Chapter 12: Transfusion Medicine

Transfusion medicine is the field of medicine that encompasses blood banking (the collection, preparation, testing, and storage of blood components and plasma derivatives) as well as the therapeutic uses of blood components, plasma derivatives, and apheresis technology. It also includes the collection, storage, and use of hematopoietic and other blood-derived cells. An overview of the steps from collection of the blood to transfusion of its components is shown in Figure 12–1. Briefly (with more complete descriptions to follow), blood is collected as whole blood or by apheresis from screened, volunteer donors, and samples of the blood are tested for infectious diseases and to determine the blood type. Whole blood may be fractionated into packed red blood cells (RBCs), platelets, and a plasma product. Alternatively, all 3 components can be collected by apheresis. Plasma can be further processed to provide albumin, clotting factor concentrates, and immunoglobulin preparations. The transfusion of blood components requires testing to be done to establish compatibility between the product and the intended recipient. Blood components may also be treated to reduce complications of transfusion (eg, remove leukocytes to prevent febrile reactions). As complex, biologically derived therapeutic agents, blood components and derivatives are responsible for a variety of potential untoward effects that must be evaluated and managed. The entire process, from blood collection to transfusion and posttransfusion evaluation, is described in this chapter (see Figure 12–1).

Blood Collection

The cornerstone of a safe blood supply is the volunteer blood donor who is motivated by altruism. In the past, the use of paid donors was associated with increased levels of transfusion-transmitted hepatitis. Concerns remain about the impact of significant financial incentives on the frank disclosure of health problems or high-risk behaviors that might disqualify a potential blood donor. In the United States, virtually all of the blood is collected from volunteer donors. Regional blood centers collect and distribute more than 90% of the US blood supply while hospital blood banks collect the remainder. The Food and Drug Administration (FDA) Center for Biologics Evaluation and Research regulates all aspects of blood collection and processing, but most blood banks and donor centers are also accredited, on a voluntary basis, by the American Association of Blood Banks (AABB). Blood donors are screened for behaviors or medical conditions that might make blood donation unsafe for them (eg, anemia, coronary artery insufficiency) or the donated blood hazardous for the transfusion recipient (eg, exposure to viral hepatitis, use of a teratogenic medication). The AABB has developed, and the FDA has sanctioned, a Uniform Donor Health Questionnaire that is in wide use in the United States and reflects the consistency of donor criteria throughout the country. To qualify for blood donation, the prospective donor must also pass a basic physical screening that includes temperature, blood pressure, pulse, and examination of the arms for signs of intravenous drug use, and have a hemoglobin level of at least 12.5 g/dL from a finger-stick or venous blood sample. The collecting agency must check its records to be sure that the donor has not previously been disqualified from donating.

In the process of blood collection, the venipuncture site is disinfected and a needle, which is connected to a collecting set, is inserted into a vein in the arm. The collecting set includes a primary collection bag and several integrally connected satellite bags that are used to make components. The primary collection bag contains a solution that includes an anticoagulant (citrate) and a variety of substances, such as phosphate, adenine, and dextrose, which improve the recovery of the RBCs when transfused and permit their storage in the refrigerator for 35 to 42 days. The typical donation is approximately 450 to 500 mL of whole blood, but may not exceed 10.5 mL/kg, and must be collected in no more than 10 minutes if a unit of platelets is to be made. This volume of blood represents approximately 10% of the total blood volume of a donor weighing 70 kg, and its loss is well tolerated by healthy individuals. Samples of blood for serologic and infectious disease testing are also obtained at the time of donation, often by collecting the first 15 to 25 mL of blood drawn, which has the advantage of diverting blood most likely to be contaminated with skin bacteria away from the collection bag. Once the collection is complete, the needle is withdrawn from the donor's arm, and the tubing connecting the needle to the primary collection bag is heat-sealed off.

Component Preparation

Almost all of the whole blood collected is separated into its components—RBCs, platelets, and plasma—in order to be able to store each under optimal conditions. This separation process entails 2 centrifugation steps and relies on the system of integrally connected satellite bags to carry out all of the preparation steps in a closed, aseptic environment (see Table 12–1 for a description of blood components, and Chapter 2 for a diagram of blood component preparation). In the procedure used in the United States, RBCs are separated from platelet-rich plasma by the first, relatively low g force, centrifugation step. The platelet-rich plasma is expressed from the primary collection bag into one of the satellite bags that is heat-sealed off from the packed RBCs in the primary collection bag. The packed RBCs remaining in the primary collection bag may be stored for up to 35 days at 1°C to 6°C (CPDA-1 RBCs). In some collection systems, additional additive–preservative solution from another satellite bag may be added to the packed RBC, creating a product with a lower hematocrit (but identical RBC mass) and an additional week of storage (42 days).

The platelet-rich plasma, which was expressed off the packed RBC after the first spin, is then separated into platelets and plasma by a second, higher g force, centrifugation step. The platelet-poor plasma is expressed into another satellite bag after this second centrifugation step and is usually frozen within 8 hours of collection (as fresh frozen plasma [FFP]) or within 24 hours of collection (24-hour plasma). The platelet pellet that remains is suspended in 40 to 60 mL of plasma and is called a platelet concentrate or whole blood-derived platelets. The term “random donor platelets,” although commonly used, is inaccurate. Platelets are stored at 20°C to 24°C for up to 5 days, whereas the various plasma-derived components are stored frozen (≤–18°C for 1 year; ≤–65°C for 7 years). Since 1 transfusion dose for an adult patient consists of 4 to 10 units of platelet concentrates, it is common to use commercially available closed systems to pool and leukoreduce them.

FFP can be used to prepare another useful component, called cryoprecipitated antihemophilic factor (or “cryoprecipitate”). When FFP is thawed at 1°C to 6°C, a precipitate forms. Most of the plasma can be expressed into a satellite bag, leaving behind this cryoprecipitate that is suspended in 10 to 20 mL of plasma, refrozen, and then stored (≤–18°C for 1 year). Cryoprecipitate contains about half of the Factor VIII, von Willebrand factor, Factor XIII, and fibrinogen that was present in the original unit of plasma, but in a much smaller volume. The plasma remaining after the cryoprecipitate has been removed may also be refrozen. “Plasma–cryoprecipitate reduced” (or cryo-poor plasma) may be used as the starting material (source plasma) for the preparation of plasma derivatives such as albumin and immunoglobulins, and occasionally for replacement during plasmapheresis for patients with thrombotic thrombocytopenic purpura (see Table 12–2). Thus, each unit of whole blood can be separated into a unit of packed RBCs, a platelet concentrate, and either FFP or cryoprecipitate plus some other plasma product. The different plasma products (absent cryoprecipitate) may all be used as source plasma for the preparation of derivatives, such as albumin or immunoglobulin.

Blood components also may be donated by a procedure known as apheresis, in which whole blood is removed from the donor, the component of interest (plasma or platelets most commonly, but RBCs as well) is removed, and the remaining blood elements are returned to the donor (see Chapter 2 for a diagram of apheresis). This procedure may be done manually, but is now usually carried out using an automated device. Using an apheresis instrument, whole blood is drawn from the donor's vein as an anticoagulant solution (usually citrate) is added, and pumped into a centrifuge where it is separated into its components. The component of interest is drawn into a collection bag, and the rest of the blood is returned to the donor via the same or a different vein. This process may be discontinuous (filling the instrument, separating the components, returning the residual blood, and repeating the cycle) or continuous (using separate lines to draw blood into the instruments and return it to the donor). The entire extracorporeal circuit is sealed except at the points of contact with the donor's vein(s). Apheresis is commonly used to obtain plasma, usually for further processing into derivatives such as albumin, clotting factor concentrates, and immunoglobulins, as well as to obtain platelets. A unit of apheresis platelets (commonly called “single donor platelets”) contains more platelets than a unit derived from a whole blood donation. Transfusion of a unit of apheresis platelets, which must contain at least 3 ×10¹¹ platelets, usually elevates an adult patient's platelet count by 30,000 to 50,000 platelets/μL. Since 1 unit of whole blood-derived platelets must only contain 5.5 × 10¹⁰ platelets, the usual adult dose is 4 to 10 units or 1 unit/10 kg. Whole blood-derived platelets are less expensive to prepare than apheresis platelets because they do not require special equipment for their isolation. However, apheresis platelets can easily be prepared in such a way that they contain very few residual white blood cells (“process leukoreduced”), which is an advantage for some patient groups.

