Pretransfusion procedures

Question 1

proper labeling of of blood from recipients

Answer 1

labels:

• labeled at bedside with 2 unique identifiers
• date and time of collection
• identity of phlebotomist
• paperwork and blood specimen must match
  
  • in case of discrepancy or doubt, a new specimen must be obtained

Question 2

If recipient has been pregnant or transfused within 3 months or if history is uncertain, what testing must be done?

What should be done in the absence of the above conditions?

Answer 2

• sample for pretransfusion testing must be obtained within 3 days of transfusion (day 0 is the date of sample collection) in recently pregnant/transfused patient
• In the absence of pregnancy or recent transfusion, ABO samples older than 3 days may be used
  
  • ABO testing, Rh testing, and antibody screening must be performed and no discrepancies or alloantibodies must be detected to use this sample for crossmatching prior to elective surgery
  • do not need to test for weak D
  • anti A reagent is blue
  • anti B reagent is yellow
• The recipient pretransfusion blood sample as well as the tube segment from any transfused blood must be retained for 7 days after transfusion

Question 3

Visual inspection of donor unit

Answer 3

• bacterial contamination may appear as purple color, zone of hemolysis, or with clots
• lipemic units should not be used

Question 4

Causes of forward/reverse typing discrepancies

Answer 4

• extremes of age
• acquired immunodeficiency affecting Ig production (hematolymphoid neoplasms and immunosuppressive agents)

Question 5

Antibody screen

Answer 5

• done on recipient blood
• uses 2-4 reagent O test cells that have differential expression of antigens
• required that 18 clinically significant antigens are represented
• patient plasma/serum is added to the test cells followed by
  
  • immediate spin
  • 37 degree incubation
  • addition of anti human globulin (AHG)
• room temperature incubation is omitted by many labs since it detects clinically insignificant alloantibodies mostly (anti M, N, Lewis, I, and P)
• tests may be performed with LISS or PEG
• if no reactions occur, cross match can proceed
• if any cells react in screen, then antibody panel is required

Question 6

antibody panel

Answer 6

• 10-14 different donor test cells, all group O
• autocontrol run in parallel (recipient serum/plasma added to recipient red cells)

Question 7

Crossmatch

Answer 7

• Immediate spin or computer crossmatch
• Computer crossmatch is ok in patients with negative antibody screen
  
  • computer system must be validated
  • 2 separate determinations of recipient’s ABO/Rh required
• The IS crossmatch
  
  • performed at room temperature
  • recipient serum/plasma mixed with donor RBCs followed by centrifugation
  • If known clinically sig alloAb present, then complete or full crossmatch with AHG phase testing is required
  • if cold reacting antibodies are present, the crossmatch may be prewarmed to prevent interference
• Infant blood up to 4 months old need not be crossmatached if no maternal antibodies are detected
  
  • the ABO/Rh is determined as for adult recipients, but must only be done once and may be omitted for the remainder of the hospitalization unless lasting beyond 4 months of age or infant leaves and is readmitted
• If alloantibody is present, the crossmatch must be carried through the antiglobulin phase

Question 8

Compatibility of granulocytes

Answer 8

Must be ABO compatible with recipient serum and must be subjected to crossmatch

Question 9

Compatibility of RBCs

Answer 9

Whole blood must be ABO identical

• If recipient is < 4 months old
  
  • first choice is O with matching Rh
  • second choice is O with opposite Rh
  • third choice is identical to patient’s blood type
  • all RBCs are CMV negative and irradiated

Question 10

Compatibility of platelets and cryo

Answer 10

• Contain plasma
• May be transfused outside of ABO compatibility unless unit is visibly bloody
• preferably, plasma in these products is compatible with recipient red cells
• preferable that Rh- recipient receive Rh- platelets
  
  • if giving Rh+ is unavoidable, Rh immune globulin should be suggested
• first choice is identical to patient type
• second choice is identical ABO and opposite Rh group
• Third choice is AB for A or B type patient or AB, A, or B for O type patient

Question 11

Plasma compatibility

Answer 11

• Must be compatible with recipient’s RBCs
• If recipient < 4 months of age:
  
  • first choice is AB
  • second choice is A or B or O
  • Rh should match the patient

