Protocol adherence for continuously titrated interventions in randomized trials: an overview of the current methodology and case study.

BACKGROUND: The standard definition for protocol adherence is the proportion of all scheduled doses that are delivered. In clinical research, this definition has several limitations when evaluating protocol adherence in trials that study interventions requiring continuous titration. DISCUSSION: Building upon a specific case study, we analyzed a recent trial of a continuously titrated intervention to assess the impact of different definitions of protocol deviations on the interpretation of protocol adherence. The OVATION pilot trial was an open-label randomized controlled trial of higher (75-80 mmHg) versus lower (60-65 mmHg) mean arterial pressure (MAP) targets for vasopressor therapy in shock. In this trial, potential protocol deviations were defined as MAP values outside the targeted range for >4 consecutive hours during vasopressor therapy without synchronous and consistent adjustments of vasopressor doses. An adjudication committee reviewed each potential deviation to determine if it was clinically-justified or not. There are four reasons for this contextual measurement and reporting of protocol adherence. First, between-arm separation is a robust measure of adherence to complex protocols. Second, adherence assessed by protocol deviations varies in function of the definition of deviations and the frequency of measurements. Third, distinguishing clinically-justified vs. not clinically-justified protocol deviations acknowledges clinically sensible bedside decision-making and offers a clear terminology before the trial begins. Finally, multiple metrics exist to report protocol deviations, which provides different information but complementary information on protocol adherence. CONCLUSIONS: In trials of interventions requiring continuous titration, metrics used for defining protocol deviations have a considerable impact on the interpretation of protocol adherence. Definitions for protocol deviations should be prespecified and correlated with between-arm separation, if it can be measured.

Brain Charts for the Rhesus Macaque Lifespan.

Recent efforts to chart human brain growth across the lifespan using large-scale MRI data have provided reference standards for human brain development. However, similar models for nonhuman primate (NHP) growth are lacking. The rhesus macaque, a widely used NHP in translational neuroscience due to its similarities in brain anatomy, phylogenetics, cognitive, and social behaviors to humans, serves as an ideal NHP model. This study aimed to create normative growth charts for brain structure across the macaque lifespan, enhancing our understanding of neurodevelopment and aging, and facilitating cross-species translational research. Leveraging data from the PRIMatE Data Exchange (PRIME-DE) and other sources, we aggregated 1,522 MRI scans from 1,024 rhesus macaques. We mapped non-linear developmental trajectories for global and regional brain structural changes in volume, cortical thickness, and surface area over the lifespan. Our findings provided normative charts with centile scores for macaque brain structures and revealed key developmental milestones from prenatal stages to aging, highlighting both species-specific and comparable brain maturation patterns between macaques and humans. The charts offer a valuable resource for future NHP studies, particularly those with small sample sizes. Furthermore, the interactive open resource (https://interspeciesmap.childmind.org) supports cross-species comparisons to advance translational neuroscience research.

The Association between Frailty and Short-Term Outcomes in an Intensive Care Unit Rehabilitation Trial: An Exploratory Analysis.

BACKGROUND: Physical therapy initiated early in an ICU stay may reduce functional deficits in critically ill patients; however, the association of frailty with outcomes in those receiving early in-ICU rehabilitation is unknown. OBJECTIVE: To estimate the association between frailty and 3 outcomes in patients enrolled in an ICU randomized clinical trial (RCT). DESIGN: Exploratory secondary analyses of the CYCLE pilot RCT (NCT02377830). SETTING: 7 Canadian ICUs. PARTICIPANTS: Previously ambulatory critically ill adults. INTERVENTION: Participants were randomized to early in-bed cycling plus routine physiotherapy versus early routine physiotherapy alone. MEASUREMENTS: Using regression analyses, we modelled the association between pre-hospital Clinical Frailty Scale (CFS) scores, Physical Function in ICU Test-scored (PFIT-s), muscle strength, and mortality at hospital discharge, adjusting for illness severity (APACHE II) and the randomized intervention. We explored the influence of imputing mean PFIT-s and strength scores for decedents, and with listwise deletion of decedents in a sensitivity analysis. RESULTS: Of 66 patients, 2 had missing data, 2 had incomplete data, and 21 died by hospital discharge. At hospital discharge for 66 patients, frailty was not associated with PFIT-s (mean difference (MD) [95% CI]=0.20, [-2.08, 2.74]) or muscle strength (1.96, [-12.6, 16.6]). A sensitivity analysis yielded consistent results. Frailty was also not associated with hospital mortality (odds ratio 0.91, [0.28 to 2.93]). CONCLUSION: We found no association between pre-hospital frailty, physical function, strength, or mortality at hospital discharge in critically ill patients enrolled in an early rehabilitation trial. Larger sample sizes are needed to further explore the association of frailty with these outcomes at hospital discharge.