Granulocytes also can be harvested by apheresis for transfusion to patients who are neutropenic and suffering from severe infection. Instruments designed to collect RBCs by apheresis (typically 2 units at a time if the donor meets the somewhat more stringent size and hematocrit requirements) or various combinations of RBCs, platelets, and plasma are also in use. Finally, apheresis is used to collect peripherally circulating hematopoietic progenitor cells (HPCs) for autologous and allogeneic HPC transplantation.

Testing of Donated Blood

Donated blood is held in quarantine following collection while a variety of laboratory tests are performed using blood specimens obtained from the donor. The ABO and Rh types are determined on an RBC sample obtained at each donation, and the donor serum or plasma is screened for the presence of unexpected RBC alloantibodies. The concern is that such alloantibodies could cause destruction of a transfusion recipient's RBCs if they express the target antigen. Plasma or platelets from a donor with an alloantibody are not used for transfusion, although RBCs are generally safe, particularly if they have been saline washed. Records from any previous donations, including the results of ABO and Rh typing, are also checked, to reduce the opportunity for donor or unit misidentification.

Infectious Disease Testing

Transmission of viruses, bacteria, and parasites by transfusion of blood components has been well documented. To minimize infectious disease transmission, blood donors are screened for evidence of infection and for participation in activities that may have exposed them to infectious agents. In addition, each blood donation is subjected to several tests for infectious agents before it is made available for transfusion. The tests required for each donation are shown in Table 12–3. Platelets, which are stored at 20°C to 24°C, must also be screened for evidence of bacterial contamination, which is currently responsible for the majority of transfusion-transmitted infections. Several commercial systems have been licensed for the testing of leukoreduced apheresis and whole blood-derived platelets. All donors must be screened once for antibody to Trypanosoma cruzi, the organism that causes Chagas disease, and deferred indefinitely if they are positive. Thereafter, donors do not need to be retested unless they have a possible exposure, that is, residence in an endemic area of South or Central America.

Pretransfusion Testing to Assess Donor/Recipient Compatibility for Blood Components Containing RBCs

Prior to transfusion, the compatibility of donor RBCs with the intended transfusion recipient must be established (see Tables 12–4 and 12–5 for RBC compatibility issues). Part of this process involves various serologic tests. However, an equally important part of this process is the proper identification of the patient when the blood bank specimen is obtained, and again when the transfusion is initiated. Misidentification of patients and mislabeling of specimens are the most common serious errors encountered in transfusion. ABO mistransfusion as a result of this kind of error is far more frequent than the transmission of HIV and all of the hepatitis viruses, combined. Compatibility testing includes:

• The identification of patient and proper labeling of the specimen for compatibility testing. The blood bank specimen (tube of blood) must be labeled at the bedside using the patient's armband for identification. The label must include 2 patient identifiers (typically name and medical record number) and the date. There must also be some means of identifying the phlebotomist (commonly, but not necessarily, by signing or initialing the tube or requisition).

• The determination of the ABO and Rh type of the donor. The collecting facility determines the ABO group (by front- and back-typing as described below) and Rh type of the donated unit (and checks prior records). The hospital transfusion service must confirm the ABO group (front type only) and the Rh type of Rh(D)-negative units that have been received from the collecting facility.

• The determination of the ABO and Rh type of the patient on a current specimen, and a comparison to previous records, if any. The ABO group (front and back types) and Rh type are determined on a current specimen. The specimen must be <3 days old for any patient who has been transfused or pregnant within the last 3 months; however, many transfusion services require new specimens every 3 days to keep things simple.

• A screen of the recipient's serum/plasma for unexpected RBC alloantibodies. If unexpected antibodies (ie, not anti-A or anti-B) are found, as described below, the antigen specificity of these antibodies must be identified to establish the risk of a hemolytic transfusion reaction (HTR) and to help identify potentially compatible donor RBCs that lack the target antigen. A record check for previously identified alloantibodies must also be made.

• The performance of a crossmatch (see Chapter 2 for illustration of the crossmatch). Several techniques for performing the crossmatch are described below.

• The identification of the patient when the transfusion is initiated. The patient must once again be properly identified using the armband to be sure that the unit is intended for the patient. The armband is the only link between the patient, the specimen, and the blood component.

ABO Grouping

After the identification of the patient and the specimen for compatibility testing, the most important step in assuring the safety of an RBC transfusion is the determination of the ABO group of the donor unit and the intended recipient. The specificity of the A and B blood group antigens lies in the presence of carbohydrate structures that are borne by membrane-associated glycoproteins and glycolipids. The A gene encodes a glycosyltransferase that attaches an N-acetylgalactosamine residue to the core structure (called “H”) while the B gene encodes an enzyme that transfers a galactose residue. These 2 different residues impart A or B serologic activity, respectively, to the glycoprotein or glycolipid core structure. The O gene, which is a phenotypic recessive, does not encode for an active enzyme, so the RBC of people who are group O are coated with an unmodified H structure. Since the A and B genes are codominant, people who inherit 1 copy of each will have both enzymes, and thus both A and B antigens will be expressed on their RBCs.

During the first year of life, individuals begin to make antibodies to whichever A and B antigens they lack. Thus, a person with A antigen on his or her RBCs (group A) has naturally occurring anti-B antibodies in the plasma (see Table 12–4). It is the presence of these antibodies (called isoagglutinins because of their ability to agglutinate RBCs in vitro) that makes ABO mistransfusion so hazardous. The isoagglutinins are largely IgM and fix complement readily. Hence, they can cause intravascular hemolysis.

Because determining the ABO group is so critical, it is required not only to test for A and B antigens on the RBCs but also to demonstrate the presence of the appropriate anti-A and anti-B isoagglutinins in the plasma or serum (see Chapter 2 for a diagram of ABO and Rh typing). The presence of A or B antigens on patient or donor RBCs is detected by combining them with reagent “anti-A” in 1 test tube and reagent “anti-B” in another test tube, and then assessing RBC agglutination. Agglutination with anti-A, for example, indicates the presence of A antigen on the RBCs. This test is called the “front” or “cell” typing. The plasma of a patient or donor is tested for the presence of anti-A or anti-B antibodies by combining the plasma with reagent RBCs known to be either group A or group B, and then assessing RBC agglutination. Agglutination of the reagent B cells indicates the presence of anti-B isoagglutinin in the plasma, which would be the expected finding in a person who is blood group A. This test is called the “back” or “serum” typing. The results of the front- and back-typing must be congruent.

Rh Typing

The second most important antigen system with respect to transfusion safety is the Rh system. Approximately 85% of Caucasians express the D (or Rh) antigen and are called D (or Rh)-positive (see Table 12–4). Rh-negative individuals, who lack the D antigen, are vulnerable to development of an alloantibody to the D antigen, the most immunogenic antigen on human RBCs, if they are exposed to D-positive RBCs by transfusion or, for a woman, by maternal–fetal hemorrhage. Anti-D is the most common cause of severe hemolytic disease of the newborn, although the frequency of this complication of pregnancy has been considerably decreased since the advent of Rh immune globulin. This product is an immunoglobulin fraction obtained by pooling the plasma of people with high-titer anti-D. When given by intramuscular injection to individuals who have been exposed to D-positive RBCs (eg, women pregnant with a D-positive fetus), it reduces the chance of sensitization presumably by binding to D-positive fetal cells, leading to their rapid clearance from the maternal circulation before an immune response can be generated (see the section “Hemolytic Disease of the Newborn [HDN]” in Chapter 10).

