bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling the growth of Yersinia enterocolitica in donated blood
Authors: Gibb A.P. , Martin K.M. , Davidson G.A. , Walker B. , Murphy W.G.
Published: March 2003

Background: Sepsis and death subsequent to the transfusion of blood containing Yersinia enterocolitica is an increasing problem. The organisms probably originate from bacteremia in the donor and can subsequently multiply at low temperature.

Study Design and Methods: Reported here are experiments with a strain of Y. enterocolitica associated with a case of transfusion‐associated bacteremia.

Results: It was found that the rapid early killing of Y. enterocolitica injected into donated blood does not require viable phagocytes and can be explained by complement‐mediated killing. Complement resistance in Y. enterocolitica is known to be plasmid‐coded. It is expressed at 37°C, but not at 20°C, and is favored by calcium‐deficient culture media. Y. enterocolitica organisms induced to express complement resistance were still killed in donated blood, though the initial rate was slower. Such organisms multiplied in plasma at 37°C, but were killed after 6 hours of incubation at 20°C, presumably because complement resistance genes are switched off at this temperature.

Conclusion: This experiment is thought to reflect the natural history of Y. enterocolitica contamination of blood, in which complement‐resistant organisms in the donor blood encounter lower temperatures after donation. These observations suggest that the practice of plasma depletion may have contributed to the increased incidence of mortality due to Y. enterocolitica contamination of donated blood.
Fatal outcome in a patient with autoimmune hemolytic anemia associated with an IgM bithermic anti‐ITP
Authors: Ramos R.R. , Curtis B.R. , Eby C.S. , Ratkin G.A. , Chaplin H.
Published: March 2003

BACKGROUND: Several cold autoantibodies (usually IgG) with IT specificity have been reported previously, as have autoantibodies with joint I and P blood group specificities (IP1, ITP1, iP1, IP). A fatal outcome associated with an IgM cold autoantibody of ITP specificity is reported. CASE REPORT: A 54‐year‐old man suffered from progressively severe cold autoimmune hemolytic anemia for 9 months. Hemoglobin concentration ranged from 6 to 7 g per dL (60–70 g/L) and reticulocytes from 3 to 5 percent (0.030‐0.050). The direct antiglobulin test was weakly positive for IgM and strongly positive for C3d. The serum contained a cold agglutinin that reacted strongest with cord i red cells (RBCs) > adult I RBCs > adult i RBCs, which is consistent with IT specificity. The Donath‐Landsteiner test was positive; the reaction was neutralized by globoside. The serum reacted weakly or was negative with RBCs from five group p blood donors, which suggests anti‐ITP specificity. Dithiothreitol treatment of the serum abolished the cold agglutinin reactivity, which suggests that the anti‐IT was IgM. The patient received > 40 RBC transfusions and failed to respond to oral steroids, oral cytoxan, high‐dose pulse intravenous steroids, and plasma exchange at room temperature and at 35 degrees C. He died of sepsis following an unsuccessful trial of chlorambucil. Autopsy revealed unsuspected disseminated non‐Hodgkin's lymphoma.

CONCLUSION: Serologic studies are consistent with our patient's having a single IgM cold autoantibody with IT and P specificities (anti‐ITP) and requiring both specificities on the same RBC to permit maximal antibody expression.
FPTT is a low‐incidence Rh antigen associated with a “new” partial Rh D phenotype, DFR
Authors: Lomas C. , Grassmann W. , Ford D. , Watt J. , Gooch A. , Jones J. , Beolet M. , Stern D. , Wallace M. , Tippett P.
Published: March 2003

BACKGROUND: Several Rh D phenotypes with partial D antigens are recognized. Some partial D antigens are associated with low‐incidence Rh antigens. New partial D antigens are revealed by an atypical pattern of reactions with anti‐D.

STUDY DESIGN AND METHODS: The reactions of D variant cells with panels of monoclonal anti‐D and with antibodies to low‐incidence antigens were compared to those of known D categories to identify a new Rh D phenotype. The inheritance of partial D antigens was studied by Rh phenotyping of the families of the probands. Standard serologic methods were used and family data were analyzed.

RESULTS: A new Rh D phenotype, to be called DFR, was identified in 17 probands, two of whom had made anti‐D. The partial D antigen carries epD3, epD4, and epD9 and lacks epD8. The presence of other D epitopes is ambiguous; different answers were obtained for the same sample with different monoclonal anti‐D of the same apparent epitope specificity. The immunoglobulin class of the anti‐D was important: IgG were more successful than IgM monoclonal anti‐D in detecting the partial D of DFR. Family studies showed that DFR traveled with Ce more frequently than with cE. The low‐incidence antigen FPTT (International Society of Blood Transfusion number 700048) was found on all DFR samples. Family studies demonstrated that FPTT is, as suspected, part of the complex Rh system.

CONCLUSION: The partial D of the Rh D phenotype, DFR, is recognized by its pattern of reactions with monoclonal anti‐D and its association with the low‐incidence antigen FPTT, FPTT has now been numbered Rh50.
Anti‐hepatitis C virus (anti‐HCV) seroconversion in patients undergoing hemodialysis: comparison of second‐ and third‐generation anti‐HCV assays
Authors: COUROUCÉ A.‐M. , MARREC N. LE , GIRAULT A. , DUCAMP S. , SIMON N.
Published: March 2003

BACKGROUND: The results obtained in sequential specimens from recently infected subjects generally provide the best means of comparing the sensitivity of assays.

