Measuring Electrical And Mechanical Properties Of Red Blood Cells With A Double Optical Tweezers

The fluid lipid bilayer viscoelastic membrane of red blood cells (RBC) contains antigen glycolproteins and proteins which can interact with antibodies to cause cell agglutination. This is the basis of most of the immunohematologic tests in blood banks and the identification of the antibodies against the erythrocyte antigens is of fundamental importance for transfusional routines. The negative charges of the RBCs creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The first counterions cloud strongly binded moving together with the RBC is called the compact layer. This report proposes the use of a double optical tweezers for a new procedure for measuring: (1) the apparent membrane viscosity, (2) the cell adhesion, (3) the zeta potential and (4) the compact layer's size of the charges formed around the cell in the electrolytic solution. To measure the membrane viscosity we trapped silica beads strongly attached to agglutinated RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. The RBC adhesion was measured by slowly displacing two RBCs apart until the disagglutination happens. The compact layer's size was measured using the force on the silica bead attached to a single RBC in response to an applied voltage and the zeta potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. We believe that the methodology here proposed can improve the methods of diagnosis in blood banks.6326Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem., 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun., 54, pp. 29-42Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force trap for dielectric particles (1986) Opt. Lett., 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Grier, D.G., A revolution in optical manipulation (2003) Nature, 424, pp. 810-816Zhu, C., Bao, G., Wang, N., Cell Mechanics: Mechanical response, cell adhesion, and molecular deformation (2000) Annu. Rev. Biomed. Eng., 2, pp. 189-226Neuman, K.C., Block, S., Optical trapping (2004) Rev. Sci. Instrum., 75, pp. 2787-2809Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-43Hochmuth, R., Worthy, P., Evans, E., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Sze, A., Erickson, D., Ren, L., Li, D., Zeta-potential measurement using the Smoluchowski equation and the slope of the current-time relationship in electroosmotic flow (2003) J. Colloid and Interface Science, 261, pp. 402-410Hunter, R.J., (1981) Zeta Potential in Colloid Science, , Academic Press, New YorkPollack, W., Hager, H.J., Reckel, R., Toren, D.A., Singher, H.O., A study of the forces involved in the second stage of hemaggltination (1965) Transfusion, 5, pp. 158-183Chelidze, T., Dielectric spectroscopy of blood (2002) J. Non-crystalline Solids, 305, pp. 285-294Hymer, W.C., Barlow, G.H., Blaisdell, S.J., Continuous flow electrophoretic separation of proteins and cells from mammalian tissues (1987) Cell Biophys., 10, pp. 61-85Hashimoto, N., Fujita, S., Yokoyama, T., Cell electrophoretic mobility and glycerol lysis of human erythrocytes in various diseases (1998) Electrophoresis, 19, pp. 1227-123

Mechanical Properties Of Stored Red Blood Cells Using Optical Tweezers

We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.593016Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Barjas-Castro, M.L., Brandão, M.M., Fontes, A., Costa, F.F., Cesar, C.L., Saad, S.T.O., Elastic properties of irradiated red blood cell units measured by optical tweezer (2002) Transfusion, 42, pp. 1196-1199Brandão, M.M., Fontes, A., Barjas-Castro, M.L., Barbosa, L.C., Costa, F.F., Cesar, C.L., Saad, S.T.O., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) European Journal of Haematology, 70, pp. 207-211Williamson, L.M., Warwick, R.M., Transfusion-associated graft-versus-host disease and its prevention (1995) Blood Rev., 9, pp. 251-261Button, L.N., Dewolf, W.C., Newburger, P.E., The effecr of irradiation on blood components (1981) Transfusion, 21, pp. 419-426Platt, O.S., The sickle syndrome (1995) Blood: Principles and Practice of Hematology, , R. I Hadlin, S. E. Lux, T. P. Stossel, J. B. Lippincott, PhiladelphiaBallas, S.K., Dover, G.J., Charache, S., Effect of hydroxyurea on the rheological properties of sickle erythrocytes in vivo (1989) Am. J. Hematol, 32, pp. 104-111Groner, W., Mohandas, N., Bessis, M., New optical technique for measuring erythrocyte deformability with the ektacytometer (1980) Clin. Chem., 26, pp. 1435-1442De Franceschi, L., Bachir, D., Galacteros, F., Tchernia, G., Cynober, T., Alper, S., Platt, O., Brugnara, C., Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease (1997) J Clin Invest, 100, pp. 1847-1852Hochmuth, R.M., Worthy, P.R., Evans, E.A., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Evans, E.A., La Celle, P.L., Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation (1975) Blood, 45, pp. 29-43Itoh, T., Chien, S., Usami, S., Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation (1995) Blood, 85, pp. 2245-2253Evans, E.A., Mohandas, N., Membrane-associated sickle hemoglobin: A major determinant of sickle erythrocyte rigidity (1987) Blood, 70, pp. 1443-1449Dong, C., Chadwick, R.S., Schechter, A.N., Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation (1992) Biophys. J., 63, pp. 774-783Suzuki, Y., Tateishi, N., Cicha, I., Decreased deformability of the X-ray irradiated red blood cells stored in manitol-adenine-phosphate medium (2000) Clin. Hemorheol. Micro-cire., 22, pp. 131-14

