BLOOD BANK

POST DONATION CARE

• MATERIAL REQUIRED:
•  Sterile Cotton.
•  Adhesive tape.
•  Leaflet for post donation instructions.

PROCEDURE:

•  To prevent adverse reactions like giddiness ask the donor not to get
up from the chair/cot for 5 minutes even if he feels perfectly all right.
•  Observe for another 10 minutes in the refreshment area whilst
having light refreshments.
•  Inspect the venepuncture site before the donor leaves the donor room.
• Apply an adhesive tape only after oozing stops.
• If there is persistent oozing at the site of venepuncute, apply pressure to
stop oozing.
• Then apply adhesive at the site of venepuncture. If oozing still purchase
apply pressure with ice.
• If there is haematoma apply ice gently over the area after 5 minutes.
Inform the donor about the expected change in skin colour.
• If still bleeds consult with doctor.
•  Instruct the donor to drink adequate fluid in the day and avoid
strenuous activities.
•  No smoking for thirty minutes.
•  No driving on that day
• DOCUMENTATION:

•  Give a leaflet of post donation instructions to the donor.

•  Record donor profile in the register.
•  Note any adverse reaction on the donor record.
• If any problem occurs after returning home, inform the Blood
Transfusion Center.
• Give thanks to the donor.
•  Observe the donor is donor for 15 minutes
•  Drink more fluids than usual in the next four hours.
•  Remove bandage after few hours.
 ABO BLOOD GROUPING

• MATERIALS REQUIRED:
• Equipment:
•  Refrigerator to store samples and reagents at 2 – 8 0 C
•  General purpose centrifuge
•  Microscope
• Specimen:
•  Blood samples of donor/ patient.
• Reagents:
•  Anti-A, anti-B, anti-AB anti-sera
•  20% A, B & O pooled cell suspension
•  0.9 % saline
• (* All reagents must be used in accordance with the manufacturer’s instructions.)
• Glassware:
•  Tiles
•  Plastic pipette
•  Glass slides for microscopic reading
• 5.5 Miscellaneous:
•  Disposal bucket with Na hypochlorite
•  Plastic beakers - 2
• 
• ROCEDURE:

• Principle:
• ABO system is the only system in which there is a reciprocal relationship between the
antigens on the red cell and the naturally occurring antibodies in the serum. Routine
grouping of donor and patient must therefore include both cell and serum tests, each
serving as check on the other. The procedure is based on the principle of
agglutination of antigen positive red cells in the presence of antibody directed
towards the antigen.
• Cell testing:
• Separate the cell & serum from blood sample
• Label the tiles with patient & donor number and test
identification
• Prepare 20 % cell suspension for red cells being tested
• Add 2 drops of anti-A, anti-B and anti-AB reagent in the
labelled areas
• Add 1 drop of 20% cell suspension of the red cells to be
tested
• Gently mix the contents
• Examine for agglutination within 2-4minutes
• Interpret & record test results
• Serum testing:
• Add 2 drops of test serum in all labelled areas
• Add 1 drop of 20% pooled A reagent cells to the area labelled
Ac
• Add 1 drop of 20% pooled B reagent cells to the area labelled
Bc
• Add 1 drop of 20% pooled O reagent cells to the area labelled
Oc
• Mix the contents gently and examine for agglutination within
2-4minutes
• Interpret & record test results.
• Results:
•  Depending on presence (+) or absence (-) of agglutination, the test
is described as positive or negative.
•  Observe anti AB as positive control and O cell as negative control.
•  Confirm the cell grouping results with those obtained in serum
grouping and vice versa.
• Interpretation:
•  Agglutination of tested red cells and serum constitute positive test
results. Positive reactions characteristically show 3+ to 4+
agglutination.
•  Absence of agglutination represents negative test result.
•  The interpretation of serum and cell tests for ABO group is as
follows:
 REACTION FOR ABO GROUP SYSTEM
(Forward and Reveres Group).
Cell Grouping Reverse B Group
Anti A Anti B Anti AB A cell B cell
+ - + A - + A
- + + B + - B
+ + + AB _ _ AB
_ _ _ O + + O
• Any discrepancy between cell and serum typing tests
should be resolved before an interpretation is
recorded for the donor/ patient.
• DOCUMENTATION:
• Enter the results of donor /patient grouping in the
grouping register and also record the following details-
•  Date on which the test is run.
•  Name of the reagents used.
•  Lot number of the reagents
•  Initials of the technologist who performed the test.
•  Initials of the Supervisor who verified the result
 CONROL OF ABO & Rh BLOOD
GROUP REAENTS
• MATERIALS REQUIRED
• Equipment:
•  Refrigerator to store samples and reagents at 2-60 C.
•  Table top Centrifuge.
•  Automated Cell Washer/Mcroscope.

