Advanced Antibody Identification: Case Studies. Justin R. Rhees, M.S., MLS(ASCP) CM, SBB CM University of Utah Department of Pathology

Transcription

1 Advanced Antibody Identification: Case Studies Justin R. Rhees, M.S., MLS(ASCP) CM, SBB CM University of Utah Department of Pathology

2 Acknowledgements Thank you to the following scientists at the ARUP Immunohematology Reference Lab (IRL) for contributing to several of the case studies used in this presentation: Rebecca Whitney Catherine Thom Noel Pusey Becky Condas 1. Sally Rudmann, Ed. Serologic Problem-Solving: A Systematic Approach for Improved Practice AABB Press. 2. Guerlain S, Smith PJ, Obradovic JH, et al. Interactive critiquing as a form of decision support: An empirical evaluation. Hum Factors 1999;41: Kanter MH, Poole G, Garraty G. Misinterpretation and misapplication of p values in antibody identification: the lack of value in a p value. Transfusion. 1997;37:

3 About me B.S. Degree Medical Laboratory Science/German Weber State University M.S. Degree Laboratory Medicine and Biomedical Science University of Utah University of Texas Medical Branch at Galveston (UTMB) Specialist in Blood Bank Technology Training Board Certification American Society for Clinical Pathology SBB(ASCP) Assistant Professor, Program Director Medical Laboratory Science Program, University of Utah Nelda the Chicken

4 Objectives 1. Describe the principles and procedures of the antibody identification tests. 2. Explain what factors make an antibody clinically significant. 3. Given patient test results, correlate knowledge of the serologic characteristics of several antibodies and work through the procedures to correctly resolve several antibody identification cases.

5 Why are there so many different procedures for ABID? The protocols that laboratories choose will affect what they detect. Protocols should be tailored to the experience of the staff and the general patient population encountered. Media needs to be taken into account Gel Solid Phase Tube Saline Albumin LISS PeG Enzymes Principles and Procedures of the tests

6 An antibody is considered significant if it has been associated with HDFN HTR Notable decreased survival of RBCs The degree of clinical significance varies among antibodies with the same specificity Most commonly identified alloantibodies: Anti-D Anti-E Anti-K Clinical Significance and Prevalence

7 Antibody Reactivity in Various Media Albumin LISS PeG Gel Solid Phase May enhance Rh and anti-p 1 antibodies during the 37ºC spin phase Some examples of anti-k do not react well in LISS Newly forming IgM antibody may not react Some examples of anti-jk a not detected Can enhance clinically benign autoantibodies Increased detection of antibodies that are not clinically significant Can enhance clinically benign autoantibodies Increased detection of antibodies that are not clinically significant Can enhance clinically benign autoantibodies Sources: Sally Rudmann, Ed. Serologic Problem-Solving: A Systematic Approach for Improved Practice AABB Press. John D. Roback, Ed. AABB Technical Manual, 17 th Edition. Denise Harmening, Ed. Modern Blood Banking and Transfusion Practices, 6 th Edition.

8 Principles and Procedures of the tests Use of sensitive media can enhance reactivity of antibodies that lack clinical significance Context Benign autoantibodies Anti-Ch, Anti-Bga, etc. A large transfusing facility with sickle patients, oncology, active transplant programs, and other multiply transfused patients A medium-sized community hospital with a busy Labor and Delivery unit A small clinic in a rural area that stocks blood for trauma and transport scenarios Staffing Level of training and experience Ratio of experienced technologists to new hires Experienced new hires vs. new MLS graduates

9 Principles and Procedures of the tests Microscopic evaluation of macroscopically negative test tube reactions? Use of the autocontrol in antibody screening and panels? Two- or three- cell screens? Screen and panel methods the same? Variations in ruling out Homozygous for C, c, E, e, Duffy, Kidd, MNSs How many strikes? Heterozygous ok? How many strikes? Variations in ruling in Etc. 2/2 rule? 3/3 rule?

