Antiglobulin

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Antiglobulin
6.1 HISTORY AND INTRODUCTION OF ANTI-HUMAN GLOBULIN
The antibody could be of two types: (a) complete or saline agglutinating type, (b) incomplete type, and
it is difficult todetect the incomplete type in saline medium. Coombs et al. observed that, if an antibody
to this particular globulin could be added at an appropriate time and biochemical environment the
agglutination could be effected. Actually they developed this idea from the principle described by Moreschi
in 1908 where it was experimentally shown that rabbit-red cells incubated with a goat anti-rabbit serum
and subsequently washed and when retreated by rabbit antigoat serum show strong agglutination.
In blood group serology, it is very important to detect the incomplete antibody particularly IgG anti-
Rh in cases of HDN and other clinical conditions. It was observed by Coombs, Mourant et al., in 1945
that anti-IgG could effect this agglutination. It was rather a rediscovery.and successful clinical application
of the test originally described by Moreschi as stated above. They applied the test to detect the antibody
coating on red cells in vivo by direct antiglobulin test or direct Coomb's test as in case of HDN and
haemolytic transfusion reaction etc. In the indirect method they exposed the normal red cells deliberately
to a serum suspected of containing antibody and subsequently test further to see whether they have
infact adsorbed the antibody.
Dacie et al., first described at 1957, that antiglobulin might react with complement components viz.
C4 and later on, it was observed that it can react with C3; Polley and Molison further detected that some
antibody could only be detected by anticomplement, fraction Polley described that IGM antibody of
incomplete variety viz. Lewis antibody could be detected by ICTIIAT. But 10M antibody is best detected
by anti-complement fraction of AHG
In view of multifarious utility of AHa, the Coomb's test has attained a status of backbone of
investigative tests connected with blood group serology. An AHG should, therefore, ideally contain
antibody for IGM, IgG, IGA and for anticornplement fraction, it contains mainly C3 and C4 in AHa, it
also contains anti-complement for Cs and C6 and depending on its contents the AHG sera is designated
polyspecific and monospecific.
AHG sera (PolyspecificJBroad spectrum)
Containing antibodies against IgM, IgG and IgA and also anti-complement C3b and C3d, C4d (Table 6.1).
126

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Antiglobulin
127
Table 6.1. Antibody titre in anti-human globulin sera
Antibody
Minimum titre
Preferable titre
anti-IgG
1 : 64
1 : 512
anti-IgM
1 : 4
anti-C3b
1 : 8
1 : 32
anti-Cjd
1 : 8
1 : 16
AHG sera (Monospecific) contains antibody specific only to immunlglobulin Igo, or IgA or IgM
and not a mixture or it contains specific anticomplement fraction. Considering the importance ofCoomb's
test in blood group serology an idea regarding preparation and standardisation ofAHG reagent will give
us a better understanding regarding the performance of the AHG reagent.
6.2 PREPARATION AND STANDARDISATION OF AHG REAGENT
Monoclonal anti-IgG by hydridoma technology does not give satisfactory result but whereas potent anti-
C] monoclonal antibodies gave result oriented performance. So to have satisfactory broad spectrum
AHG reagent still now the old procedure are followed. Animal sources mainly used for the purpose are
rabbits bigger animals such as goat, sheep, horse were also tried. But because of high heteroagglutinin,
very often the AHG reagent produced from them showed prozone phenomenon. And as such rabbits
with low titre heteroagglutinin (1 : 4-8) are commonly used source. The method described by Proom in
1943 is the commonly accepted method for preparation of AHG reagent, and briefly described below.
In this method young rabbits of 8-10 weeks are used as animal source for immunisation. The animals
preferred are with low titre heteroagglutinin as described above.
Rabbits found suitable for the purpose are bled from veins of the ear lobule or directly from heart,
veins of ear lobule are preferred and 15 to 20 ml of blood are collected on alternate day for 4 to 5 times
followed by a weeks rest and bled again. We can collect 75 to 100 ml of serum like this in a period
stretching for 2 to 3 months.
