Manual 512402

104 PART II  n  Overview of the Major Blood Groups For questions 3 through 5, use the following ABO typing results: ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 0 0 4+ 4+ 3. What is the ABO interpretation? c. group B a. group O d. group AB b. group A 4. What ABO phenotypes would be compatible if the patient required a transfusion of RBCs? a. group AB, O, A, or B c. group AB or O b. group O or B d. only group O 5. What ABO phenotypes would be compatible if the patient required a transfusion of fresh frozen plasma? a. group AB, O, A, or B c. group AB or O b. group O or B d. only group O 6. Using known sources of reagent antisera (known antibodies) to detect ABO antigens on a patient’s red cells is known as: a. Rh typing c. direct antiglobulin test b. reverse grouping d. forward grouping 7. Which result is discrepant if the red cell typing shown in the following chart is correct? ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 0 4+ 0 0 a. negative reaction with group c. negative reaction with group B cells A1 cells b. positive reaction with anti-B d. no discrepancies in these results 8. What ABO antibody is expected in this patient’s serum based on the following information? Patient Red Cells with Anti-A Anti-B 0 4+ a. anti-B c. anti-A and anti-B b. anti-A d. none 9. According to Landsteiner’s rule, if a patient has no ABO antibodies after serum testing, what ABO antigens are present on the patient’s red cells? a. A c. both A and B b. B d. none 47

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 105 10. Select the ABO phenotypes, in order from most frequent to least frequent, that occur in whites: a. A, B, O, AB c. B, A, AB, O b. O, A, B, AB d. AB, O, B, A 11. Which of the following statements is true about ABO antibody production? a. ABO antibodies are present in c. ABO antibodies are stimulated by newborns. bacteria and other environmental b. ABO titers remain at constant factors. levels throughout life. d. All of these statements are true. 12. What immunoglobulin class is primarily associated with ABO antibodies? a. IgA c. IgE b. IgG d. IgM 13. What immunodominant sugar confers B blood group specificity? a. D-galactose c. N-acetylgalactosamine b. L-fucose d. L-glucose 14. An individual has the genotype of AO, hh. What antigens would be present on the red cells of this individual? a. A only c. A and O b. A and H d. none of the above 15. What gene controls the presence of soluble H substance in saliva? a. H c. Se b. A d. B 16. Which lectin agglutinates A1 red cells? c. Dolichos europaeus a. Dolichos biflorus d. Ulex biflorus b. Ulex europaeus 17. What immunodominant sugar determines the specificity of H antigens? a. D-galactose c. N-acetylgalactosamine b. L-fucose d. L-glucose 18. Which of the following situations may produce ABO discrepancies in the serum testing? a. newborn c. cold alloantibody b. patient with d. all of the above hypogammaglobulinemia 19. What soluble antigen forms are detectable in saliva based on the following genotype: AB, HH, SeSe? a. none (nonsecretor) c. A, B, and H b. only H d. A and B 20. Which ABO discrepancy is the best explanation for the results shown in the following chart? ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 4+ 0 2+ 4+ a. an elderly patient c. deterioration of reagents b. subgroup of A d. hypogammaglobulinemia 48

106 PART II  n  Overview of the Major Blood Groups REFERENCES 1. Issitt PD, Anstee DJ: Applied blood group serology, ed 4, Durham, NC, 1998, Montgomery Scientific Publications. 2. Daniels GL, Cartron JP, Fletcher A, et al: International Society of Blood Transfusion Committee on terminology for red cell surface antigens: Vancouver report, Vox Sang 84:244, 2003. 3. Daniels GL, Anstee DJ, Cartron JP, et al: International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens, Vox Sang 80:193, 2001. 4. Landsteiner K: Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe, Zbl Bakt 27:357, 1900. 5. von Decastello A, Sturli A: Über die Isoagglutinine im Serum gesunder und kranker Menschen, Munchen Med Wochenschr 95:1090, 1902. 6. Reid ME, Lomas-Francis C: The blood group antigen facts book, ed 2, San Diego, 2004, Elsevier Academic Press. 7. Economidou J, Hughes-Jones N, Gardner B: Quantitative measurements concerning A and B antigen sites, Vox Sang 12:321, 1967. 8. Mourant AE, Kopeâc AC, Domaniewska-Sobczak K: The distribution of the human blood groups and other biochemical polymorphisms, ed 2, London, 1976, Oxford University Press. 9. Roback JD, editor: Technical manual, ed 17, Bethesda, Md, 2011, AABB. 10. Pittiglio DH: Genetics and biochemistry of A, B, H and Lewis antigens. In Wallace ME, Gibbs FL, editors: Blood group systems: ABH and Lewis, Arlington, VA, 1986, AABB. 11. Yamamoto F, Clausen H, White T, et al: Molecular genetic basis of the histo-blood group ABO blood group system, Nature 345:229, 1990. 12. Fukuda MN, Hakamori S: Structures of branched blood group A-active glycosphingolipids in human erythrocytes and polymorphism of A- and H-glycolipids in A1 and A2 subgroups, J Biol Chem 257:446, 1982. 13. Lopez M, Benali J, Bony V, et al: Activity of IgG and IgM ABO antibodies against some weak A (A3, Ax, Aend) and weak B (B3, Bx) red cells, Vox Sang 37:281, 1979. 14. Nance ST: Serology of the ABH and Lewis blood group systems. In Wallace ME, Gibbs FL, editors: Blood group systems: ABH and Lewis, Arlington, VA, 1986, AABB. 15. Carson TH, editor: Standards for blood banks and transfusion services, ed 27, Bethesda, Md, 2011, AABB. 16. Gerbal A, Maslet C, Salmon C: Immunological aspects of the acquired B antigen, Vox Sang 28:398, 1975. 17. Beck ML, Kowalski MA, Kirkegaard JR, et al: Unexpected activity with monoclonal anti-B reagents, Immunohematology 8:22, 1992. 18. Beck ML, Yates AD, Hardman J, et al: Identification of a subset of group B donors reactive with monoclonal anti-A reagent, Am J Clin Pathol 92:625, 1989. 19. Bhatia HM: Serologic reactions of ABO and Oh (Bombay) phenotypes due to variations in H antigens. In Mohn JF, Plunkett RW, Cunningham RK, et al, editors: Human blood groups: Proceedings of the Fifth International Convocation on Immunology, Basel, Switzerland, 1977, Karger. SUGGESTED READINGS Poole J, Daniels G: Blood group antibodies and their significance in transfusion medicine, Transfus Med Rev 21:58-71, 2007. 49 tahir99-VRG & vip.persianss.ir

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] Quick Response Code: Review Article Indian J Med Res 146, September 2017, pp 305-315 DOI: 10.4103/ijmr.IJMR_914_16 Evolution of technology for molecular genotyping in blood group systems Ajit Gorakshakar1, Harita Gogri1 & Kanjaksha Ghosh2 1Department of Transfusion Medicine, ICMR- National Institute of Immunohaematology, Mumbai & 2Surat Raktadan Kendra & Research Centre, Surat, India Received June 10, 2016 The molecular basis of the blood group antigens was identified first in the 1980s and 1990s. Since then the importance of molecular biology in transfusion medicine has been described extensively by several investigators. Molecular genotyping of blood group antigens is one of the important aspects and is successfully making its way into transfusion medicine. Low-, medium- and high-throughput techniques have been developed for this purpose. Depending on the requirement of the centre like screening for high- or low-prevalence antigens where antisera are not available, correct typing of multiple transfused patients, screening for antigen-negative donor units to reduce the rate of alloimmunization, etc. a suitable technique can be selected. The present review discusses the evolution of different techniques to detect molecular genotypes of blood group systems and how these approaches can be used in transfusion medicine where haemagglutination is of limited value. Currently, this technology is being used in only a few blood banks in India. Hence, there is a need for understanding this technology with all its variations. Key words ABO alleles - India - microarrays - molecular genotyping - red blood cell antigens - single nucleotide polymorphisms Introduction systems were also cloned. After studying these genes carefully, it was observed that single nucleotide Since the time Karl Landsteiner discovered ABO polymorphism (SNP) is the main cause of variation blood groups, agglutination was the method of testing in these genes. One or more SNPs in particular blood for detecting the presence of blood group antigens and group system can help to identify specific alleles of antibodies. Apart from this, adsorption-elution, serum that system. Apart from this, various other causes inhibition and anti-human globulin test are some other responsible for variation seen in different blood group techniques routinely used in transfusion medicine. The alleles at the molecular level include deletion of a gene first major breakthrough for blood group genotyping or an exon or a nucleotide(s) (e.g. whole gene deletion at molecular level occurred when GYPA, the gene for seen in case of Rh system, point deletion(s) in case of the MN blood group system was cloned in 19861. This ABO, Kell, Duffy, Dombrock blood group systems), was followed by cloning of the genes for ABO and Rh sequence duplication plus a nonsense mutation blood group systems in 1990 and 1992 respectively2,3. (e.g. inactive RHD gene), formation of hybrid genes Subsequently, the genes for the other blood group 305 50

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] 306 INDIAN J MED RES, SEPTEMBER 2017 (e.g. MNS, Rh, ABO and Ch/Rg blood group systems), Fig. 1. Schematic diagram of the ABO gene. The numbers indicate duplication of an exon (e.g. Gerbich blood group positions of exons. UTR, untranslated region. system), etc (https://www.ncbi.nlm.nih.gov/projects/ gv/mhc/xslcgi.cgi?cmd=bgmut/systems). catalyses the addition of a monosaccharide onto a carbohydrate sequence expressing the H antigen. In the ABO blood group system, initially, the N-Acetyl-galactosamine is a specific sugar molecule DNA sequence of ‘A’ group specific transferase responsible for expression of ‘A group’ whereas was partially sequenced from human lung tissue4. D-galactose is a specific sugar molecule responsible for Subsequently, various A, B and O alleles were cloned ‘B group’ expression. The entire locus spans over 18 kb and sequenced, and sequence variations among them and consists of seven exons13. Fig. 1 gives a schematic were identified5-8. These variations were based on the representation of ABO gene and Table I depicts the sizes presence of different SNPs as well as due to insertion (bp) of exons and introns. Exons 6 and 7 of the gene or deletion of single nucleotides. This prompted the encode for 77 per cent of the full coding region of the scientists to develop molecular techniques to identify glycosyltransferase and 91 per cent of the catalytically these alterations to characterize various alleles. The active soluble transferase protein13. Therefore, initially, main advantages of these techniques were: (i) small techniques were developed to identify ABO alleles by amount of DNA was required, and (ii) an individual’s screening SNPs from exons 6 and 7 of the gene. genotype could be determined without doing laborious and time-consuming family investigations or without A101 is considered as a reference allele. B101 detecting blood group specific molecules on the surface allele differs from A101 allele in seven positions. of red blood cells (RBCs). Four of these changes [nucleotide (nt) 526, 703, 796, 803] result in amino acid substitution. There are two Today, 35 blood group systems comprising common A alleles responsible for A1 group; one is of more than 300 specific antigens are known A101 reference allele and the other one is A102. The [International Society of Blood Transfusion (ISBT), latter has single nucleotide substitution at position http://www.isbtweb.org]. At molecular level, more than 467. The rest of the sequence is same as the A101. The 1200 alleles have been identified while 50 genes are A201 allele has two alterations as compared to the involved in the expression of blood group antigens9. A101: a single base substitution (nt467) and a single Several review articles on blood group genotyping base deletion (nt1061). The O alleles are divided into covering various aspects have been published so two categories: deletional and non-deletional. The far10-12. In the ABO blood group system, several alleles O01 is the most common type of deletional allele, and encoding each antigen are identifiable and this has it differs from the A101 allele by a single nucleotide tremendous application in forensic science, chimerism, deletion at nt261. The O1 variant i.e. O02 allele also etc. Similarly, such heterogeneity may also throw some from the deletional category shows nt261 deletion light on hitherto unexplainable or partially explainable phenomenon of ABO isoimmunization. For antigens Table I. Sizes of the exons and introns of the ABO gene from other blood group systems, this technology helps to identify several alleles belonging to these systems Exons Introns and type in alloimmunized patients11. No Size (bp) No Size (bp) In the present review, initial development of DNA- based technology for the detection of molecular genotypes 1 28 1 12, 982 has been discussed in the context of the ABO blood group system along with the evolution of these technologies from 2 70 2 724 low to medium to high throughput for genotyping of other blood group antigens as well. This field is currently at the 3 57 3 1451 crossroads, bringing in new perspectives and techniques to replace a century-old practice of haemagglutination- 4 48 4 1686 based cross-matching in transfusion medicine. 5 36 5 554 Structure of ABO gene and alleles 6 135 6 1052 The ABO gene is located on ‘q’ arm of chromosome 9 (9q34). It encodes a glycosyltransferase which 7 688 Source: Ref 14 51

