Unfiltered Blood Research Articles

Detection of dual-methylated BRCA1/BRCA2 cell-free DNA for ovarian cancer on an aptamer-based integrated microfluidic system

A new screening strategy has been developed to select nucleic acid aptamer probes with an “in vivo-like” systematic evolution of ligands by exponential enrichment (SELEX) process featuring positive, negative selection and in vivo-like selection steps. One methylation-specific aptamer with high affinity and specificity was selected and it could capture methylated cell-free DNA (cfDNA) from unfiltered blood of ovarian cancer (OvCa) patients. This aptamer was conjugated onto magnetic beads and integrated into a new microfluidic chip for rapid and automatic detection and quantification of two prevalent methylated tumor suppressor genes, breast cancer type 1 and 2 susceptibility protein (BRCA 1/2), in the molecular profile of OvCa. The assay was conducted under physiological conditions that mimic the microenvironment of cancer patients at 25–37 °C, with a low pH value (5.5) and/or high blood ion levels, without requiring additional sample preparation steps. The results of this study could be extrapolated to the direct detection of methylated cfDNA at levels as low as 1 pM in blood or plasma. Not only was DNA methylation observed in OvCa patients, but it was also detected in many other types of cancer; it is therefore considered an important factor in carcinogenesis. Hence, it is inferred that this screened aptamer should not only be used for OvCa diagnosis but also for multiple cancer epigenetic detection and drug development research in the future.

Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-basedtherapies.

5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates

BackgroundWhole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7–8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS.MethodsWhole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia range: 0.04–5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02–1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples.ResultsAfter applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×).ConclusionsThe novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.

Hemodialysis

Hemodialysis

Features of chromatographic separation on heteropaternal sorbents

The possibility of using a short 20x4 mm chromatographic column with a heterosurface sorbent for direct determination of theophylline and caffeine in blood plasma and a 120x4 mm column for determination of tetracycline antibiotics in cow’s milk was studied. Complete separation of tetracycline peaks was not achieved. Determination of caffeine and theophylline was performed successfully in plasma filtrates without long and time-consuming stages of preliminary preparation of the sample without protein deposition. Before backwashing, unfiltered blood plasma can be injected in an amount equivalent to 50 mg of dry matter. Retention characteristics of caffeine and theophylline are practically stable: the retention factor of theophylline for k = 4,0 + 0,1 for caffeine k = 8,5 + 0,4 eluent in composition of 0.02 M sodium hydrogen phosphate (pH = 6,0) – acetonitrile (90:10).

Predictors of red cell alloimmunization in multitransfused Egyptian patients with β-thalassemia.

Thalassemia is a major health problem in Egypt. Red blood cell alloimmunization is an important complication in transfusion-dependent patients. To determine alloimmunization prevalence in Egyptian patients with β-thalassemia and to evaluate risk factors that could influence alloimmunization, with the hope of minimizing transfusion-associated risks in those patients. Records of 272 patients with β-thalassemia who are receiving regular blood transfusions, matched for ABO-Rh(D), were analyzed. Alloantibody identification was performed by DiaMed-ID microtyping system. Autoantibodies were detected by direct Coombs test. Alloimmunization incidence was 22.8% with 123 alloantibodies detected in 62 patients. The most common alloantibody was Rh-related (37.4%; 46 of 123), comprising anti-E (14.6%; 18 of 123), anti-D (8.9%; 11 of 123), anti-C (8.9%; 11 of 123), and anti-c (4.9%; 6 of 123), followed by anti-Kell (26%; 32 of 123), → anti-MNS (9.8%; 12 of 123), → anti-Kidd (8.9%; 11 of 123) → anti-Duffy (8.1%; 10 of 123), → anti-Le (5.7%; 7 of 123), → anti-Lu (2.4%; 3 of 123), and → anti-P1 (1.6%; 2 of 123). Anti-D antibodies developed in 34.5% of all Rh-negative patients. Eighty percent of all anti-D antibodies developed in patients older than 18 years. Males had the highest alloimmunization incidence. Alloimmunization incidence increased with the number of units transfused (P = .01). Patients who received unfiltered blood had a higher alloimmunization rate than did those who always received leukoreduced blood (P < .001). Splenectomized patients had a higher alloimmunization rate (32%; 40 of 125) than did those who did not have a splenectomy (16.3%; 24 of 147; P = .003). Autoantibodies occurred in 1.5% (4 of 272) of all patients. Transfusion of leukoreduced and phenotypically matched cells for selective antigens may help reduce expenses and risks of alloimmunization in patients with thalassemia.

