ABO DISCREPANCIES

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ABO DISCREPENCIES

• Dr. BIPIN B PALLATH
• 06/09/2016

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FORWARD BLOOD GROUPING

• Can be done by Slide method, tile method, or tube
  method
• Slide or tile method is usually used in case of
  emergencies or in camps
• The most accepted method for routine grouping is
  tube method

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• Steps of tube method
• Get the sample, see for any blood lysis
• Separate Cell and Serum
• Wash the Cell 3 times with normal saline and
  clear the supernatant away.
• Make 5 cell suspension by taking 19 drops of
  normal saline and 1 drop of red cell.

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• 5. Take 4 test tubes and mark as Anti A, Anti B ,
  Anti AB. And Control
• 6 Add one drop of corresponding antisera to the
  test tubes and Normal saline to control
• 7.Add one drop of 5 Red cell suspension to the
  above test tubes

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• 8. Mix it gently and wait for 10 min, and
  centrifuge at 1000 rpm for 1 min
• 9. Then look for agglutination. Free cell
  suspension indicates negative results.
• 10. Negative cell results should be conformed
  with microscope.

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SERUM GROUPINGReverse Grouping

• A, B, O cell reagents are prepared by pooling
  three sample of each group
• Wash each with normal saline for 3 times and make
  5 solution of each by adding 19 drops of saline
  and 1 drop of red cell

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• Procedure
• Take 3 test tubes and mark A, B, O
• Place 2 drops of test serum to each test tubes
• Put one drop of A, B, O cells into corresponding
  test tubes
• Mix the test tubes and keep it in room
  temperature for 10min

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• 5. Centrifuge at 1000 rpm for 1 min
• Examine the tubes for haemolysis or agglutination
  which indicates positive results

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Grading of results

• One solid aggregate - 4
• Several large aggregate of cells - 3
• Medium sized aggregates of cells - 2
• Small aggregates in reddish back ground - 1
• Microscopic aggregates only - W
• No aggregate - 0

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DISCREPANCY

• A discrepancy occurs when the red cell testing
  does NOT match the serum testing results
• In other words, the forward does NOT match the
  reverse

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MAY BE BECAUSE

• Reaction strengths could be weaker than expected
• Some reactions may be missing in the reverse or
  forward typing
• Extra reactions may occur

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THINGS TO DO

• Identify the problem
• Most of the time, the problem is technical
• Mislabeled tube
• Failure to add reagent
• Either repeat test on same sample, request a new
  sample, or wash cells
• Some times, there is a real discrepancy due to
  problems with the patients red cells or serum

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• ERRORS

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Technical Errors

• Clerical errors
• Mislabeled tubes
• Patient misidentification
• Inaccurate interpretations recorded
• Transcription error
• Computer entry error

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• Reagent or equipment problems
• Using expired reagents
• Using an uncalibrated centrifuge
• Contaminated or hemolysed reagents
• Incorrect storage temperatures

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• Procedural errors
• Reagents not added
• Manufacturers directions not followed
• RBC suspensions incorrect concentration
• Cell buttons not resuspended before grading
  agglutination

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CLOTTING DEFICIENCIES

• Serum that does not clot may be due to
• Low platelet counts
• Anticoagulant therapy (Heparin, Aspirin, etc)
• Factor deficiencies

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• Serum that does not clot completely before
  testing is prone to developing fibrin clots that
  may mimic agglutination
• Thrombin can be added to serum to activate clot
  formation
• Tubes containing EDTA can be used

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Contaminated samples or reagents

• Sample contamination
• Microbial growth in tube
• Reagent contamination
• Bacterial growth causes cloudy or discolored
  appearance, so do not use if you see this.
• Reagents contaminated with other reagents (dont
  touch side of tube when dispensing)

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Equipment problems

• Routine maintenance should be performed on a
  regular basis (daily, weekly, etc)
• Keep instruments like centrifuges, thermometers,
  and timers calibrated
• Uncalibrated centrifuges can cause false results

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Hemolysis

• Detected in serum after centrifugation (red)
• Important if not documented
• Can result from
• Complement binding
• Anti-A, anti-B, anti-H, and anti-Lea
• Bacterial contamination