STUDY DESIGN AND METHODS: The sensitivity of second‐ and third‐generation assays for antibody to hepatitis C virus (HCV) was compared on sequential specimens, generally collected at monthly intervals from 45 patients undergoing hemodialysis who seroconverted for HCV between 1980 and 1990.

RESULTS: Fifteen patients (33%) were positive earlier in the third‐generation enzyme‐linked immunosorbent assay (ELISA), with a mean difference of 17 days (range, 7–30) between the last negative and the first positive specimens. At the first rise in alanine aminotransferase, and at its peak, 63 and 91 percent of the patients, respectively, were anti‐HCV positive in the third‐generation ELISA. Third‐generation recombinant immunoblot assay (RIBA) reacted at the same time as third‐generation ELISA. Of the first specimens that were positive in second‐generation ELISA, 44 percent reacted and 56 percent were indeterminate in third‐generation RIBA, while 10 percent reacted, 72 percent were indeterminate, and 18 percent did not react in second‐generation RIBA. From the beginning to the end of the follow‐up, antibody to c33c was the most prevalent, followed in descending order by antibody to c22‐3, antibody to c100‐3, and antibody to NS5: 56, 54, 26, and 18 percent, respectively, at time 0, and 100, 86, 83, and 31 percent, respectively, 12 months later.

CONCLUSION: Third‐generation assays (both ELISA and RIBA) were more sensitive than second‐generation assays in the diagnosis of HCV infection, in that positive results were obtained earlier and a higher proportion of specimens were confirmed positive in RIBA testing
Hemolytic disease of the newborn due to the Scianna antibody, anti‐Sc2
Authors: DeMarco Marianne , Uhl Lynne , Fields Lore , Pacini Donna , Gorlin Jed B. , Kruskall Margot S.
Published: March 2003

BACKGROUND: Alloantibodies to the low‐frequency antigen Scianna 2 (Sc2) are uncommon and not previously reported to cause immune hemolysis. CASE REPORT: A group B, Rh‐negative infant born to a group B, Rh‐ positive mother had a 2+ direct antiglobulin test, as well as modest hyperbilirubinemia and a hematocrit of 45 percent. Ongoing immune hemolysis led to a hematocrit of 17.3 percent on Day 20 of life, and the infant required hospitalization and red cell transfusions. The routine maternal antibody screen was negative, but anti‐Sc2 was detected during work‐up for a low‐frequency red cell antigen, and the father's red cells typed as Sc:1,2. CONCLUSION: Anti‐Sc2 can cause clinically significant hemolytic disease of the newborn. Although the antibody is uncommon, its frequency and hemolytic potential may be underappreciated, in part because investigations often are not carried out in the infant whose red cells are ABO‐incompatible with maternal blood.
Determination of ABO glycosyltransferase genotypes by use of polymerase chain reaction and restriction enzymes
Authors: Stroncek D. F. , Konz R. , Clay M. E. , Houchins J. P. , McCullough J.
Published: March 2003

BACKGROUND: The molecular basis of red cell ABO group antigens has been determined. The genes encoding the group A and B glycosyltransferases and a nonfunctional group O transferase have been cloned and sequenced. All three genes were similar. When compared to the nucleotide sequence of the A gene, the O gene has a one‐base deletion that leads to a frame shift and results in a nonfunctional protein. The B gene differs from the A gene at seven nucleotides. STUDY DESIGN AND METHODS: Techniques using polymerase chain reaction and restriction enzymes to determine ABO transferase genotypes from white cell DNA were modified. Nucleotide sequence differences within the genes were analyzed by the application of selected restriction enzymes. Restriction enzymes Asp718 and BstEII were used to analyze the genes at nucleotide 258, and BssHII and Kas I were used to analyze the genes at nucleotide 523. ABO red cell phenotypes were compared in 60 unrelated individuals with ABO transferase genotypes. The ABO phenotypes and genotypes of individuals from two different families were also analyzed to determine if this method could distinguish individuals who were homozygous for A or B transferase genes from those who were heterozygous. RESULTS: The phenotypes and genotypes were consistent for all unrelated individuals, and within the families, heterozygous individuals could be distinguished from homozygous individuals. Nevertheless, two individuals from one family were found to have a group A red cell phenotype, but when the transferase genes were analyzed at nucleotide 523 with enzymes BssHII and Kas I, both A and B transferase genes were detected. Further analysis of the transferase genes at nucleotide 700 by using restriction enzymes Alu I and Hpa II and those at nucleotide 793 by using enzyme BstNI found that both transferase genes in the two individuals were similar to the A transferase gene. CONCLUSION: An A allele of the group A glycosyltransferase was detected that had the same sequence as the B gene at nucleotide 523 but was identical to the A gene at positions 700 and 793. The identification of this variant gene makes genotyping at nucleotide 523 unreliable. However, analysis of the genes at other sites of nucleotide variation may accurately identify phenotypes.
Umbilical cord blood transplantation
Authors: Wagner John E.
Published: March 2003