'Procedure
1. 25 ml of serum added to 75 rnl of redistilled water.
2. 90 ml of 10% potash alum is added to above solution adjust pH to 6.5-.
3. Centrifuge at 1000 rpm for one to two minute discard the supernatant and milky white precipitate
is collected.
4. Wash the precipitate twice or thrice with large volume ofsaline containing 1 : 10,000 marthiolate.
Collect the washed precipitate.
5. Resuspend in 100 ml of marthiolate saline..
6. Storage of above mixture is done at 4°C for a maximum period of three weeks.

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Blood Banking and Transfusion
Stage II
After preparation of antigen the next step is preparation of immune serum:
1.. 5 ml of precipitate mixture which was stored at 4°C can be injected. 5 ml of this precipitate can
be injected intramuscularly as a single dose in the buttocks of each rabbit. Some prefers to
inject it in divided doses of 0, 8, 14 and 42 days.
2. In both schedule a repeat dose is given· two weeks after last injection.
3. Booster dose of 1 ml of whole serum or plain serum is administered LV. if necessary.
4~
Animals are bled ten days after last injection and serum collected.
Standardisation
The immune serum thus collected is inactivated by heating at 56°C for at least 30 minutes. Well washed
A, Band 0 cells are now taken and inactivated serum is treated with these cells. Absorption is effected
both at 37°C and 4°C.
The incomplete antibody only sensitises the red cells in saline medium and IgM, IgG l' IgG3 fixes
the complement, actual cell lysis in vivo is mediated through complement C3b andCid. And the autocold
antibody and other blood group specific antibody may belong to any of three immunoglobulin IgM, IgG,
IgA. As such a true polyspecific anti-human globulin antisera should also contain non-gamma type of
antibodies in addition to IgG, C3b, C3d. The IgG antibody should cover all sub-classes though most of
blood group antibodies belong to IgG 1 and IgG 3 but any of subclass may be present and AHG should be
able to detect them. Presence of IgM can be advantageous as it may detect non-agglutinating and non-
complement binding IgM antibodies. Anti-lewis and anti-kidd are complement binding IgM .. Presence
of IGA is preferable but not mandatory, lOA blood group antibody are very rare and if at all present, they
occur in association with IgG of same specificity, so detected along the IgG A polyvalent antisera, thus
.contains IgG, IgM, IgA (preferable) C3b and C3d and standardised accordingly.
AHG antisera is optimally diluted to avoid prozone and read instructions before doing any further
dilution. It should not contain ariti-T, anti-TK and anti-Tn also no heteroagglutinin because it may clump
normal ABO cells.
AHG antisera should be tested fOJ;" anti-lewis, anti-kidd, anti-kell and anti-duffy to see its
capacity to detect them. It shows doubtful specificity or weak specificity with -JKa, -Fy", -5.. -s, -Xga,
-yt, -Vel.
AHG antisera can detect anti-RH and minimum number of Rh 'D' antibody molecules of IgG type
that it is capable to detect ~er
red cells varies between 100 to 150 in numbers, so it is very effective in
detecting Rh 'D' antibody molecule. As such the AHG reagent should also be tested with anti-Rh while
standardisation is being done.
Common source of large scale manufacture ofAHG antisera are rabbit and sheep. Serum of both the
animal are used as source for IgG and anti-Cjb. For complement component, it has been observed that
murine monoclonal produces potent anti-C 3d- lgM .·Ablend ofabove two from different sources produces
a better result oriented AHG reagent which can be preserved for two years at a temperature of 2 to goC.
Any AHG reagent preserved continuously for a long period has to be monitored by ascertaining its
activity from time to time. AHG reagent before use has to be checked for presence of turbidity. Turbidity
is suggestiveofmicrobial contamination or denaturation of protein, such reagents should not be used for
test purpose.