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Patient Problems

• a) Failure to express ABO antigen
• 1) Age (New born and Old age)
• 2) Disease state Leukaemia, Lymphoma
• b) Aquired B antigen can occur in
• 1) Infection ( Gram negative septicemia)
• 2) Carcinoma of colon

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• c) Blood group Chimera
• 1) Artificial Chimera occurs in
• Bone marrow transplantation
• Transfusion of group O blood to A
  and B recipient
• 2) Permanent Chimera
• Exchange of blood in utero due to vascular
  anastomosis
• Due to dispermy. When two sperms fertilize one egg

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• d) Rouleaux formation
• Occurs when albumin/globulin ration is
  abnormal or occurs in cord blood samples due to
  whartons jelly contamination

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• e ) Acquired antibodies
• Anti A1 in A2 persons
• Anti H in Bombay phenotype
• Cold autoantibodies
• Unexpected alloantibodies

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• f) Absence of weakening of antibodies eg.
• Immunodeficiency state
• Agammaglobulinemia
• g) Excessive A or B substance

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GROUP I Discrepancies

• Unexpected reactions in the reverse grouping due
  to weakly reacting or missing antibodies
• The reason for missing or weak isoglutinins is
  that the patient has depressed antibody
  production or cannot produce the ABO antibodies

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• Newborns
• Elderly patients
• Patients with leukemias
• Patients using immunosuppressive drugs
• Patients with bone marrow transplantations
• Patients whose existing ABO antibodies may have
  been diluted by plasma transfusion or exchange
• ABO subgroups

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Resolution of Common Group I Discrepancies

• The best way to resolve this discrepancy is to
  enhance the weak or missing reaction in the serum
  by incubating the patient serum with reagent A1
  and B cells at room temperature for approximately
  15 to 30 minutes.

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• If there is still no reaction after
  centrifugation, the serum-cell mixtures can be
  incubated at 4 C for 15 to 30 minutes.

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• An auto control and O cell control must always be
  tested concurrently with the reverse typing when
  trying to solve the discrepancy, because the
  lower temperature of testing will most likely
  enhance the reactivity of other commonly
  occurring cold agglutinins

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Rare Group I Discrepancies

• Chimerism the presence of two cell populations
  in a single individual.

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• True chimerism is rarely found and occurs in
  twins, in utero exchange of blood occurs because
  of vascular anastomosis, two cell populations
  emerge, both of which are recognized as self, and
  the individuals do not make anti-A or anti-B .
  Therefore, no detectable isoagglutinins are
  present in the reverse grouping

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• If the patient or donor has no history of a twin,
  then the chimera may be due to dispermy (two
  sperm fertilizing one egg) and indicates
  mosaicism
• Blood transfusions (e.g., group O cells given to
  an A or B patient)
• Transplanted bone marrows or peripheral blood
  stem cells
• Exchange transfusions
• Fetal-maternal bleeding

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Group II Discrepancies

• Group II discrepancies are associated with
  unexpected reactions in the forward grouping due
  to weakly reacting or missing antigens

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• Some of the causes of discrepancies in this group
  include
• Subgroups of A (or B) may be present
• Leukemias may yield weakened A or B antigens
• Hodgkins disease has been reported in some cases
  to mimic the depression of antigens found in
  leukemia
• Acquired B phenomenon is most often associated
  with diseases of the digestive tract

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Resolution of Group II Discrepancies

• Enhanced by incubating the test mixture at room
  temperature for up to 30 minute
• If negative, incubate the text mixture at 4C for
  15 to 30 minutes.
• Include group O and autologous cells as controls.
  RBCs can also be pretreated with enzymes and
  retested with reagent antisera.

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• The acquired B antigen arises when bacterial
  enzymes modify the immunodominant blood group A
  sugar (N-acetylD-galactosamine) into
  D-galactosamine, which is suf?ciently similar to
  the immunodominant blood group B sugar (D
  galactose) to cross-react with anti-B antisera

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Acquired B Phenotype
Group A individual
N-acetyl galactosamine
D Galactosamine now resembles D-galactose
(found in Group B)
Bacterial enzyme removes acetyl group
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• Limited mainly to Group A1 individuals with
• Lower GI tract disease
• Cancer of colon/rectum
• Intestinal obstruction
• Gram negative septicemia (i.e. E. coli)

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• these acquired antigens demonstrates a weak
  reaction, often yielding a mixed-?eld appearance.
  