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] GORAKSHAKAR et al: MOLECULAR GENOTYPING IN BLOOD GROUP SYSTEMS 307 Fig. 2. Single nucleotide polymorphisms in exons 6 and 7 of the for differentiating these alleles15,16. A similar approach ABO gene describing the seven common ABO alleles. Allele names using C526G polymorphism instead of G703A to described in parentheses are as per old nomenclature. detect B alleles was used by Stroncek et al17. However, they found an anomalous A allele which showed all and nine single base substitutions as compared to polymorphisms as a normal A allele except for C526G A101 allele. The O03 allele is an allele from non- polymorphism. This clearly showed that more than one deletional category and shows four single nucleotide polymorphism were required to differentiate between substitutions when compared with A101 reference A and B alleles. In 1996, PCR-RFLP was developed allele. The detailed SNP positions occurring in the to detect polymorphisms at four sites, namely, 261, seven common ABO alleles are shown in Fig. 2. 526, 703 and 79618. This helped to identify A, B and O alleles. The use of NarI which has a cleavage site at nt Low-throughput techniques 526 helped them to differentiate between O01 and O03 alleles. A clinically applicable and simple genotype ABO genotyping: Techniques were first developed screening technique based on previously undescribed to discriminate between O and non-O groups using HpaII site in 3’ untranslated region of the ABO gene Kpn I/Bst EII enzymes to determine the presence was developed19. The polymorphism G1096A was or absence of G at nucleotide position 261 specific found in A101 and O01 alleles but not in B101 and O03 to the O allele2. Polymerase chain reaction (PCR) alleles. This polymorphism abolishes the HpaII site followed by allele-specific restriction fragment and is valuable marker in identification of ABO alleles. length polymorphism (RFLP) using BssHII/NarI and The same enzyme was found to be useful in identifying HpaII/AluI restriction enzyme pairs were used to polymorphisms associated with alleles such as A201 distinguish between A and O alleles from B alleles. (C467T), B101 (G703A) and O03 (G1096A). Direct Initially, 14 individuals of different blood groups were sequencing of the PCR amplified fragment for ABO analyzed by Southern Blot technique and the results genotyping was then developed in 199720. were compared with those of PCR-RFLP technique. This allowed the homo/heterozygous detection of SNP The second approach which was simultaneously at this position2. developed was PCR using allele-specific primers (PCR-ASP). The main advantage of this method was Later on, techniques were developed to identify that it did not require post-amplification treatment with common ABO alleles by characterizing minimum restriction enzymes thus reducing the processing time. number of SNPs. For example, normal A and B allele However, in case of ABO blood group system, more polymorphisms are present at seven positions. Of these, than one SNP is required to be identified to characterize four sites namely, 526,703,796 and 803 are crucial. the alleles. Hence, more than one set of primers are Hence, techniques were developed to identify A and B required. Initially, ASP incorporated with P32 were alleles by analyzing these four sites. G261 deletion for developed to characterize common ABO alleles21. detection of O allele and G703A substitution specific However, due to radioactivity extra care was required for B allele were taken into account by some researchers while using this technique. PCR-sequence specific oligonucleotide method developed in 1996 was based on specific sequence of the allele using an oligonucleotide probe hybridization reaction22. To detect three alleles (O01, O02 and O03) within O group, ten probes were developed. Alternatively, other researchers developed methods that required seven or eight PCR reactions to identify the common ABO alleles23,24. Later on, the reactions were multiplexed, and thus, only two PCR reactions were required to identify the common ABO alleles25. One of the drawbacks of this approach was that homo- or heterozygosity at each SNP could not be detected. Initially, all the SNPs covered by ASPs to identify common alleles were from exons 6 and 7 of the ABO 52

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] 308 INDIAN J MED RES, SEPTEMBER 2017 gene. Later on, primers were designed to screen all exons, by the inverse PCR-RFLP and inverse PCR ASP two regulatory regions and introns except intron 126. techniques. In a modified assay, labelled primers were This study revealed several unknown polymorphisms used and the alleles were identified by measuring the in coding as well as non-coding regions. Based on this, excess radioactivity present in the amplified reaction and other similar studies, various ABO alleles were mixture35. reanalyzed and renamed, and database of these alleles has been developed (dbRBC, www.ncbi.nlm.nih.gov/ A multiplexed single base primer extension projects/gv/mhc/xslcgi.cgi?cmd=bgmut/systems). reaction which allows the simultaneous determination Some investigators developed another approach which of six SNPs (nt 261, 297, 681, 703, 802, 803) has also involved combined use of PCR-RFLP and PCR-ASP been described to detect common ABO genotypes36-38. techniques to detect hybrid alleles as well as weaker The evolution of the molecular genotyping techniques variants of A and B27,28. for characterization of ABO alleles is illustrated in Table II. Due to extremely heterogeneous nature of ABO gene and the possibility of identifying new alleles The technology was developed to extract DNA based on the SNPs that were not detected earlier, some from various tissues and characterize the ABO alleles. researchers tried to use mutation scanning techniques This was a very sensitive technique as ABO genotype such as denaturing gradient gel electrophoresis was determined using only 0.1 ng of genomic DNA. (DGGE) or single strand conformation polymorphism In addition, the ABO genotype could also be detected (SSCP) to detect ABO alleles. Johnson and Hopkinson29 from the tissues obtained from bones, muscles, teeth, identified four different O alleles and two B alleles nails, semen contaminated vaginal fluid, etc. where the by amplifying 250 bp fragment of ABO gene and by conventional serological technique could not be used. running DGGE for 19 h at 61°C. Akane et al30 could Lee et al39 developed ABO genotyping technique using identify four common ABO alleles by SSCP analysis of four reactions of allele-specific multiplex PCRs to a single PCR product covering exon 6 of the ABO gene. detect five common ABO alleles. In this, whole blood Ogasawara et al31 further developed this technique and without extracting DNA was used. Here, ‘AnyDirect’ analyzed four PCR products amplified from exons PCR reaction buffer was used. It conserved the 6 and 7. Thirteen different alleles (common as well enzyme activity of DNA polymerase for effective use as rare) were identified. This approach was further in direct PCR from whole blood which contained PCR developed by multiplexing three PCRs in a single tube inhibitors40. The specificity and sensitivity of the novel and analyzing the three amplified products by SSCP in buffer used in this reaction was good. This is a rapid a single lane32. This could identify polymorphisms at and convenient technique and has many applications in nine positions in exons 6 and 7 (nt 261, 297, 467, 526, forensic medicine. ABO genotyping using fresh blood, 646, 657, 681, 1059 and 1096). Based on these SNPs, hair, body fluids, etc. without extracting DNA has also seven common ABO alleles could be differentiated been described41 where, a fast PCR instrument and using a ‘single tube-single lane format’. Initially, a optimized Taq polymerase were used. The amplified catalogue of various patterns corresponding to different products were analysed by GeneScan programme after ABO genotypes has to be prepared. For this, genotypes capillary electrophoresis. For amplifications, ASP was of the samples should be determined by PCR-RFLP used. This technique saved lot of time 41. technique. The same samples are then analyzed by SSCP to develop a catalogue. This method can also A kit based on PCR-SSP technique has been identify new alleles based on unknown SNPs in the developed for detection of common ABO alleles as three amplified fragments. This technology has been well as for some weaker variants such as A3, Ax, B3 used to identify common ABO alleles in the Indian and Bx42. The kit also contains ASP to detect common population33. alleles of Kell, Kidd and Duffy blood group systems. To genotype three major alleles (A1, B and O1) of Genotyping of Rh and other minor blood group the ABO blood group system simultaneously, inverse systems: After ABO, Rh is the second clinically PCR technique was developed34. In this technique, important blood group system. It is encoded by two sequence (about 1.7 kb) from exons 6 and 7 of each genes, namely, RHD and RHCE, which are closely allele was amplified, both the termini of the fragment linked and highly homologous and located on were then ligated and allele typing was performed chromosome 1, exact location being 1p36.1. Both these genes are inherited together. The RHD gene 53

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] GORAKSHAKAR et al: MOLECULAR GENOTYPING IN BLOOD GROUP SYSTEMS 309 Table II. Evolution of different techniques for molecular genotyping with reference to ABO blood system Year Author Method Nucleotide position analyzed No of PCR reactions Reference No. 1990 Yamamoto et al PCR‑RFLP 261, 526, 703, 796 4 2 1992 Uggozoli and Wallace PCR‑ASP using P32 261, 526, 703 1 21 labelled primers 1992 Lee and Chang PCR‑RFLP 258, 700 2 15 1992 Johnson and Hopkinson PCR‑DGGE 261, 297 1 29 1993 O’ Keefe & Dobrovic PCR‑RFLP 261, 703 1 16 1995 Stroncek et al PCR‑RFLP 261, 526 2 17 1995 Olsson and Chester PCR RFLP 261, 467, 703, 1096 1 (multiplex) 19 1996 Mifsud et al PCR‑RFLP 261, 526, 703, 796 4 18 1996 Gassner et al PCR‑ASP 261, 802, 803, 1059 2 23 1996 Mifsud et al PCR‑SSO 261, 297, 526, 802 2 22 1996 Akane et al PCR_SSCP ‑ 1 30 1996 Ogasawara et al PCR‑SSCP 261, 297, 467, 526, 703, 4 31 796, 802, 1061 1997 Nata et al Direct sequencing All positions encompassing 20 the amplified product 1997 Procter et al PCR‑ASP 261, 467, 796, 802, 1061 7 24 1998 Pearson & Hessner PCR‑ASP 261, 796, 802, 1061 2 25 2000 Yip PCR‑SSCP 261, 297, 467, 526, 646, 4 32 657, 681, 1059 and 1096 2000 Kobayashi and Akane IP‑RFLP, AISP 261, 796, 803 2 34 2001 Watanabe et al CASPA 261, 796, 803, 1065 1 35 2003 Seltsam et al PCR‑SSP 52 known polymorphisms 102 26 and remaining rare 2004 Doi et al Multiplex 261, 297, 681, 703, 802, 803 1 38 single‑base primer extension reaction RFLP, restriction fragment length polymorphism; DGGE, denaturing gradient gel electrophoresis; SSO, sequence specific oligonucleotide; SSCP, single strand conformational polymorphism; ASP, allele specific PCR; IP‑RFLP, inverse PCR‑  restriction fragment length polymorphism; AISP, allele specific inverse PCR; CASPA, consumed allele‑specific primer analysis is responsible for the expression of the D antigen. gene in that particular group or region is essential. Multiple genetic events may be responsible for the Very few studies have reported alterations in RHD at absence of D antigen on RBCs. Among Caucasians, molecular level46,48,49. As it is very difficult to design a D negativity is associated with deletion of RHD gene generalized technique to detect Rh alleles in different between upstream and downstream of Rhesus boxes, populations, only a few investigators have included while D negativity with intact RHD gene has been detection of RH alleles in genotyping platforms50-52. observed in many other populations43-45. Shao et al46 studied 76 RhD-negative cases and 26 Del cases from Subsequently, molecular basis of other blood China and found as many as five alterations responsible group systems was investigated. MNS (46 antigens), for affecting the expression of D antigen on RBCs. At Diego (22 antigens) and Kell (35 antigens) are some phenotypic level, 54 antigens have been detected while of the blood group systems where many antigens are at molecular level 493 alleles have been illustrated47. recognized phenotypically, and 59, 91 and 92 alleles Thus, Rh is a very complex system and to develop a have been identified among these blood group systems, strategy like PCR-SSP for detecting various Rh alleles respectively. In majority of the cases, alleles have been in a particular population group or geographical area, identified on the basis of SNPs. Hence, PCR-SSP knowledge about the profile of alterations in the RH technique was developed to detect these alleles. Olsson 54