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Antiglobulin
129
TEST
Fig. 6.1 depicts the different stages and principle of the test in brief:
I. Normal red cells.
2. Red cells showing antigenic sites.
3. Sensitised red cells coated with immune antibodies (IgG) and complement. This coating could
occur in vivo or in vitro when red cells are incubated at 37°C alongwith serum suspected to
contain antibody. Incomplete' antibodies usually IgG attaches to cell membrane by FAB part of
antibody molecule but fails to agglutinate and gap between the sensitised cells remains
unabridged, also the electrical charges of the cells repel each other.
4. An AHG molecule.
5. Agglutination is effected by
~ddition
of AHG serum to cell suspension of washed sensitised
cells. Please note that Fab portion of AHG molecule reacts with Fe portion of two adjacent
antibody molecule, So one AHG molecule is connecting the two adjacent antibody molecule,
the gap in between the two antibody is thus bridged and agglutination is effected.
6.3 COOMB'S TEST
Materials and reagents
1. Slide, tube or microtitre plate depending on the method whichever to be employed.
2. Pipettes, micropipettes.
3~
AHG reagent:
(a) Polyspecific reagent detects approximately 500 molecules oflgG per red cells.
(b) Monospecific reagent.
4. Sample of blood in plain vial or EDTA (if samples are to be kept more than 24 hours before test,
this is to prevent the uptake of complement).
5. Binocular or monocular microscope, other optical aid if necessary.
o
1. Red Cell
2. MembranceAntigen on Red Cell
Y...,!
'trd
~i9G
~
Molecule
3. SensitisedRed Cell, ig G Attached
4. Antihuman GlobulinMolecule
5. Agglutination Effected" by AHG
Fig. 6.1. Aggl"utination by AHG.

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Blood Banking and Transfusion
6. Tube racks.
7. Serological water bath.
8. Centrifuge-swing out rotor head.
9. Control cells for AHG test: Positive control, Negative control, Coomb's control cells.
Indication of tests
A. Direct antiglobulin testJDirect Coomb's test: This is indicated in cases where we want to detect
antibody coating of red cells in vivo and they are as follows:
(a) Haemolytic diseases of newborn (HON).
(b) Haemolytic transfusion reaction-immediate post-transfusion sample.
(c) Haemolytic anemia and diagnosis. In acquired haemolytic anemia, it is positive and negative
in congenital haemolytic anemia.
(d) Drug induced red cell sensitisation.·
B. Indirect antiglobulin test/indirect Coomb's test:
(a) To detect incomplete antibodies during pregnancy.
(b) To detect transfusion incompatibility by Coomb's compatibility test.
(c) Antibody identification.
(d) To detect DU antigens (incomplete D antigens).
(e) To detect some other blood group antigen difficult to detect by usual technique. This is
done by using antisera anti-FYA, anti-JK, anti-K, anti-Le, which works alongwith AHG
antisera to detect Lea, K, Ky", Fyb,Jka, Jkb etc.
Indirect test is thus employed to detect the presence ofincomplete antibody and complement
binding antibodies in the serum by treating with the red cells sensitised in vitro.
C. Coomb's consumption test: Fall in titre of antiglobulin suggests consumption of antiglobulin:
(a) Detection of incomplete antibody to leucocyte and platelet.
(b) Determine the presence of contaminating globulin.
(c) Thermodynamic studyantigen antibody reaction.
D. Mixed antiglobulin test: Mainly used for academic and research work to study two different
incomplete-antibody by linking up two types of sensitised cells.
From blood banking ~~1di.
of view the first two tests are considered important, so will be discussed
in details and the other two will be briefly touched in the para to follow.
The tests can be done by tile method, tube method, i.e., spin technique, microtitre plate, automatic
analyser.
nle method
Four drops of antibody containing senim with one drop of 20% cell suspension in saline is mixed, so
that serum cell ratio is lower and in this case is 20 : 1. After cells are sensitised, the AHG antisera are
added. This is done in ICTIIAT.

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Antiglobulin
131
In OCT by tile method
5 to 10% washed red cells are used. Two drops of suspension are placed adjacent to each other on a
translucent opal glass tile. To one add a drop of saline and to the other a drop of diluted AHG antisera.
Results are read after 5 minutes,Agglutination indicates positive result whereas no agglutination indicates
negative result.