Studying Red Blood Cell Agglutination By Measuring Membrane Viscosity With Optical Tweezers

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.6644Fontes, A., Fernandes, H.P., Barjas-Castro, M.L., Thomaz, A.A., Pozzo, L., Barbosa, L.C., Cesar, C.L., Red blood cell membrane viscoelasticity, agglutination and zeta potential measurements with double optical tweezers (2006) Proceedings of SPIE, 6088, pp. 296-305Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem, 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun, 54, pp. 29-42Fontes, A., Giorgio, S., de Castro Jr., A.B., Neto, V.M., Pozzo, L.Y., Marques, G.P., Barbosa, L.C., Cesar, C.L., Determination of femto Newton forces and fluid viscosity using optical tweezers: Application to Leishmania amazonensis (2005) Proceedings of SPIE, 5699, pp. 419-425Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-4

Bartonella Clarridgeiae Bacteremia Detected In An Asymptomatic Blood Donor

Human exposure to Bartonella clarridgeiae has been reported only on the basis of antibody detection. We report for the first time an asymptomatic human blood donor infected with B. clarridgeiae, as documented by enrichment blood culture, PCR, and DNA sequencing.531352356Maggi, R.G., Duncan, A.W., Breitschwerdt, E.B., Novel chemically modified liquid medium that will support the growth of seven Bartonella species (2005) J Clin Microbiol, 43, pp. 2651-2655. , http://dx.doi.org/10.1128/JCM.43.6.2651-2655.2005Drummond, M.R., Pitassi, L.H., Lania, B.G., Dos Santos, S.R., Gilioli, R., Velho, P.E., Detection of Bartonella henselae in defibrinated sheep blood used for culture media supplementation (2011) Braz J Microbiol, 42, pp. 430-432. , http://dx.doi.org/10.1590/S1517-83822011000200003Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., Basic local alignment search tool (1990) J Mol Biol, 215, pp. 403-410Dalton, M.J., Robinson, L.E., Cooper, J., Regnery, R.L., Olson, J.G., Childs, J.E., Use of Bartonella antigens for serologic diagnosis of cat-scratch disease at a national referral center (1995) Arch Intern Med, 155, pp. 1670-1676Breitschwerdt, E.B., Maggi, R.G., Chomel, B.B., Lappin, M.R., Bartonellosis: An emerging infectious disease of zoonotic importance to animals and human beings (2010) J Vet Emerg Crit Care (San Antonio), 20, pp. 8-30. , http://dx.doi.org/10.1111/j.1476-4431.2009.00496.xChamberlin, J., Laughlin, L.W., Romero, S., Solorzano, N., Gordon, S., Andre, R.G., Pachas, P., Watts, D., Epidemiology of endemic Bartonella bacilliformis: A prospective cohort study in a Peruvian mountain valley community (2002) J Infect Dis, 186, pp. 983-990. , http://dx.doi.org/10.1086/344054Maggi, R.G., Ericson, M., Mascarelli, P.E., Bradley, J.M., Breitschwerdt, E.B., Bartonella henselae bacteremia in a mother and son potentially associated with tick exposure (2013) Parasit Vectors, 6, p. 101. , http://dx.doi.org/10.1186/1756-3305-6-101Scott, M.A., McCurley, T.L., Vnencak-Jones, C.L., Hager, C., McCoy, J.A., Anderson, B., Collins, R.D., Edwards, K.M., Cat scratch disease: Detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease (1996) Am J Pathol, 149, pp. 2161-2167Slater, L.N., Welch, D.F., Min, K.W., Rochalimaea henselae causes bacillary angiomatosis and peliosis hepatis (1992) Arch Intern Med, 152, pp. 602-606Sander, A., Zagrosek, A., Bredt, W., Schiltz, E., Piemont, Y., Lanz, C., Dehio, C., Characterization of Bartonella clarridgeiae flagellin (FlaA) and detection of antiflagellin antibodies in patients with lymphadenopathy (2000) J Clin Microbiol, 38, pp. 2943-2948Kordick, D.L., Hilyard, E.J., Hadfield, T.L., Wilson, K.H., Steigerwalt, A.G., Brenner, D.J., Breitschwerdt, E.B., Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease) (1997) J Clin Microbiol, 35, pp. 1813-1818Margileth, A.M., Baehren, D.F., Chest-wall abscess due to cat-scratch disease (CSD) in an adult with antibodies to Bartonella clarridgeiae: Case report and review of the