Question 12

Platelet compatibility for < 4 month old

Answer 12

• First choice = AB and matching Rh
• Second choice = match patient’s blood type
• All platelets CMV negative and irradiated

Question 13

Blood transfusion set

Answer 13

• 170 um filter
• “microaggregate” 40 um filters are not required
• same filter can be reused for multiple transfusions for up to 4 hours
• these filters are not capable of leukoreduction
• if a leukocyte reduction filter is used, the inline filter may be omitted
• neonatal tranfusions may be filtered by the blood bank staff prior to release
• other fluids should not be run in the same line as the unit
  
  • only normal saline and other FDA approved crystalloids can be run in the same line
  • lactated ringers may cause blood clotting
  • dextrose and hypotonic solutions (0.45%) saline may cause hemolysis

Question 14

Timing of transfusion and monitoring

Answer 14

• Transfusion must be started within 30 minutes of issue and must be completed within 4 hours
  
  • if 2 units are released simultaneously then both must be infused within 4 hours
• Monitoring
  
  • baseline set of vitals should be recorded
  • observe patient for first 15 minutes
  • slow rate of infusion (2 ml/minute) is good at the beginning
  • a second set of vitals at 15 minutes
  • final set of vitals at end of transfusion
  • recipient should be observed periodically throughout transfusion and for an appropriate length of time thereafter
  • unless there are difficulties, most single unit red cell transfusions can be completed within 90 minutes (and must be completed within 4 hours)

Question 15

In an antibody panel the 10-14 cells must express what antigens?

Answer 15

1. D
2. C
3. E
4. c
5. e
6. M
7. N
8. S
9. s
10. Pl
11. Le^a
12. Le^b
13. K
14. k
15. Fy^a
16. Fy^b
17. Jk^a
18. Jk^b

Question 16

Phases of antibody panel testing

Answer 16

1. immediate spin phase
   
   • antibodies only reacting at this phase are “cold reacting”
   • usually IgM and are likely to be clinically insignificant; may be significant if high titer present (>1,000)
2. Serum/plasma mixed with RBCs and centrifuged at 37 degrees
   
   • “warm reacting” antibodies detected
   • usually IgG and potentially clinically significant
3. Serum/plasma mixed with RBCs in the presence of AHG in the AHG phase (indirect agglutination test - IAT)
   
   • IgG
   • likely to be clinically significant

Question 17

Positive reactions on antibody panel

Answer 17

• agglutination is characterized by red cell clumps
  
  • graded m+ to 4+
• hemolysis shows as a pink supernatant

Question 18

Dosage effect seen with which antigens

Answer 18

1. Kidd
2. Duffy
3. Rh
4. MNS

Question 19

Alloantibodies that are nearly always insignificant

Answer 19

1. Lewis
2. Lutheran
3. I

Question 20

Alloantibodies that are nearly always significant

Answer 20

1. Kell
2. Kidd
3. Rh
4. Duffy

Question 21

Number of cells required to establish presence of a specific antibody in clinical practice

Answer 21

• 3 positive cells with reactivity
• 1 negative cell with nonreactivity

Question 22

Direct antiglobulin test

Answer 22

1. Polyspecific
   
   • polyspecific reagent agglutinates RBC coated with IgG or C3
2. Anti IgG AHG
   
   • agglutinates red cells with bound IgG on their surface
     
     • autoantibody bound in vivo to the patient’s red cells
     • alloantibody to transfused red cells (usually mixed field)
     • maternal antibody bound to neonatal red cells
     • medications
3. Anti C3
   
   • agglutinates red cells with bound C3 on their surface
     
     • could be positive along with IgG or an IgM

Question 23

Antibodies to high incidence antigens or hight titer, low avidity (HTLA) antibodies

Answer 23

• Suspected when there is weak reactivity in AHG phase to all cells in the panel
• Continue to react at high dilutions (>1:64)
• they can mask other antibodies
• Clinically significant HTLA antibodies:
  
  • cartwright (Yt^a)
  • Holley (Hy)
  • Gregory (Gy)
• HTLA antibodies that are not significant
  
  • Chido/Rodgers (Ch/Rg)
  • Sd^a
  • Cs^a
  • Kn^a
  • York (Yk^a)

Question 24

Antibodies to test reagents

Answer 24

• autocontrol is negative and there is across the board reactivity
• test cells can be washed to remove these substances
• occurs most frequently with gel and solid phase systems
  
  • in the tube method, the panreactivity may disappear
    
    • when lot numbers of reagent are used, may also disappear

Question 25

Antibodies to low incidence antigens

Answer 25

• Wr^a and Kp^a
  
  • ^​suspected when 1 test cell is reactive within a panel
  • typically these antigens are not indicated on the panel, but the package insert for the test cells delineates a longer list of antigens for which the cell is positive

Question 26

Polyagglutination

• what is it caused by?
• consequences?