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] 310 INDIAN J MED RES, SEPTEMBER 2017 et al53 developed this technique to detect various Three studies describing molecular genotyping of alleles of Duffy blood group system, while Hessner various blood group systems and platelet antigens using et al54 designed sequence-specific primers to detect SNP platforms specific for blood group genotyping alleles of Kidd blood group system. Yan et al55 used were published simultaneously in 200550,60,61. All the this technique to identify alleles of eight blood group three groups used multiplexed PCR based assays with systems (ABO, Rh, MNS, Kidd, Duffy, Cartwright, visual endpoints. Hashmi et al60 identified 18 SNPs Scianna and Colton) among Chinese; while Touinssi describing 36 alleles of more than 11 blood group et al56 developed this approach to screen French systems. Denomme and Van Oene50 screened 372 Basques to detect the alleles of six blood group systems samples for 12 SNPs detecting several blood group (Kell, Kidd, MNS, Dombrock, Colton and Cartwright). and platelet antigens. Beiboer et al61 detected various platelet antigens in 92 blood donors. Subsequently, Medium-throughput techniques several investigators reported52,62-67 their results of molecular genotyping using various platforms Assays with medium throughput include real-time of microarray technology, the details of which is PCR, Sanger DNA sequencing and Pyrosequencing. summarized in Fig. 3. In real-time PCR, amplified DNA is detected as the reaction progresses57. Three methods are used Fluidic microarray system (Luminex xMAP) is a for the detection of the products58 (i) Non-specific microsphere-based technology used for blood group fluorescent dyes like SYBR green which intercalates genotyping. In this method, microspheres are dyed with any double-stranded DNA; (ii) TaqMan probes; with two spectrally distinct fluorochromes. Using and (iii) Hybridization probe protocol involving precise amounts of each of these fluorochromes, an fluorescence resonance energy transfer (FRET). Sanger array is created. The system detects PCR amplified DNAsequencing involves the principle of termination of targets involving various SNPs by direct hybridization growing DNAchain after inclusion of dideoxynucleotide to microspheres which are coupled to allele (SNP) triphosphates with fluorochrome labelled bases. SNPs specific oligonucleotides. Using this technology, can be identified after reading the sequence of the Karpasitou et al64 analysed alleles of seven different gene. In pyrosequencing, a pyrophosphate molecule is blood group systems. Later on, this technique was detected on nucleotide incorporation. Pyrosequencing validated by the same group using biotinylated PCR technique has been used to detect alleles of Kell, Kidd product. This method involves two multiplex PCRs and Duffy blood group systems59. for screening of 16 antigens of Kell, S, Duffy, Kidd, Lutheran and Colton blood group systems68. High-throughput techniques Nanofluidic open array system has been used to Assays with high throughput involve the use genotype 32 SNPs for 42 blood group antigens in more of microarray technology. Majority of the alleles of than 40,000 donors66. The results were confirmed by various blood group systems can be identified by phenotyping before release of blood unit. This helped detecting one or two SNPs. Microarray technology can them to get antigen-negative blood units. Hence, they identify large number of SNPs at the same time from abandoned the screening by serology. These results genomic DNA. This technology is generally used to are encouraging as these help to shift to molecular detect the extended genotype of a donor. The Blood genotyping from serological techniques, provided Chip (Progenika Biopharma, Spain), HEA BeadChip that the platform for molecular genotyping is well (IMMUCOR, USA), GenomeLab (Beckman Coulter established. USA), Progenika IDcore+ (Progenika Biopharma, Spain) and The Bead Chip (Bioarray Solutions, Other techniques of high-throughput technology USA) are some of the microarray platforms available for molecular genotyping. Glass slides or beads or Mini sequencing or the SNaPshot assay: This microtitre plates are used to attach SNP specific DNA method involves SNP analysis to detect the exact base probes. The ‘on-chip’ test is based on the hybridization and computer-assisted visualization of the specific of targets which were amplified earlier by multiplex PCR alteration/polymorphism. Fluorescently labelled followed by a detection step allowing the simultaneous dideoxynucleotides are used with multiplex PCR identification of many SNPs involved in detection of product as a template. After the hybridization and various alleles of different blood group systems which extension steps, the fluorescent signals from the array in turn help to determine the extended genotype. are measured and the genotypes are determined by 55

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] 311 GORAKSHAKAR et al: MOLECULAR GENOTYPING IN BLOOD GROUP SYSTEMS Fig. 3. Various platforms of microarray technology used for blood group genotyping. Numerals in parentheses denote reference number. LU, Lutheran blood group system; KEL, Kell blood group system; FY, Duffy blood group system; JK, Kidd blood group system; DI, Diego blood group system; YT, Cartwright blood group system; SC, Scianna blood group system; DO, Dombrock blood group system; CO, Colton blood group system; HPA, Human platelet antigens cluster analysis. This technique has been successfully SNPs need to be identified to characterize a particular used to detect the most common alleles of the ABO ABO allele. Furthermore, considering the pace of blood group system69. The SNaPshot platform has been discovery of new alleles in ABO as well as other used to detect the alleles from 12 different blood group blood group systems, it became difficult to develop a systems70. Using this platform, nine SNPs defining low-throughput method to detect all the alleles. These 16 blood group alleles from five blood group systems techniques require post-PCR analysis and may give were simultaneously identified among blood donors false- or false-negative reactions with certain hybrid from Brazil71. alleles. There is a preferential amplification of only one allele when present in heterozygous condition. Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS): This Medium-throughput techniques such as real-time technique can discriminate two DNA fragments which SNP assay and DNA sequencing are time-consuming differ in a single nucleotide. The technique involves and complex techniques. Further, in DNA sequencing, two parts: first, laser-induced desorption/ ionization of large data are generated which are difficult to store matrix molecules and second, separation and analysis and analysis of the data requires bioinformatic tools. of these molecules on the basis of their intrinsic Second, the sample to results takes days to weeks which physical properties. It is a quantitative as well as later on decreased due to automation and optimization qualitative technique and can analyse several SNPs in of workflow74. a single reaction which takes about eight hours. This technique has been used to detect some alleles of Kell High-throughput donor typing techniques vary blood group system72. Two groups from Switzerland considerably in methodology, antigen selection, also used this system to identify several blood group throughput and cost. Even though several techniques alleles among Swiss blood donors73,74. are reported, only a few report this as an ongoing activity and provide data on large number of samples51,66,75. Limitations of molecular blood group genotyping: Although these are highly efficient for testing a large Low-throughput techniques such as PCR-SSP and number of samples for multiple blood group alleles PCR-RFLP were initially developed to detect various simultaneously; but these may be suitable for screening alleles of ABO blood group system. However, many only some populations. The new alleles identified 56

[Downloaded free from http://www.ijmr.org.in on Monday, June 17, 2019, IP: 202.28.250.83] 312 INDIAN J MED RES, SEPTEMBER 2017 cannot be detected and have to be incorporated into weaker subtypes. However, complex genotyping the testing platform. It has then to be validated again strategies are required to identify correct ABO or Rh before routine use. These technologies are expensive alleles. So serology will still be required to type for and one has to consider the cost. As per the requirement these systems. Molecular genotyping will help to type of the centre, the platform with number of antigens to donors for a wider spectrum of minor blood group be screened can be designed (e.g. clinically important antigens and also genotype blood group antigens antigens, minor antigens, rare alleles, null types) which of multiply transfused patients such as sickle cell can also take into account cost per sample. anaemia or β-thalassemia or patients having positive direct antiglobulin test. Providing a donor’s blood to Molecular genotyping in India: Only one study the patient after studying extended antigen profile will has described molecular ABO genotyping in Indian help in preventing alloimmunization. Alloimmunized population33. In this study, molecular genotyping transfusion recipients will also be benefited if the was done by PCR-RFLP and PCR-SSCP techniques. donor’s blood is electronically cross matched using Totally, 13 common and rare alleles belonging to the the extended array of SNPs. This technology can be ABO blood group were identified. Considering the used to screen for uncommon or rare antigens or to heterogeneity of Indian population many more alleles look for the absence of high-frequency antigens or are likely to be detected among various population to detect the antigens where specific antisera are not groups. available. Molecular genotyping will also play an important role in non-invasive prenatal RhD typing Identification of rare donors is a critical factor in of foetus of RhD-negative pregnant woman. In short, establishing a rare donor registry76. It can be done by molecular genotyping will make transfusion medicine performing mass screening of donors for clinically more personalized and patient-oriented. important blood group antigens by serological testing or by using gel technology. However, serological Conflicts of Interest: None. typing of large cohorts of donors is labour intensive and expensive exercise and many a times hampered References by the lack of reliable antisera. To overcome this, genotyping of various blood group systems will be an 1. Siebert PD, Fukuda M. Molecular cloning of a human important aspect. glycophorin B cDNA: Nucleotide sequence and genomic relationship to glycophorin A. Proc Natl Acad Sci U S A 1987; There are many reports where healthy blood donors 84 : 6735-9. from different cities in India have been screened to look for the frequency of various blood group antigens77-82. 2. Yamamoto F, Clausen H, White T, Marken J, Hakomori S. Similarly, many studies report the prevalence of Molecular genetic basis of the histo-blood group ABO system. alloimmunization against various antigens from Nature 1990; 345 : 229-33. different parts of India83-90. Recently, in a screening among 1221 donors from Mumbai, 261 donors who 3. Le van Kim C, Mouro I, Chérif-Zahar B, Raynal V, Cherrier C, lacked a combination of clinically important common Cartron JP, et al. Molecular cloning and primary structure of antigens, were detected91. All these studies have the human blood group RhD polypeptide. Proc Natl Acad Sci used serological techniques. At National Institute of U S A 1992; 89 : 10925-9. 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Mini Review published: 04 December 2017 doi: 10.3389/fmed.2017.00219 Pathogen inactivation of Cellular Blood Products—An Additional Safety Layer in Transfusion Medicine Axel Seltsam* German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany Edited by: In line with current microbial risk reduction efforts, pathogen inactivation (PI) technolo- Christoph Niederhauser, gies for blood components promise to reduce the residual risk of known and emerging Transfusion Interrégionale infectious agents. The implementation of PI of labile blood components is slowly but steadily increasing. This review discusses the relevance of PI for the field of transfusion CRS SA, Switzerland medicine and describes the available and emerging PI technologies that can be used to treat cellular blood products such as platelet and red blood cell units. In collaboration Reviewed by: with the French medical device manufacturer Macopharma, the German Red Cross Owen McCarty, Blood Services developed a new UVC light-based PI method for platelet units, which is Oregon Health & Science currently being investigated in clinical trials. University, United States Keywords: transfusion, platelets, pathogen inactivation, ultraviolet light, red blood cells Wei Li, Marshall University, United States INTRODUCTION *Correspondence: From the late 1970s to the mid-1980s, contaminated hemophilia blood products were a serious Axel Seltsam public health problem, resulting in the infection of large numbers of hemophiliacs with the human immunodeficiency virus (HIV). If safety measures had been implemented in a timely and consist- [email protected] ent manner after identification of the acquired immune deficiency syndrome (AIDS) epidemic in 1981 and isolation of the HIV in 1983, the transmission of HIV infection by these blood products Specialty section: could have been prevented in most cases. This contaminated blood scandal made the community This article was submitted aware that new pathogens may emerge and threaten blood safety at any time. However, there was a significant delay in the introduction of HIV detection systems in some countries and in some cases, to Hematology, the detection tests that were implemented proved to be unreliable. In addition, the plasma products a section of the journal used for therapy were not even treated by heat inactivation—a pathogen inactivation (PI) method that was readily available and approved at that time. Consequently, blood and blood components Frontiers in Medicine became subject to drug law in some countries (1, 2). Received: 26 September 2017 Increasingly stringent donor eligibility criteria and more sensitive virus detection methods have Accepted: 20 November 2017 reduced the risk of transfusion-transmitted infection (TTI) by blood products significantly, but a Published: 04 December 2017 residual risk of TTI with viruses, bacteria, protozoa, and prions remains. False-negative test results due to test failures, very low-pathogen concentrations in the peripheral blood or escaped mutants can Citation: result in TTI in spite of negative screening tests (e.g., for Treponema pallidum, hepatitis B, hepatitis C, Seltsam A (2017) Pathogen and HIV). In addition, transfusion recipients may be infected by pathogens not targeted in regular Inactivation of Cellular Blood blood donor screening programs (e.g., hepatitis A and bacteria). Transfusion safety is particularly Products—An Additional Safety susceptible to pathogens that enter regions in which they are not yet endemic. The fact that viruses Layer in Transfusion Medicine. that are usually endemic in tropical regions have recently caused outbreaks in Western countries demonstrates that these pathogens can arise and threaten transfusion safety at any time (3, 4). Front. Med. 4:219. doi: 10.3389/fmed.2017.00219 Frontiers in Medicine  |  www.frontiersin.org 611 December 2017 | Volume 4 | Article 219

