9. Vigorous shaking for resuspension of cells
Resuspend the cells slowly and gently after centrifugation. Each laboratory must
calibrate its equipment at regular intervals
Problem: Hemolysis of red blood cells
Problem: Delayed or weak agglutination
1. Reagents used immediately after removing
The reagent vial must be brought to room temperature prior to starting the test. Anti-D
(IgG) type reacts at 37°C hence at low temperatures it may not react properly
2. The antigen and antibody are not present in
The sample volume and the reagent volume dispensed should be as per the
instructions given in the protocol
3. In case of weak D or partial D cells in slide
Should be confirmed by tube test or Coombs test
Anti-human Globulin (Coombs Reagent)
Sources of Error in Antiglobulin Testing—Coombs
¾ Neutralization of anti-human globulin (AHG)
1. Failure to wash cells adequately to remove all serum/
plasma. Fill tube at least ¾ full of saline for each wash.
Check dispense volume of automated washers.
2. If increased serum volumes are used, routine wash
may be inadequate. Wash additional times or remove
3. Contamination of AHG by extraneous protein. Do
not use finger or hand to cover tube. Contaminated
droppers or wrong reagent dropper can neutralize
4. High concentration of IgG paraproteins in test serum;
protein may remain even after multiple washes.
1. Bound IgG may dissociate from red cells and either
leave too little IgG to detect or may neutralize AHG
2. Agglutination of IgG-coated cells will weaken.
Centrifuge and read immediately.
1. AHG reagent may lose reactivity if frozen. Reagent
may become bacterially contaminated.
2. Excess heat or repeated freezing/thawing may cause
loss of reactivity of test serum.
3. Reagent red cells may lose antigen strength on storage.
Other subtle cell changes may cause loss of reactivity.
1. Overcentrifugation may pack cells so tightly that
agitation required to resuspend cells breaks up
agglutinates. Undercentrifugation may not be
2. Failure to add test serum, enhancement medium or
3. Too heavy a red cell concentration may mask weak
agglutination. Too light suspension may be difficult
4. Improper/insufficient serum: Cell ratios.
1. Rare antibodies may only be detected when polyspecific AHG is used and active complement is
1. Low pH of saline solution can decrease sensitivity.
Optimal saline wash solution for most antibodies is
temperature to retain antibody on cell. Use 37 or 4°C
¾ Cells agglutinated prior to washing
1. If potent agglutinins are present, agglutinates may
not disperse during washing. Observe cells prior to
addition of anti-human globulin (AHG) or use control
tube substituting saline for AHG; reactivity prior to
addition of AHG or in saline control invalidates AHG
1. Dust or dirt in glassware may cause clumping (not
agglutination) of red cells. Fibrin or precipitates in
test serum may similarly produce cell clumps that
1. Overcentrifugation may pack cells so tightly that they
do not easily disperse and appear positive.
2. Centrifugation of test with polyethylene glycol or
positively charged polymers prior to washing may
create clumps that do not disperse.
¾ Cells have positive direct antiglobulin test (DAT)
1. Cells that are positive by DAT will also be positive in
any indirect antiglobulin test.
1. Complement components, primarily C4 may bind
to cells from clots or from CPDA-1 donor segments
during storage at 4°C and occasionally at higher
temperatures. For DATs, use red cells anticoagulated
2. Samples collected in tubes containing silicone gel
may have spurious complement attachment.
3. Complement may attach to cells in specimens
collected from infusion lines used to administer
dextrose-containing solutions. Strongest reactions
are seen when large-bore needles are used to when
sample volume is less than 5 µl.
Anti-human Globulin (AHG or Coombs Reagent)
Problem: False positive results
1. Presence of colloidal silica, which is absorbed by the red
cells when saline is stored in glass bottles
Ensure that all glassware used is clean and dry and properly stored
saline is being used for the test. Use freshly prepared normal saline
2. Red cells may be agglutinated before the washing is carried
Check properly for agglutination before proceeding to the antiglobulin
3. Overcentrifugation causes tight packing of the cells that
cannot be dispersed easily and is mistaken for a positive
Ensure that centrifugation is carried out at the proper speed for the
appropriate time as per the instructions given in the package insert.
Each laboratory must calibrate its equipment at regular intervals
4. Absorption of normal cold antibody and complement onto
the cells during the refrigeration of clotted blood sample
The blood sample should be tested as soon as possible after
collection and should not be stored. For the indirect antiglobulin test,
serum not more than 48 hours old should not be used
5. Use of various drugs and certain disease conditions such
as megaloblastic anemia are known to be associated with
positive direct antiglobulin test
Check the patient’s history for disease conditions like megaloblastic
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