bluuebunny/crossref_metadata_2025_split huggingface dataset:
P22 Immunohematologic Monitoring and Blood Component Support in a Major ABO Mismatch Allogeneic Peripheral Blood Stem Cell Transplant Recipient
Authors: Verma A. , Sinha S. , Chaudhary R. , Ray V. 
Published: September 2006

Background  Close clinical and immunohematologic monitoring is essential in ABO mismatch peripheral blood stem cell transplantation (PBSCT) in order to anticipate potential complications and to plan appropriate blood component support throughout the disease course.Case Report  We describe here a first case of a ABO incompatible PBSCT from our center‐ a 19‐year‐old male patient of aplastic anemia (O Rh positive) who had a successful HLA matched but major ABO mismatch PBSCT from his sibling donor (A Rh positive). On admission patient's hemoglobin (Hb) was 4.7 g dL−1, platelet count (PC) 12 × 109 L−1, total leukocyte count (TLC) 2 × 109 L−1. Patient was typed as O Rh positive with negative direct coombs test (DCT) and IgM isohemagglutinin anti‐A titer‐512 and anti‐B‐512. In the pretransplant period total number of blood components (recipient type) transfused were 35, 1, and 10 random donor platelets (RDP), single donor platelets (SDP) and packed red cells (PRBC) respectively. On day of transplant patient had Hb 9 g dL−1, PC 20 × 109 L−1, TLC 2.6 × 109 L−1, IgM anti‐A‐32, anti‐B‐16 (due to immunosuppressive regimen) and negative direct and indirect coombs tests (ICT). Adequate dose of peripheral blood stem cells harvested from donor by a continuous cell separator was infused to patient as per the institute protocol. Engraftment occurred on 12th day of transplant. During these 12 days patient received a total of four PRBC (recipient type) and 8, 3 units of RDP and SDP (stem cell donor type) respectively. After the engraftment patient's laboratory parameters were Hb 8.2 g dL−1, PC 40 × 109 L−1, TLC 5 × 109 L−1. Patient received very few transfusions after the engraftment and was discharged with Hb of 9.2 g dL−1, PC 146 × 109 L−1, TLC 10.9 × 109 L−1 and with blood group still O Rh positive. Post‐transplant immunohematological workup was done on a weekly basis for first 3 weeks and then on each outpatient visit. On follow up DCT showed mixed field reaction from 1st week till 3rd week post transplant, which became negative by 9th week. ABO cell grouping changed from O to A and thus a cell‐serum discrepancy was noted by 9th week (cell group A Rh positive and serum group O) and by 16th week his blood group finally changed to A Rh positive with IgM anti‐A‐undetectable, IgM anti‐B‐512 along with negative DCT. Throughout the disease course the patient received irradiated blood to avoid transfusion‐associated graft‐versus‐host disease.Conclusion  Our patient had uneventful course with appropriate component support and immunohematologic monitoring during and after the transplant and ABO incompatible stem cell could be transplanted without negative outcome on stem cell engraftment.
P27 Gathering evidence for BCSH FMH guidelines ‐ counting controversies in FMH
Authors: White J. L. , Milkins C. E. , Rowley M. R. , Lubenko A. 
Published: September 2006

The BCSH guidelines for FMH first published in 1999 are now being updated. Evidence on which some of the recommendations are made is being re‐examined, e.g. the number of cells to be counted using the acid elution method (AE), to identify and quantify fetal bleeds. When using flow cytometry (FC) to measure FMH, the guidelines recommend that 500,000 events be collected, based on the statistics of rare event analysis where the CV% of the method decreases with the number of events collected (see Fig. 1). Extrapolating this statistical principle to acid elution (1 in 1000 ‘event’ equates to a 2.4ml bleed using Mollison's formula), where counting 6000 adult cells for FMH quantification is currently recommended, the CV% would be approximately 60 for a 4ml bleed. However this can be improved to approx. 25 if 10,000 cells are counted.