Open Labeled, Randomized, Controlled Trial Comparing Leukodepleted (Filtered) Blood Transfusion and Non-Leukodepleted (Unfiltered) Blood Transfusion in Cases of Severe Falciparum Malaria

Open Labeled, Randomized, Controlled Trial Comparing Leukodepleted (Filtered) Blood Transfusion and Non-Leukodepleted (Unfiltered) Blood Transfusion in Cases of Severe Falciparum Malaria

In vitro assessment of red‐cell concentrates in SAG‐M filtered through the MacoPharma™ P‐CAPT prion‐reduction filter

This study investigated whether filtration of leucodepleted red cells in SAG-M through the P-CAPT filter in order to prevent the potential risk of vCJD infection associated with prion transmission through transfusion has any deleterious effect on red-cell quality. Bottom-and-top SAG-M leucodepleted red-cell concentrates (24 units) were prion-reduction filtered on the day following collection, with half of the units undergoing irradiation on day 14. A control group (12 units) was not prion filtered. Units were sampled at 7-day intervals up to day 35 and tested using standard measures of red-cell quality as well as prothrombin content (to examine prion filter efficacy). Haemoglobin loss per unit was approximately 9 g and in some cases levels were below standard specification (40 g). Haemolysis increased significantly after filtration [0.01 (0.00-0.05) vs. 0.23 (0.07-0.52, p<0.001)]. Prothrombin levels were reduced 41.6-fold compared to leucodepleted red-cell units. Product specifications were within or close to routine acceptable levels. Owing to the reduction in haemoglobin levels below those specified, it may be preferable to reduce haemoglobin specification levels and transfuse more prion-filtered units rather than transfuse potentially unsafe blood product. The risk of transfusing more units with less haemoglobin should be offset against the risk of transfusing unfiltered blood.

Removal of T and B lymphocytes by in-line filtration: evaluation of the efficiency of a polyester filter type (Pall WBF-2) by flow cytometric counting.

The aim of this study was to investigate whether in-line filtration, using a polyester filter for the preparation of red cell concentrates (RCC) and plasma (PL), leads to an altered proportion of T and B lymphocytes in the fraction of residual white blood cells (WBC). The capacity of Pall WBF-2 in-line filters to reduce the numbers of T and B lymphocytes from red blood cell concentrates (RCC) and plasma (PL) of 22 donations was investigated by three-colour flow cytometry (FC) using the Tritest-Trucount kit. T and B lymphocytes were identified using monoclonal antibodies (mAbs) against CD3, CD19 and CD45, conjugated with fluorescein isothiocyanate, phycoerythrin or peridinin chlorophyll protein-A, respectively. As the number of B cells was below the detection limit of the FC method, WBC of the respective blood components of healthy donors were concentrated 25-fold by Percoll density-gradient centrifugation. In this fraction the absolute numbers of T and B cells, as well as their ratio, were determined using the Attractor software, which provides a discrimination of rare cell counts from FC in relation to debris. The mean numbers, as well as minima and maxima of T and B lymphocytes per unit, were as follows. T cells in RCC: 4.51 x 10(3) (1.68 x 10(2)-4.09 x 10(4)) and in PL: 1.35 x 10(3) (2.21-1.78 x 10(4)); B cells in RCC: 2.33 x 10(3) (7.10 x 10(1)-9.15 x 10(3)) and in PL: 2.33 x 102 (7.5 x 10(2)-2.8 x 10(3)). T cells were retained, on average, at a higher level than B cells: 3.01 times higher in RCC and 1.01 times higher in PL. After filtration, the ratio of T and B lymphocytes changed in RCC (1.95 : 1) compared with unfiltered blood, where it was 5.83 : 1. In PL the ratio did not change notably compared to unfiltered blood. The results of this research show that cell concentration (using gradient centrifugation) in combination with an appropriate FC acquisition and analysis procedure, allows both residual T and B lymphocytes (being under the detection limit without cell concentration) to be determined.

