Carol A Starks MS, MLS(ASCP), CLS. castarks

Transcription

1 Carol A Starks MS, MLS(ASCP), CLS

2 Brief review of the common blood groups ABO Rh Kell Duffy Kidd P Lutheran MNS Lewis

3 Basic Blood Bank workups ABO discrepancies Single antibody not demonstrating Single antibody Multiple Antibodies Cold Warm Auto Antibodies High Frequencies Low Frequencies

4 Genotype Phenotype Homozygous Heterozygous Dosage AHG (aka IAT) IS Clinically Significant (HTRs or HDFN)

5

6 ABO Antigens begin to appear on fetal RBCs at weeks gestation; reach adult levels by age 4. ABO antigens are found on endothelium, kidney, heart, lung, bowel, and pancreatic tissues.

7 CLINICALLY SIGNIFICANT Naturally occurring Appear at age 4 months; and reach adult levels by age 10. Levels may fade with advanced age.

8 Generally IgM class antibodies For Group A and Group B persons the predominant antibody class is IgM For Group O people the dominant antibody class is IgG (with some IgM) React best at room temperature (22-24 o C) or below in vitro. Activates complement to completion at 37 o C Can cause acute Hemolytic Transfusion reactions RBC Immune form: Predominantly IgG

9 They both react strongly with reagent anti-a. 80% of group A individuals phenotype as A1 20% phenotype as A2 Reagent anti-a is a mixture of two Abs ; anti-a which react with both A1 and A2 cells. anti-a1 which reacts with A cells but not with A2 cells in simple testing.

10 Differentiation between the A blood subgroups Reagent anti-a is a mixture of two Abs The two Abs can be functionally separated by adsorption with A2 cells. Anti-A1-lectin: is another source of anti-a1. lectins are seed extracts that agglutinate human cells with some degree of specificity. The seeds of the plant Dolichos biflorus serve as the source of the anti-a1 lectin; this reagent agglutinates A1 or A1B cells but does not agglutinate A2 or A2B cells.

11 Other A subgroups: RBC of the A int, A 3, Ax, A y or A cl. are only rarely seen in transfusion practice. Subgroup of B: infrequent than the weaker subgroup of A, identified by anti-b and anti-a,b. Subgroups B3, Bx, Bm and Bcl.

12 ABO discrepancies happen when there is no match in results between forward and reverse grouping. ABO discrepancies are usually technical in nature and can be simply resolved by correctly reporting the testing and carefully checking reagents with meticulous reading and recording of results.

13 There are some ABO discrepancies that can happen due to technical errors and may lead to false positive or false negative reactions. False positive reactions are due to; Un-calibrated centrifuges Contaminated reagents Dirty tubes or glassware

14 False negative reactions can be due to many causes Failure to add serum or reagents Use of incorrect reagents or samples Cell suspension is too heavy or too light Inadequate identification of samples or test tubes

15 Group I discrepancies These discrepancies are between forward and reverse grouping due to weak reaction or missing antibodies. These kind of discrepancies are the most common. The reason for the missing antibody or weak reaction is that the patient has depressed antibody production or cannot produce the ABO antibodies.

16 Resolving discrepancies Eliminate all technical errors Enhancing the reaction in reverse grouping Incubate the patient s serum with reagent cells at room temp. for 15 mins. Increase serum to cell ratio in the reverse typing.

17 Anti-A Anti-B Anti-D A cells B cells Interpretation History ? O pos This type of discrepancy can be seen in new born infants, elderly patients. Patients with lymphoma. Patients using immunosuppressive drugs. Patients with immunodeficiency disease, BM transplant.

18 Group II discrepancies These discrepancies are between forward and reverse grouping due to weak reaction or missing antigens. These discrepancies are the least frequent. Can be caused by some subgroups of A or subgroups of B or both. Also it can be present in patients with leukemia and Hodgkin's disease. To resolve the problem wash the patient s cells with saline.