The unidentified interstellar infrared bands: PAHs as carriers?

Infrared emission spectra of gas-phase naphthalene and pyrene have been measured in the range of 3 to 7.5 micrometers with ultraviolet laser desorption-excitation and a spectroscopic technique featuring single-photon counting in the infrared. The spectra were compared with the unidentified infrared emission bands that are observed in many astronomical objects. Marked discrepancies between those observations and the laboratory emission spectra in the wavelengths and relative intensities of principal spectral features led to the conclusion that small neutral unsubstituted polycyclic aromatic hydrocarbons cannot be the carriers of the unidentified infrared emission bands.

Anomaly detection using temporal data mining in a smart home environment.

OBJECTIVES: To many people, home is a sanctuary. With the maturing of smart home technologies, many people with cognitive and physical disabilities can lead independent lives in their own homes for extended periods of time. In this paper, we investigate the design of machine learning algorithms that support this goal. We hypothesize that machine learning algorithms can be designed to automatically learn models of resident behavior in a smart home, and that the results can be used to perform automated health monitoring and to detect anomalies. METHODS: Specifically, our algorithms draw upon the temporal nature of sensor data collected in a smart home to build a model of expected activities and to detect unexpected, and possibly health-critical, events in the home. RESULTS: We validate our algorithms using synthetic data and real activity data collected from volunteers in an automated smart environment. CONCLUSIONS: The results from our experiments support our hypothesis that a model can be learned from observed smart home data and used to report anomalies, as they occur, in a smart home.

Tumor necrosis factor-alpha blockade prevents neutrophil CD18 receptor upregulation and attenuates acute lung injury in porcine sepsis without inhibition of neutrophil oxygen radical generation.

Tumor necrosis factor (TNF alpha), both by direct action and by trafficking cells of the immune system, is implicated in cardiopulmonary derangements and PMN-mediated microvascular injury associated with gram-negative sepsis. We examined the effects of pretreatment with a monoclonal antibody to TNF alpha on PMN function, hemodynamic derangements, and alveolar capillary membrane damage in a septic porcine model. Anti-TNF alpha profoundly improved hemodynamic consequences in this model. Reduction in PMN CD11/18 receptor expression, lung myeloperoxidase activity, and attenuation of peripheral neutropenia (all P < 0.05) indicate that pretreatment significantly reduced lung sequestration of PMNs seen in septic controls. In contrast, PMN oxygen radical (O2-) generation was not significantly different from unprotected septic animals. Despite the presence of circulating PMNs primed for O2- burst, alveolar capillary membrane damage, assessed by bronchoalveolar lavage protein content and arterial PO2 was markedly attenuated in the treatment group (P < 0.05). We conclude that anti-TNF alpha suppresses systemic hemodynamic actions of TNF alpha. Further, it prevents upregulation of PMN adhesion receptors inhibiting PMN/endothelial cell interaction. This prevents formation of a "microenvironment," protected from circulating oxidant scavengers, into which sepsis-activated PMNs release their toxic products. Pretreatment with anti-TNF alpha monoclonal antibody thus affords global protection in porcine Gram-negative sepsis.

Attrition from heart transplant waiting list for patients on ventricular assist devices is not affected by desensitization strategies.