thoracopulmonary manifestations of CSD (1998) Clin Infect Dis, 27, pp. 353-357. , http://dx.doi.org/10.1086/514671Chomel, B.B., Mac Donald, K.A., Kasten, R.W., Chang, C.C., Wey, A.C., Foley, J.E., Thomas, W.P., Kittleson, M.D., Aortic valve endocarditis in a dog due to Bartonella clarridgeiae (2001) J Clin Microbiol, 39, pp. 3548-3554. , http://dx.doi.org/10.1128/JCM.39.10.3548-3554.2001Gillespie, T.N., Washabau, R.J., Goldschmidt, M.H., Cullen, J.M., Rogala, A.R., Breitschwerdt, E.B., Detection of Bartonella henselae and Bartonella clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease (2003) J Am Vet Med Assoc, 222, pp. 47-51. , http://dx.doi.org/10.2460/javma.2003.222.47, 35Robinson, M.T., Hillman, T., Langton, D.A., Shaw, S.E., Bartonella clarridgeiae in a cat in the UK (2009) Vet Rec, 164, pp. 58-59. , http://dx.doi.org/10.1136/vr.164.2.58Sykes, J.E., Westropp, J.L., Kasten, R.W., Chomel, B.B., Association between Bartonella species infection and disease in pet cats as determined using serology and culture (2010) J Feline Med Surg, 12, pp. 631-636. , http://dx.doi.org/10.1016/j.jfms.2010.04.003Fouch, B., Coventry, S., A case of fatal disseminated Bartonella henselae infection (cat-scratch disease) with encephalitis (2007) Arch Pathol Lab Med, 131, pp. 1591-1594Boudebouch, N., Sarih, M., Beaucournu, J.C., Amarouch, H., Hassar, M., Raoult, D., Parola, P., Bartonella clarridgeiae, B. Henselae, and Rickettsia felis in fleas from Morocco (2011) Ann Trop Med Parasitol, 105, pp. 493-498. , http://dx.doi.org/10.1179/1364859411Y.0000000038Kordick, D.L., Brown, T.T., Shin, K., Breitschwerdt, E.B., Clinical and pathologic evaluation of chronic Bartonella henselae or Bartonella clarridgeiae infection in cats (1999) J Clin Microbiol, 37, pp. 1536-1547Chomel, B.B., Carlos, E.T., Kasten, R.W., Yamamoto, K., Chang, C.C., Carlos, R.S., Abenes, M.V., Pajares, C.M., Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from the Philippines (1999) Am J Trop Med Hyg, 60, pp. 593-597Dehio, C., Bartonella interactions with endothelial cells and erythrocytes (2001) Trends Microbiol, 9, pp. 279-285. , http://dx.doi.org/10.1016/S0966-842X(01)02047-9Dehio, C., Meyer, M., Berger, J., Schwarz, H., Lanz, C., Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome (1997) J Cell Sci, 110 (18), pp. 2141-2154Braga Mdo, S., Diniz, P.P., André, M.R., Bortoli, C.P., Machado, R.Z., Molecular characterisation of Bartonella species in cats from São Luís, state of Maranhão, North-Eastern Brazil (2012) Mem Inst Oswaldo Cruz, 107, pp. 772-777. , http://dx.doi.org/10.1590/S0074-02762012000600011Eremeeva, M.E., Gerns, H.L., Lydy, S.L., Goo, J.S., Ryan, E.T., Mathew, S.S., Ferraro, M.J., Koehler, J.E., Bacteremia, fever, and splenomegaly caused by a newly recognized Bartonella species (2007) N Engl J Med, 356, pp. 2381-2387. , http://dx.doi.org/10.1056/NEJMoa065987Chomel, B.B., Boulouis, H.J., Breitschwerdt, E.B., Kasten, R.W., Vayssier-Taussat, M., Birtles, R.J., Koehler, J.E., Dehio, C., Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors (2009) Vet Res, 40, p. 29. , http://dx.doi.org/10.1051/vetres/2009011Breitschwerdt, E.B., Maggi, R.G., Duncan, A.W., Nicholson, W.L., Hegarty, B.C., Woods, C.W., Bartonella species in blood of immunocompetent persons with animal and arthropod contact (2007) Emerg Infect Dis, 13, pp. 938-941. , http://dx.doi.org/10.3201/eid1306.061337Carson, J.L., Grossman, B.J., Kleinman, S., Tinmouth, A.T., Marques, M.B., Fung, M.K., Holcomb, J.B., Djulbegovic, B., Red blood cell transfusion: A clinical practice guideline from the AABB (2012) Ann Intern Med, 157, pp. 49-58. , http://dx.doi.org/10.7326/0003-4819-157-1-201206190-00429Ramirez-Arcos, S., Goldman, M., Blajchman, M., Bacterial contamination (2012) Transfusion Reaction, 4, pp. 153-189. , Popovsky MA (ed), American Association Of Blood Banks, Bethesda, MDVamvakas, E.C., Blajchman, M.A., Transfusion-related mortality: The ongoing risks of