Answer 26

• adult plasma contains naturally occuring IgM antibodies to T, Tn, Tk, and Cad
• these are normally cryptic antigens that are expressed only when bacterial neuraminidase enzymatically alter red cell antigens (T activation)
• In adults this phenomenon is mainly a factor in pretransfusion testing
• In babies this phenomenon is significant in 2 instances:
  
  • necrotizing enterocolitis (NEC)
    
    • T activation occurs in up to 25%
    • caused by Clostridia neuramindase
  • Atypical hemolytic uremic syndrome
    
    • neuraminidase of S pneumoniae
• Babies with T activation can develop intravascular hemolysis when transfused
  
  • such babies should receive RBCs with minimal plasma or washed RBCs to minimize the amount of naturally occurring anti T antibodies made in the adult blood
• The effects of this are transient and abate following resolution of infection

Question 27

Polyagglutination testing

Answer 27

• lectin Arachis hypogaea can detect T activation
• polyagglutinable red cells are agglutinated by adult but not cord serum

Question 28

Elution

Answer 28

• removal of antibody bound to red cells
• several tenchniques
  
  • freeze thaw
  • heat
  • acid
  • digitonin
• the eluate, which contains the Ab, can then be tested against a panel of RBCs to determine its specificity

Question 29

Autoadsorption

Answer 29

• useful when there are autoantibodies potentialy masking alloantibodies
• autoantibody is adsorbed using the patient’s own red cells that have been pretreated with ZZAP to remove bound autoantibody
• the adsorbed plasma/serum is left with any alloantibodies that were present and can then be tested against a panel of red cells
• autoadsorption cannot be used if
  
  • the patient has been transfused or pregnant in the past 3 months because they may have leftover donor or fetal cells present

Question 30

Alloadsorption

Answer 30

this can be used to remove antibodies from serum in patients who have been transfused or pregnant within 3 months

Question 31

Hemagglutination inhibition (neutralizing substances)

Answer 31

• HI is the use of a substance that is known to contain or mimic a particular antigen to neutralize an antibody
• alternatively, neutralization can assist in detecting additional antibodies that may be masked
• HI also used to determine secretor status (by detecting H substance in saliva)

Question 32

Hemagglutination (lectins)

Answer 32

• lectins are reagents derived from plants that will bind to specific antigens on red cels and agglutinate them (causing agglutination)
• 2 lectins that are commonly used to resolve ABO discrepancies include
  
  • Ulex europaeus to detect the H antigen (agglutinates group O RBCs); useful in determination of secretor status
  • Dolichos biflorus to detect A1 antigen
• Other lectins
  
  • Bandeiraea simplicifolia detects B antigen
  • Lotus tetragonolobus detects H antigen
  • Arachis hypogaea detects T antigen
  • Vicea graminea detects N antigen

Question 33

Neutralizing substances below are associated with what antigens?

1. Guinea pig urine
2. Hydatid cyst fluid
3. Saliva
4. Breast milk
5. Pigeon eggs
6. Plasma

Answer 33

1. Guinea pig urine - Sda
2. Hydatid cyst fluid - P1
3. Saliva - H, Le^a
4. Breast milk - I
5. Pigeon eggs - P1
6. Plasma - Chido, Rodgers

Question 34

List enzymes that either enhance or decrease activity

Antigens that are enhanced by enzymes?

Antigens that are decreased by enzymes?
Unaffected by enzymes?