Seltsam PI Technologies for Blood Products Blood safety is still mainly based on the reactive principle of properties of amotosalen HCl (S-59), a photoactive compound introducing new test systems or new donor election criteria after which penetrates cellular and nuclear membranes and binds to a threat to transfusion recipients has been identified. In other the double-stranded regions of DNA and RNA. When activated words, infections by contaminated blood products must first by low-energy UVA light (320–400 nm), amatosalen cross-links occur before appropriate counter-measures are established. At nucleic acids and thus irreversibly blocks the replication of DNA the beginning of the last decade, a number of cases of West Nile and RNA (11). After illumination, residual amotosalen and its virus occurred in the USA through the transmission of blood photoproducts must be removed during an incubation step last- components before the first detection system for donor testing ing up to 16 h. The amatosalen/UVA procedure is not suitable for was implemented (3). The recent Zika virus outbreak on the RBCs because of UVA light absorption by hemoglobin. American continent has heightened concerns over this reactive approach to blood supply safety (5, 6). MIRASOL PRT System for Platelets and Plasma During an international consensus conference, transfusion experts and other stakeholders in the field of transfusion medi- The MIRASOL system was developed by TerumoBCT (Lakewood, cine recommended a change from the hitherto reactive strategy CO, USA). This photodynamic procedure employs riboflavin toward a proactive, preventive approach to blood safety (7). (vitamin B2) and broad spectrum UV light (mainly UVA und Recently, developed and approved PI technologies for cellular UVB, 285–365  nm). On exposure to UVA and UVB light, blood products, such as red blood cell (RBC) and platelet units, riboflavin associates with nucleic acids and mediates oxygen- are considered key measures for closing or at least reducing the independent electron transfer, causing irreversible damage to the safety gap caused by emerging pathogens. While virus reduction nucleic acids (12). Because naturally occurring vitamin B2 and procedures are an integral part of the process of manufacturing its photodegradation products are non-toxic and non-mutagenic, plasma derivatives from plasma pools, and although the methyl- they do not need to be removed prior to transfusion. In addition ene blue system has been used for PI of single donor plasma units to plasma and platelets, protocols for extension of the MIRASOL for nearly two decades (8), a new generation of PI methods for system to whole blood are now in development. platelet units have recently become available (9, 10). PI technolo- gies for the treatment of RBC units are still in development and THERAFLEX System for Platelets have not received market authorization yet. THERAFLEX UV-Platelets is a novel UVC-based PI tech- TECHNOLOGIES nology that works without photoactive substances. It is the product of a joint venture between Macopharma (Mouvaux, The use of PI technologies for blood products has a number France) and the German Red Cross Blood Service NSTOB in of advantages. Because they inactivate most clinically relevant Springe, Germany. Shortwave UVC light (254  nm) directly viruses, bacteria, and protozoa, they can help to eliminate the interacts with nucleic acids to form pyrimidine dimers that residual risk of infection during the “window period” when block the elongation of nucleic acid transcripts (13). UVC transfusion-relevant pathogens (e.g., HIV) cannot be detected irradiation mainly affects the nucleic acids of pathogens and by donor screening tests. Their broad activity against pathogens leukocytes and does not impair plasma and platelet quality. As also helps to reduce the risk of recognizable infectious agents no photoactive substances are involved, UVC treatment is just (e.g., bacteria), which still cannot be prevented completely. In as simple but faster (takes less than 1 min) than gamma irra- contrast to screening tests for transfusion-borne pathogens, PI diation, and can easily be integrated into the manufacturing proactively protects against emerging infectious agents enter- processes at blood banks (Figure 1). The THERAFLEX system ing the blood supply in a given community. was originally developed for platelets but is also suitable for plasma and RBC units. All PI methods used to treat cellular blood products work by impairing the target pathogen’s ability to replicate. When used S-303 PI System for RBCs alone or in combination, ultraviolet (UV) light and alkylating agents cause irreversible damage to the nucleic acids of patho- The S-303 PI system (INTERCEPT RBC system, Cerus gens. Therefore, they effectively eliminate classical pathogens Corporation, Concord, CA, USA) was specifically developed such as viruses, bacteria, fungi, and protozoa, but are ineffective for RBC units. S-303 is a modular compound that prevents against prions. The latter protein-based pathogenic agents can nucleic acid replication by targeting and cross-linking nucleic cause sporadic and variant Creutzfeldt–Jakob disease in humans. acids. Once added to the RBC unit, this amphipathic compound rapidly passes through cell and viral envelope membranes and The following PI technologies for cellular products are cur- intercalates into the helical regions of nucleic acids. S-300, rently available or in the pipeline. the non-reactive byproduct of this reaction, is subsequently removed by incubation and centrifugation, which can take up INTERCEPT Blood System for Platelets to 20 h (14). In contrast to the other PI technologies described and Plasma here, the S-303 system does not require UV light. However, glutathione (GSH), a naturally occurring antioxidant, must be The INTERCEPT Blood System for platelets and plasma is used to prevent non-specific reactions between S-303 and other manufactured by Cerus Corporation (Concord, CA, USA). nucleophiles present in the RBC unit. These may include small The mechanism of action of this PI technology is based on the Frontiers in Medicine  |  www.frontiersin.org 622 December 2017 | Volume 4 | Article 219

Seltsam PI Technologies for Blood Products Figure 1 | The THERAFLEX ultraviolet (UV)-Platelets pathogen inactivation system uses UVC light to induce irreversible damage to the nucleic acids of viruses, bacteria, fungi, protozoa, and leukocytes. Intense agitation of the platelet bag during UVC illumination results in efficient mixing, ensuring the uniform treatment of all blood compartments (A). For the illumination step of this simple and rapid procedure, platelet units are placed in the irradiation device for a period of less than 1 min (B). Afterward, the pathogen-reduced platelet product can be used for transfusion. Table 1 | Pathogen inactivation technologies. Mechanism of action INTERCEPT blood system Technology THERAFLEX UV-Platelets S-303 system Blood products UVA plus amotosalen (alkylating agent) MIRASOL PRT system UVC alone Alkylating agent Plasma and platelets Plasma and platelets (in RBCs UV plus riboflavin (vitamin B2 = photosensitizer) development for RBCs) Status Approved in some countries Plasma and platelets (in development for whole In clinical development In clinical blood) development Approved in some countries UV, ultraviolet light; UVA, wavelength A; UVC, wavelength C; RBC, red blood cell. molecules, such as phosphate and water, and macromolecules, CLINICAL STUDIES such as proteins. Platelets The INTERCEPT and MIRASOL systems for platelets Clinical studies show that platelets retain their hemostatic efficacy and plasma have already been approved in the USA and some after PI treatment. Following prophylactic transfusion, there was European and Asian countries, while both the THERAFLEX no difference in the ability of pathogen-reduced and untreated system and the S-303 system are still in clinical development. The platelet units to prevent severe bleeding (15). However, almost UVC-based THERAFLEX system is expected to receive market- all clinical trials demonstrated that post-transfusion survival ing authorization within the next few years (Table 1). Frontiers in Medicine  |  www.frontiersin.org 633 December 2017 | Volume 4 | Article 219

Seltsam PI Technologies for Blood Products and recovery rates were consistently lower in patients receiving Evaluation of PI technologies for platelets is under way at some platelets treated with PI technology than in those transfused blood centers in Germany. In 2011, the Swiss national authority with untreated platelets (16–19). Accordingly, the transfusion (Swissmedic) ordered the nationwide implementation of PI of of pathogen-reduced platelets resulted in lower platelet count platelet units. This measure was mainly aimed at preventing or increments (CIs), lower corrected count increments, shorter at least minimizing the risk of fatal transfusion reactions caused intervals between platelet transfusions, and a higher number of by bacterially contaminated platelet units. Analysis of Swiss platelet transfusions per patient. However, observational studies hemovigilance data revealed that without PI, one fatal case of showed no evidence of increased product consumption rates transfusion-transmitted sepsis by contaminated platelet units when pathogen-reduced platelet units were used in a routine would occur in Switzerland every 2  years. The US Food and setting (20). Drug Administration (FDA) recently recommended the use of approved PI technologies as an alternative to bacterial detection Interestingly, the rate of acute transfusion reactions may be methods in order to adequately control the risk of bacterial lower after the transfusion of pathogen-reduced versus untreated contamination of platelets (26, 27). platelets. However, there have been concerns over acute respira- tory distress associated with amatosalen/UVA-treated platelets The preventive potential of PI of cellular blood components (15). While the results of animal studies suggest that UV light- first became apparent during a chikungunya virus epidemic on treated platelets mediated a higher risk of pulmonary toxicity the French island of La Reunion in the Indian Ocean in 2006 (21), an analysis of clinical data by an expert panel does not (28). Because more than 30% of the inhabitants were infected, confirm significant differences in the rates of acute lung disorders local blood donation was suspended to prevent TTI. To sustain between PI-treated and untreated platelets (22). The results of the availability of platelet components, the French national blood ongoing large-scale phase III and hemovigilance studies will help service (Etablissement Français du Sang) implemented universal to further clarify open questions with respect to therapeutic effi- PI of platelet components on the island. The success of this meas- cacy and potential side effects of pathogen-reduced platelets (23). ure demonstrated that PI can effectively support the availability of safe labile blood components during an epidemic. Red Blood Cells The West Nile virus epidemic in the USA was the first example The S-303 system, which is in clinical development, is the only PI of a large-scale arboviral threat to the blood supply of a Western technology available for RBCs. Current studies are investigating country that required an urgent response across government the second-generation S-303 PI process. The first-generation S-303 agencies and non-governmental organizations. The dramatic procedure only marginally affected RBC quality and function, but spread of Zika virus in the Americas since 2015 has generated a after reports of immunization against pathogen-inactivated RBCs sense of public health urgency akin to AIDS, along with immedi- in transfused patients emerged, a new generation of the S-303 ate concerns over blood safety. In areas of active transmission, system had to be developed. In the second-generation S-303 “FDA guidance recommends that blood be outsourced from system, the quencher concentration of GSH was increased from 2 unaffected areas, unless there are measures to screen donations to 20 mmol/l in order to decrease the affinity of S-303 for proteins using a laboratory test, or unless the blood components are and thus to avoid the formation of neoantigens on the surface of subjected to PI technology” with an approved method (29). The erythrocytes (24). However, recent studies show that immuniza- INTERCEPT system was approved by the FDA in 2014 and has tion against S-303-coated RBCs still occurs after modification already been implemented at a number of US blood centers. of the S-303 system (25). In particular, the fact that antibodies against S-303-treated cells were also detected in healthy blood OUTLOOK donors who had never been transfused with pathogen-reduced RBCs suggests that some individuals may be immunized by Despite the increasing and profound safety and efficacy record of S-303-like substances in the environment (e.g., food or air) or pathogen-reduced blood cellular products, there are still concerns may have naturally occurring antibodies against epitopes on the that impede the introduction of PI technology in hemotherapy. S-303 molecule. These data clearly show that the use of chemical The INTERCEPT protocol includes incubation and adsorption agents for PI of cellular products increases the risk of immune steps that result in a significant loss of platelets (up to 15%) during responses against blood components in transfusion recipients. preparation and PI treatment. However, this loss could be offset Various phase III clinical trials to test the second-generation by performing PI with higher platelet counts in the starting prod- S-303 PI system for RBCs in acute and chronic anemia patients ucts. The platelet yields could be increased by using more buffy are currently ongoing or planned. coats (e.g., five instead of four) to manufacture pooled platelets, or by collecting higher numbers of platelets during the apheresis IMPLEMENTATION IN ROUTINE USE process. Moreover, this measure could compensate for reduced platelet CIs in transfusion recipients and thus lower the possible The INTERCEPT and MIRASOL PI systems for platelets and need for increased platelet unit utilization. plasma are used in some parts of Asia, Europe, and the USA. In Europe, the willingness to use pathogen-reduced platelet All PI technologies mentioned in this review exhibit units varies between countries and regions. PI technologies are gaps in their PI efficacy. The amatosalen/UVA-based system implemented nationwide in some countries (e.g., Switzerland (INTERCEPT) is ineffective for non-enveloped viruses such as and Belgium), but only regionally in others (e.g., Poland). hepatitis A, hepatitis E, and parvovirus B19 (30). The riboflavin/ UV-based system (MIRASOL) has only weakly effects against Frontiers in Medicine  |  www.frontiersin.org 644 December 2017 | Volume 4 | Article 219