白血球除去フィルターろ過後の全血に残存する白血球分画の解析

We assessed the performance of a leukocyte reduction filter, WBF2, which has been developed for preparation of blood products with pre-storage leukocyte reduction, and analyzed residual leukocyte subsets in the filtered whole blood.Whole blood was collected from six healthy volunteer donors and filtered 4 hours after collection. Leukocyte subsets of unfiltered and filtered whole blood were analyzed by flow cytometry, and absolute cell numbers of each leukocyte subset were determined.Total leukocyte counts per bag were 1.75±0.34×109 for the unfiltered blood and 2.23±1.37×104 for the filtered blood. Average log10 leukocyte reduction rate was 4.83. In the filtered whole blood, the number of granulocytes, CD4+ T lymphocytes, CD8+ T lymphocytes and NK cells was 8.67±7.66×103, 11.6±36.5×103, 4.05±0.80×103 and 1.66±1.35×103, respectively. No monocytes or B lymphocytes were detected in the whole blood after filtration.These results show that leukocyte reduction rate using the WBF2 filter varies according to leukocyte subset; in particular, monocytes and B lymphocytes are reduced more effectively by this filter.

Correlates of Change in Cytomegalovirus Viremia in Patients with Advanced Human Immunodeficiency Virus Infection Who Require Transfusion

The Viral Activation Transfusion Study compared leukocyte-reduced to unfiltered red blood cell transfusions in human immunodeficiency virus (HIV)- and cytomegalovirus (CMV)-coinfected patients. Relationships between serially measured plasma CMV load and clinical and laboratory outcomes over a median of 12 months were examined in 511 subjects. At baseline, subjects had a median of 15 CD4(+) cells/mm(3), 25% had CMV disease, and 21.5% were viremic. No relationship was found between changes in CMV viremia and changes in HIV RNA. Increased CMV viremia was associated with a concomitant fall in Karnofsky score. Highly active antiretroviral therapy (HAART) led to a decrease in CMV viremia after a 90-day delay. After adjustment for HIV load and CD4(+) cell count, CMV viremia remained associated with an increased risk of CMV disease (relative hazard, 5.78). In late-stage HIV-infected patients, CMV viremia was associated with lower functional status and increased risk of CMV disease. HAART suppressed CMV viremia only after a delay of several months.

Qualitative and quantitative PCR measures of cytomegalovirus in patients with advanced HIV infection who require transfusions.

The Viral Activation Transfusion Study (VATS) was a randomized trial that compared leukocyte-reduced transfusions with unfiltered red blood cell transfusions in HIV and cytomegalovirus (CMV) antibody-positive patients with anemia who were undergoing their first blood transfusion. The relations of the baseline qualitative and quantitative polymerase chain reaction (PCR) measures of plasma CMV viremia, HIV RNA, CD4(+) cell counts, and quality of life in these study subjects were examined. The 511 study subjects had a median CD4(+) cell count equal to 15 cells/mm3, and 110 (21.5%) had CMV viremia by qualitative assay. In multivariate models, frequency of positive qualitative CMV increased with decreasing CD4(+) cell counts (p =.04 trend), higher HIV RNA (p <.001), and a history of CMV disease (p <.001). Quantitative CMV PCR were performed on the 110 qualitative assay-positive study subjects. Median CMV viral load was 1780 copies/ml. In multivariate regression models, lower CD4(+) cell count (p =.03), and a history of CMV disease (p <.001) correlated with the level of CMV load. HIV RNA load and CMV load were not correlated. A lower Karnofsky score was associated with both the presence and quantity of CMV DNA.

Comparison of the level of cytokine mRNA in buffy coat-derived platelet concentrates prepared with or without white cell reduction by filtration.

