Transfusion Therapy Part 1

Transfusion medicine developed rapidly, owing to several key discoveries and technical advances. These include the discovery of blood group antigens and the understanding of the host immune response to these antigens, the development of methods of anticoagulation and storage of blood, and the creation of plastic bags that allow sterile fractionation of whole blood into components. The potential of blood to act as an agent of disease transmission has heavily shaped both the donation process and transfusion practice.1 Decisions about whether to transfuse must involve weighing the benefits against the risks. This topic provides a basis for these decisions, including indications for blood-component use, complications of transfusion therapy, and methods of reducing risks during the collection, processing, and preparation of blood components.

Blood Donation

The donation process for either whole blood or special products, such as single-donor platelets (SDPs) obtained by aphere-sis, is designed to protect both the donor and the recipient. For example, persons weighing less than 110 lb (49.9 kg) are excluded from donation because they have too small a blood volume to donate blood safely. Donors taking drugs that would impair recovery from a vasovagal donor reaction may also be excluded in some locales. Recipient safety is promoted by excluding donors who are at risk for viral or bacterial infections or are taking medications that could cause reactions or impair the function of donated blood products.

AUTOLOGoUS and DIRECTeD DONATIoN

Autologous donations and directed donations are two strategies adopted by patients seeking to minimize their real or perceived risk of infection from blood products.

Autologous Donation

In autologous donation, patients deposit their own blood and then receive that blood if they need transfusion therapy. This eliminates the infectious and sensitization risks associated with allogeneic blood. Absolute contraindications to autologous donation are tight aortic stenosis, unstable angina, and active bacterial infection.2 Low hemoglobin levels and poor venous access frequently limit the number of units that can be collected. With the increasing safety of allogeneic blood, the rationale for autolo-gous donation may ultimately depend on the importance of the possible modulation of recipient immune function associated with allogeneic transfusion rather than the avoidance of blood-borne infections.

Directed Donation

Directed donation is donation for a specific recipient. It usually involves donations made by friends or family members of the intended recipient. It is based on the assumption that transfusions involving donors selected by the recipient carry lower risk of infections than transfusions involving donors from the general population. However, available prevalence data show that the risk of infectious disease from directed donors is no different from that of first-time donors.3 The current risk of infection via transfusion is so low [see Table 1] that justification for directed donor programs depends primarily on patient preferences or on the need for a selected donor serving as the only source of blood products to reduce the recipient’s risk from exposure to multiple donors. The latter form of directed donation is most appropriate for neonatal transfusions, in which one of the biologic parents may provide all the needed blood products.4

SCREENInG PROCEDUReS

The combination of improved donor selection and postdona-tion testing has greatly decreased the infectious risks of allogene-ic blood [see Table 1]. Predonation donor screening to identify clinical and lifestyle characteristics associated with higher incidences of infection has produced the biggest decrease in the risk of transfusion-transmitted disease.

POSTDONaTION TESTiNG

Postdonation testing is essential in identifying donors likely to transmit blood-borne infections who are missed in the initial screening process.

Screening for Hepatitis Viruses

Hepatitis C Screening for hepatitis C began in 1990 with the availability of a single antigen-based enzyme-linked immuno-sorbent assay (ELISA). This assay, together with second- and third-generation assays, their associated confirmatory tests, and nucleic acid testing (NAT) for viral RNA or DNA, has reduced the per-unit risk of hepatitis C virus (HCV) transmission to less than 0.0001% (1 per 1.6 million to 1 per 1.935 million).56 Before these tests were available, the risk per unit was about 4%. Improved hepatitis C testing has eliminated the need for surrogate tests, such as the measurement of alanine aminotransferase (ALT) levels and testing for antibody to hepatitis B virus (HBV) core antigen. However, the test for antibody to HBV core antigen is still used to detect recently infected donors who lack measurable circulating HBV antigen.

