Manual 512402

Additional laboratory evaluation If any of the three initial checks and tests (error check, visual inspection for plasma free hemoglobin, and DAT) gives positive or suspicious results, the diagnosis of an acute HTR should be vigorously pursued. Even if no error or apparent incompatibility is found, the possibility of acute HTR should still be considered if the patient’s clinical presentation is consistent with such a reaction. The tests listed below help characterize the cause of the HTR, if one has occurred, or help clarify the immunologic and serologic status of patients in whom the diagnosis is unclear. Some or all may be performed following a written institutional protocol or at the discretion of the physician in charge of the transfusion service. 1. Perform ABO and Rh testing on the patient’s pre-reaction and post-reaction samples and on blood from the unit or an attached segment. If ABO and Rh typing on the pre-reaction and postreaction samples do not agree, there has been an error in patient or sample identification, or in testing. If sample mix-up or mislabeling has occurred, another patient’s specimen may also have been incorrectly labeled; it is important to check the records of all specimens received at approximately the same time. If blood in the bag is not of the ABO type noted on the bag label, there has been an error in unit labeling. 2. Perform antibody detection tests on the pre-reaction and postreaction samples and on the donor blood. If a previously undetected antibody is discovered, it should be identified. Once the antibody is identified, retained sample from transfused donor unit should be tested for the corresponding antigen. If a previously undiscovered antibody is present in a postreaction specimen but not in a pre-reaction sample, the reason may be 1) a sample identification error, 2) anamnestic antibody production following a recent transfusion, or, less likely, 3) passive transfer of antibody from a recently transfused component. It may be desirable to use enhancement techniques such as an increased serum-to-cell ratio, low-ionic- strength saline, polybrene, polyethylene glycol, or enzyme techniques when retesting the pre-reaction specimen. 3. Repeat crossmatch tests, with pre-reaction and postreaction samples in parallel using the antiglobulin technique. A positive crossmatch in the face of a negative antibody screening test may indicate the presence of an antibody directed against a low frequency blood group antigen, which might be missed by an immediate-spin or computer crossmatch. 4. Perform DAT and antibody detection tests on additional specimens obtained at interval after the transfusion reaction. A first postreaction sample may have serologically undetectable level of a significant alloantibody, especially if all the antibody molecules have attached to the incompatible transfused cells. In this event, antibody levels would rise rapidly, and antibody detection and identification would become possible within a few days. 5. Perform frequent checks of the patient’s hematocrit or hemoglobin values, to see whether the transfused cells produced the expected therapeutic rise, or whether a 91

decline occurs after an initial increase. In patients with sickle cell anemia, survival of transfused red cells can be followed by evaluation of the levels of hemoglobin A. in complex cases, phenotypic differences between autologous and transfused cells quantitated by flow cytometry have been used to follow survival. 6. In vivo red cell survival studies have been used to demonstrate the rare occurrence of acute HTR in the absence of detectable alloantibody. When the patient is phenotyped in preparation for such studies, it is important that the sample be one that contains only the patient’s red cells. This may be difficult if the patient has received transfusions within the previous several weeks. Method 2.15 gives a technique for obtaining autologous red cells from a patient who has been transfused. If an antigen is present on the donor’s red cells and absent from those of the patient, its presence or absence in postreaction samples indicates whether the transfused cells have survived and remained in the circulation. 7. Markers of hemolysis including lactate dehydrogenase, bilirubin, and haptoglobin levels may be useful, particularly if pre- and multiple postreaction measurements are available. 8. Examine the blood remaining in the unit and the administration tubing for evidence of hemolysis, especially if no immune explanation for hemolysis can be demonstrated. Depending on how the blood was damaged, hemolysis may be present in the container and the administration tubing, or only in the administration tubing. For example, if the unit had been inappropriately heated in the container, both the blood in the container and in the administration tubing would be hemolyzed. If a faulty infusion device had been used during blood administration, hemolysis might be present in the administration tubing, but not in the container. 9. Test the patient’s serum for presence of anti-IgA if the presentation suggests an anaphylactic reaction. Preliminary information can be obtained by quantitation of IgA, because most patients with IgA-related anaphylaxis have been IgA deficient. Note, however, that subclass or allotype-specific antibodies may develop in patients with normal IgA levels. If additional transfusions are required, cellular components can be washed; if plasma (or platelets; see above) is required, a test dose can be administered under maximal surveillance with epinephrine at the bedside. 10. Examine the returned unit for any abnormal appearance, including clots or any brownish, opaque, muddy, or purple discoloration. If the clinical presentation suggests bacterial sepsis, a Gram’s stain and bacterial cultures of the contents should be performed, even if the unit looks normal. Treatment for suspected bacterial contamination should be based on clinical consideration, as a delay in therapy may result in severe morbidity or death. Treatment includes prompt intravenous administration of antibiotics after blood and other appropriate cultures are obtained, combined with therapy for shock. 11. Examine the patient’s pretransfusion sample and a sample of the donor’s plasma for antibodies to HLA and/or neutrophil antigens if the clinical presentation suggests 92

TRALI. Crossmatching recipient lymphocytes or granulocytes with implicated donor sera can provide supportive evidence for TRALI. DELAYED CONSEQUENCES OF TRANSFUSION ALLOIMMUNIZATION TO RED CELL ANTIGENS Pathophysiology Primary alloimmunization, evidenced by appearance of newly formed antibodies to red cell antigens, becomes apparent weeks or months after transfusion. It has been estimated that alloimmunization occurs in unselected immunocompetent recipients with a risk of 1-1.6% per RBC unit, provided that D-negative recipients received D-negative cellular components. Hemolysis has been reported in cases of primary immunization, but these reports are controversial, and even if it occurs, the phenomenon must be very rare and usually subclinical. Serologic observations Once alloimmunization has occurred, antibodies may diminish to undetectable levels, especially antibodies in the Kidd system (anti-Jka and anti-Jkb). If red cells that express the antigen are subsequently transfused, however, an anamnestic response may cause the appearance, within hours or days, of IgG antibodies that react with the transfused red cells. In a prospective study, previously undetected alloantibodies were found in 58 of 2082 (2.8%). RBCs recipients within 7 days of transfusion. In two of 58, only the DAT was positive, but in all others, repeat antibody screening would have detected the new antibody. Regardless, characterization of an eluate is necessary because alloantibodies may be present on the RBCs that are not in the serum. If the clinical laboratory discovers an anamnestic response, both the transfusion service director and the patient’s clinician should be notified and the possibility of a delayed HTR (DHTR) should be investigated. Delayed reactions In most cases, anamnestic antibody production does not cause hemolysis, leading to the designation “delayed serologic transfusion reaction” (DSTR). However, in some patients, red cell destruction will result from the combination of significant levels of antibody with hemolytic potential and large numbers of transfused red cells in the circulation; in the study cited above, only one of 58 recipients with a new antibody within 7 days of transfusion was shown to have hemolysis. This translated into a DHTR rate of one per 2082 recipients or one for every 11,328 units transfused. As would be expected, retrospective studies, which would be similar to the routine experience of a transfusion service, yield lower rate of DSTRs, but the rate of clinically detectable hemolysis may be roughly equivalent. The most common presenting signs of a DHTR 93

are fever, a declining hemoglobin, and mild jaundice. Some DHTRs present as the absence of anticipated hemoglobin or hematocrit elevation after transfusion. Other clinical problems are infrequent; hemoglobinuria is occasionally noted, but acute renal failure is uncommon. If a DHT is suspected, a freshly obtained blood sample may be tested for unexpected alloantibodies, both in the serum and, by DAT, on the red cells. Discoveries of a previously undetected red cell alloantibody in a patient manifesting hemolysis strongly supported by demonstration of the corresponding antigen on the red cells from a retained segment from one or more transfused units. Antigen typing of the red cells currently circulating in the patient may also suggest whether the newly incompatible cells have been eliminated, or whether some are still circulating. Repeat antibody screening on the patient’s previous specimen will rule out technical errors. Treatment Specific treatment is rarely necessary, although it may be prudent to monitor the patient’s urine output and renal function and observe for changes in coagulation function. If transfusions are still needed, donor units should be selected that lack the antigen corresponding to the newly discovered antibody. If such units are unavailable and transfusion is necessary, the risk of a possible overt, acute HTR should be weighed against the risk of delaying transfusion. Prevention Future transfusion for the patients should lack the antigen(s) responsible for the anamnestic response, even if the antibody again becomes undetectable. Some facilities issue a “medical alert card” with this information for the patient to carry and present at the time of hospitalization or transfusion in a different facility. It is to prevent these problems that Standards for Blood Banks and Transfusion Services mandates permanent preservation of records of clinically significant antibodies, and review of previous records before red cells are issued for transfusion. POSTTRANSFUSION AUTOANTIBODY Occasionally, transfusion of allogeneic RBCs and platelets stimulates production of autoantibodies; in some of these patients, hemolytic anemia or thrombocytopenia may occur. See Chapter 20 for more details. 94

TRANSFUSION-ASSOCIATED GRAFT-VS-HOST DISEASE Transfusion-associated graft-vs-host disease (TA-GVHD) is a usually fatal immunologic transfusion complication caused by engraftment and clonal expansion of donor lymphocytes in a susceptible host. The engrafted lymphocytes mount an immunologic attack against recipient tissues, including hematopoietic cells, leading to refractory pancytopenia with bleeding and infectious complications, which are primarily responsible for the 90-100% mortality rate in afflicted patients. TA-GVHD is rare in US transfusion recipients and has been observed almost exclusively in immunocompromised patients. In contrast, over 200 cases of TA-GVHD have been described in Japan, with incidence rates reaching 1:660 patients undergoing cardiovascular surgery. Greater genetic homogeneity of the Japan population and frequent use of fresh whole blood from related donors are thought to be the primary reasons for the surprisingly frequent occurrence of TA- GVHD in that country. Pathophysiology and manifestations The pathophysiology of TA-GVHD is complex and incomplete understood. Key mechanisms include escape of donor T lymphocytes present in cellular blood components from immune clearance in the recipient and subsequently clonal expansion of these cells with immune destruction of host tissues. This results in some or all of the following clinical findings: fever, dermatitis or erythroderma, often starting on palms, soles, earlobes, and face, with additional finding ranging from edema to widespread blistering; hepatitis, with elevations in alanine and aspartate aminotransferases, alkaline phosphatase, and bilirubin; enterocolitis, with up to 3-4 liters per day of secretory diarrhea; and pancytopenia, with a hypocellular marrow and reduction in all marrow elements. Cytokine dysfunction, recruitment of recipient cells into the immune reaction, and release of biologic mediators, in particular nitric oxide, all play a role in the pathogenesis. Clinical symptoms typically appear within 10-12 days of transfusion. Factors that determine an individual patient’s risk for TA-GVHD include whether and to what degree the recipient is immunodeficient, the degree of HLA similarity between donor and recipient, and the number of transfused T lymphocytes capable of multiplication. GVHD may occur in an immunologically normal recipient if the donor is homozygous for an HLA haplotype for which the recipient is heterozygous, a so-called “one-way” HLA match, and if the component contains numbers of viable T cells. Of interest is the fact that TA-GCHD has not been reported in an AIDS patient. Treatment and prevention At present, there is no effective treatment for TA-GCHD, so prevention is necessary. Gamma irradiation of cellular blood components is the accepted standard method to prevent TA-GVHD. The dose mandated by the FDA is a minimum of 2500 cGy targeted to the midline of the container and minimum dose of 1500 cGy delivered to all other parts of the component. This renders T lymphocytes incapable of replication without substantially affecting the function of red cells, platelets, and granulocytes. 95