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[
CV% (y‐axis) vs number of events counted (x‐axis) for a Poisson distribution where approx. 1 in 1000 positive events is expected. ]These statistics can also be applied to screening by FMH, where identification of bleeds >4ml is essential since the most commonly used post‐natal dose of anti‐D immunoglobulin (500 i.u.) covers a 4mL bleed. When scanning 25 low power fields (lpf) using x10 objective and x10 eyepiece, a minimum of 50,000 cells are screened (based on data from Mollison and UK NEQAS). The inter‐laboratory CV% = 38 for bleeds of 2 ‐ 6ml (by AE) in UK NEQAS exercises and the CV% according to rare event statistics is approximately 20. These figures can be used to predict the range of FMH values observed at different bleed volumes (see table), using the formula SD = (CV x mean) / 100 and where the expected bleed volume is the mean.

mL FMH
Expected range of FMH (mL) at 95% confidence (=2SD)

CV% 20 (rare event)
CV% = 38 (UK NEQAS)

4
2.4–5.6
1.0–7.0

2
1.2–2.8
0.5–3.5

Theoretically, 2ml is a safer trigger for quantification and follow‐up, since with a 4ml ‘cut‐off’ there is a risk that FMH of >4ml will not be recognised on screening 25 lpf, even where proficiency is good. Also, given the predicted high CV% for quantification by AE, the guidelines will recommend referral of bleeds >2ml for FC and instigation of follow‐up, even where the standard anti‐D dose exceeds 500 i.u.
P03 The Effect of Apheresis on the Donors’ Platelets
Authors: Garner S. F. , Jones C. I. , Angenent W. , Bernard A. , Carr P. , Rankin A. , Stephens J. , Tom B. D. , Walton J. , Dudbridge F. , Ouwehand W. H. , Goodall A. H. 
Published: September 2006

Introduction  Many studies have investigated the effects of donation processes on the activation state of platelet concentrates. Apheresis platelets are often found to be the least activated products. However, little is known about the immediate or long‐term effects of frequent apheresis on donors’ platelets. This question was addressed in a cohort of donors in the BLOODOMICS research project, comprising 157 whole blood donors who had donated twice on average (range 1–4), and 349 apheresis donors with an average of 10 donations (range 1–25), in the previous year.Method  The activation status of the platelets in the donors was determined from the percentage on platelets with bound fibrinogen, by whole‐blood flow cytometry. The immediate effect of apheresis on circulating platelets was studied in 10 donors in samples taken from the arm, pre and post donation, measuring platelet‐bound fibrinogen in unstimulated blood samples and in response to in vitro stimulation with ADP (10‐7M). The long‐term effect of apheresis was assessed by measuring fibrinogen binding to platelets in unstimulated blood, and in response to ADP (10‐7M) and cross‐linked collagen related peptide (XL‐CRP, 0.1μg/mL) activation prior to donation.Results  Apheresis did not have a consistent immediate effect on resting platelet activation: pre‐donation bound fibrinogen levels ranged from 2.69–7.63% positive platelets (mean 4.41%) and post donation levels ranged from 1.46–9.66% (mean 4.71) (p=0.71). ADP induced fibrinogen binding levels ranged from 11.6–41.4% (mean 30.7%) prior to donation and decreased post donation in 8/10 cases, with values ranging from 6.74–43.6% (mean 23.45%) (p=0.024). The long‐term effect of apheresis was assessed by comparing the results of apheresis and whole blood donors. For all donors resting bound fibrinogen levels ranged from 0.51% to 6.96% positive, with no significant difference between the whole blood and apheresis donors (p=0.69). Responses to ADP did not differ between the two groups, but the apheresis donors showed a 4.01±1.8% (P=0.03) increase in fibrinogen binding in response to XL‐CRP.Conclusion  Apheresis does not lead to short‐ or long‐term platelet activation. However, prior to donation, apheresis donors show slightly higher responses to XL‐CRP than whole blood donors. This may reflect an increase in circulating younger, more active platelets.
P30 Removal of the Endogenous TSE Infectivity From Blood Using PRDT TSE Affinity Resin And Integration Of The Resin Into The Macopharma P‐CAPT™ Filter
Authors: Rohwer R. G. , Gregori L. , Gurgel P. V. , Lathrop J. T. , Edwardson P. , Lambert B.C. , Carbonell R.G. , Burton S. J. , Hammond D. J. 
Published: September 2006