Seltsam PI Technologies for Blood Products bacteria and some viruses (31). The UVC light-based system the implementation of PI systems for platelets and plasma as an (THERAFLEX) is highly effective against bacteria and most important step toward improving blood safety. A Canadian risk- transfusion-relevant viruses, but only moderately effective benefit analysis suggests that if a new pathogen entered the blood against HIV (32). However, when highly sensitive screening tests supply, the use of pathogen-reduced plasma and platelets would for HIV are performed, UVC-based PI could further reduce the reduce the risk of TTI by 40% (33). risk of virus transmission during the “window period” in which the pre-nucleic acid testing can be negative and in patients with The additional costs of PI implementation may be responsible occult infections. Despite these weaknesses, PI systems generally for the hesitant acceptance of this technology by hospitals and have the potential to significantly add an additional layer of safety funding agencies. Although based on assumptions and simpli- to blood transfusion. fications, the available cost-effectiveness analyses suggest that PI implementation, like other measures for the improvement Major concerns surrounding the implementation of PI have of blood safety, has an acceptable cost–benefit ratio in this to do with its impact on the integrity of blood components and specific application (34, 35). The potential cost savings from the toxicity of the chemicals used in these systems. In particular, PI implementation could offset some costs associated with acute and chronic toxicities may be caused by PI technologies that the technology (e.g., production costs); however, the amount use active chemicals. Although only small quantities of photo- of potential offsetting cost reductions may vary considerably chemical compounds are used in PI technologies and they appear between different countries and regions and must be evaluated to provide sufficient safety margins, it cannot be excluded that on an individual basis for blood centers and hospitals (36). alkylating agents such as amatosalen may be carcinogenic in the Finally, the available resources influence how politicians and long term in a subset of transfused patients. A major advantage health authorities decide on how to meet public concerns for of the THERAFLEX system is that it works without photoactive safety in transfusion medicine. If emerging evidence continues substances, thus eliminating the risk of photoreagent-related to demonstrate the efficacy of PI, it will be difficult to explain adverse events (10, 13). to individuals with severe transfusion-associated infections why this readily available risk mitigation and safety measure was not According to various stakeholders in the field of transfusion implemented. medicine, it is crucial to inactivate pathogens in all blood com- ponents in order to increase the safety margin of the entire blood AUTHOR CONTRIBUTIONS supply. As long as PI is not routinely implemented in the produc- tion of RBC units (the most commonly used blood components), The author confirms being the sole contributor of this work and PI cannot achieve its full potential to enhance blood safety. approved it for publication. Experts and health authorities are increasingly recommending REFERENCES 10. Seltsam A, Muller TH. 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This is an open-access article distributed under the terms and Drug Administration draft guidance for mitigating septic reactions of the Creative Commons Attribution License (CC BY). The use, distribution or from platelet transfusions. Blood Adv (2017) 1(15):1142–7. doi:10.1182/ reproduction in other forums is permitted, provided the original author(s) or licensor bloodadvances.2017008334 are credited and that the original publication in this journal is cited, in accordance 28. Rasongles P, Angelini-Tibert MF, Simon P, Currie C, Isola H, Kientz D, et al. with accepted academic practice. No use, distribution or reproduction is permitted Transfusion of platelet components prepared with photochemical pathogen which does not comply with these terms. Frontiers in Medicine  |  www.frontiersin.org 666 December 2017 | Volume 4 | Article 219

CHAPTER 6: NONINFECTIOUS COMPLICATIONS OF BLOOD TRANSFUSION This chapter addresses four broad categories of transfusion reactions: 1) acute immunoglogic 2) acute nonimmunologic 3) delayed immunologic 4) delayed nonimmunologic complications Manifestations All personnel involved in ordering and administering transfusion must be able to recognize a transfusion reaction so that appropriate actions can be taken promptly. List below are signs and symptoms that are typically associated with acute transfusion reactions and can aid in their recognition. In general, it may be useful to consider any adverse manifestation occurring at the time of the transfusion to be a transfusion reaction until proven otherwise. § Fever with or without chills [generally defined for surveillance purposes as a 1 oC (2 oF) increases in body temperature] associated with transfusion. Fever is the most common symptom of an acute hemolytic transfusion reaction (AHTR), but more frequently it has other causes. § Chills with or without rigors § Pain at the infusion site or in the chest, abdomen, flanks or back § Blood pressure changes, usually acute, either hypertension or hypotension. Circulatory shock in combination with fever, severe chills, and high-output cardiac failure suggests acute sepsis, but may also accompany an acute HTR. Circulatory collapse without fever and chills may be the most prominent finding in anaphylaxis § Respiratory distress, including dyspnea, tachypnea, wheezing, or hypoxemia § Skin changes, including urticaria, rash, flushing, pruritus (itching) and localized edema (angioedema) § Nausea with or without vomiting § Darkened urine. Such as a change may be the earliest indication of an acute hemolytic reaction in anesthetized patients § Bleeding or other manifestations of a consumption coagulopathy § Oliguria/anuria 67

ACUTE TRANSFUSION REACTIONS IMMUNE-MEDIATED HEMOLYSIS Pathophysiology and manifestations The most severe hemolytic reactions occur when transfused RBCs interact with preformed antibodies in the recipient. In contrast, the interaction of transfused antibodies with the recipient’s RBCs rarely causes symptoms, although there may be accelerated red cell destruction. The interaction of antibody with antigen on the red cell membrane can be initiate a sequence of complement activation, cytokine and coagulation effects, and other elements of a systemic inflammatory response that result in the clinical manifestations of a severe acute HTR, and severe symptoms can occur after the infusion of as little as 10-15 mL of ABO-incompatible blood. In anesthetized patients who cannot report symptoms, the initial manifestations of an acute HTR may be hemoglobinuria, hypotension, or diffuse bleeding at the surgical site. Such severe acute HTRs today are usually associated with ABO incompatibility, but were seen with antibodies other than ABO isoagglutinins in the sera when even potent forms of such antibodies were undetectable. In contrast, hemolysis of an entire unit of blood, can occur in the virtual absence of symptoms, and may be a relatively slow process. In such cases hemolysis is presumably due to opsonization of RBCs by antibody followed by cell-mediated destruction without generation of significant systemic levels of inflammatory mediators. Complement activation The binding of antibody to blood group antigens may activate complement, depending on characteristic of both antibody and antigen, including antibody specificity, class, subclasses, titer, and antigen density. C3 activation releases the anaphylatoxin C3a, and red cells coated with C3b are removed by phagocytes with C3b receptors. If the enzymatic cascade proceeds to completion and membrane attack complex is assembled, intravascular hemolysis results, and multiple anaphylatoxins are liberated, including C5a, which is 100 times as potent as C3a. This sequence is characteristic of ABO-associated reactions and causes the cardinal manifestations of hemoglobinemia and, if the renal threshold for hemoglobin is exceeded, hemoglobinuria. Anaphylatoxins interact with a wide variety of cells including monocytes/macrophages, granulocytes, platelets, and vascular endothelial and smooth muscle cells, the latter leading to hypotension and bronchospasm. Anaphylatoxins cause release or production of multiple local and systemic mediators including granule enzymes, histamine and other vasoactive amines, kinins, oxygen radicals, leukotrienes, nitric oxide, and cytokines. These mechanisms may cause manifestations that mimic allergy, such as flushing and rarely urticaria, wheezing and chest pain or tightness, and abdominal pain, nausea and vomiting. 68

With most non-ABO blood group antibodies, complement activation is usually incomplete; hemoglobinemia is absent or mild, but consequences of complement activation, most notably release of anaphylatoxins and opsonization of red cells, may have adverse effects. Cytokines The role of cytokines in inflammatory response, including acute HTRs, is increasingly recognized. The known activities of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1, and interleukin-6 (IL-1, IL-6), and chemoattractant cytokines, such as interleukin-8 (IL-8) and macrophage chemoattractant protein (MCP), suggest that they mediate some of the effects of alloimmune hemolysis. IL-1 and TNF cause fever and hypotension (particularly in synergy), stimulation of endothelial cells to increase expression of adhesion molecules and procoagulant activity, and recruitment and activation of neutrophils and platelets, perhaps through induction of IL-8 and MCP. If the plasma in this model system is treated with heat, cytokine production does not occur, suggesting that complement is involved in the reaction. A similar model of IgG-mediated hemolysis, in which Rh-positive RBCs coated with anti-D is incubated with washed mononuclear cells yield “high-level” responses of IL-8, and MCP, and “low-level” responses of IL-1, IL-6, and TNF. The relevance of this in vitro models to HTRs in vivo are suggested by a case in which TNF and neutrophil elastase levels were found to be elevated when a group O patient received 100 mL of group A RBCs; elevation of neutrophil elastase is consistent with IL-8 activity. These findings are exciting and may lead to new therapeutic options for patients; however, the complete role of cytokines in the consequences of immune hemolysis remains to be defined. Coagulation activation A number of mechanisms, including those listed above, may be responsible for abnormalities of coagulation in HTRs. The antigen-antibody interaction may activate the “intrinsic” clotting cascade through Hageman factor (Factor XIIa) acts on the kinin system to generate bradykinin; bradykinin increases capillary permeability and dilates arterioles, causing a decrease in systemic arterial pressure. Several factors cited above may increase expression of tissue factor by leukocytes and endothelial cells, including activated complement components, TNF, IL-1, and IL-8. Tissue factor activates the “extrinsic” coagulation pathway, and its release is associated with disseminated intravascular coagulation (DIC), which may, in turn, cause: 1) Formation of thrombi within the microvasculature and ischemic damage to tissues and organs. 2) Consumption of fibrinogen, platelets, and Factors V and VIII. 69

3) Activation of the fibrinolytic system and generation of fibrin degradation products. The outcome can be a hemorrhagic diathesis characterized by generalized oozing or uncontrolled bleeding. Shock and renal failure Considering the absolute mass of antigen and antibody, and the list of mediators that may be involved in HTRs including anaphylatoxins, vasoactive amines, kinins, and cytokines, it may not be surprising that shock can occur. Hypotension provokes a compensatory sympathetic nervous system response that produces vasoconstriction in organs and tissues with a vascular bed rich in α–adrenergic receptors, notably the renal, splanchnic, pulmonary, and cutaneous capillaries, aggravating ischemia in these sites. Renal failure is another sequel of an acute HTR. Although free hemoglobin, historically considered the cause of renal failure, does impair renal function, current thought attributes renal failure largely to hypotension, renal vasoconstriction, antigen-antibody complex deposition, and formation of thrombi in the renal vasculature, all of which comprise renal cortical blood supply. Frequency Clerical and other human errors leading to mistaken identity are the most common causes of ABO-incompatible transfusion, occurring either at sample collection or at the time of blood administration; clerical and technical errors may also occur within the transfusion service. A study of reported transfusion errors in New York State over a 10-year period (the 1990s) estimated the incidence rate of ABO-incompatible red cell transfusions at 1:38,000; correction for the expected rate of fortuitously compatible transfusion led to an estimate of the rate of mis-transfusion of 1:14,000. A survey of 3601 institutions by the College of American Pathologists found 843 acute HTRs reported over a 5-year period, of which 50 (6%) were fatal. The Serious Hazards of Transfusion (SHOT) initiative in the United Kingdom and Republic of Ireland reported 62 cases of ABO-incompatible transfusion with three deaths in its first 2 years. In the third year of the study, 35 cases were reported, with one certain and two possible related death. Although no precise denominator is available for these confidential reports, it is believed that over 90% of total transfusions were reviewed and more than 2 million units of red cells were issued during the third reporting period for a rate of no less than 1 in 70,000. However, these values may underestimate the true frequency, because some transfusion errors go unrecognized or unreported. Estimates of mortality rates from acute HTR are generally in the range of 1 in 1,000,000 transfusions. 70

Treatment The treatment of an acute HTR depends on its severity. Vigorous treatment of hypotension and promotion of adequate renal blood flow are the primary concerns; if shock can be prevented or adequately treated, progression to renal failure may be avoided. Adequacy of renal perfusion can be monitored by measurement of urine output, with a goal of maintaining urine flow rates above 100 mL/hr in adults for at least 18-24 hours. The usual first support is intravenous normal saline, but underlying cardiac and/or renal disease may complicate therapy, and it is important to avoid overhydration. Invasive monitoring of pulmonary capillary wedge pressure is recommended in guiding fluid therapy in the face of hemodynamic instability. Diuretics help to improve blood flow to the kidneys and increase urine output. Intravenous furosemide at a dose of 40-80 mg for an adult or 1- 2 mg/kg for a child not only has a diuretic effect but also improves blood flow to the renal cortex. This dose may be repeated once, and the patient should be adequately hydrated. Mannitol, an osmotic diuretic, has been used in the past, but furosemide probably does a better job of maintaining renal cortical blood flow. If no diuretic response occurs within a few hours of instituting fluid and diuretic therapy, there is a strong likelihood that acute tubular necrosis has occurred and further administration may be harmful. Treatment of hypotension of pressor agents that decrease renal blood flow, such as dopamine in higher doses, should be avoided if possible. However, in low dose (less than 5 µg/kg/minute), dopamine increases cardiac output, dilates the renal vasculature, and has been recommended in the management of acute HTRs. Consumption coagulopathy with resultant bleeding or generalized oozing may be a prominent clinical finding in some HTRs and may be the initial presentation in an anesthetized patient. Heparin has been recommended by some, both to forestall DIC when an ABO incompatibility is the first discovered and to treat the established coagulopathy. Others believe the dangers of heparin outweigh its potential benefits, especially because the immune event that provoked the DIC is self-limited. Administration of platelets, Fresh Frozen Plasma (FFP), and Cryoprecipitated AHF, a source of fibrinogen and factor VIII, may be necessary. 71