BACKGROUND: Ventricular assist device (VAD) patients, who are commonly sensitized, can be successfully transplanted using strategies aimed at diminishing antibody burden. However, the impact of these therapies on outcomes for VAD patients on the waiting list is ill-defined. The following study was conducted to ascertain the relationship between desensitization therapies and attrition rate from the waiting list for VAD patients. METHODS: The VAD patients listed between July 1996 and June 2002 were used for this report. Transplant and inpatient pharmacy databases were queried for demographics, date of transplantation, degree of allosensitization, use of desensitization therapy, immunosuppressive strategies, and specific causes of death. RESULTS: Among 232 patients listed for heart transplantation who required bridging to transplantation with a VAD, 79 (34%) died while on the waiting list. Common causes of death included multisystem organ failure in 32 (40.5%), sepsis in 19 (24.0%), and stroke in 10 (12.6%) patients. While nearly 50% of these patients were sensitized at listing, only 5 (6.3%) patients received desensitization therapy following VAD implantation. Therapies included mycophenolate mofetil in 3 (3.7%) and IVIG in 2 (2.5%) patients. Not a single patient underwent plasmapheresis or OKT3 therapy. CONCLUSION: For patients bridged to heart transplantation with a VAD, attrition from the waiting list was associated with factors other than desensitization or induction regimens.

Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s.

The monitoring of activity at home after total hip arthroplasty.

AIMS: Total hip arthroplasty (THA) has well known subjective benefits, but little is known objectively about the recovery of mobility in the early post-operative period. PATIENTS AND METHODS: A total of 33 patients aged > 60 years who underwent elective primary THA had their activity monitored for 30 days post-operatively using an at-home (Fitbit) ankle accelerometer. Their mean age was 70.7 years (61 to 86); 15 (45.5%) were female. The rate of compliance and the mean level of activity were determined. Comparisons between subgroups based on age, body mass index (BMI), surgical approach, and the destination of the patients when discharged were also performed. RESULTS: The mean compliance over the 30 days was 26.7 days (16 to 30; 89%) of use. The mean number of steps increased from 235 (5 to 1152) to 2563 (87 to 7280) (p < 0.001) between the first and the 30th post-operative day. Age < 70 years and an anterior surgical approach were significantly associated with higher levels of activity (1600 to 2400 (p = 0.016 to 0.031) and 1000 to 1800 (p = 0.017 to 0.037) more steps per day, respectively) between the second and the fourth week post-operatively. There was also a trend towards higher levels of activity in those who were discharged to their home rather than to a nursing facility (a mean of 1500 more steps per day, p = 0.02). BMI greater or less than 30 kg/m2 was not predictive of activity (p = 0.45 to 0.98). CONCLUSION: At-home remote mobility monitoring using existing commercially available technology is feasible in patients who have undergone THA. It showed a clear trend towards increased activity with the passage of time. Additionally, the remote device was able to detect differences in levels of activity clearly between patients in relation to variables of interest including age, BMI, surgical approach, and the destination of the patient at the time of discharge from hospital. Such monitoring may allow for the early identification and targeted intervention in patients who recover slowly. Cite this article: Bone Joint J 2016;98-B:1450-4.

Improving patients' safety locally: changing clinician behaviour.

Safety initiatives in hospitals should focus on common health care interventions that when used appropriately can improve important health outcomes, and when used inappropriately or not at all, result in substantial harm. We suggest that errors of omission should be a safety priority. We focus on preventive health care interventions, and describe five steps that can improve patients' safety by changing clinician behaviour. The steps are to: do an environmental scan; understand current behaviour, target behaviour for change (why, what, when, where, and who); adopt effective strategies to change behaviour; and synergise.

Neither baseline tests of molecular hypercoagulability nor D-dimer levels predict deep venous thrombosis in critically ill medical-surgical patients.

OBJECTIVE: Predicting patients who are harboring asymptomatic deep venous thrombosis (DVT), or who are at particular risk of developing DVT, is a desirable clinical goal since prevention or early treatment of DVT might reduce the risk of fatal pulmonary embolism. Thus validation of simple laboratory tests that reliably predict venous thromboembolism (VTE) would be clinically very important. Tests that might be useful for these applications include markers of hypercoagulability (predicting patients at risk of DVT) and D-dimer (predicting which patients may have acute DVT). METHODS: In a prospective cohort study we measured a panel of hypercoagulability markers at the time of ICU admission, and six commercial D-dimer assays were performed serially during the ICU stay in medical-surgical ICU patients who were screened for DVT with biweekly lower limb compression ultrasonography. Ultrasonography was also performed at the time of any clinically suspected DVT events. We matched cases with DVT with controls without DVT for length of stay in the ICU to generate receiver operating characteristics (ROC) curves. RESULTS: One hundred ninety-seven patients were enrolled. Blood was collected on a total of 763 occasions (median number of occasions per patient: 3, range 1-21). None of the assays predicted DVT, as indicated by the areas under the ROC curves, that did not differ significantly from 50%. CONCLUSION: In critically ill patients, neither tests of hypercoagulability nor D-dimer levels predict patients at risk of DVT and thus they should not be used to guide diagnostic testing for DVT.

