Is it quinine TTP/HUS or quinine TMA? ADAMTS13 levels and implications for therapy.

Thrombocytopenia with or without microangiopathy following quinine is often referred to as quinine "hypersensitivity." When schistocytes are present it is frequently termed "quinine-associated TTP/HUS." A severe deficiency of the vWF-cleaving protease, ADAMTS13, is associated with idiopathic TTP. A previous study of patients with "quinine-associated TTP/HUS" found that ADAMTS13 activities were not abnormal in 12/12 patients. A retrospective review of TTP patients with quinine-associated thrombotic microangiopathy (TMA) for whom ADAMTS13 was measured before plasma exchange was performed. Six patients were identified. All were females (age range: 43 to 73, mean = 61.7 years) and had taken quinine for leg cramps. Four of the six experienced renal failure requiring dialysis. Five of the patients had D-Dimers levels measured, all were elevated. In four patients the levels were > or = 18 times the upper limit of normal. ADAMTS13 was normal in four patients and mildly decreased in two patients. We conclude that while thrombocytopenia and schistocytosis can be seen in quinine-associated TTP/HUS, the pathophysiology seems to be distinct from that seen in most cases of idiopathic TTP (i.e., severely decreased ADAMTS13 with an inhibitor). We recommend that a TMA in association with quinine be consistently referred to as quinine-associated thrombotic microangiopathy (quinine-TMA) to better distinguish this entity from idiopathic TTP. The use of plasma exchange in quinine-TMA is called into question.

ADAMTS13 activity levels in patients with human immunodeficiency virus-associated thrombotic microangiopathy and profound CD4 deficiency.

BACKGROUND: Thrombotic microangiopathy (TMA) encompasses a number of disorders with hemolytic anemia and thrombocytopenia, including thrombotic thrombocytopenic purpura (TTP). A deficiency in ADAMTS13 enzyme levels, along with an inhibitory antibody, is found in most patients with idiopathic TTP. Patients with human immunodeficiency virus (HIV) infection can have a TTP-like illness; however, it appears to have a different etiology. METHODS: A retrospective review of patients who had an ADAMTS13 activity level performed from 2005 through 2007 was completed. Patients with a diagnosis of HIV infection with TMA were investigated. RESULTS: Two patients were identified. Case 1: a 47-year-old man with HIV infection and a CD4 count <10/microL presented with altered mental status, pneumonia, acute renal failure, thrombocytopenia, and anemia. The ADAMTS13 level was 71%. He was treated with plasma infusion. Two days after admission, he expired because of respiratory distress syndrome and metabolic lactic acidosis. Case 2: a 39-year-old man with HIV infection and a CD4 count of 9/microL presented with chest pain, acute renal failure, thrombocytopenia, and anemia. The ADAMTS13 level was 65%. He received multiple units of fresh frozen plasma without significant improvement in his platelet count. Six days after admission, the patient began highly active antiretroviral therapy, which resulted in a rapid increase in his platelet count. CONCLUSIONS: HIV-associated TMA is postulated to have a different pathophysiology than idiopathic TTP. This study supports that assumption because both patients exhibited many of the classic findings of TTP but did not have a deficiency of ADAMTS13.

Is it HIV TTP or HIV-associated thrombotic microangiopathy?

Thrombocytopenia is a common complication of Human Immunodeficiency Virus (HIV) infection. With advanced HIV disease, the presence of both thrombocytopenia and schistocytosis are frequently observed. In such cases, the diagnosis of HIV associated TTP is often considered. This article reviews emerging concepts of HIV associated microangiopathies. It concludes that the pathophysiology, in many cases seems to be distinct from idiopathic TTP (particularly with advanced HIV disease-<100 CD4/microliter). A sine que non for successful therapy of HIV-TMA appears to be the treatment of the underlying HIV infection.

The introduction of 7-day platelets: a university hospital experience.