Categories and management of adverse transfusion reaction Type Incidence Etiology Presentation Acute (<24 hours) transfusion reactions-Immunologic Hemolysis ABO Rh Red cell incompatibility § Chills, fever mismatch: 1 in § Hemoglobinuria 40,000 § Hypotension AHTR: 1 in § Renal failure w 76,000 Fatal HTR: 1 in oliguria 1.8 million § DIC (oozing fro Febrile, non- 0.1 to 1% with § Antibody to donor sites) hemolytic universal WBCs § Back pain, pain Urticaria leukoreduction 1:100-1:33 (1-3%) § Accumulated infusion vein cytokines in § Anxiety platelet unit § Fever § Chills/rigors Antibody to donor § Headache plasma proteins § Vomiting § Urticaria § Prulitis § Flushing 7

Laboratory testing Therapeutic/Prophylactic approach a § Clerical check, DAT § Keep urine output >1 with § Visual inspection (plasma-free mL/kg/hr with fluids and IV om IV diuretic (Furosemide) n along Hb or methemalbumin) § Repeat patient ABO, pre- and § Analgesics (may need morphine) posttransfusion sample § Further tests as indicated to § Pressors for hypotension (Low-dose dopamine) define possible incompatibility § Further tests as indicated to § Hemostatic components (Platelets, Cryoprecipitate, detect hemolysis (LDH, bilirubin, FFP) for bleeding etc.) § Rule out hemolysis § Antipyretic premedication (DAT, inspect for (acetaminophen, no aspirin) hemoglobinemia, repeat patient ABO) § Leukocyte-reduced blood § WBC antibody screen § Rule out hemolysis § Antihistamine (DAT, inspect for § Treatment or medication hemoglobinemia, repeat patient ABO) (PO or IV) § May restart unit slowly after antihistamine if symptoms resolve 72

Type Incidence Etiology Presentati § Hypotension Acute (<24 hours) transfusion reactions-Immunologic § Urticaria Anaphylatic 1:20,000- Antibody to donor § Bronchospasm 1:50,000 plasma proteins (includes IgA, (respiratory d haptoglobin, C4) wheezing) Cytokines § Local edema § Anxiety TRALI 1:2,000- § WBC antibodies in § Hypoxemia 1:190,000 donor (occas. in § Respiratory fa recipient) § Hypotension § Fever § Other WBC § Bilateral pulm activating agents in components edema Note: ACE: Angiotensin-converting enzyme, DIC: disseminated intravascular coagul immunoglobulin, LDH: lactate dehydrogenase, PO: per oral, RBC: red blood cell, SC 7

ion Laboratory testing Therapeutic/Prophylactic approach § Rule out hemolysis (DAT, § Trendelenberg (feet up) inspect for hemoglobinemia, position m repeat patient ABO) § Fluids distress, § Anti-IgA § Epinephrine (adult dose: 0.2- § IgA, quantitative 0.5 mL of 1:1000 solution SC or IM; in severe cases, 1:10,000 IV, initial rate 1mcg/minute) § Antihistamines, corticosteroids, b-2 agonists § IgA-deficient blood components § Rule out hemolysis (DAT, § Supportive care until recovery ailure inspect for hemoglobinemia, § Deferral of implicated donors repeat patient ABO) § Rule out cardiogenic pulmonary edema monary § WBC antibody screen in donor and recipient. If positive, antigen typing may be indicated WBC crossmatch § Chest X-ray lation, DAT: Direct antiglobulin test, Hb: hemoglobin, IV: Intravascular C: subcutaneous, WBC: white blood cell 73

Categories and management of adverse transfusion reaction (cont.) Type Incidence Etiology Presentation Acute (<24 hours) transfusion reactions-Nonimmunologic Transfusion- Varies by § Bacterial contamination § Fever associated components § Chills sepsis § Hypotension Hypotension Dependent § Inhibited metabolism of § Flushing associated on clinical bradykinin with infusion of § Hypotension with ACE setting bradykinin (negatively inhibition charged filters) or § Dyspnea Circulatory <1% activators of prekallikrein § Orthopnea overload § Cough Rare Volume overload § Tachycardia Nonimmune Rare § Hypertension hemolysis Physical or chemical § Headache Air embolus destruction of blood (heating, § Hemoglobinu freezing, hemolytic drug or § Hemoglobine solution added to blood) § Sudden short Air infusion via line breath § Acute cyanos § Pain § Cough § Hypotension § Cardiac arrhy 7

Laboratory testing Therapeutic/Prophylactic approach § Gram’s stain § Broad spectrum antibiotic (until § Culture of component § Patient culture sensitivities completed) § Rule out hemolysis (DAT, § Treat complications (i.e.. shock) inspect for hemoglobinemia, § Withdraw ACE inhibition repeat patient ABO) § Avoid albumin volume § Rule out hemolysis (DAT, inspect for hemoglobinemia, replacement for plasmapheresis repeat patient ABO) § Avoid bedside leukocyte filtration § Upright posture § Chest X-ray § Oxygen § Rule out TRALI § IV diuretic (Furosemide) § Phlebotomy (250 mL increments) n § Identify and eliminate cause uria § Rule out hemolysis (DAT, § Place patient on left side with emia inspect for hemoglobinemia, legs elevated above chest and tness of repeat patient ABO) head § Test unit for hemolysis § X-ray for intravascular vein sis ythmia 74

Type Incidence Etiology Presentation Acute (<24 hours) transfusion reactions-Nonimmunologic § Paresthesia Hypocalcemia Dependent Rapid citrate infusion § Tetany (Ionized on clinical (massive transfusion of § Arrhythmia calcium; setting citrated blood, delayed citrate metabolism of citrate toxicity) apheresis procedures) Hypothermia Dependent Rapid infusion of cold blood § Cardiac arrhy on clinical setting 7

ythmia Laboratory testing Therapeutic/Prophylactic approach § Ionized calcium § Slow calcium infusion while § Prolonged Q-T interval on EKG monitoring ionized calcium levels § Central body temperature in severe cases § PO calcium supplement for mild symptoms during apheresis procedures § Employ blood warmers 75

Categories and management of adverse transfusion reaction (cont.) Type Incidence Etiology Presentation Delayed (>24 hours) transfusion reactions-Immunologic Alloimmunization, 1:100 (1%) § Immune response § Positive blood gr RBC antigens to foreign antigens antibody screenin Alloimmunization, 1:10 (10%) on RBCs, or WBCs test HLA antigens and platelets (HLA) § Platelet refractoriness § Delayed hemolyt disease of the newborn Hemolytic 1;2,500- Anamnestic immune § Fever 1:11,000 response to RBC § Decreasing antigens hemoglobin § New positive antibody screenin test § Mild jaundice Graft-vs-host Rare Donor lymphocytes § Erythroderma disease engraft in recipient § Maculopapular ra and amount attack on host tissues § Anorexia § Nausea, Vomitin § Diarrhea § Hepatitis, Pancytopenia § Fever Posttransfusion Rare Recipient platelet § Thrombocytopen purpura antibodies (apparent purpura alloantibody, usually § Bleeding 8-10 da anti-HPA1a) destroy following transfus autologous platelets 7

Laboratory testing Therapeutic/Prophylactic approach roup § Antibody screening § Avoid unnecessary transfusions ng § Direct antiglobulin test § Leukocyte-reduced blood § Platelet antibody screen § HLA antibody screen tic § Antibody screen § Identify antibody § Direct antiglobulin test § Transfuse compatible RBCs as § Tests for hemolysis (visual needed inspection for hemoglobinemia, ng LDH, bilirubin, urinary hemosiderin as clinically indicated) § Skin biopsy § Corticosteroids, cytotoxic agents ash § HLA typing § Irradiation of blood components for § Molecular analysis for patients at risk (including related g chimerism donors and HLA-selected components) nic § Platelet antibody screen and § IVIG identification § HPA1a-negative platelets § Plasmapheresis ays sion 76

Type Incidence Etiology Presentation Delayed (>24 hours) transfusion reactions-Immunologic Immunomodulation Unknown Incompletely § Increased renal g understood survival interaction of donor § Increased infectio WBC or plasma rate factors with recipient § Postresection tum immune system recurrence rate Delayed (>24 hours) transfusion reactions-Nonimmunologic (controversial) Iron overload Typically Multiple transfusions § Diabetes after >100 with obligate iron § Cirrhosis units of load in transfusion- § Cardiomyopathy RBCs dependent patient 7

Laboratory testing Therapeutic/Prophylactic approach graft § None specific § Avid unnecessary transfusions on § Autologous transfusion mor § Leukocyte-reduced RBCs and platelets § Serum ferritin § Desferioxamine (iron chelator) § Liver enzymes § Endocrine function tests 77

Acute hemolytic reactions are very rare and few clinicians have first-hand experience with their treatment. Because medical management of an acute HTR is often complicated and may require aggressive interventions such as hemodialysis, consultation with physicians experienced in the organ systems most damaged or specialists in critical care medicine may be prudent when treating a patient with a severe acute HTR. Prevention Because clerical errors cause the majority of acute, immune-mediated HTRs, the best hope for prevention lies in preventing or deleting errors in every phase of the transfusion process. In each institution there should be systems designed to prevent or detect errors in patient and unit identification at the time of phlebotomy (sample acquisition), at all steps in laboratory testing, at the time of issue, and when the transfusions are given. The SHOT reports document multiple errors in a majority of mis-transfusion incidents and particularly emphasize the importance of the bedside check at the time of transfusion. Ensuring that all clinical staffs recognize signs of acute reactions and stop the transfusion before a critical volume of blood is administered is essential to preventing harm to the patient. Crucial in the prevention of transfusion mishaps are training and assessment of personnel performing transfusions. Active participation by physicians and management, as well as by nursing, technical, and clinical personnel, is essential. NONIMMUNE-MEDIATED HEMOLYSIS Causes Red cells may undergo in vitro hemolysis if the unit is exposed to improper temperatures during shipping or storage, or is mishandled at the time of administration. Malfunctioning blood warmer, use microwave ovens or hot water baths, or inadvertent freezing may all cause temperature-related damage. Mechanical hemolysis may be caused by the use of roller pumps (such as those used in cardiac bypass surgery), pressure infusion pumps, pressure cuffs, or small-bore needles. Osmotic hemolysis in the blood bag or infusion set may result from the addition of drugs or hypotonic solutions. Inadequate deglycerolization of frozen red cells may cause the cells to hemolyze after infusion. Finally, hemolysis may be a sign of bacterial growth in blood units. In a patient with transfusion-associated hemolysis for which both immune and nonimmune causes have been eliminated, the possibility might be considered that the patient or donor has an intrinsic red cell defect, such as glucose-6-phosphate dehydrogenase deficiency, causing coincidental hemolysis. Treatment Treatment depends on the cause. If the patient develops a severe reaction with hypotension, shock, and renal dysfunction, intensive clinical management is required even before the cause of the mishap is investigated. If the patient exhibits only hemoglobinemia and hemoglobinuria, supportive therapy may be sufficient. 78

Prevention There should be written procedure for all aspects of procuring, processing, and issuing blood, and administering transfusions. All staffs should be trained in the proper use of equipment, intravenous solutions, and drugs used during the administration of blood and blood components. Equipment must be properly maintained and records kept of how and when items are used. Intravenous medications must never be injected into blood bags, unless approved for that purpose, and care must be exercised in selection and used of intravenous access devices. TRANSFUSION-ASSOCIATED SEPSIS Fever (particularly a temperature of 38.5 oC or 101 oF) and shaking chills and hypotension during or shortly after transfusion are the most frequent presenting symptoms of transfusion- related sepsis. In severe cases, the patient may develop shock with accompanying renal failure and DIC. Differential Diagnosis The abruptness of onset and severity of the signs and symptoms associated with transfusion-related sepsis may be very similar to those AHTRs. Mild cases may be confused with FNHTRs. The key to diagnosis transfusion-related sepsis is culturing the same organism from both the patient and the remainder of the component. The returned component should be visually examined in suspected cases of posttransfusion sepsis. Particular attention should be paid to any color changes, especially brown or purple discoloration in an RBC component and bubbles/frothiness in a platelet component. A Gram’s stain should be performed on the returned components. Treatment If transfusion-related sepsis is suspected, the transfusion should be stopped immediately and supportive care of the patient should be initiated. Broad-spectrum antibodies may be indicated. FEBRILE NONHEMOLYTIC REACTIONS Pathophysiology and manifestations A febrile nonhemolytic transfusion reaction (FNHTR) is often defined as a temperature increase of >1oC associated with transfusion and without any other explanation. The 1oC definition is arbitrary; the same events might cause smaller temperature increments without altering the physiologic significance. Indeed, some authors discuss reactions characterized by rigors or other symptoms, in the absence of fever, as FNHTRs because of a presumed common mechanism. Febrile reactions complicate 0.5-6% of red cell transfusions and are often accompanied by chills and/or rigors. Previous opportunities for alloimmunization, especially pregnancies and multiple 79