Interest in umbilical cord blood as an alternative source of hematopoietic stem cells is growing rapidly. Umbilical cord blood offers the clinician a source of hematopoietic stem cells that are readily available and rarely contaminated by latent viruses. Moreover, the collection of umbilical cord blood poses no risk to the donor; there is no need for general anesthesia or blood replacement, and the procedure causes no discomfort. Whether cord blood lymphocytes are as likely to cause GVHD as lymphocytes from older individuals is unknown. Current clinical experience would suggest that the incidence may be low. Few of the patients who have thus far received umbilical cord blood, including recipients of HLA‐disparate grafts, have developed clinically significant GVHD. These results and associated laboratory findings pose intriguing possibilities for the future of umbilical cord blood stem cells in the setting of unrelated‐donor transplantation. With the marked incidence of Grade 2 to 4 acute GVHD that is currently observed after unrelated‐donor bone marrow transplantation, a reduction in incidence or severity would be a major advance in this field. In the setting of autologous marrow transplantation, there are other intriguing possibilities; for example, cord blood may be an optimal source of pluripotential stem cells for use in gene therapy. As detailed in Broxmeyer's review, the large‐scale collection and storage of cord blood stem cells is no longer just a concept; it has become a reality. Pilot programs for the banking of unrelated‐donor umbilical cord blood have already begun in the United States and Europe.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypotensive reactions to white cell‐reduced plasma in a patient undergoing angiotensin‐converting enzyme inhibitor therapy
Authors: Fried M.R. , Eastlund T. , Christie B. , Mullin G.T. , Key N.S.
Published: March 2003

Background: Hypotensive reactions to platelet transfusions performed with white cell (WBC)‐reduction filters with negatively charged surfaces have been reported recently in patients taking angiotensin‐converting enzyme (ACE) inhibitors. Experimental studies have shown that the filter material can activate bradykinin, which may cause symptoms in patients with reduced bradykinin catabolism. Symptomatic adverse reactions after the administration of fresh‐frozen plasma (FFP) through a WBC‐reduction filter have not been reported in a patient on ACE Inhibitor medication.

Case Report: A 58‐year‐old man with congenital coagulation factor V deficiency and hypertension treated with an ACE inhibitor was admitted for rehabilitation after orthopedic surgery. On 3 consecutive days, he received FFP through a WBC‐reduction filter; within minutes of the beginning of each infusion, he experienced a drop in blood pressure, facial erythema, abdominal pain, and anxiety. When the infusions were stopped, symptoms quickly abated without treatment. Multiple prior transfusions of unfiltered FFP and FFP filtered through a WBC‐reduction filter made by a different manufacturer, as well as subsequent transfusions of unfiltered FFP, had not produced such reactions.

Conclusion: Facial flushing, hypotension, and abdominal pain after FFP administration in a patient on ACE inhibitor medication appeared to be associated with a specific type of WBC‐reduction filter. This association and other reported studies suggest that special caution is warranted when patients who are treated with ACE inhibitors receive blood components administered through WBC‐reduction filters capable of generating bradykinin.
Direct extraction of A and B blood group antigens from human red cells by liposomes
Authors: Suzuki K. , Okumura Y. , Sato T. , Yasuda T. , Oki A. , Oki M. , Sunamoto J.
Published: March 2003

BACKGROUND: Some of the major blood group antigens are on lipids and proteins of the red cell membrane. Incubation of intact red cells with liposomes containing specially designed artificial lipids has been shown to result in the extraction of membrane proteins by the liposomes. The extraction of blood group structures and the retention of their antigenicity have not been reported. STUDY DESIGN AND METHODS: After the incubation of red cells with liposomes, the extraction of the antigens from human red cells by liposomes was examined by evaluation of the agglutination of the liposomes by respective antisera. RESULTS: Agglutination specific to the A and B blood group antigens was seen, which indicated that the antigenicity of the blood group antigens was retained even after the extraction by the liposomes. The presence of an artificial boundary lipid, 1,2‐dimyristamido‐1,2‐ deoxyphosphatidylcholine, in the liposome was crucial to the efficient extraction of the A and B antigens. On the other hand, the extraction of D, M, N, and P1 was not always detectable by agglutination. CONCLUSION: The A and B blood group antigens were directly extracted from red cells by liposomes without loss of antigenicity.
Icteric plasma suggests Gilbert's syndrome in the blood donor
Authors: Naiman J. L. , Sugasawara E. J. , Benkosky S. L. , Mailhot E. A.
Published: March 2003

BACKGROUND: In a recent quality assurance audit of component returns over a 6‐month period, 9 of 81 returns were due to icteric plasma. With the sensitive, new methods used to screen donors for anemia and hepatitis, it seemed likely that the icteric discoloration reflected benign unconjugated hyperbilirubinemia (Gilbert's syndrome) in the donor, rather than liver disease or hemolysis. The donors were recalled for repeat blood study to resolve this question. STUDY DESIGN AND METHODS: Seven of the nine donors could be reached, and they submitted blood samples for measurement of serum levels of conjugated (direct‐ reacting) and total bilirubin and for complete blood and reticulocyte counts. RESULTS: All seven donors had mild unconjugated hyperbilirubinemia, with total bilirubin levels ranging from 1.3 to 2.8 mg per dL. None showed evidence of overt hemolysis. CONCLUSION: All seven donors of the components with icteric plasma have Gilbert's syndrome, a benign genetic anomaly occurring in approximately 3 to 5 percent of the general population. With the sensitive screening tests for viral hepatitis used today, the presence of icteric plasma in a component suggests that the donor has Gilbert's syndrome. Policies about the acceptability of icteric components from blood donors merit reassessment.
Blood transfusion and infection after colorectal cancer surgery
Authors: Vamvakas E. C. , Carven J. H. , Hibberd P. L.
Published: March 2003