The tile methods are not used now-a-days for being less sensitive than spin tube technique.
Microtitre plate technique
U-shaped wells are advocated for antihuman globulin test. Potential disadvantages (i) pretreatment of
plate to overcome the problem of static electricity, plates are treated with albumin, (ii) to enable
centrifugation, wire slings are to be made and fix to the centrifuge so that plates can be made to spun.
Autoanalyser
It has many advantages viz. agglutination reaction can beenhanced by using antisera augmented by K90
PVP and sensitivity of anti-Rh, anti-Fy etc. are greatly increased at least more than 100 times than
manual test. But because of high cost and sophisticated technology, it can not be afforded by many
laboratory.
Commonly accepted technique is spin tube technique and is described in detail in the following
paragraph.
For this purpose we are to useall glass round bottom tube, tube rack. AHG antisera (already discussed
how to prepare) and control cells.
.
Control cells
We require both positive control and negative control and also Coomb's control cells positive control is
prepared by sensitising the ORR +ve red cells. Take four drops of weak incomplete anti-D' sera titre less
than 1 : 16 in a test tube and two drops twice or thrice. washed known ORH +ve cells and incubate at
37°C for fifteen minutes, wash again 3 to 4 times and reconstitute to make saline suspension of cells.
Negative control
1. Sensitised ORH D negative cells can be used as negative control.
2. 4 drops of unsensitised ORlI '0' +ve cells washed thrice or four times and reconstituted to make
saline suspension of cells.
Coomb's control cells
This cells are added to all non-reactive Coomb's tests to know the validity of tests performed :
1. Make a weak incomplete anti-'D' by diluting anti-'D' reagent I : 20 in isotonic saline. It, of
course depends on the titre of antibody as indicated by manufacturer.
2. Make 5% saline suspension of ORH +ve cells in isotonic saline.
3. Equalvolumes of 1 and 2 above are taken in a test tube and incubated at 37°C for quarter of an
hour. Look for agglutination and if positive, repeat procedure by making more dilution of anti-
'D' reagent, tilJ there is no agglutination microscopically.

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Blood Banking and Transfusion
4. Wash thoroughly with isotonic saline and decant and in final wash i.e, third wash decant as
completely as possible.
5. Reconstitute to make 5% saline suspension in isotonic. saline.
In case of a non-reactive AHG test, a drop of Coomb's control cell is added and a positive reaction,
i.e., agglutination indicates :
(a) The activity of AHG reagent and that it is not neutralised.
(b) Proper washing of red cells has been done and it is not a causal washing.
(c) That the AHG antisera were actually ~dde~
during test procedure.
(d) Final volume of saline did not dilute the AHG serum.
In case of no agglutination after addition of Coomb's control cells to .a non-reactive Coomb's test
indicates that the test is not valid and the process has to be repeated again.
Procedure for direct antiglobulin test/OCT
1. Take two drops of 5% cell suspension in saline in'a clean all glass round bottomed tube 10 x 75
mm tube.
2. Wash at least thrice in large volume of isotonic saline after each wash supernatant is removed.
In final wash remove supernatant as completely as possible.
3. Add two drops of broad spectrum AHG antisera immediately and resuspend the cell button in
serum by gently tapping the bottom of the tube.
4. Centrifuge for 3400 r.p.m for fifteen seconds.
5. Tap the tube gently to dislodge cell button.
6. Examine for agglutination. Take help of optical aids if necessary. Weak agglutination to be
confirmed under microscope.
7.. If no agglutination is observed leave the test tube at room temperature for five minutes, tills
sometimes becomes necessary to allow optimal sensitivity. for IgG and .complement. But
immediate reading is essential as in (6) above.
8. Run control with each test.
9. Coomb's control cell two volume is added to any non-reactive test and centrifuge for 15 seconds
at 3400 r.p.m. If agglutination is observed, test is valid and if no agglutination test is invalid and
the whole procedure is to be repeated.