Early and late rejection and HLA sensitization at the time of heart transplantation in patients bridged with left ventricular assist devices.

Over the years, the frequency of heart transplant candidates with HLA sensitization has increased as a result of the number of patients bridged to transplant using left ventricular assist devices (LVAD). Here we have examined 119 patients who were bridged to transplant with LVAD for a relationship between HLA antibodies and early (30 days) and late (2 years or more) rejection, as evidenced by endomyocardial biopsies. Both cytotoxic panel-reactive antibody reactions against a panel of T lymphocytes (T-PRA) and the percentage of transplants that occurred across a positive class I flow cross-match were examined. Biopsies were scored using ISHLT criteria. At 30 days, patients who had a biopsy grade of 0 had a mean T-PRA at transplant of 2.2%, while the mean PRAs of the other biopsy grades were significantly higher (P < .001). A similar pattern was seen with the highest biopsy results at 2 years or later (P < .001). None of the patients who had a grade 0 biopsy at 30 days posttransplant had a positive flow cytometry class I cross-match (P = .02), although the same pattern did not occur later due to a small number of patients (n = 3) who had negative biopsies. Thus, when biopsy results were examined early or late posttransplant, patients with negative biopsy results tended to have less HLA sensitization. While the methods of HLA sensitization involve humoral responses, more aggressive immunosuppression might be warranted to attempt to reduce cellular rejection posttransplant if HLA class I antibodies are present at the time of transplant.

Survival beyond 10 years following heart transplantation: The Cleveland Clinic Foundation experience.

BACKGROUND: Long-term survival after heart transplantation is a desirable although challenging goal. METHODS: We analyzed clinical outcomes in the cohort of 170 patients who have undergone heart transplantation at The Cleveland Clinic Foundation and survived >10 years. RESULTS: We found 10-year and 15-year survival rates of 54% and 41%, respectively, in these patients, but there was also a high incidence of complications, such as hypertension, renal dysfunction, transplant vasculopathy, and malignancy. CONCLUSIONS: Long-term survival following cardiac transplantation is possible although complications are frequent. Beyond 10 years, malignancy is a major cause of death.

Multidisciplinary education for oxygen prescription. A continuous quality improvement study.

OBJECTIVES: To examine the oxygen-prescribing habits and monitoring patterns on a medical teaching ward and to review the literature in this area. DESIGN: A continuous quality improvement study. SETTING: A 29-bed medical clinical teaching unit in a 453-bed university-affiliated tertiary care hospital. PATIENTS: We studied 50 consecutive patients who required 79 oxygen treatments. METHODS: We recorded the indication, prescriber, documentation of prior hypoxemia, method and mode of delivery, oxygenation assessment after initiation, and duration of therapy. RESULTS: Patients received oxygen for a mean (+/- SD) of 4.7 +/- 4.5 days. Oxygen therapy was ordered on a continuous basis 60.3% of the time. It was ordered by house staff in 54 cases (68%); nurses initiated oxygen therapy in 14 cases (18%) but discontinued it more often than any other health care workers. The most common indications for starting oxygen therapy were dyspnea and tachypnea. In 15 patients (30%), none of the American College of Chest Physicians and National Heart, Lung, and Blood Institute criteria for starting oxygen therapy were fulfilled. For 16 patients (32%), arterial blood gas values were measured within 1 hour of oxygen administration; for 29 patients, oximetry was performed. For 9 patients (18%), no testing of adequate oxygenation was performed within 24 hours. Oxygenation status was assessed daily for 23 patients (46%). CONCLUSIONS: Oxygen prescribing and monitoring practices were suboptimal on our busy medical teaching ward. Practice guidelines based on best available evidence are needed to increase the efficiency of oxygen use. A physiologic, multidisciplinary educational focus on the benefits and hazards of supplemental oxygen is necessary, and randomized trials of such educational interventions should be conducted.