Background  To date, there have been three transfusion transmitted cases of vCJD among 19 recipients that are known to have received blood from persons that later died of vCJD and lived long enough themselves to have developed detectable disease. This is a transmission rate of ∼15%. A survey of surgically removed tissues concluded that the number of incubating cases of vCJD in the UK was at least 4000. Seven percent of this group would on average be blood donors.Methods  Pathogen Removal and Diagnostics Technologies (PRDT) screened millions of compounds to obtain high affinity ligands to prion protein. A group of ligands were challenged with scrapie infected hamster brain homogenate mixed with a unit of leucoreduced human RBC. Infectivity of the challenge and the unbound were compared. To test for removal of the endogenous blood TSE infectivity, blood from scrapie infected hamsters was collected, leucoreduced and passed through the resin. Whole blood, leucoreduced blood and resin flow‐through samples were titered by the limiting dilution method. A total of 5 ml of each sample was inoculated in 50 μl aliquots into 100 hamsters. The experiment was terminated at 540 days and the brain of every animal was checked for evidence of prion amyloid.Results  The best resins reduced the titer of the brain‐derived spike into RBC of 107 ID50/ml by over 4 log10ID50, far exceeding the capacity needed to capture endogenous RBC infectivity. To address the uncertainty about the relevance of brain‐derived spikes, the lead resin was also assessed for its ability to remove the infectivity endogenously present in blood. There were no infections among the 100 hamsters inoculated with the resin treated blood. The resin removed > 1.2 log10ID of relevant TSE blood infectivity. The resin has been incorporated by MacoPharma into the P‐CAPTTM filter, a stand alone device for removing TSE infectivity from leucoreduced red blood cells.Conclusions  PRDT's lead resin adsorbs high concentrations (107 ID50/ml) of brain derived TSE infectivity with high efficiency (4 log10ID50) even in the presence of RBC, and removes the low concentrations (10 ID/ml) of endogenous infectivity that is present in leucoreduced whole blood to the limit of detection (>1.2 log10ID). Implemented as the Macopharma P‐CAPTTM filter it should significantly reduce the risk of transmission of vCJD by blood and blood products.
P36 A Novel RhD Variant
Authors: Tilley L. , Bullock T. , Mason R. , Poole J. , Daniels G. 
Published: September 2006

Introduction  Variants of the RhD antigen are often categorised into weak D and partial D phenotypes, although the distinction between the two groups is unclear. One frequently used definition is the ability to make alloanti‐D in partial D, but not in weak D phenotypes, however some phenotypes characterised as weak D are now known to be associated with alloanti‐D production. On a molecular level, weak D phenotypes usually possess RHD mutations encoding amino acid subsitutions within the cytoplasmic or membrane spanning regions of RhD, whilst partial D phenotypes result from changes in the extracellular loops. Thus, weak D red cells could be regarded as having all epitopes of D, but expressed weakly, whilst partial D cells lack some D epitopes. We investigated the red cells of a black pregnant woman because of aberrant reactions obtained in routine D typing, in order to decide if prophylactic anti‐D should be given.Methods  Routine anti‐D sera and a reagent anti‐D panel were tested serologically by their recommended techniques. Genomic DNA was extracted from whole blood and subjected to sequencing of exons 1 to 9 of the RHD gene.Results and treatment  The Rh phenotype of the patient was C‐ c+, E‐, e+, V+, VS+. Anti‐D reagents gave variable reactions and a novel pattern of reactions was seen with an anti‐D panel. Sequencing analysis revealed a single G>A mutation at position 490, within exon 4 of the RHD gene, resulting in an Aspartate to Asparagine change at position 164 of the RhD protein (D164N). This appears to be a novel RHD mutation. The patient received prophylactic anti‐D antenatally and at delivery. The maternal antibody screen and Kliehauer screen were negative and she delivered a full term D+ DAT negative infant.Conclusions  A novel D variant has been identified with an D164N mutation, predicted to lie within the third extracellular loop of the RhD protein. We propose to call this D variant DDN. The molecular basis of this novel phenotype suggests that this could be categorised as a partial D , as the mutation results in a loss of D epitopes. This would be expected to result in the ability to produce alloanti‐D although alloanti‐D production was not reported in this case.
P37 Molecular and Serologic Characterization of RhD Status
Authors: Catherwood M. , Curran M. C. , Wylie J. , Daniels G. , Martin P. , Morris K. 
Published: September 2006