AABB Standards for Blood Banks and Transfusion Services requires routine irradiation of cellular components from units collected from the recipient’s blood relatives, and donors selected for HLA compatibility by typing or crossmatching. Policies should be in place to define the other groups of patients who should receive irradiated cellular components, and there must be a process for ensuring that once a patient has been determined to at risk for TA-GVHD, all cellular components will be irradiated as long as clinically indicated. Published guidelines additionally recommend component irradiation for: 1) Immunocompromised hematopoietic progenitor cell (HPC) or organ transplant recipients (this includes allogeneic and autologous HPC transplants) 2) Patients with hematologic disorders who will be undergoing allogeneic HPC transplantation imminently 3) Intrauterine transfusions 4) Neonates undergoing exchange transfusion or use of extracorporeal membrane oxygenation 5) Patients with Hodgkin’s disease 6) Patients with congenital cellular immunodeficiencies. TA-GVHD has also been reported in chronic lymphocytic leukemia patients receiving fudarabine phosphate POSTTRANSFUION PURPURA Pathophysiology and manifestation Posttransfusion purpura (PTP) is an uncommon event, although over 200 cases have been published. It is characterized by abrupt onset of severe thrombocytopenia (platelet count usually < 10,000/µL) an average of 9 days following transfusion (range, 1-24 days). Components provoking the reaction have usually been RBCs or whole blood, but PTP has also been reported after platelet and plasma transfusion, and after transfusion of frozen deglycerolized RBCs. Most patients have previously been pregnant or transfused. “Wet purpura” is common, and fatal intracranial hemorrhage can occur. The ratio of affected patients is 5 women to 1 man, and the median age is 51 years (range, 16-83). Most cases (68%) involve patients whose platelets lack the HPA-1a (PlA1) antigen (<2% of the population), and who form the corresponding antibody, but immunization to HPA-1b is reported in 10% and other platelet antibodies have been associated with the syndrome as well. PTP is usually self-limited, with full recovery within 21 days. Historically, 10-15% of patients have been reported to die from PTP, typically from intracranial bleeding, so treatment is desirable. The reason for destruction of the patient’s own platelets by what appears to be a platelet alloantibody is controversial. Three mechanisms have been proposed including: 1) formation of immune complexes of patient antibody and soluble donor antigen that bind to Fc receptors of the 96

patient’s platelets and mediate their destruction, 2) conversion of antigen-negative autologous platelets to antibody targets by soluble antigen in the transfused component, and 3) cross- reactivity of the patient’s antibodies with autologous platelets (i.e. presence of an autoantibody component). Treatment Because PTP remits spontaneously, treatments may appear falsely efficacious. Steroids are frequently given but their role is controversial. Plasma exchange typically achieves platelet counts of 20,000/ µL in 1-2 days, but the use of high-dose. Immunoglobulin intravenous (IGIV) is now supplanting this therapy. With the use of IGIV, recovery to platelet counts of 100,000/ µL is typically achieved within 3-5 days. As it does in other disorders such as immunethrombocytopenic purpura, IGIV appears to block antibody-mediated clearance of the target cells. If randomly selected platelets are transfused, patients may experience a febrile transfusion reaction, and, in the vast majority of cases, such transfusions have not been efficacious. Antigen-negative platelets can be of benefit in PTP, and, in conjunction with IGIV, reversal of this disorder in 1 day is now possible. Unfortunately, the time necessary to procure such platelets often limits their usefulness. IMMUNOMODULATORY EFFECTS OF TRANSFUSION Transfusion has been known to modulate immune responses since the 1973 observation by Opelz and coworkers of improved renal allograft survival in transfused patients. This beneficial tolerance-inducing effect of transfusion raised concerns that transfusion may have other, adverse, effects in different clinical settings, including increased rates of postoperative solid tumor recurrence and bacterial infection. Despite numerous retrospective and several large prospective studies, the clinical significance of transfusion-associated immunomodulation, and the usefulness of preventive strategies such as leukocyte reduction of transfused components remains controversial. Nonetheless, many blood centers and transfusion services are moving toward pre-storage leukocyte reduction of all cellular blood components at least in part because of these data, IRON OVERLOAD Every unit of RBCs contains approximately 250 mg of iron. The average rate of excretion of iron is approximately 1 mg per day. As red cells are destroyed, the majority of the released iron cannot be excreted and is stored in the body as hemosiderin and ferritin. Transferrin becomes saturated after the administration of 10 to 15 RBC units to a nonbleeding patient. Chronically transfused patients, especially those with hemoglobinopathies, have progressive and continuous accumulation of iron and no physiologic means of excreting it. Storage occurs initially in reticuloendothelial sites, but when these are saturated, there is deposition in parenchymal cells. Iron deposition interferes with function of the heart, liver, and endocrine glands; hepatic failure and cardiac toxicity cause most of the morbidity and mortality. Patients who are chronically 97

transfused for diseases such as thalassemia, sickle cell disease and other chronic anemias are at greatest risk for iron overload. A cumulative dose of 50 to 100 RBC units can cause significantly greater morbidity and mortality than the underlying anemia. Treatment is directed at removing iron without reducing the patients’ circulating hemoglobin. Metered subcutaneous infusion of desferoxamine, an iron-chelating agent, can reduce body iron stores in such patients, but the regimen of nightly subcutaneous infusion by pump is arduous and expensive, and compliance is often poor. Deferiprone is more effective than deferoxamine in reducing myocardial siderosis. The newest agent, defarasirox, has a much longer half-life than deferoxamine and may be administered in one daily oral dose. In transfusion- dependent patients with hemoglobinopathies, neocytes to prolong the intertransfusion interval or red cell exchange to minimize additional iron loads have been used. Each transfusion service must maintain indefinitely the records of patients who have had transfusion complications or evidence of alloimmunization. Possible cases of blood contamination and transfusion of disease must also be reported to the institution where the blood was drawn. RECORDS OF TRANSFUSION COMPLICATIONS Records must be kept, and consulted, to prevent patients who have had a transfusion reaction from being exposed to known offending agents in subsequent transfusions. For example, patients with a history of IgA-related anaphylactic reactions should be transfused with plasma products that lack IgA. A history of repeated or severe FNHTR might prompt the use of leukocyte- reduced cellular blood components. Red cell alloantibodies may become undetectable over time as discussed above, so records should be checked and compatible blood issued in order to prevent a DHTR. Routine checking of previous results of ABO and Rh may disclose an error in testing or in the identification of a current sample. Records of patients with special needs In addition to records of transfusion reactions, transfusion services should maintain records of patients who need specially prepared or manipulated components. This is especially important in institutions where physicians rotate frequently, and the need for irradiated, leukocyte- reduced, or IgA-deficient components may not be known to a particular physician writing an individual order. Reporting transfusion fatalities Fatalities resulting directly from the effects of transfusion must be reported to the Director, Office of Compliance, Center for Biologics Evaluation and Research, FDA, within 24 hours and by written report within 7 days. Patients who are critically ill and near death often receive transfusions in close temporal proximity to death, and clinical suspicion of cause and effect may 98

occasionally be raised. The overwhelming majority of such deaths are unrelated to transfusion, but if there is a suggestion that a transfusion might have contributed to death, it may be prudent to pursue an investigation. In the absence of such errors as administration of ABO-incompatible blood or of physiologic events clearly attributable to acute hemolysis, anaphylaxis, TRALI, or sepsis, transfusion is highly unlikely to be acutely responsible for death. The review should include all available medical and laboratory records and results of an autopsy, if performed. On the other hand, if an investigation does reveal evidence or possibility of hemolysis, anaphylactic or pulmonary events, unexplained sepsis, or ambiguous identification records, the case may warrant more extensive inquiry, …………………………………………………………………… Reference 1. Adams, D. M., W. H. Schultz, et al. (1996). \"Erythrocytapheresis can reduce iron overload and prevent the need for chelation therapy in chronically transfused pediatric patients.\" J Pediatr Hematol Oncol 18(1): 46-50. 2. Alving, B. M., Y. Hojima, et al. (1978). \"Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction.\" N Engl J Med 299(2): 66-70. 3. Baldwin, M. L., C. Barrasso, et al. (1983). \"A clinically significant erythrocyte antibody detectable only by 51Cr survival studies.\" Transfusion 23(1): 40-4. 4. Bando, T., S. Kosaka, et al. (1994). \"Effects of newly developed solutions containing trehalose on twenty-hour canine lung preservation.\" J Thorac Cardiovasc Surg 108(1): 92-8. 5. Beauregard, P. and M. A. Blajchman (1994). \"Hemolytic and pseudo-hemolytic transfusion reactions: an overview of the hemolytic transfusion reactions and the clinical conditions that mimic them.\" Transfus Med Rev 8(3): 184-99. 6. Blajchman, M. A. (1994). \"Transfusion-associated bacterial sepsis: the phoenix rises yet again.\" Transfusion 34(11): 940-2. 7. Blajchman, M. A. (1997). \"Allogeneic blood transfusions, immunomodulation, and postoperative bacterial infection: do we have the answers yet?\" Transfusion 37(2): 121-5. 8. Blumberg, N. and J. M. Heal (1994). \"Effects of transfusion on immune function. Cancer recurrence and infection.\" Arch Pathol Lab Med 118(4): 371-9. 9. Boyan, C. P. and W. S. Howland (1963). \"Cardiac arrest and temperature of bank blood.\" JAMA 183: 58-60. 10. Brand, A. (1994). \"Passenger leukocytes, cytokines, and transfusion reactions.\" N Engl J Med 331(10): 670-1. 11. Brecher, M. E., S. B. Moore, et al. (1990). \"Posttransfusion purpura: the therapeutic value of PlA1-negative platelets.\" Transfusion 30(5): 433-5. 99

12. Burks, A. W., H. A. Sampson, et al. (1986). \"Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia. Detection of IgE antibodies to IgA.\" N Engl J Med 314(9): 560-4. 13. Butler, J., D. Parker, et al. (1991). \"Systemic release of neutrophil elastase and tumour necrosis factor alpha following ABO incompatible blood transfusion.\" Br J Haematol 79(3): 525-6. 14. Butler, J. C., B. Schwartz, et al. (1991). \"Severe illness associated with group A-hemolytic streptococcal infections.\" Wis Med J 90(9): 525-9. 15. Ciavarella, D., R. L. Reed, et al. (1987). \"Clotting factor levels and the risk of diffuse microvascular bleeding in the massively transfused patient.\" Br J Haematol 67(3): 365-8. 16. Collins, D. P. (1987). \"Effects of L2C leukemia on macrophage-mediated responses.\" Cancer Immunol Immunother 25(2): 75-80. 17. Collins, J. A. (1987). \"Recent developments in the area of massive transfusion.\" World J Surg 11(1): 75-81. 18. Counts, R. B., C. Haisch, et al. (1979). \"Hemostasis in massively transfused trauma patients.\" Ann Surg 190(1): 91-9. 19. Davenport, R. D., M. Burdick, et al. (1993). \"Cytokine production in IgG-mediated red cell incompatibility.\" Transfusion 33(1): 19-24. 20. Davenport, R. D. and S. L. Kunkel (1994). \"Cytokine roles in hemolytic and nonhemolytic transfusion reactions.\" Transfus Med Rev 8(3): 157-68. 21. Dzik, W. H. and S. A. Kirkley (1988). \"Citrate toxicity during massive blood transfusion.\" Transfus Med Rev 2(2): 76-94. 22. Ferrara, J. L. (1993). \"Cytokine dysregulation as a mechanism of graft versus host disease.\" Curr Opin Immunol 5(5): 794-9. 23. Ferrara, J. L. (1995). \"The febrile platelet transfusion reaction: a cytokine shower.\" Transfusion 35(2): 89-90. 24. Harke, H. and S. Rahman (1980). \"Haemostatic disorders in massive transfusion.\" Bibl Haematol(46): 179-88. 25. Harrigan, C., C. E. Lucas, et al. (1985). \"Serial changes in primary hemostasis after massive transfusion.\" Surgery 98(4): 836-44. 26. Heddle, N. M., R. L. Soutar, et al. (1995). \"A prospective study to determine the frequency and clinical significance of alloimmunization post-transfusion.\" Br J Haematol 91(4): 1000-5. 27. Howland, W. S., R. G. Jacobs, et al. (1960). \"An evaluation of calcium administration during rapid blood replacement.\" Anesth Analg 39: 557-63. 28. Hume, H. A., M. A. Popovsky, et al. (1996). \"Hypotensive reactions: a previously uncharacterized complication of platelet transfusion?\" Transfusion 36(10): 904-9. 29. Iserson, K. V. and D. W. Huestis (1991). \"Blood warming: current applications and techniques.\" Transfusion 31(6): 558-71. 30. Juji, T., K. Takahashi, et al. (1989). \"Post-transfusion graft-versus-host disease in immunocompetent patients after cardiac surgery in Japan.\" N Engl J Med 321(1): 56. 100