Answer 34

• Enzymes
  
  1. ficin
  2. papain
  3. trypsin
• Enhanced by enzymes:
  
  1. AB
  2. Le
  3. I/i
  4. P
  5. Rh
  6. Kidd
• Decreased by enzymes:
  
  1. MNSs
  2. Fy^a
  3. Fy^b
  4. Lutheran
  5. Chido
  6. Rodgers
  7. Yt^{<span>a</span>}
• Unaffected by enzymes: kell

Question 35

Likelihood of finding compatible blood

Answer 35

= 100 - antigen frequency

Question 36

frequency of Js^a

Answer 36

0.1% in whites

20% in blacks

Question 37

Causes for apparent Anti D antibody

Answer 37

1. warm autoimmune hemolytic anemia is most commonly anti-Rh
2. LW antigen is expressed more strongly in D+ cells, so the antibody may actually be to LW, not D, simulating anti-D Ab because they appear to only react with D+ cells
3. partial D patients lack epitopes of the D antigen and so develop antibodies after exposure to D

Question 38

B(A) phenotype

Answer 38

• some blood type B people have very high levels of glycosyltransferase, which produces a small amout of A antigen that reacts weakly
• the beta glycosyltransferase has an increased ability to use UDP-N-acetyl galactosamine and UDP-galactose that causes production of A antigen
• (A has N-acteyl galactosamine as its additional sugar and B has galactose)

Question 39

acquired B phenotype

Answer 39

• occurs in A1 individuals
• bacterial deacetylase acts on the A antigen (N-acetyl-galactosamine) turning it into the B antigen (galactose)
• thus, this is seen in persistent bacteremia (e.g., gram negative sepsis, colon cancer, colonic obstruction)
• there is anti B in the serum, which can result in positive DAT
• to confirm presence of acquired B
  
  • type cells with another manufacturer’s anti-B
  • reacetylate A1 in vitro with acetic anhydride, which will not be agglutinated by patient’s anti B
  • if patient is a secretor, B antigen will not be present in saliva

Question 40

anti A1 antibodies present in which phenotype

Answer 40

• 1-8% of A2 individuals have anti-A1 antibodies
• 22-35% of A2B individuals have anti-A1 antibodies
• the anti-A1 is usually IgM that reacts at room temperature or below
• compatible blood can be found with a prewarmed crossmatch
• any anti A1 reactive at 37 degrees are clinically significant and patients should receive O or A2 compatible blood

Question 41

Bombay phenotype

Answer 41

these patients have anti-H so they react with all group O screening cells

Question 42

Cases

1. all cells in panel and autocontrol positive at AHG only
2. all cells in panel except autocontrol positive at AHG only, weak
3. all cells in panel and autocontrol positive at IS only
4. all cells in panel and autocontrol positive at IS and 37 degrees, not at AHG
5. antibody screen negative, but transfusion records show previous alloantibody

Answer 42

1. warm autoantibody (can be removed with adsorption)
2. HTLA antibody (e.g., Chido, Rodgers)
3. cold autoantibody
   
   • usually these are IgM, so they are not reactive at AHG (IAT) phase
   • if antibody has broad thermal amplitude it can be positive at 37 degrees
4. describes the way an antibody to enhancement media or other reagents, such as LISS or PEG, gel or solid phase media react
5. reduced titer of alloantibody, but is still clinically significant

Question 43

ABO typing in patients with

• hematolymphoid neoplasms
• gastric adenocarcinoma

Answer 43

• often weakened expression of A or B antigens with mixed field agglutination in HL neoplasm
• gastric adenocarcinoma: excess free A or B antigens in serum may bind anti A or anti B in vitro, thus neutralizing them and giving a false impression that the patient has type O red cells

Question 44

When to suspect autoantibodies?

Answer 44

• when there is a positive DAT
• anemia with reticulocytosis or microspherocytosis
  
  • a DAT will confirm or exclude autoimmune hemolysis
• positive autocontrol in the antibody panel
  
  • a DAT will ensure that the reaction is immune mediated

Question 45

1. DAT reactive with anti IgG only or with both anti IgG and anti C3
2. DAT reactive with C3 only
3. Positive DAT on cord blood
4. what should be done with positive DAT?