transfusions, increase the frequency of FNHTRs to red cells. Those patients who receive platelets may experience such reactions more frequently (1-38%). Most FNHTRs are benign, although some may cause significant discomfort and hemodynamic or respiratory changes. The temperature rise may begin early in the transfusion or be delayed in onset for up to several hours after completion of the transfusion. Many febrile reactions to red cells are thought to result from an interaction between antibodies in the recipient’s plasma and antigens present on transfused lymphocytes, granulocytes, or platelets. More recently, evidence has accumulated that febrile reactions, particularly those due to platelets, may also be caused by infusion of biologic response modifiers, including cytokine, that accumulate in the blood bag during storage. Cytokine release in the recipient undoubtedly contributes to those reactions that begin with recipient antibody against donor leukocytes. Because fever may be an initial manifestation of an acute HTR or a reaction to transfusion of blood contaminated with bacteria, any observation of a rise in temperature associated with transfusion warrants prompt attention. The diagnosis of an FNHTR is made after excluding other possible explanations for the fever, particularly a hemolytic or septic reaction. Guidelines for evaluating a suspected acute transfusion reaction are presented later in this chapter. Treatment Traditionally, occurrence of an FNHTR has caused the transfusion to be discontinued. However, some workers believe that fever should not routinely cause discontinuation of a transfusion, depending on whether the patients has symptoms, signs, or laboratory data that suggest hemolysis or bacterial contamination. The fever of an FNHTR usually responds to antipyretics; acetaminophen is preferred to the use of salicylates because the former drug does not affect platelet function. Meperidine injection may be useful in patients with severe shaking chills. Antihistamines are not indicated because most FNHTR do not involve histamine release. Prevention Febrile reactions in an alloimmunized individual can often be prevented by transfusion of leukocyte-reduced blood components; prevention of reactions due to cytokine accumulation during storage requires that the leukocyte reduction be performed prior to storage. ALLERGY; URTICARIA (HIVES) TO ANAPHYLAXIS Pathophysiology and manifestation Allergy reactions to transfusion form a continuum with the vast majority clustered at the mild end, in the form of urticaria or “hives”—erythematous, sharply circumscribed raised lesions, most often present over the upper trunk and neck, which may itch and which are not usually accompanied by fever or other adverse findings. At the other end of the spectrum are anaphylactic reactions, in which there are systemic symptoms including hypotension, loss of consciousness, 80

shock, and, in rare cases, death. The latter may begin after infusion of only a few milliliters, but less severe reactions tend to take longer develop. The term “anaphylactoid” is used in transfusion medicine to denote reactions in between these extremes, but it is also used to denote reactions that have clinical similarities to anaphylaxis but different mechanisms. Manifestations of these reactions may involve one or several systems, notably the skin (urticaria, generalized flushing or rash, localized swelling or “angioedema”), respiratory tract (upper or lower respiratory tract obstruction with cough, hoarseness, stridor, wheezing, chest tightness or pain, dyspnea), the gastrointestinal tract (cramps, nausea, vomiting, diarrhea), or the circulatory system (tachycardia and other arrhythmias including cardiac arrest). Fever is characteristically absent, a feature that aids in differentiating these reactions from hypotension due to a hemolytic reaction or bacterial contamination, and from respiratory compromise due to transfusion-related acute lung injury (TRALI). The severity of allergic transfusion reactions may increase with successive transfusions. Allergic reactions are attributed to exposure to a soluble substance in donor plasma that binds to preformed IgE antibodies on mast cells, resulting in activation and release of histamine. Reasons for this presumption include the facts that reactions tend to recur in an affected recipient and that they can be prevented by removal of the plasma from cellular components or, in the case of urticaria, by antihistamines. Anaphylactic and anaphylactoid reactions are sometimes associated with class, subclass, and allotype-specific antibodies against IgA, and IgE anti-IgA has been demonstrated in two patients with common variable immunodeficiency having reactions to immunoglobulin preparations. However, most of the IgA antibodies to which anaphylactic reactions are attributed, are of the IgG or IgM class, and these antibodies are demonstrable in only a minority of anaphylaxis cases referred for study (17.5% in the series of Sandler et al). Moreover, IgA antibodies are common but anaphylactic reactions are not. Therefore, demonstration of anti-IgA in an individual who has not been transfused does not predict anaphylaxis. Other allergens or other mechanisms are likely. Severe allergic reactions have been reported in patients with antibodies directed against C4 determinates, as well as to elements of nonbiologic origin such as ethylene oxide used for sterilizing tubing sets. However, the causative antigens have not been identified in the vast majority of cases. Reactions due to passive transferred donor antibody have rarely been documented. Hypotensive reactions mimicking anaphylaxis have been observed in patients taking angiotensin-converting enzyme (ACE) inhibitors and receiving albumin during plasma exchange. These were thought to be due to inhibition of bradykinin catabolism by the ACE inhibitors combined with bradykinin activation by low levels of prekallikrein activator (a Hageman factor fragment) in the albumin used for replacement. Similarly, bradykinin activation by prekallikrein activity in plasma protein fraction has also been implicated in hypotensive reactions, and a similar mechanism is probably responsible for the many patients on ACE inhibitors reported to have hypotensive reactions when receiving leukocyte-reduced blood components. Similar reactions have been observed in association with the contact of plasma with charged dialysis membranes, low density lipoprotein adsorption columns, and staphylococcal protein A immunoadsorption columns. Other mechanisms that have been proposed include infusion of complement-derived 81

anaphylatoxins and histamine. The differentiation and appropriate classification of these different reactions will require additional research and refined diagnostic tools. Frequency Urticaria may complicate as many as 1-3% of transfusions, the observed frequency depends on how vigorously it is sought. The incidence of anaphylactic reactions fortunately is low, estimated 1 in 20,000 to 50,000 units. The SHOT data suggest that anaphylaxis is much more common as a complication of plasma and platelet transfusions, than of RBCs, although these reactions may have contributed to the death of a few severely ill patient, they were not a primary cause of death. The mortality rate reported to the FDA is about 1 per year. Treatment If urticaria is the only adverse event noted, the transfusion may be temporarily interrupted while an antihistamine (e.g., diphenhydramine, 25-50 mg) is administered orally or parenterally. If symptoms are mild and promptly relieved, the transfusion may be resumed, provided the interrupted infusion can be completed within the acceptable duration of transfusion. If patient develops severe urticaria, a significant local swelling, respiratory or gastrointestinal symptoms, or hypotension, transfusion should be discontinued. The immediate treatment of an anaphylactic transfusion reaction should be to stop the transfusion and treat hypotension by placing the patient in the reverse Trendelenburg (feet up) position and administering a fluid challenge. If the blood pressure does not improve immediately, epinephrine should be given. In mild to moderate cases, epinephrine (1:1000) should be delivered subcutaneously or intramuscularly in a starting dose of 0.2-0.5 mL in adults, or 0.01 mL/kg in children. This dose may be repeated a second and third time at 5- to 15-minute intervals. In severe reactions (eg, systolic blood pressure below 80 mmHg, laryngeal edema with upper airway compromise, or respiratory failure), the drug should be given intravenously (1:10,000) for the most rapid effect, because drug absorption is unreliable in hypotensive patients. Aerosolized or intravenous b-2 agonists and theophylline may be required in selected patients in whom bronchospasm is unresponsive to epinephrine treatment, or in whom epinephrine is ineffective because of preexisting b-blocker therapy. Oxygen therapy should be administered as require, with endotracheal intubation if there is significant upper airway obstruction continued hemodynamic instability may require invasive hemodynamic monitoring. Under no circumstances should be transfusion be restarted. Coincidental occurrence of myocardial infarction, pulmonary embolism, or other medical catastrophes could present with hypotension and respiratory embarrassment and should be considered. Prevention Recipients who have frequent transfusion-associated urticarial reactions may respond well to administration of antihistamine (e.g., 25 mg of diphenhydramine) one-half hour before transfusion. If antihistamine administration is insufficient, 100 mg of hydrocortisone may be useful. 82

If reactions are recurrent and severe or associated with other allergic manifestations in spite of adequate premedication, transfusion of washed red cells or platelet components, or red cells that have been frozen, thawed, and deglycerolized will usually be tolerated. Patients who have had a prior life-threatening anaphylactic reaction and who are IgA- deficient or have a demonstrated IgA antibody should receive blood components that lack IgA, either by washing or preparation of components from IgA-deficient blood donors. Severe reactions that are not caused by anti-IgA can be prevented only by maximal anti-allergy immunosuppression or washing. A need for red cells may be met by the use of washed or frozen, thawed, and deglycerolized units. Washed platelets are not available and may result in decreased platelet recovery, function, and survival; therefore, after the first reaction, if there is no evidence of IgA mediation, some centers elect to re-challenge the patient under closely controlled circumstances. Prevention of anaphylactoid reactions in patients such as those with thrombotic thrombocytopenic purpura who absolutely required plasma components may be a tremendous challenge if IgA- deficient donors will not suffice. Pretreatment with antihistamine, corticorsteroids, starting with 100 mg of hydrocortisone, and ephedrine may help. Finally, it may be possible to collect and store autologous blood components from patients known to have experienced anaphylactic reactions. TRANSFUSION-RELATED ACUTE LUNG INJURY (TRALI) Pathophysiology and manifestations TRALI should be considered whenever a transfused-recipients experience acute respiratory insufficiency and/or X-ray findings are consistent with bilateral pulmonary edema, but without other evidences of cardiac failure or a cause for respiratory failure. The severity of the respiratory distress is a usually disproportionate to the volume of blood infused. The reaction typically includes chills, fever, and hypotension, usually occurring within 1-2 hours posttransfusion. Implicated components always have contained plasma, but the volume may be as small as that of a unit of cryoprecipitate or RBCs in additive-solution. TRALI may result from multiple mechanisms. Donor antibodies to HLA or neutrophil antigens of the recipients have been demonstrated and are thought to cause a sequence of events that increase the permeability of the pulmonary microcirculation so that high-protein fluid enters the interstitium and alveolar air spaces. Rarely, antibodies in the recipient’s circulation may interact with transfused granulocytes and initiate the same events. Although one would expect causative antibodies to be far more common in recipients than donors, the rarity of TRALI due to recipient antibody might be due to the fact that the pool of target leukocytes is much smaller in a cellular blood component than in a recipient’s circulation. Because specific antibodies may be absent, some cases of TRALI appear to result from other mechanisms. Severe pulmonary reactions are reported after granulocyte transfusions, particularly in patients with known or inapparent lung infections or with conditions likely to allow prompt complement activation. Other factors may include anaphylatoxins C3a and C5a, 83

aggregation of granulocytes into leukoemboli that lodge in the pulmonary microvasculature, or transfusion of cytokines that have accumulated in stored blood components. Recently, reactive lipid products from donor blood cell membranes have been implicated in the pathogenesis of TRALI. These substances accumulate during blood bank storage and are capable of neutrophil priming. A “Two-Hit” model of TRALI is proposed, in which the first hit is a recipient condition such as sepsis or trauma that activates pulmonary capillary endothelium and primes recipient neutrophils, both resulting in sequestration of these cells in the lung vasculature. The second hit is then a donor factors either a specific antibody or another factor such as a lipid agents or cytokine, which triggers the sequestered neutrophils to damage the endothelium, leading to a capillary leak. The incidence rate of TRALI is not known but data from one institution in the 1980s suggested that this complication may occur as frequently as 1 in 5000 transfusions. The passive surveillance data of the third annual SHOT report include 16 TRALI cases (approximately 1 per 190,000 total components). Seven of ten cases studied had at least one donor with a potentially causative HLA or granulocyte antibody. In six of the seven cases, the implicated component was FFP. Treatment If any kind of acute pulmonary reaction is suspected, the transfusion should be stopped immediately and the same unit should not be restarted even if symptoms abate. Clinical management focuses on reversing progressive hypoxemia with oxygen therapy and ventilator assistance, if necessary. Treatment often includes intravenous steroid whose role is unproved. Unlike other forms of acute respiratory distress syndromes, most patients recover adequate pulmonary function within 2-4 days, and the observed mortality is less than 10%. Prevention If antibody in donor plasma can be shown to have caused an acute pulmonary reaction, blood from that donor should not be used for plasma-containing components. No special precautions are needed for the patient if the problem was donor-specific and components from other donors are available. CIRCULATORY OVERLOAD Pathophysiology and manifestations Transfusion therapy may cause acute pulmonary edema due to volume overload. Few data are available on the incidence rate of transfusion-induced circulatory overload in the general population, but young children and the elderly are considered most at risk, and incidence rate of up to 1% have been observed in a study of elderly orthopedic patients. Rapid increases in blood volume are especially poorly tolerated by patients with compromised cardiac or pulmonary status and/or chronic anemia with expanded plasma volume. Infusion of 25% albumin, which shifts large 84