The generation of proinflammatory cytokines in platelet concentrates (PCs) by white cells is thought to be implicated in febrile nonhemolytic reactions. Furthermore, other cytokines could be generated in the PCs as signs of white cell activation when PCs are prepared from a pool of buffy coats (BCs). The number of white cells in the PCs is crucial to cytokine generation. Each of the PCs (n = 12) was prepared from a pool of BCs from four donors. Before storage, half of the BC-derived PCs (BC-PCs) were white cell reduced by filtration. The BCs and the unfiltered and filtered BC-PCs were assayed for the presence of cytokine mRNA (i.e., interleukin [IL]-1beta, IL-6, tumor necrosis factor alpha [TNFalpha], IL-8, IL-2, and interferon gamma [IFN-gamma]) by the use of competitive reverse transcription-polymerase chain reaction. No mRNA of cytokines with pyrogenic activity, that is, IL-1beta, IL-6 and TNFalpha, was detected in either the filtered or the unfiltered BC-PCs. Likewise, IL-2 mRNA was not found in any of the BC-PCs. IFN-gamma mRNA, however, was detected in the unfiltered BC-PCs but not in the filtered BC-PCs. IL-8 mRNA was found in both the unfiltered and the filtered BC-PCs, but with a lower frequency in the filtered BC-PCs. The unfiltered BC-PCs produced in the top-and-bottom bag system contain traces or undetectable levels of the investigated cytokine mRNA. The results can be used in defining consensus recommendations for the use of filtered and unfiltered blood components.

Leukocytes and decreased left-ventricular contractility during endotoxemia in rabbits.

We hypothesized that leukocytes contribute to decreased myocardial contractility following endotoxin infusion. To test this hypothesis, we administered endotoxin (1 mg/kg intravenously) to intact, anesthetized rabbits whose arterial blood perfused two isolated hearts at a constant pressure (75 mm Hg). One heart was perfused with blood passed through a leukocyte filter, whereas the other received unfiltered blood. Contractility of both hearts was measured every 30 min for 6 h, using the slope of the end-systolic pressure-volume relationship (Emax) and the maximum rate of change of intraventricular pressure (dP/dt(max)). In the unfiltered hearts at 6 h, Emax decreased to 81 +/- 6% (mean +/- SEM) of baseline (p < 0.05). In the hearts perfused with leukocyte-filtered blood there was no change in Emax. Similarly, dP/dt(max) decreased 74 +/- 9% of baseline in the hearts receiving unfiltered blood (p < 0.05) but did not decrease in the hearts receiving leukocyte-filtered blood. The leukocyte filter significantly reduced the number of neutrophils in perfusing blood (p < 0.01), decreased the number of neutrophils in the heart by 77% (p < 0.01), and decreased myocardial morphometric changes (p < 0.05). A 55 +/- 18% reduction in neutrophil L-selectin expression after endotoxin infusion (p < 0.01) suggests that the neutrophils were significantly activated. We conclude that leukocytes, notably activated neutrophils, may contribute to decreased myocardial contractility during septic shock.

Prevention of transfusion-associated Chagas' disease: efficacy of white cell-reduction filters in removing Trypanosoma cruzi from infected blood.

Transfusion-associated Chagas' disease (TA-CD) is a worldwide problem. Measures adopted to prevent TA-CD include the clinical and serologic screening of blood donors and/or the inactivation of Trypanosoma cruzi present in collected blood, using gentian violet as the trypanocidal agent. This study investigated the efficacy of white cell-reduction filters in removing T. cruzi from infected blood. Human blood was contaminated with 2 or 150 T. cruzi parasites per mL and then left unfiltered or filtered with white cell-reduction filters that provided either 2, 3, or 6 log10 white cell removal. The efficacy of the parasite removal of these filters was evaluated by microscopic enumeration of active forms of T. cruzi both in vivo and in vitro. The in vivo experiments were done in Swiss mice that had been intraperitoneally inoculated with T. cruzi-infected human blood. The in vitro experiments were performed with fresh human blood that had been deliberately contaminated with T. cruzi. The number of parasites seen in mice inoculated with unfiltered blood containing 2 or 150 parasites per mL was significantly higher than the number of parasites seen in mice inoculated with blood from the same sample, but filtered with white cell-reduction filters providing 3 or 6 log10 white cell removal. Fifty to 70 percent of the mice given T. cruzi-infected (2 parasites/mL) filtered blood did not develop T. cruzi infection. In vitro, the use of white cell-reduction filters, providing 2, 3, or 6 log10 white cell removal, significantly reduced the number of parasites seen in culture. The present experimental data provide evidence that white cell-reduction filters are effective in reducing the number of parasites in T. cruzi-infected blood and that this efficacy depends, in part, on the concentration of parasites in the artificially infected blood. Properly designed clinical studies of known carriers of T. cruzi must be conducted to determine whether the use of white cell-reduction filters may be an alternative method of reducing the incidence of TA-CD.