BACKGROUND: Many observational studies have described an association between perioperative transfusion and postoperative infection. Detection of such a relationship may depend on which variables are considered as potential confounders of the association under study. However, most reports have not considered risk factors for postoperative infection at specific sites as possible explanations for the observed relationship. STUDY DESIGN AND METHODS: The records of 492 patients undergoing elective colorectal cancer resection at the Massachusetts General Hospital between January 1992 and December 1994 were reviewed. The probability of infection in association with transfusion was calculated with and without adjustment for the effects of chronic systemic illness, number of days with urinary catheter, endotracheal intubation, impaired consciousness, and specific risk factors for wound infection. Postoperative infection at any site and infections at specific sites were analyzed as separate outcomes. RESULTS: After adjustment for the effects of the variables listed above, allogeneic transfusion was not associated with postoperative infection at any site (p = 0.407). Only a specific association of transfusion with wound infection could be detected. However, in an analysis that adjusted for the effects of only the 18 confounders considered by previous authors, transfusion was the most significant predictor of infection. In that analysis, the risk of postoperative infection increased by 14 percent per unit of red cells transfused (p < 0.001). CONCLUSION: The overall adverse relationship between transfusion and infection reported by previous observational studies may have been due to an incomplete consideration of the variables that confound that association. This finding may help explain the disagreement between the conclusions of recent large, randomized, controlled trials (which failed to detect a deleterious transfusion effect) and the earlier observational studies.
Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations
Authors: Padley D. J. , Koontz F. P. , Trigg M. E. , Gingrich R. D. , Strauss R. G.
Published: March 2003

Background: The performance of cultures to assess possible bacterial contamination of bone marrow and peripheral blood progenitor cell preparations is required by the standards of the American Association of Blood Banks.

Study Design and Methods: Consecutive (n = 893) bone marrow and peripheral blood progenitor cell preparations were cultured for assessment of possible contamination by microorganisms.

Results: Consecutive bone marrow and peripheral blood progenitor cell preparations (n = 893) were cultured; the overall positive rate detected was 2.5 percent (22/893). The isolates predominantly were skin contaminants (gram‐positive cocci) and so‐called water‐borne organisms (gram‐negative rods). The 6.0‐percent rate of positivity in 317 bone marrow preparations was higher than the 0.5‐percent rate in 576 peripheral blood progenitor cell preparations (p < 10(‐6)). Culture‐ positive preparations were transfused to 16 patients at this institution; however, none of these transfusions led to documented sepsis with the contaminating organism.

Conclusion: The culture method described here complies with the standards of the American Association of Blood Banks. Contamination can be detected in both bone marrow and peripheral blood progenitor cell preparations. When contaminated preparations are transfused, there are few complications that can be attributed to the contamination.
Exchange transfusion the hard way: massive hemolysis following transplantation of bone marrow with minor ABO incompatibility
Authors: Greeno E. W. , Perry E. H. , Ilstrup S. J. , Weisdorf D. J.
Published: March 2003

Background: Bone marrow transplantation using donors with minor ABO incompatibility may result in the rapid production of donor‐derived red cell isohemagglutinins, causing hemolysis of recipient red cells.

Case Report: The transplant of sibling‐donor marrow with minor ABO incompatibility (group O donor marrow to group A recipient), using FK‐ 506 as an immunosuppressant to prevent graft‐versus‐host disease, is reported. Following early myeloid engraftment, the recipient developed hemolysis of her entire A red cell population between Day 8 and Day 11. This brisk hemolytic anemia was due to rapid donor lymphoid engraftment that resulted in the explosive production of donor‐derived anti‐A.

Conclusion: Patients undergoing the transplantation of marrow from donors with minor ABO incompatibility in which the donor cells can produce isohemagglutinins against the recipient's red cells must be kept under vigilant observation for the possible development of severe hemolysis, particularly in the setting of profound T‐cell suppression without B‐cell suppression.
Differentiation between human red cells of Pk and p blood types using Pseudomonas aeruginosa PA‐I lectin
Authors: Sudakevitz D. , Levene C. , Sela R. , Gilboa‐Garber N.
Published: March 2003

BACKGROUND: The red cells of almost all human beings bear P antigen. Type P1 cells (around 75% of the population) contain P1 antigen in addition to P, and type P2 cells (around 25% of the population) contain only P. The red cells of only a few individuals are devoid of P: these cells may be of either Pk‐positive (P1k and P2k[Pk]) or p type (the latter lack all the above‐described antigens of the P system). Differentiation between them is of clinical importance, but there is a shortage of specific reagents. This article offers reliable means for differentiation.

STUDY DESIGN and METHODS: Agglutination of washed, papain‐treated red cells of all the P types by PA‐1 and soybean lectins and adsorption of the lectins onto the red cells were examined.

RESULTS: PA‐1 strongly agglutinated papain‐treated red cells. Examination of its interactions with red cells having different P system antigens revealed that Pk (both P1k and P2k) red cells of O, A, and B blood types were agglutinated significantly faster than p red cells. The agglutination intensities of Pk red cells of types O and A (most people) was considerably stronger than those of p red cells. P1 and P2 type red cell agglutination was intermediate (P1>P2). Adsorption tests with all the red cells, exhibited the same order of PA‐1 affinities for the P system blood types: Pk>P1,>p. The soybean lectin exhibited opposite behavior (p>Pl).