The Rh type is determined by incubating the RBCs with a reagent antibody to the D antigen. Rh-positive cells expressing the D antigen are agglutinated by the reagent antibody. RBCs that do not agglutinate in the presence of the Rh antibody are incubated a second time, usually after the addition of an enhancer of agglutination. RBCs that do not agglutinate after this second step are considered to be Rh(D)-negative. A small number of people have RBCs that do not agglutinate in the first step but are agglutinated after the second, enhanced, incubation step. These individuals are considered to have the weak D (formerly D^u) phenotype. Donors, and usually patients as well, who are weak D are treated as if they are D-positive, since some weak D RBCs can elicit the formation of anti-D alloantibodies in D-negative individuals, or can be the target for anti-D alloantibodies.

The RHD gene is located on chromosome 1 immediately adjacent to the highly homolgous RHCE gene, and the 2 are inherited as a haplotype exhibiting linkage dysequilibrium. The RHD gene encodes for the D protein that expresses D (“Rh”) antigenic activity. The most common mechanism for the Rh(D)-negative phenotype (especially among people of Caucasian background) is the complete absence of the RHD gene. This phenotype is often represented as “d,” but in fact there is no “d” gene or “d” protein. These individuals lack the D protein altogether. The RHCE encodes for a protein that is structurally very similar to the D protein, but carries 2 different antigens, each of which has 2 common, codominant alleles: C/c and E/e. Since these genes are inherited as a haplotype, a shorthand nomenclature is in wide use and is shown in Table 12–5.

The Antibody Screen and the Indirect Antiglobulin Assay Used to Detect Antibodies

To determine if the patient has an alloantibody to a RBC antigen, an antibody screen is performed. In this test, the patient's serum or plasma is combined with 2 or 3 reagent RBCs that are specifically chosen because they bear a number of the antigens to which clinically significant RBC alloantibodies are made. These cells are group O so that they will not be agglutinated by the anti-A or anti-B isoagglutinins that may be present. If the patient serum does not produce agglutination of the reagent screening cells, then no unexpected RBC alloantibodies are present.

Although the anti-A and anti-B isoagglutinins are predominantly IgM and readily produce agglutination in vitro, most of the other clinically significant RBC alloantibodies are IgG and do not. To detect IgG alloantibodies, an assay called the indirect antiglobulin test (formerly the indirect Coombs test) is used in the antibody screen (see Chapter 2 for a diagram of the indirect antiglobulin test). In this technique, the patient's serum is combined with the reagent screening cells, often in the presence of an additive, such as low ionic strength saline or polyethylene glycol, which promotes binding of antibody to RBCs, and the mixture is incubated at 37°C. If an RBC alloantibody is present, it will bind to the screening cell with the target antigen. The cells are then washed with saline, and the “antiglobulin reagent” is added. Antiglobulin reagent consists of a mixture of antibodies to IgG and/or complement. These antibodies bind to any IgG or complement attached to the screening cell. By binding to IgG or complement on adjacent target cells, the anti-IgG “crosslinks” the RBCs and produces RBC agglutination in vitro. It is called the indirect antiglobulin test because it requires first an incubation with the alloantibody (the serum sample) followed by a second step when the antiglobulin reagent is added. The antiglobulin test is commonly performed in a test tube, but has also been adapted to assays based on solid phase or gel column techniques.

If 1 or more of the screening cells is agglutinated by the patient's serum, indicating the presence of an RBC alloantibody, steps must be taken to identify its specificity by determining its target antigen. This is accomplished again using the indirect antiglobulin test and adding the patient's serum to a panel of group O RBCs (typically around 10) that have been chosen to express the target antigens of the most commonly encountered clinically significant alloantibodies. The pattern of which panel cells are agglutinated in the indirect antiglobulin test can be used to determine the antigen to which the patient's alloantibody is directed. Based on the accumulated clinical experience with alloantibodies of a given specificity, it is usually possible to predict the likelihood that a particular alloantibody will cause a hemolytic transfusion reaction (HTR) or hemolytic disease of the newborn (see Table 12–6). If the alloantibody has the potential of causing hemolysis, donor RBCs that lack the target antigen must be chosen for transfusion. The typing of RBCs for specific antigens is accomplished in a manner similar to that used for the determination of the ABO and Rh type using commercial antisera directed at specific antigens. Patients who have multiple alloantibodies, or alloantibodies directed at high-frequency antigens, may pose the challenge that very few donors will lack the target antigen(s). Under these circumstances, the blood bank may have to screen the red cell inventory for antigen-negative units, request their blood supplier to do the same, or, in some instances, locate suitable units through a national rare blood registry.

The RBC Crossmatch

There are 3 crossmatch techniques in common use: the antiglobulin technique crossmatch, the immediate spin crossmatch, and the electronic crossmatch.

The antiglobulin crossmatch was the standard for years and still must be performed when a patient has an RBC alloantibody or even a history of having had one (see Chapter 2 for a diagram of the blood crossmatch). This crossmatch procedure is very similar to the antibody screen and is based on the indirect antiglobulin technique, except in this case the patient's serum is combined with RBCs from the donor unit. If the patient has an alloantibody to the donor RBCs, the antibody will become bound to the donor RBCs during the incubation step, and the cells will be agglutinated by the antiglobulin reagent added in the final step. If agglutination occurs, the crossmatch is incompatible and the unit of RBCs should not be transfused to that patient. If the RBCs from this donor were mistakenly transfused, they would be destroyed prematurely, that is, an HTR would occur. If there is no agglutination, the patient does not have alloantibodies to the antigens present on this donor's RBCs, and the crossmatch is compatible.

The immediate spin crossmatch is done by combining the patient's serum with a sample of the donor RBCs intended for transfusion, centrifuging them without incubation or the use of the antiglobulin reagent, and observing them immediately for agglutination. This technique detects ABO incompatibilities, but is not sensitive to the presence of other RBC alloantibodies. It may only be used in patients who do not have unexpected alloantibodies (ie, they have a negative antibody screen), in massive transfusion (transfusion of the equivalent of 1 entire blood volume), and in emergency circumstances when an abbreviated crossmatch procedure is imperative for providing blood rapidly.

In the electronic crossmatch, blood bank personnel rely on the computer to verify the ABO (and Rh) compatibility between donor RBCs and the patient. A number of requirements must be met by the information system and the bench procedures involved in the typing, and extensive validation must be performed. This technique is again only suitable for patients who do not have unexpected RBC alloantibodies, or in emergency situations.

Direct Antiglobulin Test (Formerly Direct Coombs Test)

This test detects the presence of IgG or complement that is bound, in vivo, to the patient's RBCs, by using antiglobulin reagent specific for IgG or various complement components including C3b, C3d, and/or C4d. In this technique, the patient's RBCs are washed with saline, and the antiglobulin reagent is then added directly (hence the name of the test) (see Chapter 2 for a diagram of the direct antiglobulin test). The cells are observed for agglutination after incubation. The presence of RBC coated with immunoglobulin and/or complement is evidence of immune-mediated hemolysis. Disorders associated with a positive direct antiglobulin test include hemolytic disease of the newborn, autoimmune hemolytic anemia, and drug-induced hemolytic anemia. A positive direct antiglobulin test result is also observed in patients experiencing an HTR where donor RBCs are circulating coated with the recipient's alloantibody. Note that most patients with positive direct antiglobulin tests do not have hemolysis. A positive direct antiglobulin test is found in many patients with lymphoproliferative and autoimmune disorders, or who are taking various medications such as procainamide, vancomycin, and drugs in the penicillin and cephalosporin families.

If a patient has an RBC autoantibody, especially if it is an IgG, it may interfere with routine serologic testing, especially any test that is based on the indirect antiglobulin technique such as the antibody screen and the crossmatch. Absorption techniques are used to remove the autoantibody from the patient's plasma, but leave any alloantibody behind. In the autologous absorption technique, the patient's RBCs (after treatment to remove any autoantibody) are incubated with the patient's plasma. The autoantibodies bind to the patient's RBC, but any alloantibodies present do not since the patient lacks those antigens, by definition. After absorbing out all of the autoantibody (which may take a few cycles with fresh batches of the patient's RBCs) the autoabsorbed plasma can be tested for alloantibodies using the conventional antibody screen described above. If the patient has been recently transfused, however, this absorption technique cannot be used, because the transfused cells might absorb some of the alloantibody as well as the autoantibody if they happen to bear the target antigen. In this case the RBCs used for the absorption may be phenotypically matched to the patient, or several cells may be chosen, each of which displays a different array of RBC antigens—hence the name, the heterologous absorption technique. The sample of patient plasma that was absorbed by the heterologous cells is then also tested for RBC alloantibodies using the conventional antibody screen described above.