The epidemiology of HCV is still poorly understood. Approximately 20% to 25% of persons found to be HCV positive have no known risk factors.8 Sexual transmission occurs with enough frequency to warrant evaluation of partners and appropriate use of methods of barrier protection.9 Heterosexual transmission of HCV may be asymptomatic; a donor who was infected via sexual contact but has not yet developed detectable antibodies is a potential risk to the blood supply.

Table 1 Estimated Risk of Infection per Transfused Blood Product^5,6

Virus	Risk in Year 2000	Risk in Year 2003
Human immunodeficiency virus type 1/type 2 (HIV-1/2)*	1 in 660,000	1 in 2,135,000
Human T cell lymphotropic virus type I/type II (HTLV-I/II)⁺	1 in 641,000	1 in 2,993,000
Hepatitis B	1 in 63,000	1 in 205,000
Hepatitis C	< 1 in 103,300	1 in 1,935,000

*Risk with p24 antigen testing and HIV antibody testing.

^Approximately 67% of infections resulting from transfused blood products are infections with HTLV-II.

Therefore, persons who are sexual partners of known HCV-infected persons may be excluded from donation. Donors found to be ELISA positive for HCV should have supplemental tests, such as the second-generation and third-generation recombinant immunoblot assays (RIBA-2, RIBA-3). Donors with positive supplemental test results are likely to have a chronic HCV infection and require further clinical evaluation.10 Donors with negative supplemental test results probably had false positive screening results and may be eligible for reentry into the allogeneic donor pool after 6 months.11 The infection status of donors with indeterminate supplemental results is best resolved by testing for HCV RNA; those with only a single band on the most sensitive supplemental test (RIBA-3) have a less than 4% chance of having circulating HCV RNA.12

Gene amplification methods for detecting HCV RNA are used on all blood products before release for transfusion. These tests directly detect the presence of virus before antibody development and are responsible for the current minuscule risk of HCV transmission.6 Correlation studies have shown that only 80% of samples with confirmed serologic positive results for HCV are also NAT positive. This is consistent with previous estimates of the prevalence of HCV-positive persons who have cleared the virus.

Hepatitis B Transmission of other forms of hepatitis by blood products is extremely rare. Modern testing methods and eliminating the practice of paying whole blood donors have reduced HBV infections to about one in 205,000 units transfused.

Hepatitis A Because the viremic phase of hepatitis A lasts about 17 days in humans before signs and symptoms develop, hepatitis A transmission from single-donor products is extremely rare. Pooled products, such as factor concentrates, however, carry a substantially higher risk.

Hepatitis D Hepatitis D is a defective virus that requires HBV to produce fulminating hepatitis; it is a concern only for patients already infected with HBV.

Hepatitis G The flavivirus hepatitis G, now shown to include several strains, is present in about 4.5% of normal donors. It is transmitted from mother to infant, sexually, and by blood.14 Circulating hepatitis G RNA is removed after the recipient develops antibodies, which makes the recipient resistant to further infection.15 HGV has not been linked to any form of clinical hepatitis in children or adults. It is an example of a blood-borne virus without known pathogenicity; as yet, there is no clear reason to remove it from the blood supply.

Screening for Retroviruses

All blood products are screened for HIV-1, HIV-2, human T cell lymphotropic virus type I (HTLV-I), and HTLV-II. Data obtained nationally from American Red Cross donors indicate that the infection risk has been reduced from two per 100 transfusions to about one per 2 million transfusions because of the exclusion of high-risk donors and the postdonation testing for HIV-1 and HIV-2 antibodies and NAT for viral RNA or DNA.5

To have predictive value, the ELISA screening test for HIV must be confirmed by Western blot assay. Studies based on polymerase chain reaction (PCR) data, culture data, and donor review all indicate that donors with negative or indeterminate Western blot results are seldom, if ever, HIV posi-tive.16 A follow-up study of donors who were ELISA positive and whose Western blot results were indeterminate demonstrated that positive ELISA results persisted in about 45% of cases. Of these, 84% still had indeterminate Western blot results, but none were shown to be HIV positive by PCR.17,18 There have been occasional false positive results of Western blot assays in low-risk donors.19 The possibility of a false positive result should be remembered when one is counseling low-risk donors who have had unexplained positive results on Western blot testing; these false positive results must always be confirmed by careful clinical follow-up. Data since the introduction of NAT show that less than 6% of confirmed serologic positive results will be NAT negative. On the other hand, only 1.5% of NAT screen reactives were Western blot indeterminate or negative. Virtually none of these NAT reac-tives were confirmed by discriminatory NAT testing or PCR.