31. Kobilka, B. K., T. Frielle, et al. (1987). \"An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins.\" Nature 329(6134): 75-9. 32. Lambin, P., P. Y. Le Pennec, et al. (1984). \"Adverse transfusion reactions associated with a precipitating anti-C4 antibody of anti-Rodgers specificity.\" Vox Sang 47(3): 242-9. 33. Leitman, S. F., H. Boltansky, et al. (1986). \"Allergic reactions in healthy plateletpheresis donors caused by sensitization to ethylene oxide gas.\" N Engl J Med 315(19): 1192-6. 34. Lim, R. C., Jr., C. t. Olcott, et al. (1973). \"Platelet response and coagulation changes following massive blood replacement.\" J Trauma 13(7): 577-82. 35. Linden, J. V., K. Wagner, et al. (2000). \"Transfusion errors in New York State: an analysis of 10 years' experience.\" Transfusion 40(10): 1207-13. 36. Liu, E. A., F. L. Mannino, et al. (1994). \"Prospective, randomized trial of the safety and efficacy of a limited donor exposure transfusion program for premature neonates.\" J Pediatr 125(1): 92-6 37. Lostumbo, M. M., P. V. Holland, et al. (1966). \"Isoimmunization after multiple transfusions.\" N Engl J Med 275(3): 141-4. 38. Mair, B. and G. F. Leparc (1998). \"Hypotensive reactions associated with platelet transfusions and angiotensin-converting enzyme inhibitors.\" Vox Sang 74(1): 27-30. 39. Miller, R. D., T. O. Robbins, et al. (1971). \"Coagulation defects associated with massive blood transfusions.\" Ann Surg 174(5): 794-801. 40. Mueller-Eckhardt, C. and V. Kiefel (1988). \"High-dose IgG for post-transfusion purpura- revisited.\" Blut 57(4): 163-7. 41. Murray, D. J., B. J. Pennell, et al. (1995). \"Packed red cells in acute blood loss: dilutional coagulopathy as a cause of surgical bleeding.\" Anesth Analg 80(2): 336-42. 42. Ness, P. M., R. S. Shirey, et al. (1990). \"The differentiation of delayed serologic and delayed hemolytic transfusion reactions: incidence, long-term serologic findings, and clinical significance.\" Transfusion 30(8): 688-93. 43. Oberman, H. A. (1994). \"Controversies in transfusion medicine: should a febrile transfusion response occasion the return of the blood component to the blood bank? Con.\" Transfusion 34(4): 353-5. 44. Ohto, H. and K. C. Anderson (1996). \"Survey of transfusion-associated graft-versus-host disease in immunocompetent recipients.\" Transfus Med Rev 10(1): 31-43. 45. Olinger, G. N., C. Hottenrott, et al. (1976). \"Acute clinical hypocalcemic myocardial depression during rapid blood transfusion and postoperative hemodialysis: a preventable complication.\" J Thorac Cardiovasc Surg 72(4): 503-11. 46. Opelz, G., D. P. Sengar, et al. (1973). \"Effect of blood transfusions on subsequent kidney transplants.\" Transplant Proc 5(1): 253-9. 47. Owen, H. G. and M. E. Brecher (1994). \"Atypical reactions associated with use of angiotensin-converting enzyme inhibitors and apheresis.\" Transfusion 34(10): 891-4. 101

48. Piirila, P. L., H. Nordman, et al. (1996). \"A thirteen-year follow-up of respiratory effects of acute exposure to sulfur dioxide.\" Scand J Work Environ Health 22(3): 191-6. 49. Pineda, A. A., V. W. Zylstra, et al. (1989). \"Viability and functional integrity of washed platelets.\" Transfusion 29(6): 524-7. 50. Pinkerton, P. H., A. S. Coovadia, et al. (1992). \"Frequency of delayed hemolytic transfusion reactions following antibody screening and immediate-spin crossmatching.\" Transfusion 32(9): 814-7. 51. Popovsky, M. A., A. M. Audet, et al. (1996). \"Transfusion-associated circulatory overload in orthopedic surgery patients: a multi-institutional study.\" Immunohematology 12(2): 87-9. 52. Popovsky, M. A. and N. R. Haley (2000). \"Further characterization of transfusion-related acute lung injury: demographics, clinical and laboratory features, and morbidity.\" Immunohematology 16(4): 157-9. 53. Popovsky, M. A. and S. B. Moore (1985). \"Diagnostic and pathogenetic considerations in transfusion-related acute lung injury.\" Transfusion 25(6): 573-7. 54. Przepiorka, D., G. F. LeParc, et al. (1996). \"Use of irradiated blood components: practice parameter.\" Am J Clin Pathol 106(1): 6-11. 55. Ramsey, G. and S. J. Smietana (1994). \"Long-term follow-up testing of red cell alloantibodies.\" Transfusion 34(2): 122-4. 56. Reed, R. L., 2nd, D. Ciavarella, et al. (1986). \"Prophylactic platelet administration during massive transfusion. A prospective, randomized, double-blind clinical study.\" Ann Surg 203(1): 40-8. 57. Routledge, R. C., D. M. De Kretser, et al. (1976). \"Severe anaphylaxis due to passive sensitisation by donor blood.\" Br Med J 1(6007): 434. 58. Sandler, S. G. and D. Mallory (1995). \"Biological functions of blood groups in health and disease.\" Haematologia (Budap) 27(1): 1-13. 59. Sandler, S. G., D. Mallory, et al. (1995). \"IgA anaphylactic transfusion reactions.\" Transfus Med Rev 9(1): 1-8. 60. Savitsky, J. P., J. Doczi, et al. (1978). \"A clinical safety trial of stroma-free hemoglobin.\" Clin Pharmacol Ther 23(1): 73-80. 61. Sazama, K. (1990). \"Reports of 355 transfusion-associated deaths: 1976 through 1985.\" Transfusion 30(7): 583-90. 62. Sazama, K. (1994). \"Bacteria in blood for transfusion. A review.\" Arch Pathol Lab Med 118(4): 350-65. 63. Sessler, D. I. (1997). \"Mild perioperative hypothermia.\" N Engl J Med 336(24): 1730-7. 64. Shiba, M., K. Tadokoro, et al. (1997). \"Activation of the contact system by filtration of platelet concentrates with a negatively charged white cell-removal filter and measurement of venous blood bradykinin level in patients who received filtered platelets.\" Transfusion 37(5): 457-62. 65. Shulman, I. A., K. Lohr, et al. (1994). \"Monitoring transfusionist practices: a strategy for improving transfusion safety.\" Transfusion 34(1): 11-5. 102

66. Silliman, C. C. (1999). \"Transfusion-related acute lung injury.\" Transfus Med Rev 13(3): 177- 86. 67. Silliman, C. C., A. J. Paterson, et al. (1997). \"The association of biologically active lipids with the development of transfusion-related acute lung injury: a retrospective study.\" Transfusion 37(7): 719-26. 68. Spanos, T., V. Ladis, et al. (1996). \"The impact of neocyte transfusion in the management of thalassaemia.\" Vox Sang 70(4): 217-23. 69. Valeri, C. R., H. Feingold, et al. (1987). \"Hypothermia-induced reversible platelet dysfunction.\" Ann Surg 205(2): 175-81. 70. Vamvakas, E. C., A. A. Pineda, et al. (1995). \"The differentiation of delayed hemolytic and delayed serologic transfusion reactions: incidence and predictors of hemolysis.\" Transfusion 35(1): 26-32. 71. Westhoff, C. M., B. D. Sipherd, et al. (1992). \"Severe anaphylactic reactions following transfusions of platelets to a patient with anti-Ch.\" Transfusion 32(6): 576-9 72. Widmann, F. K. (1994). \"Controversies in transfusion medicine: should a febrile transfusion response occasion the return of the blood component to the blood bank? Pro.\" Transfusion 34(4): 356-8. 73. Williamson, L. M., S. Lowe, et al. (1999). \"Serious hazards of transfusion (SHOT) initiative: analysis of the first two annual reports.\" BMJ 319(7201): 16-9. 74. Win, N., F. Matthey, et al. (1996). \"Blood components--transfusion support in post-transfusion purpura due to HPA-1a Immunization.\" Vox Sang 71(3): 191-3. 75. Winchester, B. G. (1996). \"Lysosomal metabolism of glycoconjugates.\" Subcell Biochem 27: 191-238. 103

CASE 1 Patient Information Three units of RBCs have been requested for a 24-year old white woman scheduled for splenectomy. One unit was to be transfused preoperatively because her hemoglobin level was only 90 g/L. Records from 2 years ago showed that her blood was group B, Rh-negative and that she had received RhIG within 72 hours of delivery of a Rh-positive infant. Results of pretransfusion testing were as follows: Serologic Testing Anti-B Anti-D Rh Con A1Cells B Cells Anti-A 4+ neg neg 4+ neg neg IS AHG CCC neg 37C/LISS 3+ NT Screening cell OI neg neg 3+ NT Screening cell OII neg neg 3+ NT Screening cell OIII neg neg neg 2+ Auto neg Unit 1, O neg neg neg 3+ NT Unit 2, O neg neg neg 3+ NT Unit 3, O neg neg neg 3+ NT An RBC panel tested with her serum revealed the presence of an antibody that reacted with all cells tested. Complete Rh phenotyping gave the following results: Reagent Patient RBCs Pos Control RBC Neg Control RBC anti-C neg 3+ neg anti-c neg 3+ neg anti-E neg 2+ neg anti-e neg 2+ neg Questions 1. What is the Rh phenotype of the patient? 2. What is the most likely specificity of the antibody produced by this patient? 3. What RBC morphologic characteristics would be expected on the peripheral smear? 4. What other laboratory results could be expected to be abnormal? 5. What are the transfusion implications for this patient? 104

CASE 2 Patient Information A 35-year-old woman was admitted with a 3-day history of fever and a cough, which produced purulent sputum. On the day of admission, she developed a rigor. She had felt tired and generally unwell for the past 2 weeks. She also reported excessive bleeding of her gums, which had developed recently, as had several bruises at sites she could not remember traumatizing. She appeared pale and mildly tachypneic with obvious ecchymoses on her arms. There were petechiae on her lower legs. Rales and rhonchi were present over the right lower lung field, but there were no obvious signs of consolidation. The rest of the examination was unremarkable. A chest X-ray showed patchy infiltrates in the lower lobe of the right lung. Temperature 103.1 oF Blood pressure 110/70 mm Hg Pulse 95 beats/min Height 5'4\" Weight 144 lbs Hemoglobin 105 g/L Hematocrit 31% WBCs 35 x 109/L 80% Blasts 10% Lymphocyte 2% Granulocyte 2% Myelocyte 2% Metamyelocyte 4% Monocyte 15 x 109/L Platelets Results of bone marrow examination confirmed the diagnosis of acute myelogenous leukemia, subtype M2. The patient was started on broad spectrum antibiotics and subsequently on antineoplastic chemotherapy. Seven units of platelets were transfused soon after admission, but the platelet count measured the next morning was only 18 x 109/L. Platelets were transfused again; on the third day, however, the platelet count was again 18 x 109/L. A third transfusion of 7 units of platelets was given: the 1-hour posttransfusion platelet count reached 48 x 109/L, but the count had declined to 20 x109/L by the following morning. 105

Questions 1. Why is the response to platelet transfusion so poor in this patient? Would you suggest that the clinical service change the patient's transfusion management in any way? 2. Calculate the percent platelet recovery and CCI for the third platelet transfusion, for both 1 hour and 24 hours, assuming 7.0 x 109 platelets in each unit of platelet concentrate. Additional Information The patient's pneumonia and fever responded to antibiotics over the course of the first week, with a corresponding increase in the magnitude and duration of her responses to platelet transfusion. Her WBC count fell, and the blasts disappeared from her peripheral blood. Platelet transfusions administered every third day maintained the platelet count above 20 x 109/L. On the eleventh hospital day, she developed hemorrhagic cystitis when her platelet count was 35 x 109/L. Questions 3. Should platelets be administered at this time? What would be the rationale for such a transfusion? Additional Information The patient continued to respond to platelet transfusion until the twenty-first hospital day when she received platelets at a pretransfusion count of 12 x 109/L. One half hour after initiation of the transfusion, the patient developed a rigor and her temperature rose to 102.2 oF. Other vital signs remained stable. The following morning the platelet count was still 12 x 109/L. Platelets were again transfused, and 1 hour later the platelet count was 15 x 109/L. Questions 4. To what do you attribute the patient's poor response to transfusion now? What test might you perform to confirm this impression? 5. What sort of transfusion reaction did this patient have? Is there any relationship between this reaction and the development of platelet refractoriness? 6. How would you manage this patient's thrombocytopenia now? 7. Are there any measures that the blood bank might have taken to prevent this sequence of events? 106