Deep vein thrombosis and its prevention in critically ill adults.

BACKGROUND: Our objective was to systematically review the incidence of deep vein thrombosis (DVT) and the efficacy of thromboprophylaxis in critically ill adults, including patients admitted to intensive care units and following trauma, neurosurgery, or spinal cord injury. METHODS: Two authors independently searched MEDLINE, EMBASE, abstract databases, and the Cochrane database. Data were extracted independently in triplicate. RESULTS: Ten percent to 30% of medical and surgical intensive care unit patients develop DVT within the first week of intensive care unit admission. The use of subcutaneous low-dose heparin reduced the rate by 50% compared with no prophylaxis. Approximately 60% of trauma patients developed DVT within the first 2 weeks of admission. Use of unfractionated heparin appears to decrease the incidence of DVT by only 20%, whereas low-molecular-weight heparin decreases the incidence by a further 30%. The estimated prevalence of DVT in neurosurgical patients not given prophylaxis is 22% to 35%. Mechanical prophylaxis is efficacious, with a pooled odds ratio in 5 randomized trials of 0.28. Use of low-molecular-weight heparin has been investigated as an adjunct to mechanical prophylaxis with a pooled odds ratio of 0.59 compared with graduated compression stockings alone. The incidence of DVT without prophylaxis in acute spinal cord injury patients is likely in excess of 50% to 80%. Studies of prophylaxis in these patients are too sparse to come to any definitive conclusion. CONCLUSIONS: Critically ill patients commonly develop DVT, with rates that vary from 22% to almost 80%, depending on patient characteristics. Methods of prophylaxis proven in one group do not necessarily generalize to other critically ill patient groups. More potent prophylactic regimens other than unfractionated or low-molecular-weight heparins alone may be needed with higher-risk groups.

Emphysematous pyelonephritis. Complicated urinary tract infection in diabetes.

Renal emphysema is rare condition that may affect diabetic patients. Radiographic demonstration of gas limited to the collecting system is often treated with antibiotics. However, intraparenchymal gas requires early nephrectomy because mortality rates are high without surgical intervention.

Fluid type and the use of renal replacement therapy in sepsis: a systematic review and network meta-analysis.

Fluid resuscitation, along with the early administration of antibiotics, is the cornerstone of treatment for patients with sepsis. However, whether differences in resuscitation fluids impact on the requirements for renal replacement therapy (RRT) remains unclear. To examine this issue, we performed a network meta-analysis (NMA), including direct and indirect comparisons, that addressed the effect of different resuscitation fluids on the use of RRT in patients with sepsis. The data sources MEDLINE, EMBASE, ACPJC, CINAHL and Cochrane Central Register were searched up to March 2014. Eligible studies included randomized trials reported in any language that enrolled adult patients with sepsis or septic shock and addressed the use of RRT associated with alternative resuscitation fluids. The risk of bias for individual studies and the overall certainty of the evidence were assessed. Ten studies (6664 patients) that included a total of nine direct comparisons were assessed. NMA at the four-node level showed that an increased risk of receiving RRT was associated with fluid resuscitation with starch versus crystalloid [odds ratio (OR) 1.39, 95% credibility interval (CrI) 1.17-1.66, high certainty]. The data suggested no difference between fluid resuscitation with albumin and crystalloid (OR 1.04, 95% CrI 0.78-1.38, moderate certainty) or starch (OR 0.74, 95% CrI 0.53-1.04, low certainty). NMA at the six-node level showed a decreased risk of receiving RRT with balanced crystalloid compared to heavy starch (OR 0.50, 95% CrI 0.34-0.74, moderate certainty) or light starch (OR 0.70, 95% CrI 0.49-0.99, high certainty). There was no significant difference between balanced crystalloid and saline (OR 0.85, 95% CrI 0.56-1.30, low certainty) or albumin (OR 0.82, 95% CrI 0.49-1.37, low certainty). Of note, these trials vary in terms of case mix, fluids evaluated, duration of fluid exposure and risk of bias. Imprecise estimates contributed to low confidence in most estimates of effect. Among the patients with sepsis, fluid resuscitation with crystalloids compared to starch resulted in reduced use of RRT; the same may be true for albumin versus starch.