allogeneic blood transfusion and the available strategies for their prevention (2009) Blood, 113, pp. 3406-3417. , http://dx.doi.org/10.1182/blood-2008-10-167643Magalhães, R.F., Cintra, M.L., Barjas-Castro, M.L., Del Negro, G.M., Okay, T.S., Velho, P.E., Blood donor infected with Bartonella henselae (2010) Transfus Med, 20, pp. 280-282. , http://dx.doi.org/10.1111/j.1365-3148.2010.01001.xMagalhães, R.F., Pitassi, L.H., Salvadego, M., De Moraes, A.M., Barjas-Castro, M.L., Velho, P.E., Bartonella henselae survives after the storage period of red blood cell units: Is it transmissible by transfusion? (2008) Transfus Med, 18, pp. 287-291. , http://dx.doi.org/10.1111/j.1365-3148.2008.00871.xLin, J.W., Chen, C.M., Chang, C.C., Unknown fever and back pain caused by Bartonella henselae in a veterinarian after a needle puncture: A case report and literature review (2011) Vector Borne Zoonotic Dis, 11, pp. 589-591. , http://dx.doi.org/10.1089/vbz.2009.0217Oliveira, A.M., Maggi, R.G., Woods, C.W., Breitschwerdt, E.B., Suspected needle stick transmission of Bartonella vinsonii subspecies berkhoffii to a veterinarian (2010) J Vet Intern Med, 24, pp. 1229-1232. , http://dx.doi.org/10.1111/j.1939-1676.2010.0563.xOhl, M.E., Spach, D.H., Bartonella quintana and urban trench fever (2000) Clin Infect Dis, 31, pp. 131-135. , http://dx.doi.org/10.1086/313890Daly, J.S., Worthington, M.G., Brenner, D.J., Moss, C.W., Hollis, D.G., Weyant, R.S., Steigerwalt, A.G., O'Connor, S.P., Rochalimaea elizabethae sp. Nov. Isolated from a patient with endocarditis (1993) J Clin Microbiol, 31, pp. 872-881Oksi, J., Rantala, S., Kilpinen, S., Silvennoinen, R., Vornanen, M., Veikkolainen, V., Eerola, E., Pulliainen, A.T., Cat scratch disease caused by Bartonella grahamii in an immunocompromised patient (2013) J Clin Microbiol, 51, pp. 2781-2784. , http://dx.doi.org/10.1128/JCM.00910-13Breitschwerdt, E.B., Mascarelli, P.E., Schweickert, L.A., Maggi, R.G., Hegarty, B.C., Bradley, J.M., Woods, C.W., Hallucinations, sensory neuropathy, and peripheral visual deficits in a young woman infected with Bartonella koehlerae (2011) J Clin Microbiol, 49, pp. 3415-3417. , http://dx.doi.org/10.1128/JCM.00833-11Raoult, D., Roblot, F., Rolain, J.M., Besnier, J.M., Loulergue, J., Bastides, F., Choutet, P., First isolation of Bartonella alsatica from a valve of a patient with endocarditis (2006) J Clin Microbiol, 44, pp. 278-279. , http://dx.doi.org/10.1128/JCM.44.1.278-279.2006Welch, D.F., Carroll, K.C., Hofmeister, E.K., Persing, D.H., Robison, D.A., Steigerwalt, A.G., Brenner, D.J., Isolation of a new subspecies, Bartonella vinsonii subsp. Arupensis, from a cattle rancher: Identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice (1999) J Clin Microbiol, 37, pp. 2598-2601Probert, W., Louie, J.K., Tucker, J.R., Longoria, R., Hogue, R., Moler, S., Graves, M., Fritz, C.L., Meningitis due to a "Bartonella washoensis"-like human pathogen (2009) J Clin Microbiol, 47, pp. 2332-2335. , http://dx.doi.org/10.1128/JCM.00511-09Kosoy, M., Morway, C., Sheff, K.W., Bai, Y., Colborn, J., Chalcraft, L., Dowell, S.F., Petersen, L.R., Bartonella tamiae sp. Nov., a newly recognized pathogen isolated from three human patients from Thailand (2008) J Clin Microbiol, 46, pp. 772-775. , http://dx.doi.org/10.1128/JCM.02120-07Maggi, R.G., Kosoy, M., Mintzer, M., Breitschwerdt, E.B., Isolation of Candidatus Bartonella melophagi from human blood (2009) Emerg Infect Dis, 15, pp. 66-68. , http://dx.doi.org/10.3201/eid1501.081080Lin, E.Y., Tsigrelis, C., Baddour, L.M., Lepidi, H., Rolain, J.M., Patel, R., Raoult, D., Candidatus Bartonella mayotimonensis and endocarditis (2010) Emerg Infect Dis, 16, pp. 500-503. , http://dx.doi.org/10.3201/eid1603.081673Breitschwerdt, E.B., Maggi, R.G., Cadenas, M.B., De Paiva Diniz, P.P., A groundhog, a novel Bartonella sequence, and my father's death (2009) Emerg Infect Dis, 15, pp. 2080-2086. , http://dx.doi.org/10.3201/eid1512.AD151