Compatibility Testing for Other Blood Components That Do Not Contain RBCs

Compatibility testing for blood components without RBCs (ie, platelets and plasma) is much less complex than it is for products with RBCs since no crossmatching must be done. ABO grouping of donor units and the patient must be performed to avoid the transfusion of plasma that is ABO-incompatible with the recipient's RBCs. The amount of anti-A and/or anti-B isoagglutinin in a unit of apheresis platelets or FFP (200-300 mL) could lead to destruction of some of the recipient's RBCs if there were an ABO mismatch. Rh-negative recipients may receive plasma products or apheresis platelets from a donor of any Rh type, since these components do not contain RBCs. Whole blood-derived platelets from Rh-negative donors may be preferentially selected for Rh-negative patients, particularly if there is visible RBC contamination of the platelet product, to avoid the possibility of alloimmunization to the D antigen.

Molecular Techniques in Immunohematology

In the last 20 years, molecular techniques have greatly increased our understanding of blood group antigen structures and their genetics, and have explained many of the serologic conundrums that baffled blood bankers for decades. Although not in routine use in the hospital transfusion service at this point, a widening array of applications has been making its way into the clinical arena. Some of these applications include:

1. Genotyping blood donors—Microarray-based platforms and mass spectrometry techniques have been used to genotype large numbers of blood donors for a number of the most common clinically significant antigens. The availability of this information facilitates the identification of donor units for patients who require RBC with a specific phenotype.

2. Genotyping patients—Similar technology can be applied to individual patients in circumstances when it is difficult to obtain a reliable phenotype by serologic methods, for example, in patients who have already been transfused or who have autoantibodies.

3. Hemolytic disease of the newborn—Detection of the RHD gene in the fetus of an alloimmunized mother can be performed using amniotic fluid or maternal plasma, thereby establishing whether or not the fetus is at risk for hemolytic disease of the newborn. The absence of the RHD gene in the fetus also obviates the need for additional, more invasive testing of the fetus, such as per-umbilical blood sampling. It is also possible to determine whether or not the father is homozygous for the RHD gene.

4. Genotyping in the absence of serologic reagents—Typing sera for some clinically significant blood group systems, such as Scianna and Dombrock, are not routinely available, and others are periodically in short supply. Genotyping has been used as an alternative in these situations.

Other potential applications:

1. Extended electronic crossmatching—The use of the electronic crossmatch to insure ABO and RhD compatibility between donor RBC and recipient is well established. The extension of this technique for matching for other clinically significant antigens would be feasible if more extended genotype information was available for donors. This approach could be used in 2 circumstances:

   1. Alloimmunized patients—For example, the database could be searched for donor RBCs that were A, R1r, Kell (K1) negative for a patient who was group A with anti-E and anti-K. Only these units would require antigen confirmation (serologically according to current regulations) and crossmatching.

   2. Multiply transfused patients—Prospective genotype matching could be performed for transfusion-dependent patients, such as those with sickle cell disease, thalassemia, or aplastic anemia, to reduce the incidence of alloimmunization and delayed HTRs, especially the hyperhemolysis syndrome.

2. Autoimmune hemolytic anemia—The evaluation of patients with autoimmune hemolytic anemia is time-consuming and technically demanding, especially if they require autologous or heterologous absorptions. The goal of identifying units suitable for transfusion could be met, and perhaps more rapidly, by extended genotype matching.

Table 12–7 is a list of indications for transfusion of RBCs, platelets, plasma, and cryoprecipitate.

Red Blood Cells

A National Institutes of Health (NIH) Consensus Conference established broad parameters for perioperative RBC transfusion. Although the conclusion of the conference was that “no single measure can replace good clinical judgment as the basis for decisions regarding perioperative transfusion,” it was suggested that patients with hemoglobin levels exceeding 10 g/dL (100 g/L) rarely require transfusion, while those with hemoglobin levels less than 7 g/dL (70 g/L) frequently do. Several professional organizations have also established guidelines for RBC transfusion. There have been a small number of randomized trials comparing the clinical outcomes of liberal and stringent RBC transfusion triggers that have consistently failed to demonstrate any benefit of transfusing patients for hematocrits of 30% (10 g/dL) compared with triggers as low as 21% (7 g/dL).

Platelets

Indications for platelet transfusion have also been addressed by an NIH Consensus Conference and by professional organizations. Of particular interest in the last several years has been a reassessment of the use of prophylactic platelet transfusions in thrombocytopenic patients with marrow failure. In general, the traditional trigger level of 20,000 platelets/μL for prophylactic transfusion has been replaced with a level of 10,000 platelets/μL. There has even been a challenge to the utility of any prophylactic platelet transfusion, including before minor procedures such as line placement and lumbar puncture. This challenge suggests that platelets should only be administered in cases of actual bleeding. Appropriate uses of platelets in other settings are included in Table 12–7.

Fresh Frozen Plasma

Clinical situations in which FFP is likely to be useful also have been established by an NIH Consensus Conference and professional organizations. FFP has been used as replacement therapy for deficiencies of clotting factors and regulatory proteins, including protein C and protein S, for which specific concentrates or recombinant products are not available. The use of FFP to reverse mild coagulation abnormalities is probably not warranted. The risk of bleeding appears to be very low when the prothrombin time (PT) and the international normalized ratio (INR) derived from it are only mildly elevated (PT is <1.5 times the control or the INR is ≤1.5). The same can be said for mild elevations of the partial thromboplastin time (PTT) associated with coagulation factor deficiencies. It is also unlikely to provide any benefit to patients with mild elevations in the PT or PTT who are undergoing minor procedures (eg, line placement). On the other hand, FFP is effective in the treatment of thrombotic thrombocytopenic purpura, reversing the effects of warfarin in an emergency situation, the treatment of the bleeding patient with disseminated intravascular coagulation, and massive transfusion cases.

Cryoprecipitate

The practice of using cryoprecipitate as a source of fibrinogen and Factor XIII is well accepted. In addition, cryoprecipitate can be mixed with thrombin to form topical fibrin “glue,” which is used to initiate anatomic connections and control bleeding over large surfaces; however, products with standardized amounts of fibrinogen that have undergone viral inactivation procedures are now commercially available and are generally preferable. The role of cryoprecipitate in the treatment of bleeding in uremic patients is controversial. Cryoprecipitate is no longer recommended for treatment of hemophilia A or von Willebrand disease because of the availability of other products.

An adverse effect of blood transfusion occurs in approximately 3% of transfusions in the United States. These complications of transfusion can be classified as immunologic, infectious, or due to the chemical or physical characteristics of blood components.

Immunologic Reactions

RBC Reactions

Hemolytic Transfusion Reactions

Although HTRs are much discussed, they are fortunately quite uncommon, reflecting the efficacy of the serologic and procedural techniques in place to prevent their occurrence. Although HTRs occur with less than 0.1% of the units transfused in the United States, they can be life-threatening. It bears noting that fatal, acute HTR due to ABO incompatibility is a more frequent adverse outcome of transfusion than infection with HIV or HCV, and it is more often the result of patient or sample misidentification than to serologic mishaps or exotic blood types.

HTRs are mediated by antibodies directed against alloantigens present on transfused RBCs. Most alloantibodies to RBC antigens, other than the AB isoagglutinins, develop in response to exposure to allogeneic RBCs by transfusion or maternal–fetal hemorrhage. There are hundreds of RBC antigens comprising more than 50 systems. Fortunately, only a small proportion of these have clinical significance. In addition to the AB isoagglutinins, antibodies to antigens in the Rh, Kell, Duffy, Kidd, and MNSs systems are responsible for the preponderance of HTRs. Identification of these alloantibodies, by the techniques discussed above, is important because the degree and severity of hemolysis differs among them.