Inference
1. Agglutination post spin as in (6) above or·post R.T. incubation indicates positive test.
2. No agglutination in either phases and confirmed under microscope and validity ascertained by
addition of Coomb's control indicates negative test.
Indirect antiglobulin test (IAT/ICT) Procedure
(a) Take two to four drops of test serum in a clean labelled 10 x 75 mm round bottomed all glass
test tube. Fresh test serum should be taken, if not, then add fresh neutral serum orAls serum.

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Antiglobulin
133
(b) Add one to two drops (if two drops of serum then add one drop cell and if four drops serum,
then add two drops of cell) of 3% suspension of washed red cells (screening cells are '0' Rh +v~.
cells or it may be donor cells or patients cells as the case may be).
(c) Mix and incubate at 37~C
for 69 minutes, look for agglutination.
(d) Centrifuge at 3400 r.p.m. for 15 seconds.
(e) Look for agglutination and agglutination indicates presence of complete antibody. If no
agglutination proceed for next step.
(0 Wash with clean and sterile isotonic saline, the cells for at least thrice. Supernatant removed
after every wash and in final wash remove supernatant as completely as possible.
(g) Add one to two drops of polyspecific AHG serum and resuspend the cell button in serum by
tapping the bottom of tube gently.
(h) Centrifuge at 3400 r.p.rn. for 1.5 seconds.
(i) Shake the tube gently to dislodge cell button and look for agglutination. Confirm the observation
under microscope or with optical aids.
(j) If no agglutination is observed, then leave the test at room temperature for five minutes.
(k) Run control (both positive and negative) withthe test also auto-control is advocated with indirect
Coomb's test.
.
(1) Add one dropof Coomb's control cells with each non-reactive test and centrifuge at 3400 r.p.m.
for 15 seconds. Agglutination indicates a valid test and no agglutination indicates that .test is
invalid and whose process it to be repeated.
Inference
1.. It is considered positive if agglutination is observed post-spin (after addition of AHG antisera)
or following incubation at room temperature.
2. No agglutination at either phase and is confirmed under microscope is considered .negative,
Coomb's control cells are added to confirm validity of test.
Sensitivity of indirect antiglobulin. test could be enhanced by addition of albumin which raises the
dielectric constant of medium and was demonstrated by Stroup and Macllroxy. Moore and Mollison
observed that use of Liss medium is more effective in accelerating the uptake of antibody as compared
to albumin.
220/0 BSA-IATnCT
1. One to two drops of test serum.
2..One to two drops of 3% suspension of red cells is added to serum, now add another two drops
of 22% BSA, mix thoroughly,
3.. Next steps are same saline IATdescribed.
LISS IAT/leT
It acts in two ways:
(i) Accelerating the uptake of antibody.

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Blood Banking and Transfusion
(ii) Reducing the incubation period:
(a) Screen red cells 'a' +ve are washed twice in isotonic saline and followed by another wash
in low ionic salt strength solution (LISS).
(b) Equal volume of test serum and 3% red cell suspension in Lissis then mixed..!t isemphasised
again that equal volumes of each are to be only taken for a result oriented performance.
(c) Incubate at 37°C for 15 minutes.
(d) Next step same as saline lAT.
GEL TECHNOLOGY DATIDCT
Recent advancement in both direct and iridirectAHG isgel technology in a card with 6 (six) microtubules
each of these containing polyspecific AHG and monoclonal anti-Cjd within gel matrix are supplied by
manufacturer with following additional reagents :
(a) Diluent - a modified LISS for red cell suspension.
(b) Test cell reagents.
Procedure requires following essentials to be observed :
(a) Identify the card with the patients or donors name and registration number.
(b) The cards should not be damaged or seals broken, the microtubules should be free of air bubbles.
(c) Dispenser, pipettor, tips, suspension tubes incubator 37°C, centrifuge for cards of 6 or its
multiples, viz. 12, 24.
(d) Sample may be collected in EDTA. Citrate or in plain tubes.
(e) Red cell suspension is prepared by putting 10JlI of packed red cell in 1ml of diluent containing
LISS, then mix gently - 0.8% cell suspension is thus made.