• Reagents:
•  Anti-A, Anti-B, Anti-AB, Anti D
•  (Monoclonal and Bioclone) Antisera.
•  Clotted or anticoagulated blood samples of random blood donors.
•  Group A,B and O pooled Cells.
•  0.9% saline.
• Glassware:
•  Serum tubes.
•  Micro tubes.
•  Pasteur pipettes.
•  Glass slides.
• Miscellaneous:
•  Rubber teats.
•  Disposal box.
•  2 plastic beakers.
•  Wooden block.
•  Aluminium racks
• PROCEDURE
• Principle:
• Test for reactivity and specificity is based on the principle of
agglutination of antigen positive red cells in the presence of
antibody directed towards the antigen.
• Quality Control Checks:
• Examine each vial carefully for precipitate, gel formation, turbidity
or change in colour.
• Reactivity and Specificity
•  Add one drop of 3- 5% suspension of the appropriate red cells to
the one drop of antiserum in a micro tube.
•  Mix well and incubate
•  Note the reactions as under:
 RED CELLS FOR TESTING RED CELLS FOR TESTING
POSITIVE REACTORS NATIVE REACTORS

Anti-A Pooled A Cells Pooled B, Pooled O Cells

Anti-B Pooled B Cells Pooled A, Pooled O Cells

Anti-AB, Pooled A Pooled B Cells Pooled O

Cells
Anti-D Bioclone RhD- positive cells (any RhD- negative cells (any
ABO group) ABO group)

Anti-D RhD- positive cells RhD- negative cells (any

Monoclonal (any ABO group) ABO group)
• Results:
• Record presence or absence of precipitate, gel
formation, turbidity or colour change.
• Reactivity and Specificity:
• Centrifuge the Tubes (as per manufacturer's
instruction). Resuspend the red cell button
and examine for agglutination / haemolysis
grade and record test results.
• Interpretation:
• The presence of precipitate, gel formation, turbidity, colour change
indicates that the reagent is contaminated and should not be used.
• The absence of all the above indicates that the reagent is 'clear' and
suitable for use.
• . Reactivity and Specificity:
• Agglutination of specific red cells is a positive reaction and indicates the
reactivity of the corresponding antibody in the reagent. The expected
agglutination reaction for positive test is +3 to +4. The absence of
agglutination / haemolysis is considered to be a negative reaction and
indicates the absence of the corresponding antibody specificity in the
reagent.
• Clear cut negative reactions with the negative reactors rules out the
presence of irregular agglutinins and haemolysis in the reagent.
DOCUMENTATION
• Enter the results in the Blood Group Register in the Red Cell Serology
Laboratory. Enter identification number of the individual donor cells used
for pooling and the reaction strengths. Sign the results as the individual
preparing the pooled cells and testing the reagent.
 Rh D TYPING

• MATERIAL REQUIRED:
• Equipment:
•  Refrigerator to store samples and reagents at 2- 80 C
•  General purpose centrifuge
•  Microscope
• Specimen:
•  Blood samples of donors/ patients
• Reagents:
•  Anti-D Blended
•  Anti-D Monoclonal
•  Rh control reagents (positive & negative)
•  0.9 % saline
• Glassware:

•  Test tubes
•  Plastic pipette
•  Glass slides

• Miscellaneous:

•  Disposal box
•  Glass beakers - 2
•  Racks to hold test tubes
• PROCEDURE:
• Principle:
• Testing with anti-D is necessary to determine if red blood cells possess or
lack Rh D blood group antigen. Presence of agglutination is a positive test
result, which indicates the presence of the D antigen on the red blood cells.
• D Typing:
• Label the tiles with patient & donor number and test identification
• Prepare 20% cell suspension for cells being tested.
• Add 2 drops of anti-D reagents (Monoclonal & Blended) in D1, D2 marked.
• Add 2 drops of positive & negative control reagents in the properly labelled
areas
• Then add 1 drop of 20% patient cell suspension to reagent & control areas
• Mix gently & wait for 2-4 minutes.
• Read, interpret and record the test and control results immediately.
• Results
•  Depending on presence (+) or absence (-) of agglutination, the
test is described as a positive or negative.

• Interpretation:
•  Agglutination in D1 & D2 indicates that the red cell under
investigation is D positive.
•  Absence of agglutination in D1 & D2 indicates D negative at
this point. All negative results must be verified under
microscope.
•  In case of any discrepancy, the result is to be confirmed by ICT
method.
•  The interpretation of Rh D type is as follows:
D1 + Pt`s Cells D2 + Pt`s Cells Positive Control Negative Control Result

+ + + - D+

_ _ + D-

+ _ + _ Conform ICT
• DOCUMENTATION:
• Enter the results of donor /patient grouping in
the grouping register and also record the
following details-
•  Date on which the test is run
•  Name of the reagents used
•  Lot number of the reagents
•  Initials of the technologist who performed the
test.
•  Initials of the Supervisor who verified the result
 ANTIBODY SCREENING
• MATERIALS REQUIRED:
• Equipment:
•  Refrigerator to store samples & reagents at 2-8 0 C.
•  Deep freezer to store enzyme, albumin & AHG
reagent.
•  Table top centrifuge.
•  Automated cell washer.
•  Microscope.
•  Water bath.
• Reagents:
•  Group O pooled cells
•  Enzyme
•  22% Bovine albumin
•  Antihuman globulin reagent
•  IgG sensitized control cells
•  0.9% saline
•  Distilled water
• Specimen:
•  Clotted blood sample of donor
• Glassware:
•  Test tubes
•  Pasteur pipette
•  Glass slides
• Miscellaneous:
•  Disposal box.
•  2 plastic beakers
•  Racks to hold sample tubes.
• PROCEDURE:
• Principle:
• The antibody screen test is used to detect unexpected antibodies. In this test, pooled
O cells are combined with serum under investigation. The addition enhancer medium
enzyme/ albumin helps to promote the interaction of red cells and antibodies
allowing antigen-antibody reactions to occur.
• Antibody screen:
• Label tubes with donor/ patient and test identification. Test tube (number)
• Add 2 drops of serum to each tube.
• Add 1 drop of enzyme to the tube labelled ‘enzyme’.
• To each of the tube labelled ‘saline’, ‘enzyme’ and ‘ICT’, add 1 drop of 5% pooled O
red cell suspension.
• Incubate the tube labelled ‘saline’ for 1hr. at room temperature.
• Place the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’ into the incubator at 370 C for
1hr. e
• After 1 hr, examine the tube labelled ‘saline’ for agglutination.
• Remove the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’
from the incubator and add 1 drop of albumin to the
tube labelled ‘albumin’.
• Again place tubes labelled ‘albumin’ and ‘ICT’ to the
incubator for ½ hr. more.
• After ½ hr. incubation, tubes are from the incubator and
tube labelled ‘albumin’ are examined for agglutination.
• Positive (IGg serum) control and Negative (AB donor
serum) control
• Add pooled cell
• Results:
• 1. Gently re-suspend the red cell button and examine for
agglutination.
• 2. Examine all negative results microscopically.
• 3. Proceed to perform indirect anti-globulin test on tube labelled
‘ICT’.
• 4. Wash the cells 3 times with saline. Decant completely after last
wash.
• 5. Add 2 drops of AHG reagent to the cell button.
• 6. After 6-8 minutes, read and record the results.
• 7. Add 1 drop of IgG sensitized cells to all negative results. This
shows a positive result.
• Interpretation:
• Haemolysis or agglutination in any test may indicate the
presence of an unexpected antibody.
• The absence of agglutination in all tests is a negative test
• After addition of IgG sensitized cells to a negative test, the
presence of agglutination indicates that the AHG added was
capable of reacting and that the negative result is valid.
• DOCUMENTATION:
• o Enter the results of donor unit antibody screen in the donor
grouping register
• o Enter the results of patient unit antibody screen in the
patient grouping register.
• o All records are initialled by the technologist who performed
the test and by the supervisor who verified the result.
 DETECTION OF INCOMPATIBILTY BETWEEN
PATIENT AND DONOR