19 42 year old man, with a historical type AB+ Anti-A Anti-B Anti-D A cells B cells Interpretation History ? AB Pos Where is the problem here? What do you think went wrong? What do you do next Missing Antigens! I made a mistake, repeat Get a history

20 History told us this patient was recently transfused with multiple units of O positive and A positive RBCs. There were a total of 12 units over the last 6 days. This would be considered indeterminate due to recent transfusions. What would future transfusions look like?

21 Group III discrepancies These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities. These can be caused by elevated levels of globulin from certain diseases such as multiple myeloma, Hodgkin's lymphoma. Some are caused by (Rouleaux formation).

22 Rouleaux or red cells result from a stacking of erythrocytes that adhere in a coin-link fashion giving the appearance of agglutination. To resolve this kind of problem, washing the patient s red cells with saline or adding a drop or two of saline to the tube in case of rouleaux formation. If the agglutination is true red cell clumping will remain. Cord blood must be washed 6-8 times in forward grouping ONLY. This is to remove the Wharton s jelly.

23 Anti-A Anti-B Anti-D A cells B cells Interpretation History ? A pos Saline Replacement Anti-A Anti-B Anti-D A cells B cells Interpretation History A pos A pos

24

25 Group IV discrepancies These kind of discrepancies are between forward and reverse groping due to miscellaneous problems. Polyagglutination can occur due to exposure of hidden erythrocyte Ag. (T antigen) in patients with bacterial or viral infection. Bacterial contamination in vitro or vivo produces an enzyme that alters and exposes the hidden Ag. on red cell leading to T activation.

26 Second most important blood group after the ABO system. Terminology Fisher Race (DCE or CDE) Wiener (R 1, r 1, etc.) D type is determined by the presence of the D antigen.

27 Require exposure Warm Reacting D induces the most antibodies, then c and E. HTR with extravascular hemolysis Severe HDFN with anti-d, anti-c, and mild HDFN with anti-c, E, e. Due to the mosaic and weak features of the D antigen, can have some weird stuff.

28 Clinically and serologically important group. Antigens in the system: K, k, Jsa, Jsb, Kpa, and Kpb. Low frequencies: K, Jsa, and Kpa High frequencies: k, Jsb, Kpb Kell antigens destroyed by Thiol reagents. DTT ZZAP

29 Require Exposure Typically transfusion Warm reacting IgG Can cause severe HTR Severe HDFN, with lower bilirubin and retic levels. Attacks early RBC precursors.

30 Consist of Fya and Fyb antigens. FYA high frequency in the Asian population FYB high frequency in the Caucasian population. Fya neg and Fyb neg most common phenotype in African Americans. (gives protection from malaria(vive))

31 Exposure required Warm reacting; IgG Decreased activity with Ficin (enzymes). Marked dosage and can show variability in reactions. Severe HTR, delayed and extravascular usually Often mild but can be severe HDFN.

32 Antigens Jka, Jkb, Jk3. Jk3 very high frequency

33 Require exposure. Warm reacting, IgG, often with an IgM component as well. Can fix complement with the IgM component. Severe acute HTN possible. Demonstrate marked dosage. Variable expression, and will disappear with time/storage. Known for delayed HTN, that can be severe and intravascular. Kidds play hide and seek.

34 Antigens P1 and Pk Antibodies Cold reacting, IgM Naturally occurring Insignificant Neutralized by H. cyst fluid, or pigeon egg whites. Auto anti-p (PCH) Biphasic IgG that binds in the cold and then hemolyzed when warmed. Give full crossmatch compatible typically.

35 Antigens Lua and Lub Lua is a low frequency Lub is a very high frequency Antibodies Uncommon, may be naturally occurring Not usually significant Decreased activity by enzymes Typically give full crossmatch compatible.

36 Antigens M, N, S, s. M roughly equals N in prevalence ( 75%) s is more frequent than S. S-s- individuals may also be U-. U is a high frequency antigen. These individuals can make Anti-U.

37 M and N are almost the opposite of S and s. Anti-M and Anti-N Naturally occurring Cold reacting, IgM Dosage Insignificant Anti-S and Anti-s Require exposure Warm IgG Minimal Dosage Significant M and N can be ignored unless they react at 37. Rarely associated with severe HDFN.