HTRs can be either acute, occurring within 24 hours of transfusion, or delayed, in a reaction that appears 5 to 7 days (range 3-21 days) after the transfusion. Acute reactions are usually more severe than their delayed counterparts, and occur in patients who already have antibodies to RBC alloantigens when they are transfused with RBCs bearing those target antigens. The most severe acute HTRs are due to ABO incompatibility because the AB isoagglutinins are present at a substantial titer and fix complement efficiently, being largely IgM. The A and/or B antigen sites are also abundant on RBCs (typically 1-2 × 10⁶ antigen sites per cell). Antibodies to antigens in the Kell, Kidd, and Duffy systems also have been responsible for acute HTR.

Patients with an acute HTR typically present with temperature elevation, an important point, because they might initially be mistaken for a febrile-nonhemolytic transfusion reaction (FNHTR; discussed later). Nausea, vomiting, hypotension, low back pain, and substernal pressure may also signal the occurrence of acute hemolysis. Hemolysis is generally intravascular in this setting. The hemoglobin released into the plasma from the lysed RBCs is apparent as hemoglobinemia (red plasma rather than yellow) and hemoglobinuria (red urine that remains red after centrifugation). Disseminated intravascular coagulation and systemic hemodynamic instability may be triggered by the hemolysis. Together with the direct toxic effects of cell-free hemoglobin on the tubular cells of the kidney, these conditions are responsible for the impaired kidney function that often accompanies acute intravascular hemolysis. Therapy is largely supportive, but preservation of renal function is critical, and is often accomplished through the use of intravenous hydration and diuretics.

Delayed HTRs occur in 2 situations. In one, the patient is exposed to a foreign RBC alloantigen by transfusion and mounts a primary immune response. As the amount of antibody in the plasma increases, hemolysis may ensue. The second situation in which a delayed response may occur is when a patient is reexposed to an alloantigen to which he or she was sensitized in the past by previous transfusion or pregnancy (anamnestic response). Even if the alloantibody to this antigen is not detectable prior to the transfusion, exposure to the alloantigen can stimulate an anamnestic response. Antibodies to Kidd and Rh antigens are frequently responsible for such reactions. Hemolysis is typically extravascular in delayed HTR with the only clinical and laboratory signs being a decrease in the hemoglobin level, a rise in the bilirubin level, a low-grade temperature, and a feeling of malaise. When no hemolysis can be detected in a delayed HTR, the reaction is called a delayed serologic (rather than hemolytic) transfusion reaction.

Reactions to Plasma Components

Hypersensitivity Reactions—Allergic and Anaphylactic Transfusion Reactions

Allergic reactions occur in approximately 1% to 3% of patients receiving blood products containing plasma. In most cases, these hypersensitivity reactions are a host response to foreign plasma proteins in the donor blood components. The vast majority of these reactions consist of hives, pruritus, and erythema, and can be managed with antihistamines or steroids. More serious responses such as bronchospasm, laryngeal edema, gastrointestinal disturbance (nausea, vomiting, cramps, and diarrhea), and hypotension (anaphylactoid reaction) are much less frequent. IgA-deficient patients with anti-IgA antibodies in their plasma are at risk for serious reactions including frank anaphylaxis if exposed to IgA in a transfused blood component. If transfusion is required, these patients should be provided with components from IgA-deficient donors, or, in an elective situation, store their own components for later use. Washing packed RBC can effectively remove IgA. Patients who are IgA deficient, but who do not have anti-IgA, do not require special preparations, but should be observed closely during transfusion.

White Blood Cell Reactions

Febrile-nonhemolytic Transfusion Reactions

These reactions are among the most common transfusion-related complications and accompany approximately 1% to 3% of transfusions of cellular components. They are more common in multiply transfused patients and with nonleukoreduced cellular components. An FNHTR usually presents with a temperature elevation of 1°C or more, during or shortly after a transfusion (usually within 1-2 hours), that is unlikely to be associated with the patient's underlying disease or therapy. The temperature elevation is often accompanied by chills, rigors, and generalized discomfort, and in some patients, nausea and vomiting as well. The majority of these reactions are mild and do not persist for more than 8 hours. Antipyretics may be administered, and occasionally meperidine may be required to treat severe rigors. These reactions have long been considered to be the product of antileukocyte antibodies present in the recipient's plasma, reacting with WBCs or WBC fragments in the transfused product. There may, however, be other etiologies for the FNHTRs, including the presence of cytokines released by lymphocytes in the donated unit during storage.

Transfusion-associated Graft Versus Host Disease (TA-GVHD)

Immunocompetent T lymphocytes present in cellular blood components may engraft in an immunoincompetent transfusion recipient, particularly if cellular immunity is compromised. The engrafted, allogeneic T cells mount an alloimmune response to cells in the skin and gastrointestinal tract, similar to what occurs in hematopoietic stem cell transplant-associated GVHD. However, in transfusion-associated GVHD, the donor T cells attack the host cells in the bone marrow as well, making this complication of transfusion lethal in most cases. Fortunately, T lymphocytes in cellular blood components can be inactivated by exposure to gamma irradiation, which effectively prevents this complication. Patients at risk for this rare complication include those undergoing hematopoietic progenitor cells (HPC) transplantation or who have hematologic malignancies. Low-birth-weight infants, infants born with hemolytic disease of the newborn, and fetuses receiving intrauterine transfusions are also at risk. Patients with congenital T-cell immunodeficiencies (eg, Wiskott–Aldrich and diGeorge syndromes) have also developed this complication. Cellular components from blood relative donors are also routinely irradiated to prevent TA-GVHD that may occur in the circumstance when the donor is homozygous for an HLA haplotype shared with the transfusion recipient. In this situation, the transfused T cells remain immunologically invisible to the otherwise immunocompetent host and, rather than being cleared, they engraft and attack the host because they recognize the mismatched host haplotype antigens as foreign. Most of the reports of TA-GVHD in other patients, such a those with solid tumors or who were undergoing surgery, predate the awareness of this 1-way haplotype match, which is the most likely explanation for the occurrence of this event in these immunocompetent patients.

Transfusion-related Acute Lung Injury (TRALI)

TRALI is characterized by the development of acute respiratory distress, hypoxia, and bilateral infiltrates on chest x-ray, often accompanied by fever and hypotension, during or within 6 hours of completion of a transfusion. To meet the current working definition of TRALI, there must be no preexisting form of acute lung injury or other risk factors such as sepsis, aspiration, or pneumonia. Most patients recover completely with supportive care, which may include mechanical ventilation, and the pulmonary infiltrates usually resolve within 2 to 4 days without long-term sequelae. However, there is a 5% mortality rate. This complication has been attributed to the presence of antileukocyte antibodies in the plasma of donor blood (often from females with a history of pregnancy) that react with the recipient's WBCs. This results in the formation of immune complexes that are trapped in the pulmonary vasculature and lead to alveolar edema. At present, various steps are being taken to reduce the incidence of TRALI, including making FFP from predominately male donors or donors with no history of pregnancy or transfusion, or by testing for HLA antibodies.

Platelet Reactions

Posttransfusion Purpura (See the Section “Bleeding Disorders” in Chapter 11)

This rare complication occurs in patients who lack a common platelet antigen (often HPA-1A) and have developed an alloantibody by exposure through prior transfusion or pregnancy. When reexposed to HPA-1A by transfusion of a platelet product or an RBC product containing contaminating platelets, these patients appear to develop an anamnestic response and become severely thrombocytopenic 7 to 10 days later. Paradoxically, the patient's own platelets, which are HPA-1A negative, are also cleared. Several explanations have been offered including the observation that there is an initial IgM response that reacts with GP IIb–IIIa (essentially a platelet autoantibody) but then “matures” with the production of an IgG with anti-HPA-1A specificity. The reaction is self-limiting, but may be complicated by severe hemorrhage. Steroids and intravenous immunoglobulin have been used successfully to manage this immunologic reaction.