The prevalence of HTLV-I and HTLV-II in United States donors was about 0.03% in 1995. Data from 2001 suggest that the prevalence has been reduced to about 0.01%; about two thirds of these HTLV-positive patients have HTLV-II.5 Several longitudinal studies have defined the clinical consequences of HTLV-I/II infection20-22; they are useful in advising donors who have had positive or indeterminate test results. In a prospective, longitudinal study comparing seropositive blood donors with seronega-tive blood donors, both viruses were associated with an increase in the incidence of some infectious diseases. No cases of adult T cell leukemia or lymphoma were identified; myelopathies, though rare, were associated with both HTLV types.22 The risk of HTLV-I/II transmission by blood products is one per 2,993,000.5 As with HIV, laboratory studies and epidemiologic investigations of HTLV-I/n indicate that patients with positive screening-test results and negative or indeterminate supplemental-test results are unlikely to have clinical sequelae and that the positive results are most likely false positives.

False Positive Test Results during Donor Screening

The causes of false positive test results for HCV and retro-viruses are poorly understood. Flu vaccines administered in 1992 were associated with an increase in false positive results for these viruses.24 However, the proteins responsible for cross-reactivity have not yet been identified. Tests for low-prevalence infections, even tests with excellent specificity and sensitivity, will always be associated with a substantial proportion of false positive results. Consequently, test characteristics, as well as culture and PCR results, can provide reassurance for donors who are not at risk but who have had positive screening-test results and negative or indeterminate confirmatory-test results. As PCR technology improves, it will probably become the most reliable means of establishing whether a positive result represents infection or is a false positive result.

Emerging Infectious Diseases

Until either screening tests or sterilization procedures become available, epidemiologic considerations are the only possible protective strategy against newly recognized infections.25 For example, transmission of West Nile virus by blood products has led to new donor questions to eliminate donors at risk for this disease, and a nucleic acid-based test for all donated units was introduced in June 2003.26 In the case of prion diseases such as variant Creutzfeldt-Jakob disease (vCJD), the restrictions put in place have led to a loss of 4% to 5% of active blood donors and caused transient shortages of certain products such as albumin and immune globulin. In Great Britain, the first case of possible transfusion-transmitted vCJD was reported in December 2003.27 The report identified 48 recipients of blood from a total of 15 donors who had developed vCJD. In one recipient, symptoms of the disease developed 6.5 years after the possible exposure, a time frame consistent with human-to-human vCJD transmission. The authors estimated the chance of this patient having contracted vCJD independent of the transfusion to be between one in 15,000 and one in 30,000. Recent estimates of the incidence of new vCJD diagnoses in the United Kingdom suggest that there will be fewer than 200 cases from 2001 to 2005 and fewer than 100 cases from 2006 to 2010.28 Thus, although the current donor restrictions seem prudent, it is important that donors rejected because of epidemiologic risk for vCJD understand the low probability that they actually have a health problem.

Pretransfusion Testing

Antigen Phenotyping

Blood recipients are routinely tested to establish their ABO phenotype and Rh type. Establishing ABO type is essential because isoagglutinins (antibodies) against A or B antigens not present on a person’s red cells are acquired during the first 2 years of life. These IgM antibodies will cause an immediate hemolytic reaction if ABO-incompatible red cells are transfused.

