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Tech Tips: Titration - serological and clinical applications

A Blog from Eric Ching:
 
Brief Overview

Please read the titration procedure in the AABB Technical Manual if you are not familiar with it.

Titration is semiquantitative at best because there are many variables in the procedure:
  1. Drop size can be influenced by:
    • angle of pipette
    • bore size variation of disposable pipettes
    • viscosity of plasma if disposable glass pipettes are still being used (may be still the case in developing countries)!
  2. Variations of indicator cells:
    • dosage
    • age
    • % cell suspension (2% vs 4%)
  3. Subjectivity of end point:
    • incubation time and temperature
    • degree of agitation before reading
    • macroscopic agglutination (not everyone has 20/20 vision!)
In order to minimize variation and to prevent carryover, a master dilution can be prepared using a calibrated electronic pipetter or disposable precision pipette tips. Automation can potentially increase precision.

Serial dilutions

Plasma/saline diluent ratio: 2 fold: 1/1,/1/2,1/4,1/8 ….1/512 or 10 fold:1/1,1/10,/1/100…

A two fold serial dilution is generally used.

For titrating a strong cold agglutinin, I prefer doing a ten fold serial dilutions:
  1. Using a precision pipette, pipette 0.1 ml of sample into a 100 ml measuring cylinder, qs to 50 ml and mix thoroughly.
  2. Set up a master dilution using 1 ml saline as diluent. 1/500, 1/1,000,1/2,000,1/4,000 10 tubes to 1/256,000
A 1+ macroscopic reading in the highest dilution is the end point of titration.

Report: Titre 16 which means patient’s serum or plasma diluted to 1 in 16 (V/V) gives a 1 + macroscopic reading, because titre is defined as the reciprocal of the highest dilution giving a positive test result.

Notes:
  1. 6% BSA or LISS should not be used as diluent even though these reagents are being used for antibody screen. Potentiating media will augment reactivities of both warm reacting and much more on cold reacting cold agglutinins. 
  2. If a glass disposable pipette is used, mark the tip to contain 4 drops and two drops to be consistent.
  3. The higher the transfer volume, the better the reproducibility will be.  I prefer 0.5 ml unless is limited by sample availability.
  4. A 0.5% BSA in saline (v/v) will not affect result but help stabilize antibodies in weaker solutions. (Canadian Red Cross Technical Manual. 7th ed. 1972.)
  5. In the same manual, mixing of diluent and sample must be done at least six times!
  6. Consistency is key to the reliability of titration results:
    • continual staff training on agitation and grading
    • use of indicator cells of the same phenotype etc.
 
Clinical Applications
  1. To monitor prenatal antibody level: in most institutions with prenatal service, a titre 16 or 32 is considered as the indicator to switch to Doppler ultrasonography or amniocentesis to assess severity.
  2. To aid in the diagnosis of cold agglutinin syndrome which is associated with atypical pneumonia and lymphoproliferative disorders.
  3. To determine the level of isohemagglutinins in ABO mismatch bone marrow/progenitor blood cell and organ transplants in neonates.
  4. In autoimmune hemolytic anemias (AIHA), titration can be used to determine various specificities of cold agglutinins in Cold Agglutinin Syndrome or the “relative Rh specificity” in the warm AIHA. Such determinations are usually of academic interests.
 
Serological Applications
  1. To identify, in most cases, the antibody(ies) at higher concentration(s), when present in a mixture:​
    • Titrate patient's plasma using the strongest reactive panel red cells, it is particularly useful when all panel cells are reactive at 4+!
    • Dilute patient's serum accordingly.
    • Perform antibody identification on the diluted serum.
    • Uniform panagglutination indicates a single antibody to high incidence antigen. Distinct reaction pattern may reveal one or more specificities.
    • In case of a mixture of antibodies, the weaker antibodies may be identified by using a panel of red cells lacking the antigen(s) of the stronger antibody(ies) and with appropriate enhancement or use of a reagent which is known to inhibit or destroy certain high incidence antigens. For example, a combination of anti-k and anti-c, ZAAP or DTT can be used to inhibit anti-k to reveal the hidden anti-c.
  2. To detect the presence of the so-called high-titred low avidity (HTLA) antibody such as anti-Bg’s,  -York, -McCoy’s, -JMH etc.
  3. To detect the presence of soluble blood group antigens such as ABH, Lewis and Rogers and Chido in inhibition studies.
  4. To compare antigen expressions using a known antibody for storage stability, dosage determination.
  5. To reveal stronger alloantibody(ies) in the presence of a weaker autoantibody.
  6. To differentiate IgG or IgM or admixture of both in DTT inhibition study.
Interpretations