38 Coded for one gene Le. (like D) Le will add a fucose to the GlcNAc resulting in Lea. Secretors will add another fucose resulting in Leb Formation of Leb is preferential in secretors. Antigens are not tightly bound, rather they are absorbed onto the service of the RBCs.

39 Pregnancy Antigen levels decrease due to change of composition of patients plasma. Can give rise to transient Lewis antibodies Lea-Leb+ do not develop Anti-Lea Children s Lewis typing will change as chains are converted. Lea+Leb+ will change to Lea-Leb+ Associated Infections: H Pylori attaches via Leb Lea-Leb- more susceptible to Candida and E coli.

40 Antibodies are typically insignificant. Rare HTR Minimal to no HDFN (does not cross placenta)

41

42 When do we perform an antibody identification. When testing suggests a new antibody (warm autos) To confirm a previously identified antibody (per facility SOP)

43 Basically just a larger antibody. Uses reagent O cells from 8 20 donors Results are documented on a sheet called an antigram. The antigram is a roadmap that outlines every cell s phenotype.

44 Antigram Geography

45 Having a CONSISTENT approach to antibody workups minimizes error. Most workups are simple but cutting corners increases risk for dumb mistakes. Use this or your own system, but to the same approach EVERY TIME!

46 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

47 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

48 History can give you a clue and keep you from doing something dumb. Up to 70% of cases are impacted by history.

49 Anti-D in a pregnant patient Recent bacterial infection treated with antibiotics that cause warm auto Recent transfusion ITP Racial significance African Americans lack Duffy Ags Asians: RARELY D- Caucasian: May lack high freq. Ags Previous Ab and/or phenotyping.(kidds) Transplants

50 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

51 Ask 2 questions: Is DAT positive What is the patient s history Possibilities include: Autoantibodies (warm or cold) Recent transfusion; delayed hemolytic reaction Drug induced Passively acquired antibodies

52 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

53 Are the reactions. Uniform or variable Uniform reactions suggest a single antibody Variable reactions suggest multiple antibodies or a single antibody with dosage. Against all, most, or rare cells A mixture of positive and negative cells suggest a single antibody or multiple alloantibodies(auto neg) A single alloantibody cells suggest a low frequency. All cells reactive suggests multiple alloantibodies or a high frequency. Present at what phases

54 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

55 First approach to the interpretation of the panel results. Once results have been recorded on the antigram, the antigen profile of the first negative cell is examined. If the antigen is present on the reagent red cell and the serum/plasma did not react; presence of the antibody can be excluded. This approach often identifies simple antibodies, and should be considered only as a provisional step; particularly if the rule outs are performed on heterozygous expression. This gives explanation why many SOPs will ask for homozygous expression for rule outs when possible. If not possible use 3 heterozygous cells. From the AABB Technical Manual 17 th ed. Page 470.

56 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

57 First try for a single antibody that will explain all the reactions. If that does not work Hypothesize Two antibodies in the same phase. One warm and one cold antibody Additional reagent cells from another panel maybe ran to help with multiple antibodies and solidifying hypothesis.

58 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

59 Providing your patient has not been transfused in the last 90 days you can phenotype the patient to help provide some resolution. Some other special techniques performed at reference labs typically: Adsorption Elution Proteolytic enzymes (DTT, ZZAP)

60 Check history Check Autocontrol Look at general pattern Look at what is NOT there(cross outs)(rule outs) Look at what is there Use special techniques if needed Ensure statistical significance.

61 Traditional Interpretation: Rule of 3s Ag present and 3 positive reactions Ag absent and 3 negative reactions 95% confidence If you bump this up to 3 and 4 your confidence is 98% This may also require additional cells to be performed to achieve confidence. Has to be done for each individual Ag.

62 Antigen type your patient for the antigen corresponding to the Ab identified. Your patient should be negative. Antigen type most units to be transfused. Insignificant Abs typically only require a full crossmatch. Refer to your facility SOP. If you are low on anti-sera for Ag typing. You can use your patient s plasma/serum to perform the full crossmatch first, then Ag type.

63

64