CONCLUSION: The galactose‐binding lectins PA‐1 and soybean may facilitate the determination of Pk and p red cells.
Incidence of the Nak(a)‐negative platelet phenotype in African Americans is similar to that of Asians
Authors: Curtis B.R. , Aster R.H.
Published: March 2003

Background: About 5 to 10 percent of Asians have platelets that lack the major membrane glycoprotein (GP) IV (CD36, GPIIIb) that carries the isoantigen Naka. The GPIV‐negative platelet phenotype is extremely rare among whites, but its frequency in persons of African ancestry has not yet been determined. Isoimmunization against GPIV can occur in GPIV‐ negative persons and can lead to platelet transfusion refractoriness. Therefore, the expression of GPIV on platelets from unrelated African Americans was studied.

Study Design and Methods: Platelets were obtained from 250 African American and 280 white blood donors. Flow cytometry was used to determine the ability of these platelets to bind a monoclonal antibody that reacted with GPIV. Platelets that failed to react with this probe were tested with other GPIV‐specific monoclonal antibodies and with anti‐Naka, an isoantibody that recognizes an epitope on GPIV.

Results: Platelets from 6 of the 250 African American donors (2.4%) lacked GPIV and failed to bind anti‐Naka, whereas platelets from all of the white donors were GPIV positive (p>0.05). No platelet‐reactive antibodies were identified in the serum of the GPIV‐ negative donors.

Conclusion: The frequency of the GPIV‐negative platelet phenotype in African Americans is comparable to that in Asians and much greater than that in whites. Studies are needed to determine the frequency with which African Americans become isoimmunized to GPIV by transfusions and the possible contribution of this isoimmunization to platelet transfusion refractoriness in this population.
Neutralization of Knops system antibodies using soluble complement receptor 1
Authors: Moulds J. M. , Rowe K. E.
Published: March 2003

BACKGROUND: Antibodies of the Knops system have been referred to as nonneutralizable because they cannot be inhibited with serum, saliva, or urine. Because the Knops system antigens have been located on complement receptor 1 (CR1), the question of whether the antibodies could be neutralized with soluble CR1 (sCR1) produced by recombinant DNA techniques was studied. STUDY DESIGN AND METHODS: First, radiolabeled immunoprecipitation techniques were used to test sCR1 for the expression of the high‐incidence Knops system antigens. Then, a total of 45 antibodies were neutralized with sCR1, including the following: one each of anti‐Cr(a), ‐Dr(a), ‐Do(b), ‐Hy, ‐Ge, ‐Jr(a), ‐ Sc1, ‐Jk(a), ‐Cs(a), and ‐Kp(b); two each of anti‐Lu(b), ‐Yt(a), and ‐ JMH; three each of anti‐McC(a), ‐Rg, and ‐Sl(a); and four each of anti‐ Ch, ‐Kn(a), ‐Yk(a), ‐Kn/McC. In addition, two examples of anti‐Kn(a) + K, one example of anti‐Sl(a) + K + Fy(a), and one example of anti‐Yk(a) + E were tested. The sCR1 was added to each test serum and 6‐percent albumin was added to the control; this was followed by neutralization incubation for 5 minutes at 25 degrees C. The antibody samples were then tested by a low‐ionic‐strength solution, anti‐human globulin technique. RESULTS: The sCR1 expressed Kn(a), McC(a), Sl,a and Yk(a). All Knops system antibodies (n = 22) were neutralized by the sCR1, but none of the other 23 alloantibodies decreased in reactivity. The samples containing antibodies of two specificities showed inhibition of the Knops system antibody but not of the second antibody. CONCLUSION: This neutralization method, in which recombinant protein is used, provides an expedient and definitive method of identifying Knops system antibodies.
Concentration of citrate anticoagulant in peripheral blood progenitor cell collections
Authors: Burger S.R. , Fautsch S.K. , Stroncek D.F. , McCullough J.
Published: March 2003

Background: Peripheral blood progenitor cell (PBPC) collection by hemapheresis has become widely used in recent years. For anticoagulation during cytapheresis, citrate solutions, commonly ACD‐A, are used, at a recommended anticoagulant‐to‐whole blood ratio of 1:11 to 1:12. Although the apheresis procedure is generally well tolerated, the most common patient complaints are attributable to transient hypocalcemia, which is a side effect of the citrate anticoagulant. Patients experiencing discomfort due to hypocalcemia are sometimes managed by a decrease in the flow rate of the anticoagulant.

Case Reports: Two cases are reported in which seemingly minor reductions in the anticoagulant: whole blood ratio appeared to cause gelation of freezing solution prepared from plasma that was collected in addition to PBPCs for use in the cryopreservation of cells. In both cases, the final ratio of citrate anticoagulant to whole blood was less than 1:12. Gelation occurred when plasma collected under these conditions was used to prepare freezing solution.

Conclusion: The addition of heparin to this plasma, or the addition of ACD‐A to correct the anticoagulant:whole blood ratio, prevented the gelation of freezing solution, which suggests that coagulation activation in the autologous plasma specimen was implicated in the subsequent gelation. During cytapheresis for PBPC collection, citrate‐containing anticoagulants should be used at the recommended ratio of 1:12, or with more anticoagulant than usual. Tolerance for a reduced concentration of citrate may be more limited than is generally appreciated. When plasma is collected in addition to PBPCs, heparin should be added to both the cells and the plasma as soon as possible after the collection. Patients undergoing PBPC and stem cell collection should be given supplemental calcium, rather than less anticoagulant, to alleviate the discomfort associated with citrate.
Comparison of COBE white cell‐reduction and standard plateletpheresis protocols in the same donors
Authors: Zingsem J. , Zimmermann R. , Weisbach V. , Glaser A. , van Waeg G. , Eckstein R.
Published: March 2003