INTRODUCTION: Recently, the FDA approved the Post Approval Surveillance Study of Platelet Outcomes, Release Tested protocol which allows participating institutions to utilize 7 day platelets following guidelines. As one of the first hospitals to implement a 7-day protocol, we reviewed our hospital experience with 7-day Gambro apheresis platelets to determine the impact on inventory. METHODS: A review of apheresis platelet transfusions and outdate records was performed. Data were collected prospectively from March to August 2006. This data were compared with a retrospective review for the same time period in 2005. RESULTS: For the 1,503 platelets transfused from March-August 2005, the mean day of issue was 3.44 (SD = 1.060). During the same time period of 2006, 1,688 platelets were transfused with a mean day of issue of 4.02 (SD = 1.083). This difference was statistically significant (P < 0.001). The outdate rate dropped from 2.9% (44/1,547) to 1.3% (22/1,710, P < 0.001). During the study period, approximately 59.7% of the platelets were 7-day platelets. DISCUSSION: Over the 6-month period, we noted a decrease in outdates from 2.9% to 1.3%. There was a shift toward older platelets (from a mean of day 3.4 to day 4). During the study period, 139 platelets were transfused on days 6 or 7 of storage. Overall, the implementation of 7-day platelets in a university hospital setting was easily accomplished and has resulted in benefits to our institution by decreasing our outdate rate and to our patients by providing an additional 139 days 6 and 7 apheresis platelets with a potential cost savings of $78,952 (over the 6-month study).

Investigation of an isolate of Staphylococcus lugdunensis implicated in a platelet fatality: a possible advantage of the use of an anaerobic bottle.

BACKGROUND: Cases have been reported in which detection with an early culture failed to interdict the transfusion of a bacterially contaminated platelet. One fatal case involved Staphylococcus lugdunensis, missed with a 4-mL aerobic (BPA) bottle (BacT/ALERT, bioMérieux). This report noted "deviation from culture methods that meet manufacturer's recommendations (e.g., decreased blood volume) can result in reduced sensitivity and produce false negatives." The manufacturer's package insert "strongly" recommends that more than one type of culture bottle be utilized. The utility of an anaerobic (BPN) bottle compared to a BPA bottle was investigated for the detection of S. lugdunensis. STUDY DESIGN AND METHODS: This isolate was subjected to a series of spiking experiments designed to assess the effect of sample volume and recovery of BPA and/or BPN bottles over a range of concentrations (0.1-6 colony-forming units/mL). RESULTS: With low levels of contamination, larger volumes cultured were associated with higher recovery. With the lowest inoculation, such that only one to two organisms were present in a bottle, reactivity was accelerated in the BPN bottle compared to the BPA bottle. At higher concentrations, 10 mL in the BPA bottle yielded equivalent time to reactivity as 4 mL in the BPN bottles. CONCLUSION: These data support the use of larger inocula; however, when only one to two organisms were present in a bottle, time to reactivity was accelerated in a BPN bottle compared to a BPA bottle. The accelerated growth was not observable with higher inoculum (e.g., more than six organisms per bottle). Quicker time to reactivity might make the difference between interdiction or transfusion of the product.

Successful in vivo recovery and extended storage of additive solution (AS)-5 red blood cells after deglycerolization and resuspension in AS-3 for 15 days with an automated closed system.

BACKGROUND: Previously, cryopreserved red blood cell (RBC) units derived from CPD/AS-5 whole-blood (WB) collections have been limited to 24 hours postthaw storage (1-6 degrees C). STUDY DESIGN AND METHODS: Sixty-four leukoreduced (LR) and 54 nonleukoreduced (NLR) AS-5 (n = 118) RBC units from 500-mL WB collections were stored for 6 days, glycerolized, frozen (-70 +/- 5 degrees C) for at least 14 days, thawed, deglycerolized, and stored (1-6 degrees C) for 15 days resuspended in AS-3, using an automated closed-system cell processor (ACP 215, Haemonetics). Frozen units were stored in either ethylene vinyl acetate (EVA) or polyvinylchloride (PVC) bags. In vitro parameters were tested in all units 15 days after deglycerolization. In vivo 24-hour recovery was measured in 77 of 118 donors. RESULTS: Postdeglycerolization in vitro RBC mass recoveries (mean +/- SD) were 96.8 +/- 5.7 and 94.7 +/- 5.6% for EVA LR and NLR units, respectively, and 97.3 +/- 6.2 and 94.7 +/- 6.2% for PVC LR and NLR units, based on unit weight and hematocrit after sampling for in vitro testing, immediately before glycerolization. Hemoglobin content (g/unit, mean +/- SD) after deglycerolization was 40.4 +/- 5.6 and 42.6 +/- 6.0 for EVA LR and NLR units, respectively, and 40.7 +/- 4.8 and 43.0 +/- 7.7 for PVC LR and NLR units. Hemolysis was 0.61 +/- 0.23 and 0.54 +/- 0.16% for EVA LR and NLR units, and 0.47 +/- 0.14 and 0.43 +/- 0.12% for PVC LR and NLR units. In vivo 24-hour recoveries on Day 15 were 83.0 +/- 6.7% (PVC NLR) up to 86.2 +/- 5.7% (EVA NLR). CONCLUSION: With processing on the ACP 215 system, CPD/AS-5 LR and NLR thawed RBC units can be stored for up to 14 days after frozen storage at -65 degrees C or colder in EVA or PVC bags with acceptable in vivo and in vitro RBC quality.