The Frequency Of The New Platelet Antigen Cab In The Brazilian Population

[No abstract available]1003343343Metcalfe, P., Watkins, N.A., Ouwehand, W.H., Nomenclature of human platelet antigens (2003) Vox Sang, 85, pp. 240-245Bertrand, G., Jallu, V., Saillant, D., The new plateloantigen Cab a: a single point mutation Gln 716 His on the alpha 2 integrin (2009) Transfusion, 49, pp. 2076-2083Castro, V., Origa, A.F., Annichino-Bizzacchi, J.M., Frequencies of platelet-specific alloantigen systems 1-5 in three distinct ethnic groups in Brazil (1999) Eur J Immunogenet, 26, pp. 355-360Castro, V., Kroll, H., Origa, A.F., A prospective study on the prevalence and risk factors for neonatal thrombocytopenia and plateloimmunization among 9332 unselected Brazilian newborns (2007) Transfusion, 47, pp. 59-66Davoren, A., Curtis, B.R., Aster, R.H., Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia (2004) Transfusion, 44, pp. 1220-122

Electrical Properties Of The Red Blood Cell Membrane And Immunohematological Investigation

Hemagglutination is widely used in transfusion medicine and depends on several factors including antigens, antibodies, electrical properties of red blood cells and the environment of the reaction. Intermolecular forces are involved in agglutination with cell clumping occurring when the aggregation force is greater than the force of repulsion. Repulsive force is generated by negative charges on the red blood cell surface that occur due to the presence of the carboxyl group of sialic acids in the cell membrane; these charges create a repulsive electric zeta potential between cells. In transfusion services, specific solutions are used to improve hemagglutination, including enzymes that reduce the negative charge of red blood cells, LISS which improves the binding of antibodies to antigens and macromolecules that decrease the distance between erythrocytes. The specificity and sensitivity of immunohematological reactions depend directly on the appropriate use of these solutions. Knowledge of the electrical properties of red blood cells and of the action of enhancement solutions can contribute to the immunohematology practice in transfusion services.334297301Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl Immun., 54 (1), pp. 29-42Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J Biol Chem., 237, pp. 1992-2000Abbas, A.K., Lichtman, A.H., (2005) Imunologia celular e molecular, p. 580. , 5th ed. Rio de Janeiro: Saunders Elseviervan Oss, C.J., Absolom, D.R., Zeta potential, Van der Walls Forces and hemagglutination (1983) Vox sanguinis., 44 (3), pp. 183-190Parslow, T.G., Stites, D.P., Terr, A.I., Imboden, J.B., (2004) Imunologia Médica, , 10th ed. Rio de Janeiro: Guanabara KooganAtkins, P., Paula, J., Interações moleculares (2004) Atkins Físico-química, pp. 408-426. , In:, 7th ed. Rio de Janeiro: Livros Técnicos e CientíficosMollison, P.L., (2005) Blood transfusion in clinical medicine, p. 891. , 11th ed. Oxford: BlackwellAmerican Association of Blood Banks: Technical manual, , 14th edHunter, R.J., (1981) Zeta potential in colloid science: Principles and applications, p. 386. , London: AcademicNi, H., Amme, R.C., Ion redistribution in a electric double layer (2003) J Colloid Interface Sci., 260 (2), pp. 344-348Sze, A., Erickson, D., Ren, L., Li, D., Zeta-potential measurements using Smoluchowski equation and slope of the current-time relationship in electrosmotic flow (2003) J Colloid Interface Sci., 261 (2), pp. 402-410Pollack, W., Hager, H.J., Reckel, R., Toren, D.A., Singher, H.O., A study of the forces involved in the second stage of hemagglutination (1965) Transfusion, (5), pp. 158-183Rouger, P., Salmon, C., (1981) La pratique de l'agglutination des érythrocytes et du test de coombs, , Paris: MassonStratton, F., Rawlinson, V.I., Gunson, H.H., Phillips, P.K., The role of Zeta Potential in Rh agglutination (1973) Vox Sanguinis., 24, pp. 273-279Luner, S.J., Sturgeon, P., Szklarek, K., McQuiston, D.T., Effects of proteases and neuramidase on RBC surface charge and agglutination (1975) Vox Sang., 28 (3), pp. 184-199Baxbaum, K., Evans, E., Brooks, D.E., Quantitation of surface affinities of red blood cells in dextran solutions and plasma (1982) Biochemistry., 21 (13), pp. 3235-3239Löw, B., Messeter, L., Antiglobulin test in low ionic strength salt solution for rapid antibody screening and cross-matching (1974) Vox Sang., 26 (1), pp. 53-61Daniels, G.L., Bromilow, I., (2007) Essential guide to blood groups, , Blackwell publishingBarret, A.J., Rawlings, N.D., Woessner, J.F., (1998) Handbook of proteolytic enzyme, p. 1666. , San Diego: AcademicBeynon, R.J., Bond, J.S., (1989) Proteolytic enzymes: A practical approach, p. 259. , Oxford: IRL: Oxford UnivOmi, T., Kaji, E., Ikemoto, S., The electrokinetic behavior or red blood cells from a patient with Tn syndrome by dopller electrophoretic light scattering analysis (1994) Tohoku J Exp Med., 174 (4), pp. 369-377Barshtein, G., Tamir, I., Yedgar, S., Red blood cell rouleaux formation in dextran solutions: Dependence on polymer conformation (1998) Eur Biophys J., 27 (2), pp. 177-181Chien, S., Jan, K.M., Ultrastructural basis of the mechanism of rouleaux formation (1973) Microvasc Res., 5 (2), pp. 155-166Chien, S., Sung, L.A., Simchon, S.S., Lee, M.M., Jan, K.M., Skalak, R., Energy balance in red cell interactions (1983) Ann NY Acad Sci., 416, pp. 190-206Evans, E.A., Parsegian, V.A., Energetics of membrane deformation and adhesion in cell and vesicle aggregation (1983) Ann NY Acad Sci., 416, pp. 13-33Brooks, D.E., Evans, E., Rheology of blood cell (1987) Clinical Hemorheology, pp. 73-96. , In: Chien S, Dormandi J, Ernst E (eds)., Nijhoff, AmsterdamHarris, J.M., (1992) Poly (ethylene glycol) chemistry: Biotechnical and biomedical applications, p. 385. , New York, London: Plenumde Man, A.J., Overbeeke, M.A., Evaluation of the polyethylene glycol antiglobulin test for detection of red blood cell antibodies (1990) Vox Sang., 58 (3), pp. 207-21