volumes of extravascular fluid into the vascular space, may also cause circulatory overload. Hypervolemia must be considered if dyspnea, cyanosis, orthopnea, severe headache, hypertension, or congestive heart failure occurs during or soon after transfusion. Treatment Symptoms usually improve when the infusion is stop and the patient is placed in a sitting position. Diuretics and oxygen are often indicated and, if symptoms are not relieved, multiple medical interventions may be required, including phlebotomy. Prevention Except in conditions of ongoing, rapid blood loss, anemic patients should receive blood transfusion slowly. Administration of diuretics before and during the transfusion may be helpful. METABOLIC REACTIONS Among the numerous complications that may accompany massive transfusion metabolic abnormalities and coagulopathy are a matter of concern. Patients who are losing blood rapidly may have preexisting or coexisting coagulopathies or develop coagulopathies during resuscitation. Some of all of the following metabolic derangements can depress left ventricular function: hypothermia from refrigerated blood; citrate toxicity; lactic acidosis from systemic underperfusion; and tissue ischemia, often complicated by hyperkalemia. Hemostatic abnormalities may include dilutional coagulopathy, DIC, and liver and platelet dysfunction. CITRATE TOXICITY Pathophysiology and manifestations When large volume of FFP, whole blood, or platelets are transfused rapidly, particularly in the presence of liver disease, plasma citrate levels may rise, binding ionized calcium and causing symptoms. Citrate is rapidly metabolized, however, so these manifestations are transient. Hypocalcemia is more likely to cause clinical manifestations in patients who are in shock or are hypothermic. Prolonged apheresis procedures put patients and occasionally blood donors at some risk. Exchange transfusion, especially in tiny infants who are already ill, requires carefully attentions to all electrolytes. A fall in ionized calcium increases neuronal excitability leading, in the awake patient or apheresis donor, to symptoms of perioral and peripheral tingling, paresthesia, shivering, and lightheadness, followed by a diffuse sense of vibration, tetanic symptoms such as muscle cramps, fasciculations and spasm, and nausea. In the central nervous system, hypocalcemia is thought to increase the respiratory center’s sensitivity to CO2, causing hyperventilation. Because 85

myocardial contraction is dependent on the intracellular movement of ionized calcium, hypocalcemia also depresses cardiac function. Treatment and prevention Massively transfused patients, particularly those with severe liver disease or those undergoing rapid apheresis procedures such as peripheral blood progenitor cell collections, may benefit from calcium replacement. It should be noted, however, that empiric replacement therapy in the era before accurate monitoring of ionized calcium was available, was associated with iatrogenic mortality. Usually, however, unless a patient or donor has a predisposing condition that hinders citrate metabolism, hypocalcemia due to citrate overload requires no treatment other than slowing the infusion. Calcium must never be added directly to the blood container, as the blood will clot. HYPOTHERMIA Pathophysiology and manifestations Ventricular arrhythmias may occur in patients who receive rapid infusions of large volume of cold blood and can be prevented by even crude blood warmers. This effect is presumed to be more likely if the blood is administered via central catheters positioned close to the cardiac conduction system. Hypothermia increases the cardiac toxicity of hypocalcemia or hyperkalemia and can result in poor left ventricular performance. Other complications of hypothermia in clued impaired hemostasis and higher susceptibility to wound infections. Transfusion of a blood volume over 2 hours may double the caloric requirement of a trauma patient. Blood warming is “a must” during massive transfusion of cold blood. Treatment and prevention Hypothermia-induced arrhythmias are reduced by avoiding rapid infusion of cold blood into the cardiac atrium. Generalized effects of hypothermia can be prevented by using blood warmers. AABB Standards for Blood Banks and Transfusion Services mandates that warming does not cause hemolysis and that warming be done only by a device cleared for the purpose by the FDA. Attention to proper protocol is critical during the use of blood warming devices, as overheating of blood can cause hemolysis, which has resulted in fatalities. 86

HYPERKALEMIA AND HYPOKALEMIA Pathophysiology When red cells are stored at 1-6 oC, the potassium level in the supernatant plasma or additive solution increased. Although the concentration in the plasma/anticoagulant portion of a unit of RBCs may be high, because of the small volume, the total extracellular potassium load is less than 0.5 mEq for fresh units and only 5-7 mEq for units at their outdate. This rarely causes hyperkalemic problems in the recipient because rapid dilution, redistribution into cells, and excretion blunt the effect. Hypokalemia is probably more often observed, as potassium-depleted red cells re-accumulate this intracellular ion, and citrate metabolism causes movement of potassium into the cells in response to consumption of protons. Hyperkalemia may be a problem in premature infants and newborns receiving relatively large transfusions, such as in cardiac surgery or exchange transfusion; otherwise it can only be demonstrated a transient effect in vary rapid transfusion. Treatment and prevention No treatment or preventive strategy is usually necessary, provided the patient is adequately resuscitated from whatever condition required the massive transfusion. For large- volume transfusion to sick infants, many workers prefer red cells that are no more than 7-10 days old, but for small-volume transfusions infused slowly, units may be safely used until their expiration date. There is no evidence that routine RBC transfusions require manipulation to lower potassium levels, even in patients with no renal function. COAGULOPATHY IN MASSIVE TRANSFUSION Pathophysiology Of greater concern is the occurrence of coagulopathy during massive transfusion. Classically, this coagulopathy is ascribed to dilution of platelets and clotting factors, which occurs as patients lose hemostatically active blood. The lost blood is replaced with RBCs and asanguinous fluids, or, in the past, by stored whole blood deficient in platelets and, to a variable degree, clotting Factor VIII and V. classic studies of military and civilian trauma patients demonstrated a progressive increase in the incidence of “Microvascular bleeding” (MVB) characteristic of a coagulopathy which increasing transfusion, typically occurring after replacement of 2-3 blood volumes (20-30 units of whole blood). Although platelet counts, coagulation times, and levels of selected clotting factors all correlated with volume transfused, contrary to expectation from a simple dilutional model the relationship was marked by tremendous variability. Inspection of laboratory parameters in the patients developing a bleeding diathesis, as well as the response to various hemostatic components, suggested platelet deficits were more important in causing the bleeding than were coagulation factor deficiencies. MVB typically 87

occurred when the platelet count fell below 50,000 to 60,000/µL. on the other hand, no simple relationship could be determined between a patient’s clotting times and the onset of bleeding. Subsequent studies have refined these observations. Significant platelet dysfunction has been demonstrated in massively transfused trauma patients. In the studies of Counts and coworkers, low fibrinogen and platelet levels were better predictors of hemostatic failure than elevations of prothrombin time (PT) and partial thromboplastin time (PTT), suggesting that consumption coagulopathy was an important factor in addition to dilution. A similar conclusion was reached by Harke and Rahman, who showed that the degree of platelet and clotting abnormalities correlated with the length of time the patient was hypotensive in groups of patient receiving similar transfusion volumes, also suggesting that the most important cause was DIC due to shock. Taking these data together, Collins concluded that “…coagulopathy in heavily transfused patients was due to hypoperfusion, not transfusion”. These data may not be generalizable to patients receiving massive transfusion in the “clean” setting of the operating room, where hypotension due to volume loss is prevented. In this setting coagulation factor levels may indeed have primacy over platelet problems. Treatment and prevention The dilutional model of coagulopathy in massive transfusion would suggest that prophylactic replacement of hemostatic components based on the volume of RBCs or whole blood transfused would prevent development of bleeding diathesis. However, prospective studies have consistently shown that such regimens do not work, perhaps due to patient variability. Instead, replacement of platelets and coagulation factors in the massively transfused trauma or surgical patients should be based on characterization of the specific abnormality by use of platelet counts, the PT and PTT, and fibrinogen and fibrin degradation product levels. In such critical cases, it is imperative that the laboratory rapidly complete testing. AIR EMBOLISM Air embolism can occur if blood in an open system is infused under pressure of if air enters a central catheter while containers or blood administration sets are being changed. It has been reported in association with intraoperative and perioperative blood recovery systems that allow air into the infusion bag. Symptoms include cough, dyspnea, chest pain, and shock If air embolism is suspected, the patients should be placed on the left side with the head down, to displace the air bubble from the pulmonic vein. Aspiration of the air is sometimes attempted. Air embolism was more of a threat when blood came in glass bottles than it is with plastic collection and administration systems. Proper use of infusion pumps, equipment for blood recovery or apheresis, and tubing couplers is, however, still essential to prevent this transfusion complication. 88

EVALUATION OF A SUSPECTED ACUTE TRANSFUSION REACTION The role of clinical personnel attending the patient Medical personnel attending the patient are generally the first to suspect that a transfusion reaction has occurred and the first to take action. The appropriate actions should be specified in the institution’s patient care procedures manual, and transfusion service personnel should be prepared to act as consultants. 1. If a transfusion reaction is suspected, the transfusion should be stopped to limit the volume of blood infused. 2. All labels, forms, and patient identification should be checked to determine whether the transfused component was intended for the recipient. 3. An intravenous line should be maintained with normal saline (0.9% sodium chloride), at least until an evaluation of the patient has been completed. 4. The transfusion service and the patient’s physician should be notified immediately. A responsible physician should evaluate the patient to determine whether a transfusion reaction is a possibility, what kind it might be, and what immediate actions should be undertaken. The possibilities of acute hemolytic reaction, anaphylaxis, transfusion- induced sepsis, and TRALI should be kept in mind, because these conditions require aggressive medical management and must be reported promptly to the laboratory. 5. If the observed events are limited to urticaria or circulatory overload, the transfusion service need not evaluate postreaction blood samples. If there are signs and symptoms other than urticaria or circulatory overload, particularly if there is any possibility of acute HTR, anaphylaxis, TRALI, transfusion-induced sepsis, or other serious problem, a postreaction blood sample(s) should be sent to the laboratory for evaluation. The specimen(s) must be carefully drawn to avoid mechanical hemolysis and be properly labeled. In addition, the transfusion container with whatever contents remain, the administration set (without the needle), and the attached intravenous solutions should be sent to the laboratory, following standard precautions. In some cases, a postreaction urine sample will be useful. The role of the laboratory The laboratory should perform three steps as soon as possible after receiving notification and clinical material, regardless of the symptoms and the type of component(s) implicated: check for clerical errors, check for hemolysis, and check for evidence of blood group incompatibility by performing a direct antiglobulin test (DAT) Check for clerical errors The identification of each patient’s sample and the blood component(s) must be checked for errors. If an error is discovered, the patient’s physician or other responsible health-care professional must be notified immediately, and a search of appropriate record should be initiated 89

to determine whether misidentification or incorrect issue of other specimens or components has put other patients at risk. Once the acute crisis has passed, each step of the transfusion process should be reviewed to find the source of error. Visual check for hemolysis The serum or plasma in a postreaction blood specimen must be inspected for evidence of hemolysis and compared with a pre-reaction sample, if available. Pink or red discoloration after, but not before, the reaction suggests destruction of red cells and release of free hemolglobin. Intravascular hemolysis of as little as 5-10 mL of red cells may produce visible hemoglobinemia. Hemolysis resulting from poor collection technique or other medical interventions can cause hemoglobinemia; if faulty sampling is suspected, examination of a second specimen should resolve the question. Myoglobin, released from injured muscle, may also cause pink or red plasma and might be suspected if a patient has suffered severe trauma of muscle injury. If the sample is not drawn until 5-7 hours after an episode of acute hemolysis, hemoglobin degradation products, especially bilirubin, may be in the blood stream and cause yellow or brown discoloration. An increase in bilirubin may begin as early as 1 hour after the reaction, peak at 5-7 hours, and disappear within 24 hours if liver function is normal. In examining a postreaction urine specimen, it is important to differentiate among hematuria (intact red cells in the urine), hemoglobinuria (free hemoglobin in the urine), and myoglobinuria (free myoglobin in the urine). In acute HTRs, free hemoglobin released from damaged cells can cross the renal glomeruli and enter the urine, but hematuria and myoglobinuria would not be expected. Urine examination should be done on the supernatant fluid after centrifugation of a freshly collected specimen; misleading free hemoglobin may be present if previously intact red cells in a specimen undergo in vitro hemolysis during transportation or storage. Serologic check for incompatibility A DAT must be performed on a postreaction specimen, preferably one anticoagulated with a chelating agent (such as EDTA) to avoid in vitro coating of red cells by complement proteins. If the postreaction DAT is positive, a DAT should be performed on red cells from the pre-transfusion specimen (unless this had already been done as part of pretransfusion testing) and compared. If transfused incompatible cells have been coated with antibody but not immediately destroyed, the postreaction specimen DAT is likely to be positive, often with a mixed-field agglutination pattern. If the transfused cells have been rapidly destroyed, the postreaction DAT may be negative, particularly if the specimen is drawn several hours later. Nonimmune hemolysis, e.g. from thermal damage or mechanical trauma, causes hemoglobinemia but not a positive DAT. 90