CASE 3 Patient Information A 57-year-old white woman was transfused with 1 unit of RBCs to replace blood lost during a total right hip replacement. Her pretransfusion hemoglobin and hematocrit levels were 89 g/L and 28%, respectively. According to the patient's physician, the blood was administered to \"make her feel better prior to discharge to home\" Questions 1. What are appropriate indications for single-unit transfusion in adults? Addition Information After completion of the transfusion, the patient complained to her nurse of uncontrollable itchiness. On examination, the nurse observed numerous red wheals developing on the patient's face, arms, and trunk. Nurse contacted the orthopedic surgery resident on call to examine the patient. The following vital signs were recorded on the transfusion reaction form: Clinical Information Pre-transfusion Post-transfusion Temperature (oF) 98.8 98.6 Blood pressure (mm Hg) 116/78 120/84 Pulse (beats/min) 74 72 Respirations (per min) 14 14 Hemoglobin (g/L) 89 100 Hematocrit 27% 30% WBCs (109/L) 8.4 8.6 Platelets (109/L) 175 168 Questions 2. Assuming that the observed signs and symptoms are related to the transfusion, what type of reaction is this? 3. What do the vital signs indicate? 4. What is the pathophysiology of this transfusion reaction? 5. How should this transfusion reaction be treated? 6. What is the transfusion service's responsibility in investigating this type of reaction? 107

CASE 4 Patient Information Two units of blood were requested for a 35-year-old white woman scheduled for a colon resection. Her diagnosis is carcinoma of the colon. She had no history of transfusions. Results of presurgical compatibility testing were as follows: Serologic Testing Anti-A Anti-B Anti-D Rh Con A1Cells B Cells 4+ 2+ 3+ neg neg 3+ Screening cell OI IS 37C/LISS AHG CCC Screening cell OII neg neg neg 2+ Screening cell OIII neg neg neg 2+ Auto neg neg neg 2+ neg neg neg 2+ No crossmatches were done because the ABO grouping was in question. Question 1. Is the ABO discrepancy in the patient's forward (RBC) grouping or her reverse (serum) grouping test? Additional Information Washing the patient's red cells with saline and retesting with another lot number of anti-A and anti-B yielded the same results. Two additional group B cell samples tested with the patient's serum gave the same results. Therefore, the antibody reacting with the B cells was confirmed as anti-B Questions 2. Given the results of the serologic testing and the patient's diagnosis, what is the most likely explanation for the ABO discrepancy? 3. If the patient appears to have both the B antigen and the anti-B, why is the autocontrol negative? 108

Additional Testing When the patient's cells were checked for polyagglutinability, the following reactions were noted: AB serum Patient's RBCs Cord AB serum 2+ Arachis hypogaea (T) neg Bandeiraea simplicifolia (Tk) 2+ 2+ Questions 4. What do the above test results indicate? 5. Blood of what ABO and Rh should be crossmatched for the patient 6. Will the survival of the transfused donor RBCs be reduced because of this problem? 7. Is this condition transient or permanent? 109

CASE 5 Patient Information A 21-year-old woman was referred for evaluation by her gynecologist who had admitted her to the hospital because of menorrhagia. Her menstrual period had lasted for 12 days. In addition to heavy periods, she complained of easy bruisability. After extraction of one of her third molars 1 year previously, she reportedly bled for 2 days and required a suture to stop the bleeding. Physical examination was remarkable only for scattered bruises on her extremities. Her mother required a transfusion after the patient was born. Hematocrit 0.33 (MCV = 70) WBCs 7.1 x 109/L Platelets 425 x 109/L PT 12.5 sec (normal <14 sec) PTT 44 sec (normal <40 sec) Questions 1. What is the differential diagnosis? What test should be performed next? 2. Assuming that the results of the screening test selected in the last question were abnormal, what tests should be performed next? Additional Information The following results were obtained on coagulation tests: Bleeding time 12 min (normal <7 min) Ristocetin cofactor assay 25% Factor VIII: c level 30% Factor VIII:ag level 30% Questions 3. What is the diagnosis? What pattern of inheritance does this abnormality follow? Is any further testing required? 4. Is any treatment or therapeutic trial indicated for this acute episode? What long term management is indicated? Additional Information Administration of DDAVP corrected the bleeding time completely to 6 minutes. Six months after the diagnosis, the patient was advised to have the other impacted think molar removed. Questions 5. Is prophylactic treatment required? If so, what would you suggest? 110

CASE 6 Patient Information A 89-year-old Asian man came to the emergency department with a complaint of severe substernal chest pain of 3 days' duration associated with shortness of breath. He had a 40-lb weight loss over the previous 3 months. On examination he appeared cachectic, lethargic, and tachypneic with significant respiratory distress. His vital signs were as follows: Temperature 99.1 F Blood pressure 116/70 mm Hg Pulse 180 beats/min Respirations 32 per min Hemoglobin 123 g/L Hematocrit 39.3% WBCs 19.1 x 109/L Platelets 187 x 109/L After further examination and testing, diagnoses of acute myocardial infraction and congestive heart failure were made. Chest X-ray showed patchy, bilateral infiltrates with a consolidated right lower lobe and pulmonary vascular engorgement. The patient had a hypochromic/microcytic anemia later shown to be due to iron deficiency. An occult malignancy was suspected; however, the work-up for this possibility had to await treatment of the acute problems. The patient was admitted to the cardiac surveillance unit where a balloon flotation catheter was inserted to assess cardiac filling pressure and cardiac output. Streptococcus pneumonia was identified in cultures of purulent sputum. The patient was treated with Bactrim and clindamycin. Over the next 3 days, the chest X-ray showed only right lower lobe consolidation. His cardiac index improved to 4.3 L/min/m2, PaO2 values averaged 55 mm Hg, and O2 saturation was 84%. Hydration results in a drop in the hemoglobin and hematocrit levels to 84 g/L and 26.9, respectively. The staff physician ordered 2 units of RBCs for transfusion. Questions 1. What was the identification for transfusion of RBCs? 111

Addition Information Over a 2.5-hour period, the first 150 cc of the first unit of RBCs was administered. The patient's cardiorespiratory status changed as follows: Time Temp (F) HR/min RR/min BP (mm Hg) Progression 2:15 97.5 82 22 147/47 Transfusion begin: resting comfortably 3:15 98.1 90 26 185/66 Restlessness begins: no cardiac Ectopic; rebreather mask affixed (FIO2 = 0.60) 4:15 98.2 88 24 179/58 Anxiety, coughing and restlessness continues: 40 mg furosemide given 5:15 98.4 97 26 200/60 Patient very restless, pulling at Foley catheter, climbing out of bed, morphine sulfate given 5:30 99.3 105 40 215/80 Transfusion discontinued; diuresis good with clear, pale yellow urine CVPs and PCWP were 9 to 10 and 10 to 13 mm Hg, respectively, throughout this time period. Following the 4:15 vital signs, the nurse notified the resident about the patient's changing status. At 5:30 the resident ordered the transfusion to be stopped. Questions 2. What should be done next? Additional Information There was no evidence of chills, allergy, anaphylaxis, or change in urine color. The laboratory reported no evidence of hemolysis, a negative posttransfusion DAT result, and no clerical errors. Chest auscultation revealed diffuse rales and rhonchi bilaterally. A second chest x-ray showed diffuse alveolar infiltrates without pulmonary vascular engorgement. ABGs demonstrated that the patient was in respiratory failure. Repeat ECG and serial cardiac enzymes did not support progressive myocardial infarction. Questions 3. In this patient, what other clinical problems must be ruled out? 4. Assuming that the clinical signs and symptoms were transfusion-associated, what kind of adverse reaction might have occurred? 5. What is the pathophysiology of this type of transfusion reaction? 6. What is the recommended therapy and expected outcome for this type of reaction? 112

Additional Information The patient was given furosemide for diuresis, IV morphine for sedation, and IV nitroprusside for hypertension. Since the patient's respiratory status was deteriorating, he was intubated, mechanically ventilated, and transferred to the ICU. In the ICU, his pulmonary, cardiac, and neurologic systems all deteriorated, associated with irreversible hemodynamic instability. Asystole and death occurred approximately 33 hours after the start of the transfusion. Questions 7. At this time, what medicolegal responsibility does the medical director of the transfusion service have? 8. If an autopsy had been performed, what would the gross and histologic findings in the lungs have been? Additional Information The patient was given furosemide for diuresis, IV morphine for sedation, and IV nitroprusside for hypertension. Since the patient's respiratory status was deteriorating, he was intubated, mechanically ventilated, and transferred to the ICU. In the ICU, his pulmonary, cardiac, and neurologic systems all deteriorated, associated with irreversible hemodynamic instability. Asystole and death occurred approximately 33 hours after the start of the transfusion. Questions 9. At this time, what medicolegal responsibility does the medical director of the transfusion service have? 10. If an autopsy had been performed, what would the gross and histologic findings in the lungs have been? 113

CASE 7 Patient Information A 28-year-old male police officer was brought to the emergency room for gunshot Serologic Testing Anti-D Rh Con A1Cells B Cells Anti-A Anti-B 3+ 1+ neg 4+ 3+ neg Because of the weakness of the Rh result and the urgency of the situation, the technologist selected 4 group O, Rh-negative units and simultaneously set up the antibody screening test, crossmatches, and a \"D\" test. Du test IS 37C/LISS AHG CCC Du control 1+ NT Screening cell OI neg 2+ Screening cell OII neg neg 2 NT Screening cell OIII neg neg 2 NT Auto neg neg neg 2+ neg neg neg 2+ Unit 1, O neg neg neg neg 2+ Unit 2, O neg neg neg neg 2+ Unit 3, O neg neg neg neg 2+ Unit 4, O neg neg neg neg 2+ Questions 1. What is the Rh type of the patient? 2. Should the units be released for transfusion? Additional Testing The technologist immediately set up an antibody identification panel, the results of which are shown in Table 2-1. Questions 3. How can the apparent discrepancy between the Rh type and the panel results be explained? 4. What further studies can be done to confirm this explanation? 5. The AABB standards state that persons who are DU positive may receive Rh-positive blood. In this case, should the policy be honored? 114

CASE 8 Patient Information A 22-year-old woman, G2P1, at 28 weeks' gestation called her obstetrician because of severe abdominal pain and was told to come to the hospital. On admission, she stated that she had been awakened by painful tightening of her abdomen, which did not relax. She also noted minimal vaginal bleeding. She had a history of chronic hypertension and smoked on-half pack of cigarettes a day. She was afebrile without obvious respiratory distress. Bruises or petechiae were not noted, and examinations of the heart and lungs revealed only a systolic ejection murmur. The fundal height was 28 cm, and the uterus was rigid and tender. There were no fetal heart tones. On vaginal examination, the cervix was completely dilated with a bulging bag of waters. Ultrasound confirmed intrauterine fetal demise and revealed a large retroplacental blood clot. The clinical impression was that there was placental abruption at 28 weeks' gestation with intrauterine fetal demise. Blood pressure 150/100 mm Hg Pulse 105 beats/min Hematocrit 35% WBCs 9.5 x 109/L Platelets 120 x 109/L PT 13 sec APTT 38 sec Fibrinogen 1.05 g/L The amniotic sac was ruptured artificially, releasing bloody fluid. The patient delivered a dead, non- macerated fetus, followed immediately by the placenta. After this delivery, the patient bled profusely. The uterus was atomic. Fluid resuscitation was begun. Coagulation studies were repeated, and 2 units of RBCs were ordered stat. Thirty minutes after delivery, the following data were obtained: Blood pressure 80/50 mmHg Pulse 120 beats/min Platelets 35 x 109/L PT 16 sec (control, 11.1 sec) PTT 61 sec (control, 24.7 sec) Fibrinogen 0.30 g/L Fibrin degradation products >1:6 An RBC transfusion was started. The patient continued to bleed from her vagina, and blood began to ooze from her 2 IV sites. Large ecchymoses were noted on her arms. 115