Measuring Red Blood Cell Aggregation Forces Using Double Optical Tweezers

Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10-3 (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10-3 (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction. © 2013 Informa Healthcare.733262264Acronym; Sponsor: UM; National Science and Technology Development AgencyEylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) JBiol Chem, 237, pp. 1992-2000Jaime, J.C., Immune-medicated agglutination of cytoskeletonfree RBC microvesicles (2000) Transfusion, 40, pp. 566-570Van Oss, C.J., Absolon, D.R., Zeta potential van der walls forces and hemagglutination (1983) Vox Sang, 44, pp. 183-190Pollack, W., Hager, H.J., Reckel, R., Toren, D.A., Singher, H.O., Astudy of the forces involved in the second stage of hemagglutination (1965) Transfusion, 5, pp. 158-183Storry, J.R., Areview: Modifi cation of the red blood cell membrane and its application in blood group serology (2000) Immunohematology, 16, pp. 101-104Fernandes, H.P., Cesar, C.L., Barjas-Castro, M.L., Electrical properties of the red blood cell membrane and immunohematological investigation (2011) Rev Bras Hematol Hemoter, 33, pp. 297-301Neu, B., Wemby, R., Meiselman, H.J., Effects of Dextran molecular weight on red blood cell aggregation (2008) Biophys J, 95, pp. 3059-3065Silva, D.C., Jovino, C.N., Silva, C.A., Fernandes, H.P., Filho, M.M., Lucena, S.C., Costa, A.M., Fontes, A., Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells (2012) PLoS One, 7, pp. e317781-e317786Brandão, M.M., Fontes, A., Barjas-Castro, M.L., Barbosa, L.C., Costa, F.F., Cesar, C.L., Saad, S.T., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) Eur J Haematol, 70, pp. 207-211Fontes, A., Fernandes, H.P., Thomaz, A.A., Barbosa, L.C., Barjas-Castro, M.L., César, C.L., Measuring electrical and mechanical properties of red blood cells with double optical tweezers (2008) J Biomed Opt, 13, pp. 0140011-0140016Fontes, A., Fernandes, H.P., Thomaz, A.A., Barbosa, L.C., Barja-Castro, M.L., Cesar, C.L., Studying red blood cell agglutination by measuring electrical and mechanical properties with a double optical tweezers (2007) European Conferences on Biomedical Optics, , Munich. Biophotonics 2007 : Opt Life Sci 200766330R.1-10(2011) American Association Of Blood Banks (AABB, , Technical Manual 17th Edition. Bethesda MA USA: AABBHochmuth, R.M., Worthy, P.R., Evans, E.A., Red cell extensional recovery and the determination of membrane viscosity (1979) J Biophys, 26, pp. 101-11

Platelet Antibody Detection By Flow Cytometry: An Effective Method To Evaluate And Give Transfusional Support In Platelet Refractoriness