Introduction  Developments in technology and knowledge have allowed the elucidation of many of the blood group systems at the molecular level, much of this work is still mainly confined to larger centres and reference facilities. The D antigen is highly complex and of enormous clinical significance causing both haemolytic disease of the newborn and haemolytic transfusion reactions. D typing is routinely performed using serological techniques but examples of weakened expression can be difficult to resolve. Molecular techniques have permitted a greater understanding of these problems and can also be applied to predicting the D type of the foetus using a maternal sample which has obvious benefits to mother and baby.Aims  The development of a molecular method for RhD typing and compare this to serological typing.Study Design and Methods  Multiplex PCR was used to analyze RhD status in DNA from 127 randomly selected blood donors from the Northern Ireland Blood Transfusion Service. This method was compared to the serological assessment of RhD typing using the Ortho AutoVueTM System.Results  Serological results showed that 93 samples (73%) were RhD+ and 34 (27%) were RhD− which is expected for a Caucasian population. 75 samples (50%) were RhC+ and 52 (41%) were RhC−. 111 samples (87%) were Rhc+ and 16 (13%) were Rhc−. Positive and negative molecular results were compared to the corresponding serological results. A concordance rate of 78.7% existed for the comparison of both methods for RhD status. 65.4% of samples were concordant for both methods when assessing RhC status. The concordance rate was 73.2% for Rhc.Conclusion  We describe a multiplex PCR method for the assessment of RhD status and compare this to results obtained using standard serology. Further development of this molecular method is required to obtain a higher concordance rate. Molecular typing has potential future benefits, along with serological methods, in adding to our understanding of the Rh blood system and identifying RhD variants.
P42 A Simple Test for the Differential of Maternal and Fetal Bloods
Authors: Thomas E. 
Published: September 2006

The structure of fetal haemoglobin (HbF) differs to that of adult haemoglobin (HbA) and protects it from denaturing when subjected to a pH change. With this knowledge, Apt et al1 adjusted the pH of the meconium, in melena neonatorium, by the addition of NaOH, and noted that a change of colour to green from pink occurred only if HbA was present. By utilising the same principle, the process can be modified to confirm the source of blood samples requested for the investigation of a possible fetomaternal haemorrhage, post delivery, for the prevention of Rhesus haemolytic disease of the newborn. 10 adult male bloods and six cord samples were used as the test material for the investigation. The rate at which the colour changed from pink to green of the test material was measured over a range of variables. The haematicrit was artificially adjusted between 30% and packed cells, with a permutation of volumes from 10 to 500 μL added to NaOH at concentrations between 0.05 M and 3 M.Results The optimum NaOH concentration was 0.1 M, equating to a pH 13.0 The haematicrit showed no influence on the reaction, but packed cells were chosen as the easiest to reproduce for consistency. The rate of colour change from pink to green took a maximum 1 min. Contamination of maternal with fetal bloods, and visa versa, at a low ratio is undetectable and at a high ratio gives a visual chimera of colour and intact erythrocytes.Method Using the above data a simple routine laboratory method of adding 100 μL of packed cells to 0.5 mL of 0.1 M NaOH, rapid mixing, and observing for a colour change between 1 min and a maximum of 10 mins. Pink to green show the blood to maternal i.e. HbA, but remaining pink indicative of HbF. The simultaneous testing of blood from and adult male and known cord blood, provides a positive and negative control respectively. This simple test has been extensively used to verify which are maternal and cord samples, when both express the same ABO and Rh(D) blood groups.1. Apt L, Downey WS Jr. (1953) Melena neonatorium: the swallowed‐blood syndrome. AMA Am J Dis Child.; 86(5): 639; discussion, 639–40.
P51 Variation in the Ex Vivo Response to LPS Stimulation in Healthy Volunteers: Identification of Extremes of Monocyte Function
Authors: Farrugia R. , Garner S. F. , Gray E. , Watkins N. A. , Goodall A. H. , Ouwehand W. H. 
Published: September 2006