The terminal carbohydrate on these antigens determines specificity in the ABO system, with type A being associated with N-acetylgalactosamine and type B being associated with a terminal galactose. Persons with type O lack both of these terminal sugars. These residues are added by a glycosyltransferase, which was thought to be either nonfunctional or absent in type O persons. Yamamoto and colleagues29 used molecular techniques to prove that glycosyltransferase in type O persons is very similar to the transferase in type A persons. The type O glycosyltrans-ferase is nonfunctional because of a single base deletion that produces a frameshift and a downstream stop codon.

All methods of ABO typing depend on demonstrating that the antigens found on the red cells are consistent with the expected isoagglutinins [see Table 2]. Molecular methods to determine ABO genotype are available.30 D antigen specificity typing in the Rh system is done because of this antigen’s potency as an immunogen. Antibodies to the D antigen are the most important cause of isoimmune hemolytic disease of newborns. Rh antigens are membrane glycolipids or glycoproteins. Antibodies against antigens of this class, which includes the Rh, Duffy, Kell, Kidd, and Lutheran systems, will usually cause shortened red cell survival. In contrast to antigens with carbohydrate-mediated specificity, glycolipid and glycoprotein antigens do not stimulate antibody formation unless the transfusion recipient was previously exposed to allogeneic red cells either from transfusion or from fetal red cells during pregnancy or delivery.

D antigen typing is also done using agglutination techniques. In some cases, less antigenic forms of the D antigen, called weak D, require an antiglobulin reagent to enhance detection. Structural studies of the complementary DNA associated with the major Rh antigens (D, Cc, and Ee) have provided probes for direct geno-typing.31 Molecular methods of prenatal Rh type determination have revealed that most Rh-negative persons lack the D gene.

Some persons with the weak D phenotype have mosaic D genes because of exchange with some of the exons of the CcEe gene.

Because the genotypes of many of the clinically relevant red cell antigens are now known, it should now be possible to predict red cell phenotype by DNA analysis. Reid and colleagues32 were able to correctly predict the red cell phenotype in 60 multi-transfused patients by DNA analysis of each patient’s white blood cells. This approach, although not yet generally available, will be useful for recently transfused patients, for whom circulating allogeneic red cells complicate antigen phenotyping.

Screening For Antibodies

In addition to identifying patient ABO and D red cell pheno-types, blood banks must screen serum for red cell-specific antibodies, which can cause serious reactions with transfused red cells. Screening involves testing serum against indicator type O red cells displaying all the clinically important red cell antigens. Positive reactions are detected by adding an antiglobu-lin reagent (Coombs reagent) to the incubated mixture of type O red cells after it has been washed free of serum. Any observed agglutination is from the reaction of the antiglobulin reagent with antibody adsorbed on the surface of the indicator red cells [see 5:IV Hemoglobinopathies and Hemolytic Anemias]. Agglutination of the indicator red cells indicates the presence of other antibodies, which require identification. The absence of agglutination excludes all antibodies except those against antigens so rare that they are not displayed on the indicator red cells.

Use of type-specific blood removes the risk of ABO incompatibility. There is, however, a residual risk of an immunologic reaction from the antibodies to other red cell antigens; such antibodies are present in about 3% to 5% of a random population and in 10% to 15% of persons who were recently transfused or women with a history of pregnancy. Screening for antibodies reduces the frequency of reactions to about 0.06%. Performing a full cross-match, in which the recipient’s serum is tested against the red cells actually being transfused, is of negligible additional benefit, because it excludes only technical errors and the rare antibody that is not detected by the screening. Therefore, a full crossmatch is performed only for persons already known to have made antibodies, because such persons are more likely to form additional antibodies if they are further stimulated by red cell transfusion.

Patients who may receive allogeneic red cells who either have had a transfusion or have become pregnant within the past 3 months must be tested for new antibodies every 3 days. There is no consensus concerning how long the interval should be between patient specimen collection and use of the specimen in pretransfusion testing for patients not recently exposed to red cells. Commonly, specimens are accepted 14 to 28 days before the date for use. However, one study showed that no new antibodies appeared in paired specimens collected up to 1 year apart, suggesting that a longer acceptance interval may be possible.