Background: Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80- 90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. Objective: The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. Methods: A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. Results: Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. Conclusion: This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.354252255Rebulla, P., Refractoriness to platelet transfusion (2002) Curr Opin Hematol., 9 (6), pp. 516-520Vassallo, R.R., New paradigms in the management of alloimmune refractoriness to platelet transfusions (2007) Curr Opin Hematol., 14 (6), pp. 655-663Pavenski, K., Freedman, J., Semple, J.W., HLA alloimmunization against platelet transfusions: Pathophysiology, significance, prevention and management (2012) Tissue Antigens., 79 (4), pp. 237-245Doughty, H.A., Murphy, M.F., Metcalfe, P., Rohatiner, A.Z., Lister, T.A., Waters, A.H., Relative importance of immune and non-immune causes of platelet refractoriness (1994) Vox Sang., 66 (3), pp. 200-205Legler, T.J., Fischer, I., Dittmann, J., Simson, G., Lynen, R., Humpe, A., Frequency and causes of refractoriness in multiply transfused patients (1997) Ann Hematol., 74 (4), pp. 185-189Ferreira, A.A., Zulli, R., Soares, S., Castro, V., Moraes-Souza, H., Identification of platelet refractoriness in oncohematologic patients (2011) Clinics (Sao Paulo)., 66 (1), pp. 35-40He, Y., Zhao, Y.X., Zhu, M.Q., Wu, Q., Ruan, C.G., Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array (2013) Clin Chim Acta., 415, pp. 176-180van Velzen, J.F., Laros-van Gorkom, B.A., Pop, G.A., van Heerde, W.L., Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers (2012) Thromb Res., 130 (1), pp. 92-98Armitage, P., Berry, G., Mathews, J.N.S., (1987) Statistical methods in medical research, p. 559. , 2nd ed, Boston, MA: Blackwell ScienceAltman, D., (1990) Practical statistics for medical research, p. 624. , London: Chapman and HallKerkhoffs, J.L., Eikenboom, J.C., Schipperus, M.S., van Wordragen-Vlaswinkel, R.J., Brand, R., Harvey, M.S., A multicenter randomized study of the efficacy of transfusions with platelets stored in platelet additive solution II versus plasma (2006) Blood., 108 (9), pp. 3210-3215Sacher, R.A., Kickler, T.S., Schiffer, C.A., Sherman, L.A., Bracey, A.W., Shulman, I.A., Transfusion Medicine Resource Committee. Management of patients refractory to platelet transfusion (2003) Arch Pathol Lab Med., 127 (4), pp. 409-414. , College of American PathologistsDuquesnoy, R.J., Structural epitope matching for HLA-alloimmunized thrombocytopenic patients: A new strategy to provide more effective platelet transfusion support? (2008) Transfusion, 48 (2), pp. 221-227. , Comment in: Transfusion. 2008;48(2):204-6Heal, J.M., Blumberg, N., Masel, D., An evaluation of crossmatching, HLA, and ABO matching for platelet transfusions to refractory patients (1987) Blood., 70 (1), pp. 23-30. , JulMickey, R., Donor pool sizes for HLA matching (1989) Transfusion., 29 (4), pp. 285-286O'Connell, B.A., Lee, E.J., Rothko, K., Hussein, M.A., Schiffer, C.A., Selection of histocompatible apheresis platelet donors by cross-matching random donor platelet concentrates (1992) Blood., 79 (2), pp. 527-531Petz, L.D., Garratty, G., Calhoun, L., Clark, B.D., Terasaki, P.I., Gresens, C., Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity (2000) Transfusion, 40 (12), pp. 1446-1456. , Comment in: Transfusion. 200040(12):1425-6Hod, E., Schwartz, J., Platelet transfusion refractoriness (2008) Br J Haematol., 142 (3), pp. 348-360Brooks, E.G., McPherson, B.R., Fung, M.K., Validation of HLAMatchmaker algorithm in identifying acceptable HLA mismatches for thrombocytopenic patients refractory to platelet transfusions (2008) Transfusion., 48 (10), pp. 2159-216

Fetal Genotyping For Platelets Antigens: A Precise Tool For Alloimmune Thrombocytopenia: Case Report And Literature Review