Introduction  Numerous epidemiological and clinical studies have established a firm link between infection, the inflammatory response and cardiovascular disease. Furthermore, it is known that individuals differ in their responses to immune stimuli such as Lipopolysaccharide (LPS) that stimulates monocytes through Toll‐like receptor 4 (TLR4). In this study we set out to identify healthy individuals with extremes of monocyte response to LPS stimulation. Once identified these individuals will be studied further to identify genes responsible for the observed differences.Methods  Small volumes of whole blood from 208 healthy blood donors were incubated with 0, 1 or 5 ng mL−1E. coli LPS for 4 hr at 37°C. Cell free plasma was collected both pre‐ and post‐incubation and the production of the pro‐inflammatory cytokine IL‐6 measured by ELISA. In addition, TaqMan genotyping was performed on two TLR4 SNPs, (rs4986790 and rs4986791, encoding D299G and T399I respectively) to determine whether these SNPs correlated with the observed functional responses.Results  The range of IL‐6 produced was 0–26 ng mL−1 in response to 1 ng mL−1 LPS, and 2–56 ng mL−1 in response to 5 ng mL−1 LPS. Frequency distributions of IL‐6 levels show one‐tailed distributions for both stimuli with more outlying high extremes than low extremes. Quality control assays showed that both inter‐ and intra‐assay variability had a CV <10% and that the response correlates weakly with monocyte counts. Furthermore, repeat testing on 20 individuals demonstrated that the response to LPS is reproducible over time. No correlation was observed between TLR4 genotype and IL‐6 production following LPS stimulation.Conclusions  Our stimulation results demonstrate that there is substantial inter‐individual variation in response to both 1 ng mL−1 and 5 ng mL−1 doses of LPS. We now have identified a number of individuals with either a high or low response to LPS stimulation. These individuals will be recalled for further functional testing and gene expression profiling to identify differences between the two extremes of monocyte function.
P53 Electron Microscopical Localisation of CD61 (Beta3 Integrin) on Placental Syncytiotrophoblast Microvilli
Authors: Kumpel B. , Jackson D. , Sibley K. , White G. 
Published: September 2006

Introduction  Neonatal alloimmune thrombocytopenia (NAIT) due to maternal antibodies to the human platelet antigen (HPA)‐1a can occur in first pregnancies. The HPA‐1 polymorphism is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin). It is hypothesised that besides fetal platelets, an additional source of fetal CD61 could cause alloimmunisation early in pregnancy. CD61 was earlier identified on placental villous trophoblasts by immunocytochemistry.Methods  Whole‐mount immuno‐electron microscopy was used to study the ultrastructural localisation of CD61 on placental tissue. Fragments of first trimester (n = 5, 8–12 weeks gestational age) and term (n = 1) placental chorionic villi were incubated with anti‐CD61 or IgG1 isotype control, washed, incubated with 18 nm colloidal gold‐conjugated anti‐mouse IgG, washed, fixed in phosphate‐buffered glutaraldehyde and processed through to embedding in epoxy resin. 100 nm sections were prepared from the EM blocks; these were stained with uranyl acetate and lead citrate, and examined with a Phillips CM10 electron microscope. Positive control was term tissue stained with anti‐placental alkaline phosphatase (PLAP).Results  Microvilli were observed on the apical membrane of some but not all of the villous syncytiotrophoblast (STB) surfaces. Formation of this brush border occurs during the first trimester to increase the surface area of these cells for optimal transfer of nutrients and gases from the adjacent maternal blood to the fetus. All specimens had some microvilli. After staining with anti‐CD61, gold particles were present on and between some microvilli of all specimens, either singly or in small groups. Stronger staining with more frequent occurrence of groups of gold particles was evident on microvilli of the term tissue incubated with anti‐PLAP. No gold particles were seen on the isotype control specimens.Conclusion  From early on in pregnancy, CD61 is present on the surface of fetal STB in contact with maternal blood. This protein carrying the HPA‐1 polymorphism might be the cause of the initial immunising event that leads to NAIT.