Platelet concentrates promote procoagulant activity: evidence from experimental studies using a perfusion technique.

Evaluation of the hemostatic effectiveness of platelet transfusions is difficult. Perfusion methods have been employed to test the quality and function of platelet concentrates, allowing differentiation between platelet-platelet and platelet-surface interactions. A study was performed to investigate platelet adhesive and cohesive properties as well as the formation of fibrin when aliquots of platelet concentrates were added to thrombocytopenic blood. Blood previously anticoagulated with low-molecular-weight heparin (20 U/mL) underwent platelet and white cell reduction by filtration. Perfusates were prepared by adding to filtered blood platelets obtained from standard concentrates (stored for 1, 3, and 5 days). The final platelet count in these perfusates was standardized at 80,000 per microL. After perfusions, platelet-subendothelium interaction and fibrin formation were analyzed morphometrically. Results were always compared with those obtained in unfiltered blood (> 150,000 platelets/microL). A slight impairment in the ability of stored platelets to interact with the subendothelium was noticed during the storage period. However, the presence of fibrin was significantly greater than that observed in studies with unfiltered blood (Day 1 = 23.48 +/- 9.43%*; Day 3 = 26.99 +/- 6.74%*; Day 5 = 17.95 +/- 9.06% vs. unfiltered blood = 12.60 +/- 3.08%; *p < 0.05). The lower platelet counts (80,000/microL) in the perfusates containing platelets from concentrates could account for the reduced platelet-subendothelium interactions, but they cannot explain the increments in fibrin formation. While the preparation and storage of platelets have a detrimental effect on platelet adhesiveness, such procedures can positively influence the platelet procoagulant activity necessary to platelet hemostasis.

Acquired cytomegalovirus infection and blood transfusion in preterm infants

The urinary excretion of cytomegalovirus (CMV) DNA, amplified by polymerase chain reaction using two pairs of primers for late antigen (LA) and major immediate-early antigen (MIE), and serum CMV IgM were examined in 85 pre-term infants (birth-weight less than 2000 g) on admission and monthly until 6 months after birth. Of these 85 infants, 27 had blood exchange transfusions (BET) and 28 had bolus blood transfusions two to nine times. Fifteen of 27 infants underwent BET with blood that had been filtered through Pall RC100 leukocyte removal filter; the other 12 with unfiltered blood. Neither urinary CMV DNA nor serum CMV-specific IgM was detected at birth in any of the 85 pre-term infants; during the first 6 months after birth urinary CMV DNA, for both MIE and LA, appeared in 22 of the 85 infants (25.9%) and CMV IgM was positive in 14 of the 85 (16.5%). Nine of the 12 (75%) infants who received BET of unfiltered blood showed a significantly higher prevalence of urinary CMV DNA compared to the infants in the other three groups (i.e., those who received no blood transfusion, those who had bolus blood transfusions, or those who received BET of filtered blood; P < 0.01 in each instance). In a logistic regression model, CMV DNA urinary excretion was significantly associated with the mode of blood transfusion (unfiltered BET), and the Odds ratio was 38.9 (95% confidence interval, 9.4-160). There was no significant association with other independent variables such as gender, mother's seropositivity, gestational age, birth-weight or delivery mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil Depletion Attenuates Human Intestinal Reperfusion Injury