BACKGROUND: It is necessary to protect patients from white cell (WBC)‐ caused side effects of platelet transfusion by reducing the WBC contamination in single‐donor platelets. STUDY DESIGN AND METHODS: A new COBE Spectra WBC (leuko)‐reduction system (LRS) was compared to the COBE standard plateletpheresis (standard) procedure. Each of 62 donors underwent plateletpheresis under the two protocols (LRS and standard). The collection efficiency and WBC contamination in the components collected using the techniques were compared. Platelets were counted in a cell counter and WBCs were counted using two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers for red cells, WBCs, and platelets as well as the number of collected platelets were not different in the two techniques. Collection efficiency in the LRS procedures was 96.2 +/− 13.0 percent of that in the standard procedures. Median WBC contamination in the platelet components was 10,160 per LRS procedure and 56,500 per standard procedure. The purity of the LRS components was significantly improved (p = 0.001), as seen in a comparison of the WBC numbers in components per procedure after log10 transformation (LRS: 0.096 +/− 0.195 × 10(6); standard: 0.390 +/− 1.075 × 10(6)). CONCLUSION: These data suggest that the LRS procedure produces platelet concentrates with a collection efficiency that is comparable to that obtained with the standard technique and with a residual WBC content that satisfies even the most stringent criteria for filtered platelets. As this purity can be achieved without platelet loss or alteration, conventional fiber filtration no longer seems necessary or useful in this type of single‐donor platelet component.
Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4‐34 heavy chain variable gene segment
Authors: Thorpe S.J. , Boult C.E. , Stevenson F.K. , Scott M.L. , Sutherland J. , Spellerberg M.B. , Natvig J.B. , Thompson K.M.
Published: March 2003

BACKGROUND: The V4‐34 gene segment is commonly used by human monoclonal IgM alloantibodies against blood group antigens and by cold‐reactive red cell autoantibodies with anti‐I or anti‐i specificity. This study was conducted to determine whether cold agglutinin activity is found among the V4‐34‐encoded alloantibodies. STUDY DESIGN AND METHODS: Fifty‐ four human IgM monoclonal antibodies (MoAbs) against Rh system antigens were tested for cold agglutinin activity against red cells lacking the relevant Rh system antigen and for reactivity with tissue I and/or i antigens using immunohistochemistry. The findings were correlated with the utilization of the V4‐34 segment as determined in an enzyme‐linked immunosorbent assay with an antibody (9G4) that is specific for this gene product and were also correlated with other serologic properties. RESULTS: Of the MoAbs, 59 percent were 9G4‐positive. Of the 9G4‐ positive subset, 16 and 44 percent agglutinated native adult (express I) and cord (express i) cells, respectively, at 4 degrees C; these levels rose to 84 and 94 percent, respectively, with the use of papain‐ treated cells. The red cell antigens recognized at 4 degrees C were cleaved by endo‐beta‐galactosidase, which is consistent with their being I and i. Of the 9G4‐positive subset, 53 percent bound to tissue i antigen. These reactivities were not found among 9G4‐negative MoAbs. Endo‐beta‐galactosidase treatment of red cells enhanced Rh system antibody agglutination by 9G4‐negative MoAbs. CONCLUSION: Anti‐I/i reactivity is common among IgM Rh system MoAbs and is shown only by the V4‐34‐encoded subset. This finding has implications for the use of MoAbs for Rh system typing of blood.
Involvement of Gly96 in the formation of the Rh26 epitope
Authors: Faas B.H.W , Ligthart P.C. , Lomas‐Francis C. , Overbeeke M.A.M. , von dem Borne A.E.G. , van der Schoot C.E.
Published: March 2003

BACKGROUND: The Rh system, a complex blood group system, comprises at least 45 antigens. Red cells expressing c usually express Rh26. Rare cells that are c+ Rh:‐26 give variable reactions with anti‐c and may have weak expression of f (ce). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed with red cells from persons with the c+ Rh:‐26 phenotype occurring in two unrelated Dutch families. Red cells of 11 members of these two families were typed for Rh26, for c (with monoclonal and polyclonal reagents), and for f (ce). The cDNA of three donors was sequenced, while restricted DNA analysis was carried out on material from available members of the two families. RESULTS: Serologic tests showed that the rare c+ Rh:‐26 phenotype was associated with a weak expression of c and a normal expression of f. The cDNA analysis of three members of one family revealed a single‐point mutation (G286A) in exon 2 of the ce allele. Allele‐specific primer amplification, polymerase chain reaction followed by allele‐specific restriction analysis, and single‐strand conformation polymorphism showed the same polymorphism in all other members of both families, whereas it was absent in 80 control donors. CONCLUSION: The c+ Rh:‐26 phenotype, identified in two families, is associated with a single‐ point mutation at nucleotide 286 (G286A) in the ce allele, which predicts a Gly96Ser amino acid substitution. This substitution also affects c, because all anti‐c reagents reacted more weakly. Other polymorphic sites apparently are involved in the formation of the Rh26 epitope as well, because Rh26 is expressed only on the c polypeptide, whereas Gly96 is expressed on all polypeptides.
Automatic volumetric capillary cytometry for counting white cells in white cell‐reduced plateletpheresis components
Authors: Adams M.R. , Johnson D.K. , Busch M.P. , Schembri C.T. , Hartz T.P. , Heaton W.A.
Published: March 2003

BACKGROUND: As the benefits of white cell (WBC)‐reduced blood components become increasingly apparent, the need has arisen for a simple, automated WBC‐counting technique that is sensitive to low WBC concentrations. Automated volumetric capillary cytometry was evaluated for its ability to quantify residual WBCs in WBC‐reduced plateletpheresis components.