Questions 1. What complication occurred in this patient? What is the pathogenesis of this patient's disorder? 2. What clinical manifestations suggest that the process was disseminated? 3. What is the priority for replacement of blood elements by transfusion therapy? How might this best be accomplished? 4. What local measures might help control hemorrhage? 5. After the initial replacement of coagulation factors, what therapeutic monitoring should the patient receive? 116

CASE 9 Patient Information A 28-year-old woman, G7P6, presented at 33 weeks’ gestation without having received and prenatal care. A previous infant had required transfusion at birth but she was unaware of the indication. She was found to be group O, Rh-positive with a positive antibody screen. Antibody identification demonstrated anti-Kell at a titer of 16. She was referred for amniocentesis, at which time the ultrasound demonstrated a small amount of fluid in the fetus’s pericardial sac and abdomen as well as slight thickening of the scalp. The delta OD 450 of the amniotic fluid was 2.0, and the L/S ratio was 1.7. Questions 1. What is the diagnosis? 2. What management approach is appropriate? Additional Information The mother was admitted to the hospital and labor was induced. Pre-transfusion testing on the mother demonstrated a positive antibody screen, and anti-Kell was again identified. A 4-lb infant was born with Apgar scores of 5 and 8 at 1 and 5 minutes, respectively. The cord blood RBCs typed as group A, and had a strongly positive DAT result (3+). The Rh typing was 4+ with anti-d and 2+ with the Rh control. Kell typing could not be performed due to the positive DAT result. Cord blood testing revealed a hemoglobin level of 100 g/L and a total bilirubin level of 73.51 µmol/L (4.3 mg/dL). Questions 3. What is the baby’s Kell type? How can this be confirmed? 4. What management is appropriate now? What is the therapeutic goal? How does this form of treatment accomplish this goal? 5. What are the specifics of the selected treatment? 117

CASE 10 Patient Information Four units of blood were requested for a 57-year-old white woman undergoing open heart surgery. Previous records indicated that she was group A. Rh-positive and that she had received 8 units of blood 2 years previously for another surgical procedure. Results of pretransfusion testing were as follows: Serologic Testing Anti-A Anti-B Anti-D Rh Con A1Cells B Cells 4+ neg 3+ neg neg 3+ Screening cell OI IS 37C/LISS AHG CCC Screening cell OII 1+ neg neg 2+ Screening cell OIII w+ w+ 2+ NT Auto w+ w+ 2+ NT neg neg neg 2+ Unit 1, A pos w+ neg neg 2+ Unit 2, A pos 1+ w+ 2+ NT Unit 3, A pos neg w+ 2+ NT Unit 4, A pos w+ w+ 2+ NT The results of an RBC reagent panel tested with the patient's serum are shown in Table. Questions 1. After ruling out using cells that are nonreactive in all test phases, which antibody specificities are left? 2. Does the pattern of reactivity match that of any single antigen? 3. Which of the remaining are not like to be present because they are directed against low-incidence antigens? 4. On the basis of the differences in reaction phase and strength, what combination of antibodies appears to be present? 5. What results would be expected if the same panel were retested after enzyme treatment? 6. Additional panel cells antigen-negative for the antibodies believed to be present were tested. What additional testing could be performed to eliminate these antibodies as possibilities? 7. If the additional testing done in question 6 did not eliminate anti-E and K, what implication does this have in selecting blood for transfusion? 8. How many units should be crossmatched to find 4 compatible units for surgery? Show your calculators? 9. Should testing be restricted to ABO identical blood? 118

Antibody identification for case 10 1. r'r Rh MNSs P Lewis 2. R1R1w D C E c e M N S s P1 Lea Le 3. R1R1 0+0++++0+ + 0 0 4. R2R2 ++00+0+0+ + + 0 5. r''r ++00++00+ 0 0 + 6. rr, Kpa +0++0+0+0 + 0 + 7. rr, K 00+++++0+ 0 0 + 8. rr, Jsa 0 0 0 + + + 0 + + w+ 0 + 9. rr 000++++0+ + 0 0 10. rr, Lua 000++++0+ 0 + 0 Autologous 000++0+0+ + 0 + 000+++++0 0 + 0 SC OI + + 0 0 + + 0 + 0 0 + 0 SC OII + 0 + + 0 + + 0 + 0 0 + SC OIII 0 0 0 + + + + + 0 + 0 + 11

Kell Duffy Kidd eb K k Fya Fyb Jka Jkb IS 37C AHG CCC 0 0 + 0 0 + 0 w+ w+ 2+ NT 0 0 + + 0 0 + neg neg neg 2+ + 0 + 0 + + + 1+ neg neg 2+ + + + + + 0 + 1+ w+ 2+ NT + 0 + + + + + 1+ w+ 2+ NT + 0 + 0 + + 0 1+ w+ 2+ NT 0 + 0 + 0 + + w+ w+ 2+ NT 0 0 + + 0 + + w+ w+ 2+ NT + 0 + + + 0 + neg w+ 2+ NT 0 0 + 0 + + 0 w+ w+ 2+ NT neg neg neg 2+ 0 + + 0 + 0 + 1+ neg neg 2+ + 0 + + + + 0 w+ neg neg 2+ + 0 + + 0 0 + w+ neg neg 2+ 19

CASE 11 Patient Information Several weeks before admission, a 21-year-old athletically active black male college student had an upper-respiratory viral syndrome, which resolved in 5 days. Nine day before admission, the patient began experiencing \"pins and needle\" sensations in his toes and feet, sometimes associated with numbness and hypersensitivity. These sensations progressed in severity and duration and were associated with progressive ascending weakness of his leg, which made walking difficult. When he could no longer climb stars and his hands and forearms began to weaken, he sought medical assistance. Physical examination revealed symmetrical quadriparesis and hypotonia, which were worse in the leg, affecting the distal musculature more than the proximal musculature. Truncal strength, breathing, and results of cranial nerve examinations were within normal limits; however, all the deep tendon reflexes were absent. The patient was afebrile, had normal mental status examination results, and had no focal or lateralizing neurologic deficits. The patient denied use of any medications or illicit drugs, or exposure to organic chemicals, heavy metals, or known toxins. His medical history was significant only for tonsillectomy in childhood. The family history was noncontributory. The CBC, platelet count, electrolytes, random serum glucose levels, coagulation times, liver and renal function test results, serum protein electrophoresis results, and chest X-ray were all within reference ranges. Questions 1. What clinical conditions should be considered in the differential diagnosis? 2. What additional tests are necessary to narrow the diagnostic possibilities? Additional Information On the basis of the above results, acute Guillain Barre' syndrome (GBS) was diagnosed. Questions 3. What clinical forms does GBS take? What is the pathogenesis of the acute form? 4. What is the role of PEX in GBS? What are the expected benefits? 5. When should therapeutic PEX be started in acute GBS? With what frequency? 6. What is the appropriate replacement fluid for PEX in GBS? 120

CASE 12 History The alleged father and his wife have 2 children. The alleged father has filed for divorce and claims that the children born in wedlock are not his. The court has ordered a paternity study. All parties are white, and the children are ages 1 and 3. System Mother Child 1 Child 2 Alleged father ABO O A O A Rh cde cde MN MS Ccde CcDe Kell K-k+ MNS MNSs MNSs Duffy a+b- K-k+ K-k+ K-k+ Kidd a+b- a+b- a+b- a+b- a+b- a+b- a+b- Questions 1. Fill in the possible genotypes for the phenotypes. Once the possible genotypes are filled in, determine what markers would be required of the father of each child. Marker Mother Child 1 Child 2 Alleged Father Marker required for System Child 1 Child 2 ABO Rh MN Kell Duffy Kidd 2. Is the alleged father excluded from paternity of either child? 121

CASE 13 Patient Information Six units of blood were ordered for a 60-yrar-old black man needing extensive colon surgery. No records were found for the patient. The results of presurgical compatibility testing were as follows: Serologic Testing Anti-A Anti-B Anti-D Rh Con A1Cells B Cells 4+ neg 3+ 2+ neg 3+ Screening cell OI IS 37C/LISS AHG CCC Screening cell OII neg neg 4+ NT Screening cell OIII neg neg 2+ NT Auto neg neg 2+ NT neg neg 4+ NT Unit 1, A pos neg neg 2+ NT Unit 2, A pos neg neg 4+ NT Unit 3, A pos neg neg 4+ NT Unit 4, A pos neg neg 3+ NT Unit 5, A pos neg neg 3+ NT Unit 6, A pos neg neg 3+ NT Because of the strongly positive autocontrol and the positive Rh control, a DAT was performed on RBCs from an EDTA sample: AHO (Polyspecific) 4+ Anti-IgG 3+ Anti-C3 neg Additional Information The technologist called the patient's nurse to ask for a complete list of medications that the patient was taking or had recently took and an accurate transfusion history, particularly for the last 3 to 4 months. The patient had been taking Aldomet for hypertension for 2 years. He remembered being transfused many years previously. but not within the prior 3 months 122

Results of the repeat Rh testing with chemically modified Rh antisera were as follows: Reagent Patient Rh Control Positive Control Negative Control anti-D 3+ neg 3+ neg anti-C neg neg 3+ neg anti-c 3+ neg 3+ neg anti-E 2+ neg 2+ neg anti-e 1+ neg 2+ neg A warm autoadsorption was performed. The results of testing on the absorbed serum are shown in Table 13-1. Results of testing on the eluate prepared from the patient's RBCs shown in Table 13-2. Questions 1. What criteria are needed to distinguish between warm AIHA, a delayed hemolytic transfusion reaction, and a methyldopa-induced warm autoantibody without hemolysis? 2. If warm autoadsorption had not been possible because the patient had been transfused within the last 3 months, what techniques could be used to investigate underlying alloantibodies? 3. Why can patients have these serologic results and not be anemic? 4. If the patient had a clinical case of warm AIHA, would any alteration in therapy be indicated if the anti-C3 results of the DAT had also been positive? Would a difference in the clinical response of the patient be expected? 5. Considering the results of the antibody panel, what might be the consequences of transfusing incompatible donor units? 6. If the patient were having elective surgery, what course of action should be considered if the patient's hemoglobin level were above 110 g/L? 123

Table 13-1 Rh MNSs P Lewis D C E c e M N S s P1 Lea Le 1. r'r 0+0+++00+ + 0 0 2. R1R1w ++00+++0+ + + 0 3. R1R1 ++00++00+ 0 0 + 4. R2R2 +0++0+0+0 + 0 + 5. r''r 00+++0+0+ 0 0 + 6. rr, Kpa 0 0 0 + + + 0 + + w+ 0 + 7. rr, K 000++++0+ + 0 0 8. rr, Jsa 000+++00+ 0 + 0 9. rr 000++0+0+ + 0 + 10. rr, Lua 000+++++0 0 + 0 Autologous SC OI + + 0 0 + + 0 + 0 0 + 0 SC OII + 0 + + 0 + + 0 + 0 0 + SC OIII 0 0 0 + + + + + 0 + 0 + Table 13-2 Rh MNSs P Lewis D C E c e M N S s P1 Lea Le 1. r'r 0+0+++00+ + 0 0 2. R1R1w ++00+++0+ + + 0 3. R1R1 ++00++00+ 0 0 + 4. R2R2 +0++0+0+0 + 0 + 5. r''r 00+++0+0+ 0 0 + 6. rr, Kpa 0 0 0 + + + 0 + + +w 0 + 7. rr, K 000++++0+ + 0 0 8. rr, Jsa 000+++00+ 0 + 0 9. rr 000++0+0+ + 0 + 10. rr, Lua 000+++++0 0 + 0 Autologous 12