Intestinal ischemia/reperfusion injury (I/R) results from reactive oxygen metabolites generated by the xanthine oxidase system and activated neutrophils (PMN). In animal models, removing PMN from initial reperfusate has consistently decreased tissue injury. This experiment was designed to test this potential clinical treatment in human bowel subjected to I/R. The extent of reperfusion injury was assessed by measuring the activity of mucosal alkaline phosphatase (Aφ), which is a specific marker of reperfusion injury. Human small intestine (n = 13) obtained at the time of organ harvest for transplantation was perfused for 60 min on an ex vivo perfusion circuit. Reperfusate consisted of autologous blood passed through a leukocyte filter (n = 6) or unfiltered blood (n = 7). Control intestine was sampled at harvest, after transport to the lab on ice (cold ischemia), and after 60 min warm ischemia. Mucosa was homogenized and assayed for Aφ activity by cleavage of p-nitrophenyl phosphate. Aφ activity (nmole/mg/min) was not decreased after either cold (774 ± 37) or warm (753 ± 40) ischemia compared to freshly harvested bowel (770 ± 51). Both reperfused segments showed a significant decrease in Aφ activity compared to controls (P < 0.05); however, reperfusion with leukocytefiltered blood attenuated the decrease in enzyme activity compared to unfiltered blood (327 ± 30 vs 506 ± 25, P < 0.05), constituting an apparent reduction in injury of 35%. The observation that the severity of reperfusion injury was decreased by removal of PMN from the reperfusate demonstrates the efficacy of this strategy in human intestine for the first time.

PREVENTION OF TRANSFUSION-ACQUIRED CYTOMEGALOVIRUS INFECTION IN INFANTS BY BLOOD FILTRATION TO REMOVE LEUCOCYTES

A multicentre, controlled trial was carried out to determine whether removal of leucocytes from blood by means of 'Imugard IG500' (Terumo) filters would prevent transfusion-acquired cytomegalovirus (CMV) infection in newborn infants. 72 infants whose mothers were seronegative and who received some seropositive blood were followed for 6 months for evidence of CMV infection. There were no significant differences between the groups who received filtered and unfiltered blood in median gestation, birthweight, or amount of seropositive blood received (median volume 32·5 ml and 34·5 ml, respectively). 9(21%) of the 42 infants who received unfiltered blood and none of the 30 who received filtered blood were infected with CMV. All infected infants weighed less than 1500 g at birth; they represented 31% of very low birthweight (VLBW) infants at risk of CMV infection. None of 24 VLBW infants who received filtered seropositive blood was infected. 1 infected infant died and 5 had clinical features consistent with CMV infection. The results show that transfusion-acquired CMV infection is preventable by filtration of blood through a leucocyte filter. This method has advantages over other methods of removing leucocytes or the use of only seronegative blood for newborn infants.

SERUM CONCENTRATION OF HUMAN CHORIONIC GONADOTROPIN AND ITS ALPHA AND BETA SUBUNITS. 2. TROPHOBLASTIC TUMOURS

Using specific homologous radioimmunoassays of native hCG and its alpha and beta subunits, we determined the levels of these glycoproteins in unfiltered maternal blood serially obtained in five non-invasive hydatidiform moles before and after evacuation. Some of these samples were assayed after gel filtration chromatography on Sephadex G 100. Twelve samples, obtained in cases of invasive trophoblastic tumour after ablative surgery and chemotherapy, were also assessed for their hCG, hCG alpha and hCG beta content. In unaborted moles, mean circulating levels of native hCG and free hCG beta were considerably increased (seven and thirteen times, respectively) as compared to normal pregnancies of the same age, whereas levels of free hCG alpha were either normal or slightly elevated. Chromatographic analyses of molar sera confirmed the presence of free circulating subunits, and separated hCG beta in its monomeric form from its higher molecular weight form, the latter being in greater quantity than in normal pregnancy sera. In contrast, the elution profile of serum native hCG was comparable in cases of normal and molar pregnancy. Successful curettage was accompanied by a return to normal levels of the native hCG and its alpha and beta subunits in 40-90 days. Persistence of tumour tissue was indicated by a slight increase in levels of native hCG and the beta subunit. Determination of alpha subunit level was less useful for the detection of any relapse.