STUDY DESIGN AND METHODS: The volumetric capillary cytometry system evaluated uses a laser to excite fluorescent dye‐labeled nucleated cells. The number of nucleated cells per μL is reported. Four studies were performed: linearity, precision of results near the value of 5 × 106 WBCs per unit, the limit of detection, and correlation to the Nageotte manual counting method.

RESULTS: Assay values correlated to expected values (range, 0–125 WBC/μL) with an r2 > 0.99. In the range of 5 × 106 WBCs per unit the CV was 8.5 percent, and concentration differences of 0.15 log10 were detectable. The limit of detection was 1.0 WBCs per microliter (95% upper confidence limit). The assay correlated to the Nageotte method with an r2 of 0.98, slope of 1.0, and y‐intercept of 2.0 WBCs per μL. Assay results were 10 to 15 percent higher than Nageotte results, in samples with values near 5 × 106 WBCs per unit. Technician time per sample was 2 to 3 minutes.

CONCLUSION: Volumetric capillary cytometry is precise and sensitive to small differences in WBC concentration in the range of clinical interest. The device provides an efficient new method for quality assurance and control of WBC‐reduced plateletpheresis products.
Expression of blood group i antigen and fetal hemoglobin in paroxysmal nocturnal hemoglobinuria
Authors: Navenot J.‐M. , Mullet J.‐Y. , Blanchard D.
Published: March 2003

BACKGROUND: The mechanism by which the paroxysmal nocturnal hemoglobinuria (PNH) clone progressively takes over normal hematopoietic cells remains unknown. The respective in vivo differentiation of normal and PNH erythroid progenitors was investigated through the expression of two fetal erythroid markers (i antigen and fetal hemoglobin [HbF]) whose expression in adult red cells is associated with altered erythropoiesis.

STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against HbF and i and CD59 antigens were used to phenotype red cells of 10 PNH patients. A multiparametric flow cytometry assay of red cells and reticulocytes was designed to assess a possible association of i and HbF with PNH or normal red cells.

RESULTS: Most patients exhibited greater expression of i and HbF than did normal controls. In each case, the percentages of i‐positive or HbF‐positive cells within CD59‐deficient and CD59‐positive red cells were very close, clearly showing a lack of preferential association of these markers with normal or PNH cells.

CONCLUSION: In PNH patients, normal and PNH erythroid progenitors have the same ability to promote HbF and i antigen expression, which suggests that normal and PNH erythroid progenitors (burst‐forming units–erythroid, colony‐forming units–erythroid, erythroblasts) behave similarly in response to bone marrow stress.
Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat, and chloroquine elution methods
Authors: des Roziers N. Burin , Squalli S.
Published: March 2003

BACKGROUND: To accurately phenotype red cell from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody‐coated red cells without altering red cell antigen expression.

STUDY DESIGN AND METHODS: Antibodies coating red cells that were sensitized in vivo (warm‐reactive autoantibodies: 8 patients) or in vitro (42 alloantibodies) were eluted by using glycine‐HCl and EDTA (acid/EDTA), heat (56°C, 10 min), or chloroquine method.

RESULTS: Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p < 0.0001). With the gel low‐ionic‐strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p < 0.001). Overall, acid/ EDTA elution dissociated more antibodies than heat (p < 0.0001), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low‐ionic‐strength saline indirect antiglobulin tests or tube agglutination methods.

CONCLUSION: Although heat elution may be used on a limited basis, the acid/EDTA method appears to be the procedure of choice for typing red cell coated with warm‐reactive IgG alloantibodies or autoantibodies.
Detection and differentiation of platelet‐specific antibodies by flow cytometry: the bead‐mediated platelet assay
Authors: Helmberg W. , Fölsch B. , Wagner T. , Lanzer G.
Published: March 2003

BACKGROUND: Platelet‐reactive antibodies cause a number of clinical disorders. The detection and differentiation of these antibodies are prerequisites for the adequate treatment of these disorders. The bead‐ mediated platelet assay described here enables the detection and differentiation of platelet‐bound antibodies by the use of flow cytometry.

STUDY DESIGN AND METHODS: The bead‐mediated platelet assay is based on the isolation of human platelet glycoproteins by using flow cytometric standardization beads after the incubation of typed platelets with human sera. The specificity and sensitivity of this assay were tested with five sera, each containing a known platelet‐ reactive antibody. The monoclonal antibody‐specific immobilization of platelet antigens assay was used as a reference test.

RESULTS: The bead‐ mediated platelet assay was able to determine the glycoprotein specificity of the antibody without cross‐reactions in every case. In serial dilution tests, the bead‐mediated platelet assay was able to detect the antibodies at higher dilutions than the monoclonal antibody‐ specific immobilization of platelet antigen assay. Total test time was 3.5 hours.

CONCLUSION: The bead‐mediated platelet assay is a fast and reliable method for the detection and differentiation of platelet‐ reactive antibodies.
Factors affecting mobilization of CD34+ cells in normal donors treated with filgrastim
Authors: Anderlini P. , Przepiorka D. , Seong C. , Smith T.L. , Huh Y.O. , Lauppe J. , Champlin R. , Körbling M.
Published: March 2003

BACKGROUND: Multiple days of apheresis are required for some normal peripheral blood progenitor cell (PBPC) donors, to ensure a sufficient collection of CD34+ cells for allografting. It would be of practical value to be able to identify the patients with poor mobilization on the basis of simple pretreatment clinical or hematologic variables.