Refractoriness to Platelet Transfusions

Patients may become sensitized to leukocyte and platelet antigens through transfusion or pregnancy. Transfused platelets may be cleared rapidly when given to a patient who has preformed antibodies directed at foreign platelet antigens or HLA Class I molecules, which are also expressed on the platelet membrane. As a result, it may be extremely difficult to elevate the platelet count in such patients. A patient is considered to be refractory to platelet transfusion if the increment measured between 15 and 60 minutes after the platelet transfusion is lower than expected on 2 occasions. Note that counts done several hours afterward are not useful for determining which patients are immunologically refractory. The posttransfusion count may be corrected for the number of platelets administered and the patient's body surface area (the “corrected count increment”) as follows:

Here the default for number of platelets transfused is: 1 unit whole blood-derived platelets = 5.5 ×10¹⁰ platelets; 1 unit apheresis platelets = 3 ×10¹¹ platelets.

A corrected count increment of <7500 is a strong evidence of immunologic refractoriness. Note that other causes of refractoriness should be ruled out, among them: active bleeding, fever, sepsis, splenomegaly (splenic sequestration), disseminated intravascular coagulation, marrow transplantation, antibiotics (eg, vancomycin), IV amphotericin B, thrombotic thrombocytopenic purpura, idiopathic or immune thrombocytopenic purpura, and heparin-induced thrombocytopenia.

Patients with immunologic refractoriness may respond well to platelets from donors who lack the HLA antigens corresponding to the patient's HLA alloantibodies (or to platelets that are HLA matched) or to platelets that have been chosen by platelet crossmatching.

Leukocytes in the transfused unit appear to be necessary for stimulating the immune response to both platelet and leukocyte antigens. Alloimmunization may be prevented by transfusion of cellular components from which leukocytes have been removed, usually by passage of the product through a filter that retains the leukocytes. Patients who are likely to need extensive platelet transfusion support (eg, for HPC transplants or hematologic malignancies) often receive leukoreduced cellular components to reduce the likelihood of alloimmunization.

Nonimmunologic Reactions

Complications Created by the Physical Characteristics of Blood

Hypothermia

Transfusion of small volumes of cold blood may be associated with minor discomfort. This complication can be averted by using blood warmers or blankets. In the setting of massive transfusion, however, the rapid transfusion of large amounts of blood that is at 1°C to 10°C contributes to hypothermia. Hemostasis is impaired when the circulating blood is below 37°C and in extreme situations, cardiac dysrhythmias and arrest may occur. In this setting, the use of high-throughput blood warmers is warranted.

Transfusion-associated Circulatory Overload (TACO)

Volume overload is a relatively common but often overlooked complication of transfusion. Patients with congestive heart failure or renal failure, the very young, and the very old are particularly at risk. It should be suspected in a patient who complains of dyspnea, orthopnea, cough, or chest pain, during or soon after transfusion, particularly if there are signs of hypoxia, rales, tachycardia, or hypertension. Supplemental oxygen and diuresis may be required. Future transfusions should be carried out slowly and perhaps with the aid of a diuretic.

Chemical Complications

Iron Overload

Each unit of packed RBC contains approximately 200 mg of iron. Chronic RBC transfusion can overwhelm the body's mechanisms for eliminating excess iron, resulting in iron accumulation in various tissues. An individual who has received 100 or more units of RBCs (20 g of iron) is at risk to develop various complications of iron overload including cardiac dysrhythmias, pancreatic failure (“bronze diabetes”), and liver function abnormalities. Tissue iron can be mobilized and excreted using chelating agents such as desferroxamine or deferasirox. Chelation therapy is a slow process and is more effective if deployed well before tissue accumulation of iron is extensive.

Potassium Toxicity

Potassium leaks out of RBCs during storage as ATP levels decline and the ATPase-dependent Na⁺/K⁺ pump activity diminishes. Once the banked RBCs are transfused, they transport glucose, restore their ATP levels, and take up the K⁺ that was lost during storage. In the short term, however, each unit of RBCs might contain as much as 6 mmol of extracellular K⁺ at the time of expiration. There have been a handful of reports of neonates, or patients with renal failure receiving large volumes of banked blood, who have developed life-threatening cardiac dysrhythmias. Neonates usually receive RBC units that have been stored for less than 1 week and have not yet accumulated much extracellular K⁺. Washing RBC is also an effective means of removing extracellular K⁺, although it is very rarely required.

Citrate Toxicity

Citrate is the anticoagulant used in the collection of all blood products and is therefore transfused with the blood product into the patient. It is present in the plasma. Hence, most of it ends up in platelet and plasma products while there is relatively little in RBC products. Citrate is metabolized by every nucleated cell of the body, but in circumstances where large volumes of banked blood are being infused rapidly, as in massive transfusion, the rapid influx of citrate may overwhelm the body's metabolic capacity, leading to an accumulation in the patient's plasma. Most patients can receive up to 1 unit of FFP every 6 minutes without evidence of citrate toxicity. Patients with liver failure metabolize citrate more slowly, however, and are particularly susceptible. The accumulating citrate chelates calcium, causing the ionized calcium levels to drop and producing perioral tingling and extremity paresthesias. In extreme circumstances, it may produce severe hypo(ionized)calcemia that can lead to cardiac dysrhythmias.

Depletion of 2,3-Diphosphoglycerate (2,3-DPG)

With increasing storage time of RBCs, the intracellular level of 2,3-DPG decreases, producing a left shift of the oxyhemoglobin dissociation curve. Once banked RBCs are transfused, they restore the levels of 2,3-DPG over a period of 24 to 48 hours. It has been suggested that the high oxygen affinity of the hemoglobin in the 2,3-DPG-deficient banked RBCs might impair oxygen delivery, particularly to neonates. As a result, it has become a general practice to transfuse neonates with RBCs that have been banked less than 1 week. However, most of the literature demonstrating unfavorable outcomes for neonates receiving older units was based on studies with RBC storage systems in which maintenance of 2,3-DPG levels was not as effective as it is using the current systems.

Infectious Complications (See the Section “Infectious Disease Testing”)

The Classic Pathogens

Transfusion transmission of the hepatitis viruses and the retroviruses has been substantially reduced through the interventions of donor education, screening on the basis of medical history and risk behaviors, and testing, including the use of highly sensitive techniques based on amplification of viral genetic nucleic acids. The residual risk of HIV or HCV infection through transfusion is in the range of 1 event per 1 to 2 ×10⁶ units transfused. Viral transmission by pooled plasma products has also been largely eliminated by the use of robust viral inactivation techniques or replacement with recombinant proteins.

The Current Significant Pathogens

At the present time, bacterial contamination of blood components is the most significant infectious complication of transfusion in developed countries, in terms of both the number of transmitted infections and the number of fatalities. It has been estimated that in the United States, approximately 1 in 500,000 units of RBCs, or 1 in 10,000 to 20,000 units of platelets, is associated with transfusion-transmitted sepsis. The organisms most frequently associated with septic RBC transfusions are psychrophilic gram-negative bacteria such as Yersinia enterocolitica and Pseudomonas spp., as well as Enterobacter spp. and Serratia spp. Platelet units have been reported to transmit gram-positive cocci (Streptococcus aureus, S. epidermidis, and Staphylococcus spp.) as well as gram-negative organisms (Klebsiella spp., Serratia spp., Salmonella spp., and Enterobacter spp.). The sources of these bacteria are thought to be skin commensals picked up and introduced into the blood donation with the venipuncture, or less commonly, cryptic bacteremia in clinically healthy donors. Even if the inoculum is quite small, blood provides a superb culture medium, particularly when stored at room temperature, as is the case for platelets. Although donors are now questioned specifically about antibiotic use, the health history is neither a sensitive nor a specific screening tool. The implementation of tests to screen platelet products for evidence of bacterial contamination was discussed above and is now routine.