Frontal lobe epilepsy: clinical seizure characteristics and localization with ictal 99mTc-HMPAO SPECT.

We evaluated ictal 99mtechnetium hexamethyl propylene-amine-oxime single-photon emission computed tomography (SPECT) in 22 children with electroclinical features of frontal lobe epilepsy (FLE). Ictal SPECT demonstrated unilateral frontal hyperperfusion in 20 of 22 children (91%) (one lobar, two frontocentral, six dorsolateral, six frontopolar, three orbitofrontal, one medial frontal, and one insula), concordant with electroclinical lateralization in 19 of 20 (95%). Hyperperfusion was evident in the ipsilateral basal ganglia in 16 of 22 (73%) and the contralateral cerebellum in 14 of 22 children (64%). Interictal SPECT showed unilateral, localized frontal hypoperfusion concordant with electroclinical lateralization in only two of 22 children (9%). Ictal SPECT localization to the frontocentral, media frontal, or dorsolateral regions was associated with asymmetric tonic posturing, contralateral head/eye deviation, and unilateral clonic jerking (p < 0.01). Ictal SPECT localization to the frontopolar or orbitofrontal regions was associated with vocalization, hyperventilation, truncal flexion, and complex gestural automatisms (p > or = 0.05). Ictal SPECT has the potential to (1) localize seizures in patients with intractable FLE, and (2) advance understanding of the in vivo anatomico-clinical relationships of frontal lobe seizures.

Stress ulcer prophylaxis in the critically ill: a meta-analysis.

PURPOSE: To examine the differential effect of stress ulcer prophylaxis on overt bleeding, clinically important bleeding, and mortality in critically ill patients. DATA IDENTIFICATION: Computerized bibliographic search of published and unpublished research. STUDY SELECTION: Independent review of 168 articles identified 42 relevant randomized trials for inclusion. DATA ABSTRACTION: The validity, population, intervention, and outcomes of each trial were evaluated. RESULTS: Stress ulcer prophylaxis with antacids (odds ratio 0.40 [95% confidence interval (CI) 0.20 to 0.79]) or histamine-2-receptor antagonists (odds ratio 0.29 [95% CI 0.17 to 0.45]) decreases the incidence of overt gastrointestinal bleeding. Histamine-2-receptor antagonists are more effective than antacids at reducing overt hemorrhage (odds ratio 0.56 [95% CI 0.33 to 0.97]). A significant reduction in clinically important gastrointestinal hemorrhage is evident only with histamine-2-receptor antagonist therapy. There is a trend favoring antacids over sucralfate in the outcome of clinically important bleeding (odds ratio 0.65 [95% CI 0.16 to 2.49]); however, there are insufficient data to evaluate histamine-2-receptor antagonists versus sucralfate. No difference in mortality between treated and untreated patients was found. CONCLUSIONS: Overt gastrointestinal bleeding in critically ill patients is reduced by prophylaxis with antacids or histamine-2-receptor antagonists. Histamine-2-receptor antagonists are more effective than antacids at decreasing overt bleeding and are more effective than no treatment at reducing the incidence of clinically important bleeding. Mortality rates in the intensive care unit are not decreased by stress ulcer prophylaxis.

Serum antibody reactivity of broadly sensitized patients with HLA-matched peripheral blood T lymphocytes.

The sera of 45 hypersensitized patients with more than 90% panel reactive antibodies were tested for reactivity against peripheral blood T lymphocytes from 7 donors matched for HLA-A,B antigens. The target cells were phenotypically homozygous HLA-A1,B8 or A2,B44. Tests for lymphocytotoxic antibodies were essentially negative except for one target cell in which 10 of 25 sera were positive. More positive reactions were detected by flow cytometry, and the proportion of positive specimens ranged from 15% to 48% with the different target cells. Most positive reactions involved the HLA-A2 and B44 antigens, in which heterogeneity within the serologically defined specificities has been described. The positive reactions were further evaluated by testing the effect of purified soluble HLA molecules on the binding of antibodies to the HLA-marched targets. Purified HLA produced a detectable inhibition of antibody binding in the majority of the serum-target combinations studied. These results document that anti-HLA antibodies can still react with HLA-matched lymphocytes, which may be due to the recognition of subtypes of the known serologically defined specificities.