10 Principles and Procedures of the tests Gather relevant patient information Observe and evaluate results Phase of reactivity: immediate spin, 37C incubation, AHG Incompatible crossmatches? Strength Hemolysis? Pattern Most or all cells positive, autocontrol negative 1 or 2 cells positive, autocontrol negative Panreactivity Variability an antibody showing dosage effect, multiple antibodies, or antigen showing variable expression from one panel cell to another Weak, variable reactivity Physical Appearance

11 Knowledge of Antibody Specificities Anti-D, -E, and -K antibodies most common in U.S. Anti-C, -c, -e, -Jk a, -Jk b Fy a, -S, -s sometimes seen Anti-Fy a and anti-fy b rarely exist as single alloantibodies Temperature (IgM = cold reactive, usually not clinically significant) Lemon Pie is best served cold Lewis M, N, P 1

12 Knowledge of Antibody Specificities Antibodies to high incidence antigens are rarely seen (few people lack the antigen and can therefore form the antibody): k (Cellano), Kp b, Js b, P, P k, U, Lu b, Vel, etc. Antibodies to low incidence antigens are rarely seen (although most people can form the antibody, the antigens are rarely found on donor blood antibodies usually formed through HDFN) C w, Kp a, Js a, Lu a, etc.

13 Knowledge of Antibody Specificities Usually clinically significant: ABO, Rh, Kidd, Duffy, S, s, U, P Rarely (if ever) cause clinically obvious symptoms: Bg (HLA), Ch/Rg (C4), Le b, JMH, Xga Sometimes: Cartwright (Yt), Lutheran (Lu), Gerbich (Ge), Dombrock (Do), M,N, Lea, Vel, LW, Ii, H, At a, In b, Mi a, Cs a

14 Ruling Out A tool in the process, not infallible It is always preferable to rule out an antibody specificity on a homozygous cell It is better to rule out specificities with two unique cells rather than one. There is no reason to routinely rule out antibodies to low-incidence antigens. Screening cells may not detect these Patients rarely form these antibodies Transfusion probability Ethnicity/Geography matters: Dia 10% in Asians, 36% South American Indian Even if a specificity is ruled out by the laboratory s SOP, it does not mean the antibody is not present

15 Ruling In p value is a calculation of the number of antigen-positive cells that react and the number of antigen-negative cells that do not react. 1,3 AABB s IRL Standards require two antigen-positive cells that are reactive and two antigen-negative cells that are nonreactive Donor Cell K+ Patient Reaction + K+ K- K

16 Ruling In with Multiples Multiple specificities must be ruled in independently of each other: Donor Cell E-K- E+K- E+K- E-K+ E-K+ Patient Reaction E-K- 0 0

17 Procedure Procedure Go to the first panel cell with a negative reaction, rule out or exclude the specificities of antibodies directed against antigens present on the cell. (Rule out when the antigen is positive and the patient did not react) Some antibodies demonstrate dosage.

18 Dosage Dosage Some antibodies may react so weakly with antigens with heterozygous expression, they might not be detected. For antibodies in the following blood groups, it may be prudent to rule out with panel cells that have a homozygous expression of antigen: Rhesus (C, c, E, e) Kidd Duffy MNSs

19 Dosage Dosage Mother Father

20 Dosage Dosage Mother Father

21 Dosage Dosage Anti-Jka may not react with a heterozygous single dose cell It may only react with a cell that has double the dose of Jka antigens

22 Allelic Pairs Allelic pairs Rh System C, c E, e Duffy System Fya, Fyb Kidd System Jka, Jkb MNSs System M, N S, s

23 Ready to go? Lederhosen c. 1981

24 Step 1. Gather all relevant data 37-year-old male patient Received 4 units of prbcs during previous hospital admission under a trauma name, 3 months ago Scheduled for surgery tomorrow

25 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

26 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

27 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

28 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

29 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

30 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

31 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

32 Case 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

33 The next step What alloantibody or alloantibodies have not been ruled out? Anti-E Anti-Fya Which of the following is or are most likely? Look closely at the pattern of reactivity.