Answer 45

1. caused by warm autoantibody (IgG)
2. caused by cold autoantibody (IgM)
3. maternal alloantibody to RhIg
4. elution should be performed on positive IgG DAT

Question 46

Antithymocyte globulin

Answer 46

can cause positive DAT (autoimmune)

Question 47

Benign cold agglutinins

Answer 47

• react most strongly at 4 degrees, but can react up to 22 degrees
• Most are IgM and can activate complement in vitro
  
  • reactions may be seen at AHG phase using polyspecific antisera
  • cells agglutinate with anti C3d, but not anti IgG
• Antibody specificty is most commonly anti I
  
  • I is strongly expressed on adult red cells but weakly on cord red cells
• less common anti i reacts with cord blood but not with adult blood
• anti H reacts best with group O and A1 cells
  
  • not to be confused with anti H in Bombay, that latter causes severe hemolysis

Question 48

Pathologic cold agglutinins

Answer 48

• reactive over broad range, up to 32-37 degrees
• cause spontaneous agglutination in anticoagulated blood at room temp
• titer > 1000 at 4 degrees
• Types
  
  • idiopathic cold autoimmune hemolytic anemia (CAIHA) or cold agglutinin syndrome
    
    • chronic condition in older people with acrocyanosis and Raynaud phenomenon
    • moderate intravascular hemolytic anemia
    • almost always IgM with anti I or anti i specificity, monoclonal
  • secondary CAIHA
    
    • transient cold agglutinin, IgM anti I seen in M pneumonia infection
    • resolves within 2-3 weeks
    • more persistent anti I may be seen in lymphoproliferative disorder
    • EBV can cause IgM anti i

Question 49

Mixed type autoantibodies

Answer 49

• cold reacting IgM and warm reacting IgG autoantibodies
• no particular antigen specificity
• present with acute hemolytic anemia (idiopathic or with lupus)

Question 50

Paroxysmal cold hemoglobinuria

• presents in what patients?
• presenting symptoms
• treatment
• caused by what antibody
• lab findings

Answer 50

• least common type of AIHA
• most commonly presents in children with bacterial infections (otitis media) or viral infection (URI, measles, mumps, chickenpox, mono)
• originally described in patients with congenital or tertiary syphilis
• presents with paroxysmal episodes of hemoglobinuria provoked by cold exposure, sudden fever, chills, abdominal and back pain, jaundice (hgb < 5)
• treatment = keep patient warm and transfuse as necessary
• Caused by IgG biphasic hemolysin with anti P specificity (Donath-Landsteiner antibody)
  
  • in vitro hemolysis only when incubated at 2 different temperatures
  • DAT positive with polyspecific AHG, negative with anti IgG, and positive with C3
  • confirm diagnosis with Donath-Landsteiner (DL test)
    
    • direct DL test performed on adults with 2 vials of blood at 4 and 37 degrees
      
      • positive test if only incubation of patient’s red cells at 4 degrees then 37 degrees leads to hemolysis
    • indirect DL test uses patient plasma with reagent red cells

Question 51

How to find masked alloantibodies when cold autoantibody is present

Answer 51

• keep blood at 37 deegres and perform all tests at that temperature
• use monospecific IgG AHG reagent
• consider cold autoantibody adsorption with autologous red cells

Question 52

Mechanisms of drug induced positive DAT

Answer 52

1. drug adsorption (hapten) mechanism
   
   • noncovalent coating of RBCs
   • penicillin
   • extravascular hemolysis
   • dose dependent
2. drug dependent antibody mechanism
   
   • drug absorbed to red cell membrane and becomes coated with Ab that activates complement
   • intravascular hemolysis, renal failure
   • takes small dose to initiate, but upon reexposure brisk hemolysis occurs
   • piperacillin and cephalosporins
   • lab confirmation with positive DAT with C3 and possibly IgG
3. autoimmune induction mechanism
   
   • drug induces red cell autoimmunity that persists after withdrawal of drug
   • methyldopa, levodopa, procainamide, fludarabine, 2nd and 3rd gen cephalosporins
   • drug stimulates formation of autoantibody directed against innate component red cell membrane
   • in the lab it is indistinguishable from those that cause idiopathic warm autoimmune HA
4. nonimmune protein adsorption mechanism
   
   • drug induced nonspecific, nonimmune, binding of Ig to red cell surface
   • cephalothin
   • meds cause nonspecific adsorption of albumin, IgG, IgA, IgM, and complement to RBC
   • no antibody production
   • antibody screens and DATs positive, but eluates are negative even when drug coated RBCs are used