Life, death, and the risk of transfusion: a university hospital experience.

BACKGROUND: Frequently one encounters donor recruitment statements like ". . . about 90 percent of American adults will need at least one blood transfusion in the course of a lifetime." To assess the validity of such statements, the lifetime transfusion rate of patients dying in our tertiary-care hospital was determined. It would be expected that patients dying in a tertiary-care, Level 1 trauma center university hospital would have one of the highest transfusion rates. STUDY DESIGN AND METHODS: A retrospective review of blood bank and electronic clinical records was conducted for all patients who expired at our institution from January 1, 2004, through December 31, 2004. Stillborn infants were excluded from analysis. The rate of transfusion was stratified by age and sex. RESULTS: A total of 906 patients expired in 2004 at our institution. Of these, 35 were excluded (stillborn infants), leaving 871 patients for analysis. After review of blood bank records, 591 patients received blood products (67.9%). An additional 27 patients (3.1%) had a possible transfusion at another institution as the clinical record indicated complex surgical history. Only 253 patients (29.0%) did not have a documented transfusion event. Individuals who died before the age of 31 had the highest rates of transfusion (p < 0.001, Fisher's exact test, two-sided). CONCLUSIONS: The majority of patients who expire at our institution have a history of transfusion (67.9%-71.0%). Patients who die before the age of 31 have the highest rate of transfusion.

Patterns of platelet response in idiopathic TTP/HUS: frequency of declining platelet counts with plasma exchange and the recognition and significance of a pseudo refractory state.

For thrombotic thrombocytopenic purpura (TTP), daily plasma exchange (TPE) is typically discontinued when the platelet count normalizes (>150 x 10(9)/L). We observed a decline in platelet count during TPE and in patients who appeared pseudo-refractory because of a platelet count plateau (100-150 10(9)/L range). In the present study, we evaluated platelet count trends in TTP patients. Retrospective review of TTP patients from 01/1999 to 12/2004 was completed. Patients were categorized based on platelet count trends: Group I, counts rose then decreased to levels <100 x 10(9)/L; Group II, counts declined following TPE initiation; Group III, counts rose continuously; Group IV, counts decreased after the count was >100 x 10(9)/L. Additionally, we identified pseudo-refractory patients caused by a platelet count plateau (>100 x 10(9)/L but <150 x 10(9)/L). We identified 60 TTP patients. Within Group I (17 patients/17 series/19.1% of total), the mean decrease in platelet count was 67.3% +/- 22.1% following initial rise. Within Group II (24 patients/25 series/28.1% of total), the mean decrease was 28% +/- 5.3% following presentation. Group III included 31 patients/39 series (43.8% of the total). Within Group IV (seven patients/eight series/9.0% of total), the mean decrease was 17.4% +/- 12.6% following a sustained rise >100 x 10(9)/L. With a declining platelet count and daily TPE, it is generally sufficient to stay the course and the decline will reverse. Our limited experience with pseudo-refractory patients supports discontinuing TPE when counts plateau between 100 and 150 x 10(9)/L when a therapy goal is a platelet count of 150 x 10(9)/L. Recognition of this pseudo-refractory state can minimize the risks of prolonged TPE and the risks of adjunct interventions.

Bacterial contamination of blood components.

Blood for transfusion is a potential source of infection by a variety of known and unknown transmissible agents. Over the last 20 years, astounding reductions in the risk of viral infection via allogeneic blood have been achieved. As a result of this success, bacterial contamination of blood products has emerged as the greatest residual source of transfusion-transmitted disease. This paper summarizes the current status of detection, prevention, and elimination of bacteria in blood products for transfusion.