Kell Duffy Kidd eb K k Fya Fyb Jka Jkb IS 37C AHG CCC 0 0 + 0 0 + 0 neg neg 2+ NT 0 0 + 0 + 0 + neg neg 3+ NT + 0 + 0 + + + neg neg 3+ NT + + + + + 0 + neg neg neg 2+ + 0 + + + + + neg neg neg 2+ + 0 + 0 + + 0 neg neg neg 2+ 0 + 0 + 0 + + neg neg neg 2+ 0 0 + + 0 + + neg neg neg 2+ + 0 + + + 0 + neg neg neg 2+ 0 0 + 0 + + 0 neg neg neg 2+ neg neg neg 2+ 0 + + 0 + + + neg neg 3+ NT + 0 + + + 0 + neg neg neg 2+ + 0 + + 0 + 0 neg neg neg 2+ Kell Duffy Kidd eb K k Fya Fyb Jka Jkb IS 37C AHG CCC 0 0+ 0 0 + 0 2+ NT 0 0+ 0 + 0 + 2+ NT + 0+ 0 + + + 2+ NT + ++ + + 0 + 2+ NT + 0+ + + + + 2+ NT + 0+ 0 + + 0 2+ NT 0 +0 + 0 + + 2+ NT 0 0+ + 0 + + 2+ NT + 0+ + + 0 + 2+ NT 0 0+ 0 + + 0 2+ NT NT NT 24

CASE 14 Patient Information A 70-year-old white woman, admitted with a previously known adenocarcinoma of the stomach proven by radiographic and endoscopic test results, had a history of multiple transfusions and pregnancies. Three units of RBCs were ordered for transfusion before surgery. Pretransfusion compatibility testing showed the patient to be group AB, Rh-positive with a negative antibody screen. She had a history of an anti-E, which was not demonstrable at this time. Three units of E-negative RBCs were crossmatched and found to be compatible. After 75 cc of the second unit was administered, the patient experienced chills with temperature and vital signs as follows: Temperature (oF) Pretransfusion Posttransfusion Blood pressure (mm Hg) 98.8 100.6 Pulse (beats/min) 115/48 121/42 Respirations (per min) 83 102 Hemoglobin (g/L) 18 22 Hematocrit 83 NA 24% NA Questions 1. What actions should the nurse take? Addition Information The staff physician suspected a transfusion reaction and requested that a transfusion-reaction investigation be initiated. Questions 2. What is the differential diagnosis for fever at the time of transfusion? 3. What steps should be followed by the clinical service to investigate the transfusion reaction? 4. After receipt of the posttransfusion specimens and the proper transfusion reaction documentation, what steps must the laboratory take to rule out an acute hemolytic transfusion reaction (AHTR)? Additional Information Based on the results of the investigation, an AHTR was ruled out. Because the initial number of RBCs requested had not been transfused, the physician requested a crossmatch for 2 more units of RBCs the next morning for transfusion that same day. Using the post-transfusion reaction specimen, the technologist obtained the following serologist data: 125

Serologic Testing Anti-A Anti-B Anti-D Rh Con A1Cells B Cells 4+ 4+ 3+ neg neg neg IS 37C/LISS AHG CCC Screening cell OI neg neg neg 2+ Screening cell OII neg neg neg 2+ Screening cell OIII neg neg w+ NT Auto neg neg 0 2+ Unit 1, E-negative neg neg w+ NT Unit 2, E-negative neg neg neg 2+ The results of an antibody identification panel are shown in Table 14-1. Questions 5. Do the results correspond to that of any other clinically significant antibody? 6. What is the significance of weak reactions with Bg-positive cells? 7. List and discuss the ways an adverse transfusion reaction of the type experienced by this patient can be treated and/or prevented in the future. 126

Antibody identification for case 14 Rh MNSs P Lewis D C E c e M N S s P1 Lea Le 1. r'r 0 + 0 + + + 0 0 + + 0 0 2. R1R1w ++00+++0+ + + 0 3. R1R1 ++00++00+ 0 0 + 4. R2R2 +0++0+0+0 + 0 + 5. r''r 00+++0+0+ 0 0 + 6. rr, Kpa 000+++0++ w 0 + 7. rr, K 000++++0+ + 0 0 8. rr, Jsa 000+++00+ 0 + 0 9. rr 0 0 0 + + 0 + 0 + + 0 + 10. rr, Lua 000+++++0 0 + 0 Autologous SC OI + + 0 0 + 0 + 0 + 0 + 0 SC OII + 0 + + 0 0 + 0 + 0 0 + SC OIII 0 0 0 + + + 0 + 0 + 0 + 12

Kell Duffy Kidd eb K k Fya Fyb Jka Jkb IS 37C AHG CCC 0 0 + 0 0 + 0 neg neg w+ NT 0 0 + 0 + 0 + neg neg neg 2+ + 0 + 0 + + + neg neg neg 2+ + + + + + 0 + neg neg neg 2+ + 0 + + + + + neg neg w+ NT + 0 + 0 + + 0 neg neg neg 2+ 0 + 0 + 0 + + neg neg neg 2+ 0 0 + + 0 + + neg neg neg 2+ + 0 + + + 0 + neg neg neg 2+ 0 0 + 0 + + 0 neg neg w+ NT neg neg NT 2+ 0 + + 0 + 0 + neg neg neg 2+ + 0 + + + + 0 neg neg w+ NT + 0 + + 0 0 + neg neg neg 2+ 27

CASE 15 Patient Information Paternity testing has been requested as part of a divorce suit in which the woman is asserting that her husband is the father of her 6-year-old daughter. All parties are white. Question 1. What could you tell the woman’s lawyer about the type of tests available and the chances of ruling out her husband’s parentage if he were not the father of her daughter? Testing To maximize the chance of exclusion, the full extent of testing possible was performed with the exception of DNA probes Since HLA typing is not routinely performed by the laboratory performing the serologic testing, the HLA typing was sent to an out-of-state reference lab, which also performs parentage testing. The only results of interest are as follows: System Mother Child Alleged Father HLA A locus 3,29 3,11 2,11 HLA B locus 7,35 7,49 12,50 Additional Information Once the mother received the results of the testing and learned that her husband had been excluded, she contacted the director of the laboratory to insist that a mistake must have been made in the parentage testing. Question 2. What do you advise? Explain Additional Information On request of the mother after a discussion with the director of the HLA laboratory, the specimens for the HLA testing were redrawn and shipped overnight express to the same laboratory. The repeat testing indicated that the typing previously reported on the alleged father were HLA B7, 21. Questions 3. Based on the repeat testing, the alleged father cannot be excluded from paternity. What additional calculations are indicated? 4. Can all of the systems for which testing is performed be included in the calculation? Explain the significance of a high paternity index 5. If the HLA testing had not been repeated, would the apparent exclusion in this system have been sufficient to conclusively exclude the alleged father? Explain. 6. What other testing might be useful? 128

CHAPTER 8 Adsorption and Elution techniques Adsorption แอนติบอดสี ามารถถูกดูดซบั (adsorption) ออกจากซีรมั ได้โดยนําซีรมั ทําปฏิกิรยิ ากบั เม็ดเลือดแดงทOมี ี แอนตเิ จนทOตี รงกนั หลกั การสําคญั คอื ต้องทราบว่าแอนตบิ อดที Oตี ้องการดูดซบั ออกจากซีรมั นันU เป็น cold หรอื warm antibody ซOงึ ทราบได้จากระยะทOเี กดิ ปฏกิ ริ ยิ าในขนัU ตอน การตรวจกรองแอนตบิ อดี (antibody screening) เช่น ทอOี ุณหภูมหิ อ้ ง หรอื อุณหภูมริ ่างกาย หรอื ขนัU แอนตโิ กลบูลนิ ในการดูดซบั จงึ ตอ้ งทําปฏกิ ริ ยิ าทอOี ุณหภูมนิ ันU เพอOื เพมOิ ปฏกิ ริ ยิ าระหว่างแอนตบิ อดใี นซรี มั กบั แอนตเิ จนบนผวิ เมด็ เลอื ดแดง หลงั จากทแOี อนตบิ อดจี าํ เพาะเกาะตดิ กบั แอนตเิ จนบนเมด็ เลอื ดแดงแล้ว ปันO แยกซีรมั ออกจากเซลล์และนําไปทดสอบความจําเพาะอกี ครงัU หนOึง ส่วน แอนตบิ อดที จOี บั กบั แอนตเิ จนบนเมด็ เลอื ดแดง สามารถแยกออกจากเมด็ เลอื ดแดงโดยใชเ้ ทคนิค elution และ eluate ทแOี ยกออกมาสามารถนํามาตรวจสอบความจาํ เพาะได้ โดยใชเ้ ซลลท์ ทOี ราบแอนตเิ จนแน่นอนแลว้ ประโยชน์ของการ ทาํ adsorption คอื 1. แยกแอนตบิ อดหี ลายชนิดทมOี อี ยใู่ นตวั อยา่ งเดยี วกนั ออกจากกนั 2. แยก autoantibody ออกจากตวั อย่างทมOี ี auto- และ alloantibodies เพOอื ใหก้ ารตรวจหา alloantibody ทาํ ไดง้ า่ ยยงOิ ขนUึ 3. แยกแอนตบิ อดที ไOี ม่ต้องการ (anti-A และ/หรอื anti-B) ออกจากซรี มั เพOอื ทดสอบหาแอนตบิ อดอี Oนื ใน ซรี มั โดยใช้ reagent จาํ เพาะ 4. ยนื ยนั การมแี อนตเิ จนทจOี ําเพาะบนเมด็ เลอื ดแดง โดยอาศยั หลกั การว่าหากเมด็ เลอื ดแดงสามารถดูด ซบั แอนตบิ อดที จOี าํ เพาะชนิดใด แสดงวา่ มแี อนตเิ จชนิดนนัU บนผวิ เซลล์ 5. ช่วยทําแอนตบิ อดใี ห้บรสิ ุทธิt โดยการดูดซบั แอนตบิ อดที Oไี ม่ต้องการออกด้วยเมด็ เลอื ดแดงทOที ราบ แอนตเิ จนอยแู่ ลว้ วธิ กี าร adsorption มอี ยู่ 3 วธิ ี คอื 1. cold autoadsorption เพอOื กาํ จดั cold reactive autoantibody ในซรี มั ซงOึ ใหผ้ ลบวกกบั screening cells และ autologous control ทอOี ุณหภมู หิ อ้ งหรอื ตOาํ กวา่ 2. warm autoadsorption เพOอื กําจดั warm reactive autoantibody ออกจากซีรมั ทOีให้ผลบวกกบั screening cells และ autologous control ทอOี ุณหภมู ิ 37 oC 3. alloadsorption เพอOื กําจดั cold หรอื warm reactive alloantibody ออกจากตวั อย่างซรี มั ทใOี หผ้ ล บวกกบั screening cells และ autologous control ทอOี ุณหภมู หิ อ้ งหรอื อุณหภมู ิ 37 oC การดูดซบั แอนตบิ อดใี ชเ้ มด็ เลอื ดแดงเขม้ ขน้ ทOลี ้างกําจดั โปรตนี ออกแล้ว (washed packed red cells) กบั ซรี มั ในอตั ราสว่ น 1:1 ทาํ ปฏกิ ริ ยิ าในอุณหภมู ทิ เOี หมาะสมกบั แอนตบิ อดที ตOี อ้ งการดดู ซบั อาจจาํ เป็นตอ้ งทาํ ซUาํ ในเมด็ เลอื ดแดงชุดใหม่ หากยงั ดูดซบั ไม่หมด ซOงึ สามารถตรวจสอบได้จากการทํา indirect antiglobulin test ใน post- adsorbed serum หรอื ทาํ direct antiglobulin test ใน post-adsorbed red cells 129