Table 2 ABO Typing

Blood ^Type	Erythrocytes plus Anti-A Serum	Erythrocytes plus Anti-B Serum	Antibodies in Patient’s Serum
A	+	0	Anti-B antibodies
B	0	+	Anti-A antibodies
AB	+	+	No antibodies
O	0	0	Anti-A and anti-B antibodies

+—agglutination

0—no agglutination

Blood Components

Most blood donations undergo a fractionation process that allows each component to be used for specific indications. Whole blood can be fractionated into red cells (which contain most of the leukocytes), platelet concentrates (which contain some leukocytes), and plasma [see Table 3]. Plasma can be further subdivided into coagulation components and albumin. Each whole-blood unit can potentially support many recipients and clinical needs, maximizing use of each donation.

After 24 hours’ storage, whole blood contains no active platelets, and after 2 days, it is deficient in factors V and VIII. Therefore, except for some autologous blood programs that use whole blood rather than packed red cells, use of whole blood has now been almost completely supplanted by therapy employing specific blood components.

Red Blood Cells

The anticoagulant used determines the shelf life of red cells [see Table 3]. Citrate-phosphate-dextrose (CPD) with the addition of adenine (CPDA-1) increases storage time from 28 days to 35 days. Most red cells are now stored in CPD to which extra nutrients have been added, which increases storage time to 42 days. This additive solution sometimes contains additional saline, which can be removed if units with very high hematocrits (~70%) are needed.

To prevent transfusion reactions or to delay alloimmuniza-tion, red cells are further processed by leukocyte reduction (see below) or washing to remove plasma proteins. Current filter technology reduces white cell counts to less than 5 x 106 cells per unit, a concentration that is sufficient to reduce febrile transfusion reactions and delay alloimmunization and platelet refractoriness. Washing red cells removes the plasma, leaving less than 0.5 ml per unit, a degree of plasma depletion usually effective in treating allergic transfusion reactions. Leukocyte filtration and washing red cells usually shorten the product shelf life to 24 hours, because these procedures require breaking the seal on the plastic bag that contains the red cells, thereby increasing the risk of bacterial contamination. Leukocyte reduction can be accomplished during collection, immediately after collection in the blood bank, or at the bedside during product infusion. Prestorage or laboratory filtration is preferred to bedside filtration.34 Universal leukoreduc-tion has been implemented in Canada and Europe, but it is not yet required in the United States because of concerns regarding cost-effectiveness.

Table 3 Characteristics of Blood Products and Indications for Use

Product	Volume (One Unit)	Hematocrit (Hct) or Platelet Count	White Cell Count	Shelf Life	Donors per Unit	Storage Outside Blood Bank	Indication
Whole blood	450-500 ml (± 10%)	Hct 35-45	3-5 x 10⁹	With additive solution, 42 days; with CPDA-1, 35 days; with CPD, 28 days	1	2°-6°C	Massive transfusion if available; exchange transfusions in newborns younger than 3 days
Red cells	With additive solution, 350 ml; with CPD, 250 ml	If additive solution used, Hct 55; if CPD used, Hct 70	1-2 x 10⁹	Same as whole blood	1	Same as whole blood	To increase oxygen-carrying capacity; to maintain volume and oxygen-carrying capacity when bleeding
Platelet concentrates	40 ml	8-9 x101°	2-6 x10⁷	5 days	1/U; given as pool of 5-6 U	Room temperature	For major bleeding or surgical procedures, when platelet count is < 50,000-100,000 ^l; for prophylaxis in nonbleeding patients, when platelet count is < 10,000 ^l; for bleeding that is refractory to SDPs, when HLA-matched or crossmatched platelets are not available
Single-donor platelets	200-250 ml	3-5 ml x 10¹¹	Depends on method of collection	5 days	1	Room temperature	Same as platelet concentrates, but SDPs are preferred because of lower donor exposure
Fresh frozen plasma (FFP)	200-250 ml	—	< 1 x 10⁵	1 yr; 24 hr when thawed	1	2°-6°C	Multiple coagulation factor deficiency from bleeding or DIC; reversal of warfarin therapy; factor XI deficiency when factor XI concentrates are unavailable; treatment of TTP
Cryoprecipitate	10-20 ml; pool of 10 U ~ 200 ml	—	< 1 x 10⁵	1 yr; 24 hr when thawed	1/U; given as pool of 10 U	2°-6° C wet ice	Replacement of fibrinogen when acutely depleted or when patient cannot tolerate volume load of equivalent amount of FFP (1 pool of cryoprecipitate = 4 FFP); fibrin glue (usually only 1 unit); replacement of von Willebrand factor if concentrate is not available; replacement of factor XIII; replacement for qualitatively abnormal fibrinogen