A.  A prenatal sample showing Anti-D, titre 128:
 
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B.  Neutralization Tests of an antibody showing HTLA characteristics
 
Titre 1 2 4 8 16 32
Ch- plasma W w w w w -
Rg- plasma - - - - - -
Pooled plasma - - - - - -
Saline control W w w w w -
 
Known Chido negative plasma failed to neutralize patient’s plasma antibody, therefore, patient has anti-Childo.  Both Rg- and pooled plasma contain Chido substance, therefore patient’s anti-Chido had been neutralized. Saline Control indicates that patient’s anti-Childo was not diluted enough to cause a negative reaction.

C.  Determination of antibody class in patient serum PS; DTT= 0.1M dithiothreitol
 
Titre 1 2 4 8 16 Interpretation
PS+DTT 3+ 2+ 1+ 1+ 0  
PS+Saline 3+ 2+ 1+ 1+ 0 IgG
PS+DTT 0 0 0 0 0  
PS+Saline 3+ 2+ 1+ 1+ 0 IgM
PS+DTT 2+ 1+ 0 0 0  
PS+Saline 3+ 2+ 1+ 1+ 0 IgG+IgM
 
 
 
 
 
 
 


D.  Inhibition test on saliva to determine secretor statusGroup B serum diluted 1 in 32 as initial dilution
 
Saliva    /     Titre
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A1 secretor
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A2 secretor
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A1 non secretor
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Notes:
  • Monoclonal antiserum can not be used for saliva inhibition test
  • As secretors have less A substance in saliva when compare with A1 secretor
E.  Falsely high titre due to HTLA on indicator cells 
                
Titre 1 2 4 8 16 32 64 128 256
D+ Bga+ 3+ 2+ 1+ 1+ 1+ 1+ 1+ w 0
D+ Bga- 3+ 2+ 1+ 0 0 0 0 0 0

Unnecessary prenatal management can be avoided if one recognizes such interference!
 
F.  Titration of a cold agglutinin showing prozone
Titre 1 2 4 8 16 32 64 128 256
I+ 0 1+ 2+ 2+ 3+ 4+ 4+ 4+ 3+
 
G.  “Relative anti-e “ in warm AIHA
Titre 1 2 4 8 16 32 64 128 256
R1R1 4+ 4+ 4+ 4+ 3+ 2+ 1+ 1+ 0
R2R2 2+ 2+ 1+ 0 0 0 0 0 0
rr 4+ 4+ 4+ 3+ 3+ 2+ 1+ 0 0
 
 
Possible investigations for our curious colleagues or as student projects:
  1. In Warm AIHA, patients are usually given steroid therapy instead of transfusion; clinical management entails repeat reticulocyte counts and hemoglobin determinations to see if patient is responding to treatment in 4-7 days. If patient’s DAT is 4+ through out the initial treatment, I wonder if we can titrate the Anti-IgG to react with patient’s red cells to see if there is any correlation between the titre and retic/HB.
  2. One can purchase a standard anti-D with the antibody concentration as in microgram per ml. Ask regional CBS to provide 10-20 samples of anti-D all at titre 16 to establish a range of anti-D concentrations in ug/ml. Then set up a study to see if the prenatal samples can be quantified using titration, ELAT or Flowcytometry to determine which assay correlates best with the ultrasound technology in prenatal care.
 Your comments are encouraged!!

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