Maternal-fetal alloimmune thrombocytopenia complicates about 0.1% of all pregnancies and is associated with major fetal and neonatal morbidity and mortality, especially spontaneous central nervous system bleeding leading to death and neurological handicaps. Successful prevention and treatment depend on the identification of at-risk possible carriers of anti-platelet antibodies. Case report: We report a case of a mother with a previous child that developed neonatal hemorrhage; HPA-5b anti-platelet antibodies were detected post-natally. During the next pregnancy, fetal genotyping confirmed the presence of HPA-5b antigen; she was treated with weekly intravenous human immunoglobulin and oral prednisone. Pregnancy evolved without remarkable features and a full-term baby was delivered, with normal platelet counts. Conclusion: Fetal alloimmune thrombocytopenia is a potentially lethal condition, but early detection and prevention lead to successful outcome in most cases. © 2010 Springer-Verlag.2825573575Baujat, G., Legeai-Mallet, L., Finidori, G., Cormier-Daire, V., Le Merrer, M., Achondroplasia (2008) Best Practice and Research: Clinical Rheumatology, 22 (1), pp. 3-18. , DOI 10.1016/j.berh.2007.12.008, PII S1521694207001416Kaplan, C., Platelet alloimmunity: The fetal/neonatal alloimmune thrombocytopenia (2002) Vox Sang, 83, pp. 289-291. , 1:CAS:528:DC%2BD38XovVWrs7k%3D 12617155Garratty, G., Arndt, P., Applications of flow cytofluorometry to transfusion science (1995) Transfusion, 35 (2), pp. 157-178. , 1:STN:280:DyaK2M7isFOguw%3D%3D 10.1046/j.1537-2995.1995.35295125739.x 7825212Kiefel, V., Santoso, S., Weisheit, M., Mueller-Eckhardt, C., Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies (1987) Blood, 70 (6), pp. 1722-1726. , 1:STN:280:DyaL1c%2FltFarug%3D%3D 2445398Metcalfe, P., Watkins, N.A., Ouwehand, W.H., Kaplan, C., Newman, P., Kekomaki, R., De Haas, M., Santoso, S., Nomenclature of human platelet antigens (2003) Vox Sanguinis, 85 (3), pp. 240-245. , DOI 10.1046/j.1423-0410.2003.00331.xDavoren, A., Curtis, B.R., Aster, R.H., McFarland, J.G., Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia (2004) Transfusion, 44 (8), pp. 1220-1225. , DOI 10.1111/j.1537-2995.2004.04026.xCastro, V., Origa, A.F., Annichino-Bizzacchi, J.M., Soares, M., Menezes, R.C., Goncalves, M.S., Costa, F.F., Arruda, V.R., Frequencies of platelet-specific alloantigen systems 1-5 in three distinct ethnic groups in Brazil (1999) European Journal of Immunogenetics, 26 (5), pp. 355-360. , DOI 10.1046/j.1365-2370.1999.00174.xRozman, P., Platelet antigens: The role of human platelet alloantigens (HPA) in blood transfusion and transplantation (2002) Transpl Immunol, 10 (23), pp. 81-165Castro, V., Kroll, H., Origa, A., Falconi, M., Marques, S.B.D., Marba, S., Passini Jr., R., Arruda, V., A prospective study on the prevalence and risk factors for neonatal thrombocytopenia and platelet alloimmunization among 9332 unselected Brazilian newborns (2007) Transfusion, 47 (1), pp. 59-66. , DOI 10.1111/j.1537-2995.2007.01064.xMueller-Eckhardt, C., Grubert, A., Weisheit, M., Mueller-Eckhardt, G., Kiefel, V., Kroll, H., Schmidt, S., Santoso, S., 348 cases of suspected neonatal alloimmune thrombocytopenia (1989) Lancet, 1 (8634), pp. 363-366Williamson, L.M., Hackett, G., Rennie, J., The natural history of fetomaternal alloimmunization to the platelet- specific antigen HPA-1a (PlA1, Zwa) as determined by antenatal screening (1998) Blood, 92 (7), pp. 2280-2287. , 1:CAS:528:DyaK1cXmsVKgu7k%3D 9746765Kaplan, C., Fetal, neonatal alloimmune thrombocytopenias (2009) Transfus Clin Biol, 16 (2), pp. 214-217. , 1:STN:280:DC%2BD1Mzos1SnsA%3D%3D 10.1016/j.tracli.2009.03.013 19409828Bussel, J.B., Primiani, A., Fetal and neonatal alloimmune thrombocytopenia: progress and ongoing debates (2008) Blood Reviews, 22 (1), pp. 33-52. , DOI 10.1016/j.blre.2007.09.002, PII S0268960X07000537Kaplan, C., Neonatal alloimmune thrombocytopenia (2008) Haematologica, 93 (6), pp. 805-807. , http://www.haematologica.org/cgi/reprint/93/6/805, DOI 10.3324/haematol.13160Povoa, A.M., Ramalho, C., Machado, A.P., Matias, A., Montenegro, N., Congenital posthemorrhagic hydrocephalus: A case of fetomaternal alloimmune thrombocytopenia (2007) Fetal Diagnosis and Therapy, 22 (5), pp. 321-324. , DOI 10.1159/000103288Arnold, D.M., Smith, J.W., Kelton, J.G., Diagnosis and management of neonatal alloimmune thrombocytopenia (2008) Transfus Med Rev, 22 (4), pp. 255-267. , 10.1016/j.tmrv.2008.05.003 18848153Kjeldsen-Kragh, J., Killie, M.K., Tomter, G., Golebiowska, E., Randen, I., Hauge, R., Aune, B., Husebekk, A., A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia (2007) Blood, 110 (3), pp. 833-839. , http://bloodjournal.hematologylibrary.org/cgi/reprint/110/3/833, DOI 10.1182/blood-2006-08-040121Bussel, J.B., Sola-Visner, M., Current approaches to the evaluation and management of the fetus and neonate with immune thrombocytopenia (2009) Semin Perinatol, 33 (1), pp. 35-42. , 10.1053/j.semperi.2008.10.003 19167580Killie, M.K., Husebekk, A., Kjeldsen-Kragh, J., Skogen, B., A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn (2008) Haematologica, 93 (6), pp. 870-877. , http://www.haematologica.org/cgi/reprint/93/6/870, DOI 10.3324/haematol.12515Rayment, R., Brunskill, S.J., Stanworth, S., Soothill, P.W., Roberts, D.J., Murphy, M.F., Antenatal interventions for fetomaternal alloimmune thrombocytopenia (2009) Cochrane Database of Syst Rev, (3). , CD004226.pub1Berkowitz, R.L., Kolb, E.A., McFarland, J.G., Parallel randomized trials of risk-based therapy for fetal alloimmune thrombocytopenia (2006) Obstet Gynecol, 107 (1), pp. 91-96. , 16394045Ringwald, J., Schroth, M., Faschingbauer, F., Strobel, J., Strasser, E., Schild, R.L., Goecke, T.W., Intrauterine use of hyperconcentrated platelet concentrates collected with Trima Accel in a case of neonatal alloimmune thrombocytopenia (2007) Transfusion, 47 (8), pp. 1488-1493. , DOI 10.1111/j.1537-2995.2007.01288.xYinon, Y., Spira, M., Solomon, O., Weisz, B., Chayen, B., Schiff, E., Lipitz, S., Antenatal noninvasive treatment of patients at risk for alloimmune thrombocytopenia without a history of intracranial hemorrhage (2006) American Journal of Obstetrics and Gynecology, 195 (4), pp. 1153-1157. , DOI 10.1016/j.ajog.2006.06.066, PII S000293780600822