Cytomegalovirus (CMV) is a ubiquitous member of the herpes virus family to which approximately 30% to 60% of adults in developed countries have been exposed. CMV can be transmitted by transfusion of blood components that contain leukocytes, such as packed RBCs and platelets. Although primary infection rarely produces serious disease in immunologically intact hosts, it is associated with systemic infection in immunocompromised patients who are CMV-seronegative. The following groups of patients have been shown to be susceptible to transfusion-transmitted CMV primary infection and disease and should receive CMV reduced-risk cellular blood components:

1. Premature, low-birth-weight (<1200 g) neonates

2. CMV-seronegative pregnant women (including those undergoing intrauterine transfusions)

3. CMV-seronegative recipients of, or candidates for, hematopoietic or solid organ transplants

4. CMV-seronegative, HIV-infected patients

CMV reduced-risk blood components can be obtained by screening donors for CMV antibody (IgG) that indicates past exposure, or by removing the leukocytes that contain latent CMV by filtration with leukocyte reduction filters. These 2 approaches have been shown to be equally effective in preventing transfusion-transmitted CMV infection. Only cellular components need to be CMV reduced-risk, since intact mononuclear cells are required to transmit CMV.

Emerging Pathogens

The blood supply will always be vulnerable to the introduction of new pathogens into the donor population. In some instances, the pathogen may truly be a new organism, or one that has recently acquired the ability to infect humans, such as the SARS virus, various strains of avian flu, and the bovine prion responsible for variant Creutzfeldt–Jakob disease. Population shifts in response to natural or man-made catastrophes, or simply travel for business or pleasure, spread pathogens from 1 part of the world to another, such as the West Nile virus, Plasmodium spp., and Trypanosoma spp. In some circumstances, questioning donors about exposure to a pathogen or a history of a characteristic illness, or the rapid development of a screening test has been an effective means of interdicting transfusion transmission of a new infectious agent. However, an effective response is more difficult in the circumstance where the organism has not been identified, its biology is unique, the routes of transmission are not well understood, or the clinical effects are not well defined. As a result, work continues to develop pathogen inactivation technology that would be suitable for cellular blood components.

Transfusion Reaction Workup

If a reaction is suspected, the transfusion must be stopped immediately while maintaining venous access, and the patient must be assessed. Emergent airway and hemodynamic issues should be dealt with immediately and appropriate measures taken to alleviate the patient's major symptoms and concerns. If the assessment reveals that the patient's only symptoms are cutaneous manifestations of hypersensitivity (flushing, pruritus, and urticaria), then the transfusion may be resumed under careful observation. In all other situations, the transfusion of that unit should be stopped and a clerical check should be performed to verify that the correct unit (ie, one labeled for that patient) has been administered. A transfusion reaction form should be filled out and a new blood bank specimen should be drawn from the patient. The transfusion reaction form, the unit involved, and the new specimen should be returned to the blood bank for evaluation. A posttransfusion urine specimen should also be obtained and sent for urinalysis.

The blood bank will perform a clerical check, and compare the posttransfusion specimen with the pretransfusion specimen used for compatibility testing for the appearance of hemolysis or hyperbilirubinemia. The ABO and Rh type of the posttransfusion specimen will be determined to confirm that the pretransfusion specimen was indeed from this patient and that the ABO and Rh type of the unit that was being transfused was appropriate. A direct antiglobulin test is also performed on the posttransfusion specimen looking for antibody-coated RBC (ie, donor cells coated with recipient alloantibody) indicating an immune-based HTR. Any findings suggestive of an HTR trigger a more extensive investigation in the blood bank. If the workup rules out a hemolytic reaction, transfusion may resume.

The 1980s saw the considerable development of techniques to avoid allogeneic transfusion (transfusion with someone else's blood), particularly in elective surgery. The major driver was concern about the infectious complications of transfusion. Before the development of a screening test in 1985, HIV transmission rates may have been as high as 1 in 10,000 units transfused, while as many as 5% to 10% of transfusion recipients developed what was then called non-A, non-B hepatitis, and is now known to have been due primarily to HCV, which was only identified in 1989. Although demand for these blood-sparing techniques is not as great as it was 20 years ago, they are still in use and continue to be helpful for patients with unusual blood types or multiple alloantibodies for whom it is difficult to find compatible blood. In addition, the drive to avoid allogeneic blood exposure has reinforced common sense measures: treatment of medically correctable anemia, greater physician tolerance of asymptomatic anemia, meticulous surgical hemostasis, and the wider use of hemostatic medications. The licensing of recombinant erythropoietin also reduced the dependence of patients with renal failure, malignancies, and HIV infection on regular RBC transfusion.

Four techniques in particular were developed to reduce the dependence of surgical patients on banked RBC: preoperative autologous blood donation (PABD), acute normovolemic hemodilution (ANH), intraoperative blood recovery and reinfusion, and postoperative blood recovery and reinfusion.

PABD is suitable for patients undergoing elective surgical procedures for which RBC transfusion is commonly required, and in this setting can reduce allogeneic blood use. Since the blood may only be used by the donor/recipient, donor qualifications are simple and no testing (other than ABO/Rh typing) is required. Note that mistransfusion, bacterial contamination, and volume overload are just as likely to occur with an autologous unit as with an allogeneic unit. Since the hazards averted (especially infection) are very small, donors who might be put at even a small risk by donation (eg, mild anemia and coronary artery insufficiency) should be discouraged from PABD.

ANH is a technique whereby several units of blood are removed from a patient in the operating room immediately before a procedure. The volume is replaced with crystalloid. The blood is returned if bleeding occurs, or at the end of the procedure. It has the advantage that little advance planning is necessary, but it is not very efficacious at reducing allogeneic RBC transfusions for patients with moderate anemia from whom few units can be withdrawn at the beginning of the procedure.

Blood recovered from the operative field can be collected, processed in some manner, and reinfused. Shed blood is collected by suction into a reservoir, usually with heparin or citrate, and then usually washed in a centrifugal device specially designed for this purpose. The washed RBCs are suspended in normal saline and pumped into a bag suitable for reinfusion to the patient. The washing procedure removes materials that might cause reactions such as cell debris, activated clotting factors, and complement. A similar process can be carried out manually. This technique is particularly helpful in procedures where large volumes of blood are lost. Although somewhat expensive, the recovery of 3 to 4 units of RBCs is usually adequate to recover the costs. This technique is suitable for elective as well as emergency procedures during which blood loss is extensive.

Devices are also available for collecting blood shed in the postoperative period. Many of them rely on filtration of the shed blood. The filtration technique is not adequate to remove materials that can provoke a reaction and is generally not worth risking for the small amounts of blood that can be recovered. A small, centrifugal device that washes the blood collected postoperatively is also available. Although it provides a much cleaner product, the small volumes of blood recovered in this manner do not make it very cost-effective.

Cellular therapies encompass the collection, processing, storage, and therapeutic use of hematopoietic cells, most commonly HPCs. In addition, mononuclear cell fractions from HPC donors have been used to enhance the graft versus tumor effect of allogeneic transplantation, and dendritic cells sensitized to tumor antigens have been used to treat solid tumors. Allogeneic HPCs have the advantage that they are free of malignancy and may have a significant graft versus tumor effect; they are preferred for most forms of leukemia, Hodgkin disease, and the myelodysplastic syndromes. Allogeneic transplantation has also occasionally been used to treat certain genetic disorders of the hematopoietic system, such as sickle cell disease and thalassemia. Autologous HPC transplants are not complicated by rejection and have a lower incidence of GVHD. They are performed in patients with some forms of non-Hodgkin lymphoma and multiple myeloma, and as rescue therapy after intensive chemotherapy for some solid tumors (eg, testicular, breast, and ovarian cancer).