(0 Bring the card out and let it attain room temperature.
The test for DATIDCT can now be started:
(i) Identify the appropriate microtubules with the respective patient or donor name and registration
number,
(ii) Break the seal by removing aluminium foil as per requirement and rest of microtubules can be
left with seal intact for future use.
(iii) Pipette 50 J.11 of 0.8% red cell suspension into the appropriate microtubules,
(iv) Centrifuge the card for ten minutes in the card centrifuge.
(v) Read and interpret the result: Negative indicates absence of detectable IgG and anti-c.d on the
red cell The positive indicates red cells are sensitised and coated.
Enzyme IAT
Enzymes acts by :
(i) reducing electric charges on cell surface.
(ii) cell surface configuration gets altered by enzyme action.
(iii) enhances the mobility of antigen. Enzyme method was described by Unger in 1951 and Van der
Hart and Longman in 1953. It is suitable for anti-JKa and Lea antigen.

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Antiglobulin
135
Enzyme commonly in use is papain cystein to avoid complicacy of procedure involved with other
enzymes:
(a) Take one to two drops of test serum.
(b) Add one to two drops of 3% cell suspension, serum cell mixture is mixed thoroughly, and one
drop' of papain cystein solution is added.
(c) Incubation is 30 minutes at 37°C.
(d) Next steps same as saline lAT.
All procedures described are one stage method of IATbut difficulty was faced with serum stored for
considerable period. at 4°C or stored at -20°C for several weeks. Because serum in such cases may
become anticomplementary. In such cases two stage IATIICT described by Polley and Mollison in 1961
gives' satisfactory result.
Two stages IATnCT
1. Take test serum and treat it with EDTA @ 4 mg per ml.
2. Take one drop of above.
3. One drop of 3% red cell suspension is now added to above and mixed.
4. Incubate serum cell mixture at 37°C for 5 minutes.
5. Cells are now washed at least thrice with isotonic saline. Supernatant removed after every
wash. In final wash remove supernatant as completely as possible.
6. One drop of fresh normal AB serum is added.
7. Again incubate at 37°C for five minutes.
8. Centrifuge at 3400 r.p.rn for 15 seconds, observe for haemolysis.
9. Cell are again washed at least thrice with isotonic saline supernatant removed after every wash
and in final wash remove supernatant as completely as possible.
10. Now add two drops of anticomplement globulin serum or polyspecific serum.
11. Centrifuge at 3400 r.p.m. for 15 seconds.
12. Look for agglutination and confirm under microscope. If no agglutination is observed incubate
for 5 minutes at R.T. and again look for agglutination.
13. Run control (both positive and negative) throughout the procedure.
14. Validity of test is to be ascertained as described earlier.
Coombts consumption test
In this test sensitised cells are mixed with AHG reagent and later on after the test content of antiglobulin
is compared to its initial content prior to addition of cells. The fall in titre shows the amount ofantiglobulin
consumed were due to the antibody coated cells added during test procedure.
Rh sensitised cells are used in this test for detection incomplete antibody to leucocyte and platelet.
The antiglobulin reagent is treated with sensitised cells and fall in titre of AHG is calculated at the end
of test. The fall in titre suggests that during test procedure the AHG reagent has been consumed by the
sensitised cells (lucocytes and platelets) indicates the presence of incomplete antibody.
Similarly contaminated protein can be detected by above method, e.g. say yg is mixed with ym; to
determine yg we can employ the above method.

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Blood Banking and Transfusion
Gamma globulin neutralisation test: In this purified gamma globulin is mixed with AHG antisera,
this will neutralise anti-IgG. Now antiglobulin test, when carried out, if fails to show positive reaction,
indicates that antibody present is non-complement binding incomplete type IgG.
Mixed antiglobulin test
In this test the antiglobulin reagent is used for linking sensitised cell that has absorbed two different
antibody of incomplete type. White cells after treatment with serum suspected to contain incomplete
antibody is mixed with anti-D' Rh sensitised red cells in presence of AHG reagentPositive result is
confirmed under microscope, shows both white cells and red cells.