• Blood group reagents of a monoclonal anti-B
  clone (ES4) strongly agglutinate cells with the
  acquired B antigen.

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• Testing the patients serum or plasma against
  autologous RBCs gives a negative reaction because
  the anti-B in the serum does not agglutinate
  autologous RBCs with the acquired-B antigen

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Group III Discrepancies

• These discrepancies are between forward and
  reverse groupings caused by protein or plasma
  abnormalities and result in rouleaux formation or
  pseudo agglutination

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May be due to

• Elevated levels of globulin
• Elevated levels of ?brinogen
• Plasma expanders, such as dextran and
  polyvinylpyrrolidone
• Whartons jelly in cord blood sample

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Resolution of Group III Discrepancies

• washing the patients RBCs several times with
  saline. In true agglutination, RBC clumping will
  still remain after the addition of saline.

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• Washing cord cells six to eight times with saline
  should alleviate spontaneous rouleaux due to
  Whartons jelly, a viscous mucopolysaccharide
  material present on cord blood cells, and should
  result in an accurate RBC grouping.

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Group IV Discrepancies

• These discrepancies between forward and reverse
  groupings are due to miscellaneous problems and
  have the following causes
• ? Cold reactive autoantibodies in which RBCs are
  so heavily coated with antibody that they
  spontaneously agglutinate, independent of the
  speci?city of the reagent antibody

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• ? Patient has circulating RBCs of more than one
  ABO group due to RBC transfusion or marrow
  transplant
• ? Unexpected ABO isoagglutinins
• ? Unexpected non-ABO alloantibodies

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Resolution of Group IV Discrepancies

• Potent cold autoantibodies can cause spontaneous
  agglutination of the patients cells, may yield a
  positive direct Coombs or antiglobulin test If
  the antibody in the serum reacts with all adult
  cells

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• To resolve this discrepancy, the patients RBCs
  could be incubated at 37 C for a short period,
  then washed with saline at 37 C three times and
  retyped.

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• If this is not successful, the patients RBCs can
  be treated with 0.01 M dithiothreitol (DTT) to
  disperse IgM related agglutination.
• As for the serum, the reagent RBCs and serum can
  be warmed to 37 C, then mixed, tested, and read
  at 37 C

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• If the reverse typing is negative (and a positive
  result was expected), an autoabsorption could be
  performed to remove the autoantibody from the
  serum, and the absorbed serum can then be used to
  repeat the serum typing at room temperature.

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• Unexpected ABO isoagglutinins in the patients
  serum react at room temperature with the
  corresponding antigen present on the reagent
  cells.

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• Examples of this type of ABO discrepancy include
  A2 and A2B individuals who can produce naturally
  occurring anti-A1, or A1 and A1B, individuals who
  may produce naturally occurring anti-H.
• Serum grouping can be repeated using at least
  three examples of A1, A2, B cells, O cells, and
  an autologous control.

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Rare Group IV Discrepancies

• Antibodies other than anti-A and anti-B may react
  to form antigen-antibody complexes that may then
  adsorb onto patients RBCs

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• For eg. Some individuals have antibodies against
  acri?avine, the yellow dye used in some
  commercial anti-B reagents the
  acri?avin-antiacri?avin complex attaches to the
  patients RBCs, causing agglutination in the
  forward type

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• Washing the patients cells three times with
  saline and then retyping them should resolve this
  discrepancy.

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SUMMARY

• Weak/missing red cell reactivity
• ABO subgroup
• Leukemia/malignancy
• Intrauterine fetal transfusion
• Transplantation
• Excessive soluble blood group substance

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• Extra red cell reactivity
• Autoagglutinins / excess protein coating red
  cells
• Unwashed red cells plasma proteins
• Transplantation
• Acquired B antigen
• B(A) phenomenon
• Out-of-group transfusion

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• Mixed-field red cell reactivity
• Recent transfusion
• Transplantation
• Fetomaternal hemorrhage
• Twin or dispermic chimerism

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• Weak/missing serum reactivity
• Age related (lt4-6 months old, elderly)
• ABO subgroup
• Hypogammaglobulinemia
• Transplantation

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• Extra serum reactivity
• Cold autoantibody
• Cold alloantibody
• Excess serum protein
• Transfusion of plasma components
• Transplantation
• Infusion of intravenous immune globulin

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