34 Antibody Identification Panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

35 Antibody Identification Panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

36 Confirmation steps Anti-E is the most likely antibody reacting However, we still have not ruled out anti-fya The patient could have anti-fya underlying the reactions of anti-e We need to select another cell that is E antigen negative, and Fy(a+b-) HOMOZYGOUS for Duffy A

37 Selected Cell from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

38 Selected Cell from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

39 Selected Cell from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AH G CC AC

40 Selected Cell from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AH G CC AC

41 Rule of 3 Criteria: At least 3 panel cells with E antigen reacted (positive result) with patient s sample At least 3 panel cells lacking E antigen did not react (negative result) with the patient s sample Does our example fulfill these criteria?

42 Antibody Identification Panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

43 Rule of 3 At least 3 true positives and 3 true negatives: Following this rule gives us a P value of % chance that the antibody we have identified is correct.

44 Rule of 2 Clinical utility of P value in ABID 1,3 At least 2 true positives and 2 true negatives: AABB IRL Standards Confirmation that the antibody(ies) identified are present All other clinically significant alloantibodies are ruled out

45 Before reaching a final conclusion Is the final answer a unicorn? Are there extra reactions not explained by the final answer? Is the result consistent with the available data? Have all of the alternatives not included in the final result been ruled out? Has enough evidence been collected to establish a high degree of confidence?

46 Result Anti-E identified. All other clinically significant alloantibodies have been ruled out. Donor units lacking E antigen should appear crossmatch compatible through the indirect antiglobulin test (IAT).

47 Case 2 Gather data 48 year old female patient with lymphoma transfused 6 months ago. O positive, previous antibody screen negative.

48 Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

49 Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

50 Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

51 Option 1 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

52 Option 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

53 Is the answer a unicorn? Alice came to a fork in the road and saw a Cheshire cat in a tree. Which road do I take? She asked. Where do you want to go? was his response. I don t know. Alice answered. Then, said the cat, it doesn t matter. --Lewis Carroll Photo Cred: Rhees, Yorgo the Destroyer

54 Anti-E and anti-k are more common. Examples of anti-fy b as a single antibody specificity are rare. When choosing selected cells: Anti-E and anti-k need to be proved independently. All other clinically significant alloantibodies need to be ruled out.

55 Selected Cells from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

56 Selected Cells from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

57 Selected Cells from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

58 Selected Cells from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC w+ AC

59 Selected Cells from a different panel Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC w+ AC

60 Now what? Has the patient been transfused recently? The lifespan of an RBCs is ~120 days Since the patient has not recently been transfused, antigen typing was performed: O positive, R1r (DCe/ce) C c E e K Fyb New hypothesis: patient has anti-e, anti-k and anti-fyb. Has each specificity been proved independently?

61 Additional Selected Cells Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s AC AH G CC

62 Additional Selected Cells Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC

63 Additional Selected Cells Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC

64 Review the workup Is the final answer a unicorn? Are there extra reactions not explained by the final answer? Is the result consistent with the available data? Have all of the alternatives not included in the final result been ruled out? Has enough evidence been collected to establish a high degree of confidence?

65 Original Panel Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

66 Original Panel Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

67 Selected Cell Panel 1 Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC w+ AC

68 Selected Cell Panel 2 Case 2 Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC

69 Additional work required Have proved anti-fy b independently of anti-e and anti-k (3) Need to find additional E- K+ Fy(b-) cell(s). Need to find additional E+ K- Fy(b-) cell(s). 2/2 vs. 3/3 Rule?