Potential allogeneic donors must in general meet the criteria for blood donation, including infectious disease testing, although some of these criteria may be waived if an alternate suitable donor cannot be found. Potential donors must be typed for HLA Class I and II antigens using molecular techniques. Class I mismatches pose an increased risk for rejection and failure to engraft, whereas Class II mismatches are associated with increased incidence of GVHD. A single Class I or II mismatch usually has little impact on survival. Two Class I mismatches, or a Class I and a Class II mismatch, are usually associated with poorer outcomes. Haploidentical sibling donors have been used successfully. If a suitable family member donor cannot be found (and only 1 in 4 siblings is likely to be a 2 haplotype match), a donor may be sought through the National Marrow Donor Program, a registry of people who have been HLA typed and have expressed a willingness to donate HPCs. The search may take a few months and is less likely to be successful for patients with unusual phenotypes. There has been considerable effort in the last few years to register donors from previously underrepresented minority populations. ABO or Rh matching is not necessary since the transplant recipient will convert to the donor type if engraftment is successful, although RBC engraftment may be delayed if donor RBCs are incompatible with the recipient's anti-A or anti-B isoagglutinins. Donor isoagglutinins may also cause hemolysis of residual recipient RBCs, or at least a positive direct antiglobulin test. The conversion from recipient to donor blood type does pose problems for the transfusion service that must provide blood that is compatible with both donor and recipient until the recipient's original RBCs and isoagglutinins are undetectable.

HPCs may be collected from peripheral blood by apheresis, from bone marrow by aspiration, or from cord blood. Apheresis collection now accounts for 90% of autologous transplants, and 50% of allogeneic transplants, a fraction that is increasing. Bone marrow aspiration is performed with multiple punctures and aspirations of the posterior iliac crest and must be performed in the operating room with the donor under general anesthesia. The aspirates are anticoagulated (heparin or citrate), filtered, and pooled into a bag that is then usually stored frozen until the time of the transplant.

Collection by apheresis is less invasive and less likely to recover residual malignant cells. In addition, it has been shown to be associated with quicker engraftment, although chronic GVHD is somewhat more likely to occur than with marrow transplantation. The number of HPCs in the peripheral blood is ordinarily very low, so donors are prepared by the administration of granulocyte colony-stimulating factor or granulocyte–macrophage colony-stimulating factor at the point when their marrow is rebounding from a cycle of chemotherapy. Under these circumstances, the levels of HPCs (which can be determined by measuring the number of CD34-positive cells in the peripheral blood by flow cytometry) may be elevated 200- to 1000-fold. The pheresis instrument is configured to collect the mononuclear cell fraction. Large volume collections (with 3 blood volumes processed) are typically performed, which reduces the number of procedures needed to collect the targeted number of CD34-positive cells (typically 2-4 ×10⁶ per kg patient weight). Large volume collection may also have the effect of recruiting HPCs from the marrow during the collection.

The HPC product undergoes extensive quality control testing including ABO and Rh type, RBC and WBC counts (and differential), CD34 cell count, an assay to enumerate colony-forming units in vitro, cell viability, and testing for bacteria, fungi, and mycoplasma. Products are frozen (usually at a controlled rate) in the presence of 10% dimethylsulfoxide and 10% protein (plasma or albumin) as cryoprotectants, and stored in mechanical freezers or liquid nitrogen tanks. At the time of transplant, the units are thawed at 37°C, usually at the patient's bedside, and then administered intravenously, much like a conventional transfusion.

Umbilical cord blood contains high levels of circulating HPCs, and this observation has led to the development of cord blood banking. If a mother meets the criteria for allogeneic blood donation (except for hemoglobin level and recent pregnancy because she has just delivered) including the usual infectious disease testing, and there is no history of genetic diseases in the family of either parent, she may give consent for the blood to be drained from the placenta via the umbilical cord (after it has been severed or clamped off from the neonate) and then stored frozen. In addition to the usual quality control testing of the cord blood, HLA Class I and II typing is performed as well as ABO/Rh typing.

Over 5000 related and unrelated (but HLA matched) cord blood transplants have been performed since the technique was first developed in 1988. Cord blood HPCs home readily to the host bone marrow and do not seem to be as alloreactive to recipient antigen-presenting cells as HPCs from adults. In addition, the large numbers of HLA-typed cord blood samples may improve the chances of finding unrelated matches. Cord transplants are also less likely to be complicated by GVHD and infection with CMV. However, the total number of HPCs in each cord blood sample is small and engraftment is slower. This has led to the use of double cord transplants that accelerate engraftment, even though eventually 1 of the donor's HPCs dominates.

Therapeutic apheresis is the process of withdrawing blood from the body, selectively removing 1 particular element (ie, plasma, leukocytes, platelets, or RBCs), and returning the remaining elements along with a replacement solution (crystalloid and/or colloid) to maintain isovolemia. There are several different therapeutic apheresis procedures that are designed to remove, or treat, specific components of the blood. These are described below.

Indications for Therapeutic Apheresis

Indications for therapeutic apheresis have been classified according to the quality of the evidence demonstrating efficacy or the lack thereof (Table 12–8). The specific disorders for which therapeutic apheresis has been evaluated as a treatment are shown in Table 12–9.

Therapeutic apheresis has been used as a treatment for numerous disorders. While it is clearly effective in some diseases, such as thrombotic thrombocytopenic purpura, the therapeutic benefit of apheresis in many other disorders is much less clear, because many of them are uncommon, and therefore it is extremely difficult to obtain information about efficacy based on large-scale, prospective, randomized clinical trials.

Plasmapheresis

In plasmapheresis (plasma exchange; see the figure in Chapter 2), blood is withdrawn from a patient and the plasma is separated from the cellular components by centrifugation or, less commonly, by filtration, in an apheresis instrument. The plasma is discarded and the cellular components are returned to the patient. Liters of abnormal plasma can be removed from the patient and replaced by saline, albumin, starch solutions, FFP, or combinations of these. This technique is used to remove autoantibodies, immune complexes, paraproteins, and protein-bound toxins.

Cytapheresis

Cytapheresis is the removal of one of the cellular elements of the blood. Leukapheresis is occasionally indicated for patients with acute myelogenous leukemia or chronic myelogenous leukemia in the accelerated phase with a high level of circulating blasts and evidence of leukostasis with pulmonary or CNS involvement. Myeloid blast forms adhere to the vascular endothelium and can impede blood flow in the lungs and the brain. The collection of peripheral HPCs and granulocytes is a variation of leukapheresis.

Plateletpheresis may be indicated in patients with myeloproliferative disorders, such as essential thrombocytosis, who develop platelet counts that exceed 1 ×10⁶/μL and also show signs of hemorrhage or thrombosis.

Erythrocytapheresis (RBC Exchange)

Although most sickle crises are managed with hydration, pain medication, and supplemental oxygen, RBC exchange is occasionally performed for patients who are experiencing a severe infarctive crisis complicated by stroke, acute chest syndrome, retinal infarction, or priapism. Exchange is performed less commonly to prepare patients for surgery. In the exchange replacement of sickle RBCs with normal RBCs, the usual goals are to reduce the hemoglobin S concentration to less than 30% of total hemoglobin, and to increase the hematocrit to 30%. Red cells chosen for exchange are often screened for hemoglobin S (since donors with sickle trait may be unaware of it and have a normal hemoglobin level) and may be partially phenotype matched (eg, for Kell and the Rh antigens) to prevent alloimmunization. Red cell exchange also has been used to treat patients with malaria or babesiosis who have a high percentage (eg, >10%) of RBCs infected with organisms despite adequate medical therapy, and signs of decompensation such as marked hemolysis, pulmonary involvement, CNS involvement, renal failure, or disseminated intravascular coagulation. Patients who are immunosuppressed, asplenic, or elderly seem to be particularly at risk to develop complications from infection with Babesia.

Photopheresis

In this apheresis procedure, the patient's leukocytes are separated from whole blood and exposed to ultraviolet light, usually after the patient has ingested psoralen. The psoralen/ultraviolet light-treated leukocytes are then returned to the patient. Photopheresis has been used to treat cutaneous T-cell lymphoma and has been shown to increase patient survival when compared with conventional chemotherapy. Photopheresis has also been used to treat GVHD and cardiac allograft rejection.

A portion of this chapter related to complications of transfusion appeared previously in Clinical Laboratory Reviews (a newsletter for physicians at the Massachusetts General Hospital, Boston, 1995;4:2).