3400 rpm

9. Vigorous shaking for resuspension of cells

after centrifugation

Resuspend the cells slowly and gently after centrifugation. Each laboratory must

calibrate its equipment at regular intervals

Problem: Hemolysis of red blood cells

Possible causes Solutions

1. Use of wet slides and tubes Wet glassware can cause hemolysis of RBC’s. Ensure that only dry glassware is used

for testing

Problem: Delayed or weak agglutination

Possible causes Solutions

1. Reagents used immediately after removing

from the refrigerator

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

optimal concentrations

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

test

Should be confirmed by tube test or Coombs test

Contd...

General Instructions for

Anti-human Globulin (Coombs Reagent)

Sources of Error in Antiglobulin Testing—Coombs

Cells

False Negative Results

¾ Neutralization of anti-human globulin (AHG)

Reagent

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

serum prior to washing.

3. Contamination of AHG by extraneous protein. Do

not use finger or hand to cover tube. Contaminated

droppers or wrong reagent dropper can neutralize

entire bottle of AHG.

4. High concentration of IgG paraproteins in test serum;

protein may remain even after multiple washes.

¾ Interruption in testing

1. Bound IgG may dissociate from red cells and either

leave too little IgG to detect or may neutralize AHG

reagent.

2. Agglutination of IgG-coated cells will weaken.

Centrifuge and read immediately.

¾ Improper reagent storage

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.

¾ Improper procedures

1. Overcentrifugation may pack cells so tightly that

agitation required to resuspend cells breaks up

agglutinates. Undercentrifugation may not be

optimal for agglutination.

2. Failure to add test serum, enhancement medium or

AHG may cause negative test.

3. Too heavy a red cell concentration may mask weak

agglutination. Too light suspension may be difficult

to read.

4. Improper/insufficient serum: Cell ratios.

¾ Complement

1. Rare antibodies may only be detected when polyspecific AHG is used and active complement is

present.

¾ Saline

1. Low pH of saline solution can decrease sensitivity.

Optimal saline wash solution for most antibodies is

pH 7.0 to 7.2.

378 Concise Book of Medical Laboratory Technology: Methods and Interpretations 2. Some antibodies may require saline to be at specific

temperature to retain antibody on cell. Use 37 or 4°C

saline.

False Positive Results

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

reading.

¾ Particles or contaminants

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

mimic agglutination.

¾ Improper Procedures

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.

¾ Complement

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

with EDTA, ACD or CPD.

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

Possible causes Solutions

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

out

Check properly for agglutination before proceeding to the antiglobulin

phase

3. Overcentrifugation causes tight packing of the cells that

cannot be dispersed easily and is mistaken for a positive

control

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

can give false positive results. Anticoagulants have anticomplement activity. Refrigerated ACD blood also gives false

positive results at times

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

anemia

6. In diseases such as pernicious anemia and multiple myelomatosis, autoagglutination takes place (all erythrocytes are

agglutinated non-specifically)