CPD—citrate-phosphate-dextrose CPDA-1—CPD with adenine DIC—disseminated intravascular coagulation FFP—fresh frozen plasma HLA—human leukocyte antigen SDPs—single-donor platelets TTP—thrombotic thrombocytopenic purpura

Freezing is an alternative method for storing red cells. Red cells can be kept in a cryoprotectant (usually glycerol) for 10 years. Freezing is therefore ideal for storing rare units or autolo-gous units from persons with rare blood types, for whom it is difficult to find compatible allogeneic red cells. When a unit is at the end of its liquid storage shelf life, the cells can be rejuvenated with fresh media and nutrients; they can then be frozen and stored. To be used, frozen red cells must be thawed and the glyc-erol removed, so preparation time for this product is longer than for products stored in the liquid state. Thawed, deglycerolized red cells generally must be transfused within 24 hours.

Platelets

Platelets can be provided either as platelet concentrates from a number of blood donors or from a single donor. SDPs are collected by a continuous apheresis process that removes platelets and returns all other blood components. A single transfusion of platelet concentrates usually consists of platelets derived from four to six units of donated whole blood, which is about the same number of platelets contained in one SDP product. The advantage of SDP therapy is the reduced risk of blood-borne infection and antigen exposure, because the product is from one donor rather than from four to six; disadvantages are a longer collection time, greater cost, and often limited supply. The potential advantages of each of these products have been summarized in a review.35 ABO Rh-compatible platelets should be used when possible, because studies have shown significantly better therapeutic results from compatible transfusions.36

Plasma

Fresh plasma, frozen within 8 hours of collection (FFP), contains all the procoagulants at normal plasma concentrations. After thawing, it can be kept for 24 hours at 2° to 6° C and will retain 3 to 4 mg/ml of fibrinogen and 1 IU/ml of all the other coagulation components.

Solvent/detergent-treated plasma (S/D plasma) was formerly available as an alternative to FFP with lower infectious risks. However, in 2002 this product was withdrawn from the market by the manufacturer, presumably because of lack of demand for the product and evidence of selective inactivation of certain plasma components. Alternative methods of postcollection sterilization of single units of plasma are becoming available. The main advantage of a postdonation sterilization process is that it protects patients from blood-borne infections, including ones that are not yet recognized. Potential disadvantages are cost and less effectiveness than FFP.

Cryoprecipitate consists of the cryoproteins recovered from FFP when it is rapidly frozen and then allowed to thaw at 2° to 6° C. These cryoproteins include fibrinogen, factor VIII, von Willebrand factor, factor XIII, and fibronectin. About 40% of the components in FFP are recovered. The cryoproteins are suspended in a small amount of plasma that contains ABO isoag-glutinin at the concentration found in normal plasma. A pool of 10 units of cryoprecipitate (each derived from one unit of FFP) contains an amount of fibrinogen equivalent to four units of FFP but in one fourth to one fifth the volume. Consequently, a cryo-precipitate pool permits more rapid replacement of fibrinogen than FFP but has the disadvantage of more donor exposures. After the cryoprecipitate is removed from FFP, the residual product is known as cryopoor plasma. Once frozen, cryopoor plasma has the same shelf life as FFP.