STUDY DESIGN AND METHODS: Clinical characteristics and laboratory data for 119 normal PBPC donors who underwent apheresis on Days 4 to 6 of treatment with granulocyte‐colony‐stimulating factor (filgrastim) were analyzed for correlations with CD34+ cell yield from the first day of apheresis.

RESULTS: The CD34+ cell yield was significantly lower in donors who were more than 55 years of age, who underwent apheresis on Day 4 of filgrastim therapy, or who were not obese. There were weak direct correlations between CD34+ cell yield and the baseline white cell count, preapheresis white cell count, and preapheresis mononuclear cell count, and there was a weak inverse correlation with age. Twenty‐ one donors (18%) were considered to have poor mobilization (< 20 × 106 CD34+ cells/L blood processed). In the multivariate analysis, the only significant factor was age greater than 55 years, which conferred a 3.8 times greater risk (95% CI, 1.1–13.7) of poor mobilization (p = 0.04). However, poor mobilization occurred in all age groups, so the predictive value of the model was low.

CONCLUSION: Donor variables correlated with CD34+ cell yield only weakly, so no particular clinical characteristic can be used to exclude an individual as a PBPC donor if he or she is otherwise suitable for the apheresis procedure.
A prospective study of a serum‐pooling strategy in screening blood donors for antibody to hepatitis C virus
Authors: Liu Pei , Shi Zhixu , Zhang Yanchao , Xu Zhongcai , Shu Huasong , Zhang Xingying
Published: March 2003

BACKGROUND: To examine the feasibility and to perform a cost‐benefit analysis of a pooling protocol of enzyme immunoassay (EIA) screening for antibody to hepatitis C virus (anti‐HCV) under real conditions, a prospective study was carried out using sera from 1875 local blood donors. STUDY DESIGN AND METHODS: In the absence of knowledge of the anti‐HCV reactions, the donor's sera were pooled into groups of five consecutive samples for testing by EIA. The dilution and final volume of the serum pool were adjusted to equal those recommended for single‐ serum EIA by the manufacturer of the test kit. The results obtained were compared with those of single‐serum EIA to assess the feasibility of the pooling protocol. By applying probability theory, the percentage of reduction in the number of tests performed (L value) when the serum‐ pooling strategy was used was calculated for several anti‐HCV seroprevalences and for varied sizes of pool. The calculations were performed on a computer using a program compiled by the authors. RESULTS: The results showed that seroprevalence was 2.24 percent (95% CI, 1.57‐2.91%); the rate of false negativity was 0 (95% CI, 0–8.4%), the sensitivity of the pooling protocol was 100 percent (95% CI, 91.6‐ 100.0%), the rate of false positivity was 0.8 percent (95% CI, 0–1.8%), and the specificity of the pooling protocol was 99.2 percent (95% CI, 98.2‐100.0%). Cost‐benefit analysis showed that the pooling protocol could save 69.3 percent of the cost. A table of L values can be used conveniently by serologists to determine the optimum pool size if estimates of seroprevalence are available. CONCLUSION: The pool EIA did not perform worse than individual EIAs, and the pooling strategy was markedly less expansive. The pooling protocol was recommended for screening of anti‐HCV‐positive subjects from large populations with low seroprevalence.
Quantification of CD34+ cells: comparison of methods
Authors: Fritsch Gerhard , Printz Dieter , Stimpfl Margit , Dworzak Michael Norbert , Witt Volker , Pötschger Ulrike , Buchinger Petra
Published: March 2003

BACKGROUND: Quantification of CD34+ stem and progenitor cells is predominantly performed by flow cytometric analysis of cells prepared by whole blood staining and red cell lysis. This method also includes cell washing, which is thought to cause the destruction and loss of some of the nucleated cells (NCs). To address this cell loss and its influence on the outcome of enumeration, three techniques for preparing cells for quantification of CD34+ cells were compared. STUDY DESIGN AND METHODS: Blood (n=179), bone marrow (n=60), and leukapheresis components (n=64) were examined by the use of density separation of mononuclear cells (MNCs) and two red cell‐lysis procedures (wash and no‐ wash). Cell counts were determined in the original materials and after cell preparation. Absolute CD34+ cell counts were calculated using the flow cytometry‐analyzed proportions of CD34+ cells and the various white cell counts. RESULTS: Depending on the cell source and the cell preparation chosen, the loss of NCs ranged between 12 percent and 89 percent of the original white cell number. This loss of NCs was exclusively due to cell washing and predominantly affected granulocytic cells. Analysis of the flow cytometry data revealed that the relative CD34+ values in blood and bone marrow were roughly threefold higher in density separated MNCs than in those that underwent the lyse‐and‐wash procedure. Calculation of absolute CD34+ cell counts confirmed that the MNC procedure underestimated the CD34+ cell content by a median of 26 percent (blood), 21 percent (bone marrow), and 5 percent (leukapheresis component) when compared with the median yield from analysis and cell counting performed after the lyse‐and‐wash procedure. On the other hand, the conventional lysis procedure, which applies the original white cell counts for CD34+ quantification, was shown to overestimate the CD34+ cell content by a median of 1.2‐fold, 1.33‐fold, and 1.13‐ fold, respectively. CONCLUSION: Neither density separation nor the whole‐blood lysis procedure seems appropriate for optimal CD34+ quantification.