The mixed antiglobulintest and gamma globulin neutralisation test are not much clinically significant
and more required in research work.
6.4 FACTORS INFLUENCING THE TEST AND SOURCE OF ERRORS
The sensitivity of the test can be affected by following factors:
1. Temperature :Optimal temperature is 37°C lower temperature will reduce the uptake and higher
'temperature may damage the antibody. Higher temperature may also damage the antigen.
2. Suspending 111ediu111 : As has been stated earlier the sensitivity of indirect antiglobulin test
increases with addition of 22% BSA, enzyme and Liss.
3. Preparation ofserum to cell: Serum cell ratio is directly proportional to antibody coating ofRh
cells.
If the serum to red cell ratio is increased, the antibody coating over red cell increases. The
commonly used ratio is one drop of 2-5% cell suspension is used with two drops of serum;
whereas, in Liss medium, equal volume of serum and cells are to be mixed.
4. Incubation time : It is from the accumulated experience of various workers that an incubation
time of30 to 40 minutes is sufficient to detect clinically significant antibody, but weaker antibody
may require time up to 60 minutes for detection. This incubation time is dependent on various
other factors, some of which are given below:
(i) A study of antibody uptake by red cell shows that at the beginning, there is high association
and low dissociation rate and with passage of time, the point of equilibrium is reached
followed by less uptake whether it is due to reversal of initial process or due to break down
of Ag-Ab cornplex is yet to be established. However, reading at equilibrium point is
advocated as ideal.
(ii) Suspending medium also influences the incubation time, e.g., Liss medium requires 10 to
15 minutes incubation time.
(iii) Serum to cell ratio for 1 drop of 2 to 50/0 cell suspension requires two drops of serum to
exhibit reaction within-30 to 40 minutes provided all other conditions are within normal
limir In liss medium serum to cell ratio is equal to each other arid produces reaction within
10 tc, 15 minutes.
(iv) Various workers have found that at least 500 molecules of IgG should be present to get
detected by AHG within the advocated incubation period. In some cases of AIHA the IGg
molecules are found to be less than 500.

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137
(v) Prolonged incubations more than 60 minutes does not have any added advantage since
with the passage of time and after the equilibrium point is reached, there is a gradual fall in
antibody uptake.
(vi) Again, it depends on type of antibody, we are dealing with, e.g., in antibody detection
various type ofantibody are involved, where problems are usually faced. But for all practical
purposes, the time of detection for most of clinically significant antibody at recommended
temperature is set at 30 to 40 minutes in saline, medium and 10 to 15 minutes in Liss
medium. For some weaker antibody allowance may be given upto 60 minutes.
5. Washing ofcells: Cell washing is a very important step in AHG test. There are many globulins
and complement molecules present in human serum which, if remains attached to the cells may
interfere with proper functioning of AHG reagent. By washing successively with isotonic saline
this unbound protein molecules are removed and after final wash the cell button at the bottom
of tube should preferably be dry. Automaticcellwashers have replaced manual washing which
definitely serves the purpose more efficientlyas compared to manual washing.
SOURCE OF ERROR
A. False positive result
1. External factors :
(i) Contamination with dust, detergent etc. may cause agglutination of cells, and if this is not
noticed may be interpreted as positive antiglobulin test. Proper washing of all glassware is
the" remedy.
.
"
"
(ii) Saline may contain silica and other panicles which may also give false positive reaction.
(iii) Wharton Jelly in cord blood may cause false positive reaction.
(iv) Blood sample collected from a tube through which 5 to 10% dextrose is administered.
(v) Over centrifugation and failure to disperse the cell button.
(vi) Long storage of blood (sample clotted) at recommended temperature for preservation may
result in absorption of cold antibody and complement on to cells, thus clotted blood sample
may give false positive result. So clotted sample should be used immediately and for storage
purpose, the sample c~llected
in EDTA is recommended.
2. Internal factors :
(i) Heteroagglutinin, if present in AHG reagent may cause clumping of A, B, 0 cells may be
falsely interpreted as positive reaction.