70 Case 3 M. Martinez, 73 y/o male A positive, R1r (DCe/ce) No transfusion or drug history available

71 Case 3 LISS Rh-Hr Kell Duffy Kidd P MNSs Results D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 AH G CC AC 0 0

72 Case 3 Solid Phase Rh-Hr Kell Duffy Kidd P MNSs Res ults D C c E e K k Fya Fyb Jka Jkb P1 M N S s IgG AC

73 Additional Testing Extended phenotype C c E e K Fya Fyb Jka Jkb S s s Direct Coombs I.S. 5 CC Saline Con 0 DAT Poly 0 0 DAT IgG 0 DAT C3 0 0

74 Case 3 Cold Panel # IS RT 16C 4C I II III 0 0 r A1 Cell s A2 Cell 0 0 r Cord I 0 0 r 1+ w 2+ s Cord II 0 0 r 1+ w 2+

75 High incidence antigens absent in Antigen negative certain ethnic populations Population Di b South Americans > Native Americans > Japanese Ge: -2, -3 Jr a PP1Pk (Tja) Mexicans > Israelis > Mediterranean > Any Japanese > Mexicans > Any Japanese > Swedes > Isreali > Amish > Any JMH Autoanti-JMH is often found in, but not restricted to, elderly persons with an acquired absent or weak expression of JMH; the DAT may be positive Blood Group Antigens & Antibodies ME Reid, C Lomas-Francis SBB Books

76 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s JMH Di(b-) PP1PK Ge: -2, Jr(a-)

77 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s JMH Di(b-) PP1PK Ge: -2, Jr(a-) 1+

78 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Ch-, Yka U- McC(a-) Lan Do(b-) I Co(a-)

79 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Ch-, Yka- 1+ s U- McC(a-) Lan Do(b-) I- 1+ w Co(a-) 1+ 1+

80 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Cs(a-) Kn(a-) H- Co(b-) Sc: Vel Rh Null U-

81 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Cs(a-) Kn(a-) H- Co(b-) Sc: Vel s Rh Null U- 1+

82 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Vel Vel Vel Vel-

83 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Vel Vel Vel Vel- 0

84 Case 3 Special Selected Cell Panel Rh-Hr Kell Duffy Kidd P MNSs Additional Antigens PEG CC D C c E e K k Fya Fyb Jka Jkb P1 M N S s Vel Vel Vel Vel- 0 Antigen testing: The patient appears to be negative for the Vel (VEL1) antigen when using two examples of unlicensed human antisera.

85 Anti-Vel Anti-Vel is a clinically significant antibody capable of causing transfusion reactions. Anti- Vel is directed against a high incidence antigen present in over 99.9% of the random population. Transfusion needs for this patient may be met by autologous donation, evaluating siblings for suitable donors, and the American Rare Donor Program (ARDP).

86 Vel Vel-negative RBCs have been found in 1: 4,000 people and approximately 1: 1,500 Norwegians and Swedes

87 Case 3 Review of the workup Is the final answer a unicorn? Are there extra reactions not explained by the final answer? Is the result consistent with the available data? Have all of the alternatives not included in the final result been ruled out? Has enough evidence been collected to establish a high degree of confidence?

88 Photo cred: Cathy Thom, IRL Justin the Nerd On freezing rare cells A Guide to Droplet Freezing, National Institutes of Health Frozen RBCs: Rare Phenotype

89 Case 4 28-year-old male with sickle cell anemia. First visit to your hospital system no record. Has received many transfusions throughout his lifetime, including about 3 weeks ago. Admitted due to sickle cell crisis triggered by the high altitude. Solid phase screen was positive reflexed to antibody ID in tube using LISS.

90 Case 4

91 Case 4

92 Case 4 Selected Cells

93 Case 4 Selected Cells

94 Case 4 Review of the workup Is the final answer a unicorn? Are there extra reactions not explained by the final answer? Is the result consistent with the available data? Have all of the alternatives not included in the final result been ruled out? Has enough evidence been collected to establish a high degree of confidence?

95 Thank you! Questions? My parrot Icarus