เมด็ เลอื ดแดงทOใี ช้ต้องมแี อนตเิ จนทOตี รงกบั แอนตบิ อดที Oตี ้องการดูดซบั มหี มู่เลอื ดตรงกบั แหล่งของซีรมั ต้องการดูดซบั หากไม่ทราบควรตรวจชนิดของหมู่เลอื ดในตวั อย่างของแหล่งซีรมั บางกรณีอาจใช้ proteolytic enzymes ทาํ ปฏกิ ริ ยิ ากบั เมด็ เลอื ดแดงก่อนเพอOื ใหแ้ อนตเิ จนหมเู่ ลอื ดบางชนิดแสดงออกไดม้ ากขนUึ สามารถดดู ซบั แอนตบิ อดไี ด้ดขี นUึ และเป็นการลดจํานวนครงัU ในการดูดซบั อย่างไรก็ตาม การใช้เอนไซม์จะไม่ได้ผลในกรณีทOี แอนตเิ จนหมเู่ ลอื ดถกู ทาํ ลายโดยเอนไซม์ เชน่ แอนตเิ จน K Elution เป็นเทคนิคการแยกแอนตบิ อดอี อกจากผวิ ของเมด็ เลอื ดแดง (sensitized red cells) ส่วนใหญ่แอนตบิ อดที Oี หลุดออกมาสามารถนํากลบั ไปทาํ ปฏกิ ริ ยิ ากบั แอนตเิ จนได้ เทคนิค elution สามารถแบง่ ออกเป็น 2 วธิ ใี หญ่ๆ คอื 1. หลกั การทางกายภาพ (physical elution) เป็นการทาํ ลายพนั ธะระหว่างแอนตเิ จนและแอนตบิ อดี เป็นผลใหแ้ อนตบิ อดหี ลุดจากแอนตเิ จนบน ผวิ ของเมด็ เลอื ดแดงโดยการเปลยOี นแปลงทางกายภาพ เช่น ความรอ้ น (Heat elution) ซงOึ สามารถแยกได้ ทงัU IgM และ IgG คลนOื เสยี ง (ultrasound elution) และการแชแ่ ขง็ และละลายอยา่ งรวดเรว็ (freeze and thaw elution) ซOงึ ใชแ้ ยก IgG ออกจากผวิ เซลล์โดยการทําลายผนังของเมด็ เลอื ดแดง eluate ทแOี ยกออกมาไดจ้ ะ นํามาทดสอบกบั เมด็ เลอื ดแดงททOี ราบแอนตเิ จนแล้วเพOอื หาชนิดและความจําเพาะของแอนตบิ อดี การใช้ หลกั การทางกายภาพมีการนํามาใช้มาก ในการช่วยวินิจฉัย ABO hemolytic disease of the newborn (HDN) ในตวั อยา่ งทใOี หผ้ ลบวกกบั วธิ ี direct antiglobulin test 2. หลกั การทางเคมี (chemical elution) เป็นการใชส้ ารเคมที าํ ลายพนั ธะระหวา่ งแอนตเิ จนและแอนตบิ อดี ไดแ้ ก่ ether elution, chloroform, xylene และ methylene chloride เหมาะสําหรบั การแยก warm reactive autoantibody และ alloantibody ซOงึ ส่วนใหญ่เป็น IgG ส่วน cold-acid elution และ digitonin-acid elution เป็นการแยกแอนตบิ อดอี อกจาก แอนตเิ จนโดยการลด pH ซงOึ ใชใ้ นการแยกแอนตบิ อดชี นิด IgG เชน่ กนั ประโยชน์ของเทคนิค elution ไดแ้ ก่ 1. ใชส้ าํ หรบั ตรวจสอบตวั อยา่ งทใOี หผ้ ลบวกกบั วธิ ี direct antiglobulin test 2. ทาํ ใหแ้ อนตบิ อดบี รสิ ทุ ธแิt ละเขม้ ขน้ ขนUึ เพอOื ใชต้ รวจหาแอนตเิ จน 3. ใชย้ นื ยนั การมแี อนตเิ จนทแOี สดงออกอ่อนๆ (weak antigens) โดยการใชแ้ อนตบิ อดที ทOี ราบชนิดแลว้ ทดสอบกบั เมด็ เลอื ดแดง จากนนัU แยกแอนตบิ อดอี อกมา ทาํ การทดสอบกบั เซลลท์ ทOี ราบชนิดของ แอนตเิ จนแลว้ อกี ครงัU หนOึงเพอOื ยนื ยนั ผล 4. ใชเ้ ตรยี มเซลลท์ ไOี มม่ แี อนตบิ อดเี กาะอยเู่ พอOื นําไปทดสอบชนิดของแอนตเิ จนบนเมด็ เลอื ดแดงต่อไป 130

Heat elution ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test บวก 2. นUําเกลอื (0.85% NaCl) 3. 6% bovine albumin ในนUําเกลอื 4. Screening cell (O1 และ O2) สาํ หรบั ทดสอบกบั eluate 5. Anti-human globulin (polyspecific) 6. หลอดทดลองขนาด 12x75 mm 7. หลอดทดลองขนาด 13x100 mm 8. Transfer pipette 9. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง •,••• xg 10. เครอOื งอุน่ (water bath) อุณหภมู ิ 56 oC วิธีทาํ 1. ลา้ งตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จาํ นวน 6 ครงัU ในหลอด ทดลองขนาด 13x100 mm จากนนัU เกบ็ supernatant จากการลา้ งครงัU สดุ ทา้ ย (เกบ็ บรเิ วณทใOี กลส้ ว่ นบน ของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. นําเมด็ เลอื ดแดงอดั แน่นจากขอ้ 1 ผสมกบั 6% bovine albumin ในนUําเกลอื ในอตั ราสว่ น 1:1 ในหลอด ทดลองขนาด •‘x100 mm ผสมใหเ้ ซลลก์ ระจายตวั ดี 3. นําไปอุน่ ทOี 56 oC นาน •• นาที โดยเขยา่ หลอดทดลองเป็นครงัU คราว 4. นําหลอดทดลองไปปันO ทOี 1,000xg นาน 2 นาที (หากเป็นไปไดใ้ หป้ ันO ใน heated centrifuge) 5. ดดู เกบ็ supernatant สแี ดงใสสว่ นบน (eluate) ใสล่ งในหลอดทดลองใหม่ ขนาด 12x75 mm ทนั ที 6. ทดสอบ eluate ดว้ ยวธิ ี indirect antiglobulin test กบั screening cell (O1 และ O2) หรอื กบั เซลลท์ มOี ี แอนตเิ จนตรงกบั แอนตบิ อดใี น eluate โดยใชน้ Uําเกลอื จากการลา้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ย (last wash) เป็น eluate control ซงOึ ตอ้ งใหผ้ ลลบจงึ จะสามารถแปลผลการทดสอบนUีได้ 131

Lui Freeze-Thaw elution เมด็ เลอื ดแดงในสภาวะแช่แขง็ จะเกดิ สภาวะเหยOี วเนOืองจากนUําในตวั กลางกลายเป็นนUําแขง็ ทาํ ใหส้ มดุลของ osmolarity ระหว่างภายในและภายนอกเซลลเ์ สยี ไป นUําภายในเซลลซ์ งOึ ประกอบดว้ ยโปรตนี มากมายจะแขง็ ตวั ชา้ กว่าภายนอกเซลล์ ดงั นันU จงึ เกดิ การสูญเสยี นUําจากภายในออกสู่ภายนอกเซลล์ เซลล์จะเหOยี วและผนังเซลล์ถูก ทําลายปรากฎการณ์เช่นนUีจะทําใหแ้ อนตบิ อดที จOี บั แอนตเิ จนบนผวิ เซลล์หลุดออกไปดว้ ย เทคนิคนUีเหมาะสาํ หรบั การตรวจพสิ จู น์ภาวะ hemolytic disease of the newborn ทเOี กดิ จากแอนตบิ อดใี นระบบ ABO ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test บวก 2. นUําเกลอื (0.85% NaCl) 3. Screening cell (O1 และ O2) สาํ หรบั ทดสอบกบั eluate 4. Anti-human globulin (polyspecific) 5. หลอดทดลองขนาด 12x75 mm 6. หลอดทดลองขนาด 13x100 mm 7. transfer pipette 8. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง •,••• xg 9. ตเู้ ยน็ อุณหภมู ิ -20 oC วิธีการ 1. ลา้ งตวั อย่างเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จํานวน 6 ครงัU ในหลอด ทดลองขนาด 13x100 mm จากนนัU เกบ็ supernatant จากการลา้ งครงัU สุดทา้ ย (เกบ็ บรเิ วณทใOี กลส้ ว่ นบน ของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. ผสมเมด็ เลอื ดแดง (washed packed red cells) ปรมิ าตร 0.5 mL กบั นUําเกลอื 3 หยด 3. ปิดฝาหลอดทดสอบ หมนุ หลอดทดสอบเพอOื ใหเ้ มด็ เลอื ดแดงเคลอื บบรเิ วณผนงั หลอดทดสอบ 4. วางหลอดทดสอบในตเู้ ยน็ อุณหภมู ิ -20 oC ในแนวนอน นาน 10 นาที 5. ครบเวลานําหลอดออกจากชอ่ งแชแ่ ขง็ และละลายทนั ทดี ว้ ยนUําอุน่ (Running tap water) 6. นําหลอดทดลองไปปันO ทOี 1,000 xg นาน 2 นาที 7. ดดู เกบ็ supernatant สแี ดงใสสว่ นบน (eluate) ใสล่ งในหลอดทดลองใหม่ ขนาด 12x75 mm ทนั ที 8. ทดสอบ eluate ดว้ ยวธิ ี indirect antiglobulin test กบั screening cell (O1 และ O2) หรอื กบั เซลลท์ มOี ี แอนตเิ จนตรงกบั แอนตบิ อดใี น eluate โดยใชน้ Uําเกลอื จากการลา้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ย (last wash) เป็น eluate control ซงOึ ตอ้ งใหผ้ ลลบจงึ จะสามารถแปลผลการทดสอบนUีได้ 132

Cold-acid elution ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test บวก 2. นUําเกลอื (0.85% NaCl) ปรบั อุณหภมู ใิ หเ้ ยน็ ก่อนใชง้ าน 3. Screening cell (O1 และ O2) สาํ หรบั ทดสอบกบั eluate 4. Anti-human globulin (polyspecific) 5. หลอดทดลองขนาด 12x75 mm 6. หลอดทดลองขนาด 13x100 mm 7. Transfer pipette 8. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง •,••• x g 9. 0.1 M Glycine-HCl, pH 3.0 เตรยี มโดยละลาย Glycine 3.75 g, NaCl 2.933 g ในนUํากลนัO ปรบั pH เป็น 3.0 ดว้ ย 12 N HCl และปรบั ปรมิ าตรใหเ้ ป็น 500 ml จากนนัU เกบ็ ในตเู้ ยน็ อุณหภมู ิ 4 oC 10. 0.8 M Phosphate buffer, pH 8.2 เตรยี มโดยละลาย Na2HPO4 จาํ นวน 109.6 g และ KH2PO4 จาํ นวน 3.8 g ในนUํากลนัO ประมาณ 600 mL ปรบั pH เป็น 8.2 ดว้ ย 1 N HCl หรอื 1 N NaOH จากนนัU ปรบั ปรมิ าตรใหค้ รบ 1 ลติ รเกบ็ ในตเู้ ยน็ อุณหภมู ิ 4 oC วิธีการ 1. ลา้ งตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จาํ นวน 6 ครงัU ในหลอด ทดลองขนาด 13x100 mm จากนนัU เกบ็ supernatant จากการลา้ งครงัU สดุ ทา้ ย (เกบ็ บรเิ วณทใOี กลส้ ว่ นบน ของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. ดดู เมด็ เลอื ดแดงอดั แน่นแลว้ ปรมิ าตร 1 mL ใสห่ ลอดทดสอบขนาด 13X100 mm จากนนัU นําไปแชใ่ นอา่ ง นUําแขง็ (ice bath 0 oC) นาน 5 นาที 3. ครบเวลาเตมิ นUําเกลอื เยน็ (chilled saline) ปรมิ าตร 1 mL ตามดว้ ย Glycine-HCl เยน็ (chilled glycine- HCl) ปรมิ าตร 2 mL 4. ผสมใหเ้ ขา้ กนั และปลอ่ ยใหท้ าํ ปฏกิ ริ ยิ าในนUําแขง็ (ice bath 0 oC) นาน 1 นาที 5. นําหลอดทดลองไปปันO ทนั ทที Oี 1,000 xg นาน นาน 3 นาที 6. เกบ็ สว่ นใสขา้ งบนซงOึ เป็น eluate ใสใ่ นหลอดทดลองใหมท่ แOี หง้ และสะอาด เตมิ PBS, pH 8.2 ปรมิ าตร 0.1 mL 7. นําหลอดทดลองไปปันO ทนั ทที Oี 1,000 xg นาน นาน 3 นาที 8. เกบ็ สว่ นใสขา้ งบนซงOึ เป็น eluate ใสใ่ นหลอดทดสอบใหมท่ แOี หง้ และสะอาดและนําไปตรวจหา แอนตบิ อดี และทดสอบความจาํ เพาะ โดยใชน้ Uําเกลอื ทลOี า้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ยเป็น negative control หมายเหตทุ ีdสาํ คญั 1. Glycine-HCl ทใOี ชต้ อ้ งเกบ็ อยใู่ นทเOี ยน็ ตลอดเวลา เพอOื รกั ษา pH ไวท้ Oี 3.0 2. Phosphate buffer ทเOี ตรยี มและเกบ็ ไวใ้ นตเู้ ยน็ 40C อาจมผี ลกึ เกดิ ขนUึ ได้ กอ่ นนําไปใชง้ าน หากพบผลกึ ควร นํามาอุน่ ทอOี ุณหภมู ิ 37 oC ใหผ้ ลกึ ละลายหมด 133