(ii) AHG reagent should not contain anti-T, anti-TK and anti-Tn which may cause T activation.
(iii) AHG reagent should be free from anti-transferrin, reticulocytes are reach in transferrin.
Anemia patient with high reticount may be reported positive in DCT, if AHG reagent
contains anti-transferrin,
(iv) AHG reagent should be free from agglutinins against enzyme treated red cells which was
observed by Beck et aI., in 1971 in commercial antiglobulin reagent. False positive reaction
may be exhibited when enzyme treated red cells are used with AHG reagent containing
antibodies against the enzyme treated cells.

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138
Blood Banking and Transfusion
B~ False negative result
1. External factors :
(i) Cells are not washed properly.or improper storage oftest cell may result in loss of sensitivity.
(ii) Under centrifugation and inaccurate temperature due to faulty waterbath or incubator.
(iii) Too heavy red cell suspension to permit proper antibody coating.
(iv) Serum to cell ratio may not be proportionate.
(v) Incorrect temperature and incubation time.
(vi) Ca'" and Mg'" may be chelated in stored plasma and may not react with complement
binding antibody.
2. Internal factors :
(i) Omission of AHG reagent in test.
(ii) AHG reagent contaminated with human globulin.
(iii) Addition of AHG reagent, if not done in immediate succession after washing, similarly,
the 'centrifugation has to bequickly followed after addition ofAHG
(iv) AHG reagent should be able .to detect at least 150 to 500 molecules of IgG for exhibiting
the reaction.
(v) Prozone in AHG serum.
(vi) AHG reagent should be stored between 2°C to SOC and not frozen.
fmmunoconglutlnin, conglutinin and AttG reagent
Conglutinin - A P-globulin having mol. wt. of 75,00,00 described by Lachman and Coombs in 1965 and
was discovered by Bordet and Streng in 1909. They observed clumping of red cell sensitised with
antibody and complement. On treatment with bovine serum. The clumping they called conglutination
and the substance present in Bovine serum as conglutinin subsequently in 1930, Streng produced an
antisera which, he preferred to name Immunoconglutinin (l-K). Heproduced this immunoconglutinin,
which is an immunoglobin by immunising rabbits with human complement coated cells for bacteria.
This IgM immunoglobulin that reacts with activated complement is available in traces in normal serum,
but has been observed at a higher titre in patients of rheumatic arthritis. Hashimoto's disease, malaria
and kalaazar.
Since I-K and conglutinin sera does not act on native C3 and.C4 and acts on activated complement,
component irrespective of species barrier, it was found advantageous over AHG reagent respectively in
(a) test with unwashed cells, (b) they can even detect complement activity when the source of complement
in AHG is from animal. Preparing of species specific reagent is not required. Molison and Polley in
1960 observed that l-K serum are less sensitive then the AHG antisera. AHG reagent, considered as
backbone of serological investigations in blood bank, which helpsto detect majority of antibodies,
chance with a less sensitive reagent is not recommended to be taken even though, it is advantageous
over AHG So the use of 1-K serum in place ofAHG was never encouraged and AHG reagent is still the
sheet anchor of serological investigations related to blood bank.

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139
Books recommended for further reading
1. A Handbook ofMedical Laboratory Technology, l{H. Talib, WHO sponsored, C.B.S. Publication, 1990.
2. BloodTransfusion in Clinical Medicine, PoL. Mollison, 7th ed., 1983,Blackwell Scientific Publication:
3. Procedure in BloodBanking and Immunohaematology, H.M. Bhatia, 1977.
4. Technique in Blood Banking, H.M. Bhatia (I.e.M.R.), Seth as. Medical College, Mllmbai.
5. Transfusion Medicine Technical Manual, R.K. Saran and R.N. Makroo, 1991,DGHS(Ministry ofHealth
and Family Welfare), sponsored by WHO.
6. Introduction to Transfusion Met/,icine, Bharucha and Chauhan, 1990, D.K. 'Publishers, Mumbai.