Frequency and significance of schistocytes in TTP/HUS patients at the discontinuation of plasma exchange therapy.

INTRODUCTION: Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is characterized by thrombocytopenia, a microangiopathic hemolytic anemia (presence of schistocytes) and elevated LDH without another likely explanation. Standard of care is daily plasma exchange, which is typically discontinued when the platelet count exceeds 100-150 x 10(9)/L for 2 days. However, residual schistocytosis, the presence of schistocytes at the time of discontinuation of plasma exchange therapy, is often disconcerting. We evaluated the frequency and significance of residual schistocytosis in TTP/HUS patients when the patients' platelet counts returned to normal levels (e.g., 100-150 x 10(9)/L). METHODS: Retrospective review in our institution from 01/1999-03/2004 of all patients treated with plasma exchange for TTP/HUS with at least 2 months of follow-up for relapse was completed. Patients were excluded if the clinical course was complicated by HIV, stem cell/bone marrow and solid organ transplant, pregnancy and auto-immune disease. Schistocytes were documented on day of presentation and on the day the platelet count reached 150 x 10(9)/L. Grading scale (using 100 x objective-a high power field, with approximately 100 red blood cells per field) for schistocytes was as follows: rare for 1 schistocyte per every other other field, 1+ for 1-5%, 2+ for 6-15%, and 3+ for >15%. The frequency of schistocytes was compared to frequency of relapse within 2 months, using Fisher's exact test. RESULTS: We identified 57 patients with TTP/HUS who received plasma exchange therapy. Of these patients, 12 did not have a follow-up microscopic examination of a peripheral blood smear at discontinuation of plasma exchange therapy and were excluded from further analysis. Of the remaining 45 patients, 16 had residual schistocytosis (35.6%). There was no statistically significant difference in relapse rate with or without residual schistocytosis (P = 1.00, Fisher's Exact test, 2 sided). CONCLUSIONS: In this study, we found that the presence of residual schistocytosis is common (35.6%). The presence of residual schistocytosis, however, was not predictive of relapse.

Extended storage of AS-1 and AS-3 leukoreduced red blood cells for 15 days after deglycerolization and resuspension in AS-3 using an automated closed system.

BACKGROUND: The utilization of cryopreserved red blood cell (RBC) units had been limited by a maximum postdeglycerolization storage of 24 hours at 1 to 6 degrees C until the recent development of a closed system for the glycerolization and deglycerolization process. STUDY DESIGN AND METHODS: Sixty leukoreduced additive solution (AS), AS-1 (n = 30) and AS-3 (n = 30) RBC units from 500-mL whole blood (WB) collections were stored for 6 days, glycerolized, frozen at -70 +/- 5 degrees C for at least 14 days, thawed, deglycerolized, and stored for 15 days at 1 to 6 degrees C. Glycerolization and deglycerolization were performed with the ACP 215. In-vitro variables were tested before glycerolization, on Day 0, and Day 15 after deglycerolization storage. Forty donors were assessed for double-label 24-hour percent recovery, and T1/2 survival time was measured for 20 donors. RESULTS: Postdeglycerolization mean +/- standard deviation in-vitro RBC mass recoveries were 93 +/- 5 percent for AS-1 and 95 +/- 4 percent for AS-3. Mean hemoglobin +/- standard deviation after deglycerolization was 50.5 +/- 5.5g for AS-1 and 50.1 +/- 3.5g for AS-3. Mean hemolysis (Day 15) was 0.36 +/- 0.11 percent for AS-1 and 0.38 +/- 0.13 percent for AS-3. Double-label 24-hour in-vivo recoveries were 82.5 +/- 7.8 percent for AS-1 and 81.4 +/- 7.1 percent for AS-3. The 51Cr T1/2 value was 41.8 +/- 3.97 for AS-1 and 40.6 +/- 7.11 for AS-3. Other in-vitro variables were as expected. CONCLUSION: Leukoreduced AS-1 and AS-3 RBCs after frozen storage at -70 +/- 5 degrees C can be stored for up to 14 days when processing is performed with the ACP 215 system with resuspension of deglycerolized RBCs in AS-3.