3. ขนัU ตอนการเตมิ Phosphate buffer เพOอื ช่วยปรบั ความเป็นกรดของ Glycine-HCl ลงใหเ้ ป็นกลาง เพราะใน ภาวะกรดจะทําใหเ้ มด็ เลอื ดแดงแตกมาก หากพบว่าวธิ กี ารขา้ งต้นทําให้ เมด็ เลอื ดแดงแตกมาก ใหเ้ ตมิ 22% BSA ไปใน eluate ทแOี ยกได้ โดยเตมิ ในอตั ราสว่ น 1:4 โดยปรมิ าตร (22% BSA: eluate = 1:4) จะชว่ ย ลดการแตกของเมด็ เลอื ดแดงได้ 134

Ether elution ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test เป็นบวก 2. นUําเกลอื (0.85% NaCl) 3. Diethyl ether (reagent grade) 4. Screening cell (O1 และ O2) 5. Anti-human globulin (polyspecific) 6. หลอดทดลองขนาด •šx75 mm 7. หลอดทดลองขนาด •‘x100 mm 8. Transfer pipette 9. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง •,••• xg 10. เครอOื งอุน่ (water bath) อุณหภมู ิ 37 oC วิธีทาํ 1. ลา้ งตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จาํ นวน › ครงัU ในหลอด ทดลองขนาด 13x100 mm จากนนัU เกบ็ supernatant จากการลา้ งครงัU สดุ ทา้ ย (เกบ็ บรเิ วณทใOี กลส้ ว่ นบน ของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. ผสมเมด็ เลอื ดแดงอดั แน่นในขอ้ 1 ดว้ ยนUําเกลอื ในอตั ราสว่ น 1:1 ผสมใหเ้ ขา้ กนั 3. ผสม 50% เมด็ เลอื ดแดงทไOี ดจ้ ากขอ้ š กบั diethyl ether ในอตั ราส่วน 1:1 ใชห้ ลอดทดลอง ฝาเกลยี ว ขนาด 13x100 mm 4. ปิดหลอดทดลองดว้ ยจุกเกลยี วหรอื จุกคอรก์ แลว้ เขยา่ หลอดอยา่ งแรงนาน 1-2 นาที (ห้ามใชพ้ าราฟิลม์ เพราะ ether ทาํ ลายพาราฟิลม์ ) 5. ค่อยๆ คลายจุกเกลยี วหรอื เปิดจุกคอรก์ เพOอื ลดความดนั ภายในหลอด แลว้ นําหลอดไปอุ่นทOี 37 oC นาน 30 นาที 6. ครบเวลา ปันO ทOี 1,000 xg 10 นาที จะไดส้ ารละลายเรยี งตวั กนั 3 ชนัU - ชนัU บนสดุ เป็น ether - ชนัU กลางเป็นเศษเซลล์ (red cell stroma) - ชนัU ลา่ งสดุ คอื eluate ซงOึ เป็น hemolysate สนี Uําตาลแดง 7. ดดู ชนัU บนสดุ ซงOึ เป็นชนัU ของ ether ทงUิ 8. เปลยOี น transfer pipet เพอOื ดดู เกบ็ eluate ใสใ่ นหลอดทดลองหลอดใหม่ ขนาด 12x75 mm 9. อุ่น eluate ทOี 37 oC ประมาณ 15 นาที แลว้ เขยา่ เป็นครงัU คราวจนกวา่ กลนิO ของ ether หายไป (หากกาํ จดั ether ออกไมห่ มด จะทาํ ใหเ้ มด็ เลอื ดแดงทใOี ชท้ ดสอบในขนัU ตอนต่อไปแตกได)้ 10. ครบเวลาปันO ทOี 1,000 xg นาน 10 นาที 11. ทดสอบ eluate ด้วยวธิ ี indirect antiglobulin test กบั screening cell (O1 และ O2) หรอื กบั เซลล์ทOีมี แอนตเิ จนตรงกบั แอนตบิ อดใี น eluate โดยใชน้ Uําเกลอื จากการลา้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ย (last wash) เป็น eluate control ซงOึ ตอ้ งใหผ้ ลลบจงึ จะสามารถแปลผลการทดสอบนUีได้ 135

Chloroform elution ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test เป็นบวก 2. นUําเกลอื (0.85% NaCl) 3. Chloroform (reagent grade) 4. Screening cell (O1 และ O2) 5. Anti-human globulin (polyspecific 6. หลอดทดลองขนาด 12x75 mm 7. หลอดทดลองฝาเกลยี วขนาด 13x100 mm 8. Transfer pipette 9. ไมจ้ มUิ ฟัน (applicator stick) 10. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง 1,000 xg 11. เครอOื งอุน่ (water bath) อุณหภมู ิ 56 oC วิธีทาํ 1. ลา้ งตวั อย่างเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จํานวน › ครงัU ในหลอด ทดลองขนาด •‘x100 mm จากนนัU เกบ็ supernatant จากการลา้ งครงัU สุดทา้ ย (เกบ็ บรเิ วณทใOี กลส้ ว่ นบน ของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. ผสมเมด็ เลอื ดแดงอดั แน่นในขอ้ 1 ดว้ ยนUําเกลอื ในอตั ราสว่ น 1:1 ผสมใหเ้ ขา้ กนั 3. ผสม 50% เมด็ เลอื ดแดงทไOี ดจ้ ากขอ้ š กบั chloroformในอตั ราสว่ น 1:1 ใชห้ ลอดทดลองฝาเกลยี วขนาด 13x100 mm 4. ปิดหลอดทดลองดว้ ยจกุ เกลยี วแลว้ เขยา่ หลอดอยา่ งแรงนาน 10-15 วนิ าที (ห้ามใชพ้ าราฟิลม์ เพราะ chloroform ทาํ ลายพาราฟิลม์ ) จากนนัU ผสมอกี ครงัU เบาๆ ดว้ ยเทคนิค inversion (end-to-end) นาน 1 นาที 5. ค่อยๆ คลายจุกเกลยี วเพOอื ลดความดนั ภายในหลอด อุ่นหลอดทดลองทOี 56 oC นาน • นาที ระหว่างนUีให้ ผสมตวั อยา่ งเลอื ดและนUํายาเป็นครงัU คราวดว้ ยไมจ้ มUิ ฟัน หรอื ใชว้ สั ดอุ นOื ททOี ดแทนกนั ได้ 6. ปันO ทOี 1,000 xg นาน 5 นาที เกบ็ eluate ทอOี ยใู่ นชนัU บนสดุ 7. ดดู eluate ใส่หลอดทดลองหลอดใหม่ ขนาด 12x75 mm จากนันU นําไปทดสอบ หรอื หากตอ้ งการเกบ็ ให้ แชแ่ ขง็ ไวไ้ ด้ 8. ทดสอบ eluate ดว้ ยวธิ ี indirect antiglobulin test กบั screening cell (O1 และ O2) หรอื กบั เซลลท์ มOี ี แอนตเิ จนตรงกบั แอนตบิ อดใี น eluate โดยใชน้ Uําเกลอื จากการลา้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ย (last wash) เป็น eluate control ซงOึ ตอ้ งใหผ้ ลลบ จงึ จะสามารถแปลผลการทดสอบนUีได้ 136

Xylene elution ตวั อย่างเลือดและวสั ดอุ ปุ กรณ์ 1. ตวั อยา่ งเมด็ เลอื ดแดงทใOี หผ้ ล direct antiglobulin test เป็นบวก 2. นUําเกลอื (0.85% NaCl) 3. Xylene (reagent grade) 4. Screening cell (O1 และ O2) 5. Anti-human globulin (polyspecific 6. หลอดทดลองขนาด 12x75 mm 7. หลอดทดลองฝาเกลยี วขนาด 13x100 mm 8. Transfer pipette 9. ไมจ้ มUิ ฟัน (applicator stick) 10. เครอOื งปันO (serofuge) ทมOี แี รงเหวยOี ง •,••• xg 11. เครอOื งอุน่ (water bath) อุณหภมู ิ 56 oC วิธีทาํ 1. ลา้ งตวั อย่างเมด็ เลอื ดแดงทใOี หผ้ ลบวกกบั direct antiglobulin test ดว้ ยนUําเกลอื จํานวน › ครงัU ในหลอด ทดลองขนาด •‘x100 mm จากนันU เก็บ supernatant จากการล้างครงัU สุดท้าย (เก็บบรเิ วณทOีใกล้ สว่ นบนของชนัU เมด็ เลอื ดแดง) ไวเ้ ป็นชดุ ควบคมุ 2. ผสมเมด็ เลอื ดแดงอดั แน่นในขอ้ 1 ดว้ ยนUําเกลอื ในอตั ราสว่ น 1:1 ผสมใหเ้ ขา้ กนั 3. ผสม 50% เมด็ เลอื ดแดงทOไี ด้จากขอ้ š กบั xylene ในอตั ราส่วน 1:1 ใช้หลอดทดลองฝาเกลยี วขนาด 13x100 mm 4. ปิดหลอดทดลองดว้ ยจกุ เกลยี วแลว้ เขยา่ หลอดอยา่ งแรงนาน 1-2 นาที (ห้ามใชพ้ าราฟิลม์ เพราะ xylene จะทาํ ลายพาราฟิลม์ ) จากนนัU ผสมอกี ครงัU เบาๆ ดว้ ยเทคนิค inversion (end-to-end) นาน • นาที 5. คอ่ ยๆ คลายจกุ เกลยี วเพอOื ลดความดนั ภายในหลอด แลว้ นําหลอดไปอุ่นทOี 56 oC นาน 10-1• นาที ระหวา่ ง นUีใหผ้ สมตวั อยา่ งเลอื ดและนUํายาเป็นครงัU คราวดว้ ยไมจ้ มUิ ฟันหรอื วใั ชว้ สั ดอุ นOื ททOี ดแทนกนั ได้ 6. ครบเวลาปันO ทOี 1,000 xg นาน •• นาที - ชนัU บนสดุ เป็น xylene - ชนัU กลางเป็นเศษเซลล์ (red cell stroma) - ชนัU ลา่ งสดุ คอื eluate ซงOึ เป็น hemolysate สนี Uําตาลแดง 7. ดดู ชนัU บนสดุ ซงOึ เป็นชนัU ของ xylene ทงUิ 8. เปลOียน transfer pipet ดูดเก็บ eluate ใส่หลอดทดลองหลอดใหม่ ขนาด 12x75 mm จากนันU นําไป ทดสอบ หรอื หากตอ้ งการเกบ็ ใหแ้ ชแ่ ขง็ ไวไ้ ด้ 9. ทดสอบ eluate ดว้ ยวธิ ี indirect antiglobulin test กบั screening cell (O1 และ O2) หรอื กบั เซลลท์ มOี ี แอนตเิ จนตรงกบั แอนตบิ อดใี น eluate โดยใชน้ Uําเกลอื จากการลา้ งเมด็ เลอื ดแดงครงัU สดุ ทา้ ย (last wash) เป็น eluate control ซงOึ ตอ้ งใหผ้ ลลบจงึ จะสามารถแปลผลการทดสอบนUีได้ 137