• MATERIAL REQUIRED:
• Equipment:
•  Refrigerator to store samples at 2-80 C
•  General purpose centrifuge
•  Microscope
• Specimen:
•  Clotted blood sample of donors/patients
•  Blood sample with anticoagulant of donor/patients
•  0.9% saline
• Glassware:
•  Tiles
•  Plastic pipette
•  Glass slides
• Miscellaneous:
•  Disposal bucket with Na hypochlorite
•  2 plastic beakers
•  Racks to hold donor and patient sample tubes
• PROCEDURE:
Principle:
• The cross-match is used to detect unexpected blood group antibodies in
patient’s serum against antigens on donor cells and to detect antibodies in
donor serum against antigens on patient’s cells which are complete antibody
active at room temperature and cause direct agglutination. Positive reaction
in any test indicates incompatibility.
Cross-match:
• Label areas with donor/ patient and test identification
• Prepare 20% cell suspension from donor and patient red cells
• Add 2 drops of patient serum to area of major cross matching
• Add 2 drops of donor serum to area of minor cross matching
• Add 1 drop of 20% donor red cell suspension in area of major cross matching
• Add 1 drop of 20% patient’s red cell suspension in area of minor cross
matching
• Lightly mix the contents and examine for agglutination.
• Read, interpret and record the results
• Interpretation:
•  Hemolysis or agglutination in any test
indicates incompatibility.
•  Absence of hemolysis/ agglutination in all
tests indicates compatibility.
•  Examine all visually negative reactions under
microscope.
• DOCUMENTATION:
•  Enter results in cross-match register and
compatibility report form.
•  All records are initialled by Medical
Technologist (Lab) who performed and
checked the results and signed by Medical
officer.
 ANTIGLOBULIN CROSS-MATCH

• MATERIAL REQUIRED:
• Equipment:
•  Refrigerator to store samples & reagents at
2-60C.
•  Table top centrifuge.
•  Microscope.
• Specimen:
•  Clotted blood sample of patient.
•  Segment from donor unit.
•  Donor red cells suspended in saline.
• Reagents:
•  Antihuman globulin reagent (anti-lgG+anti-C3d).
•  lgG sensitised control cells.
•  0.9% Saline.
•  Distilled water.
• Glassware:
•  Serum tubes.
•  Pasteur pipettes.
•  Glass slides.
• Miscellaneous:
•  Rubber teats.
•  Disposal box.
•  2 plastic beakers.
•  Aluminum racks to hold test tubes.
• PROCEDURE:
• Principle:
• The cross match through the anti-globulin
phase permits detection of clinically significant
incompatibilities caused by incomplete
antibodies that sensitized cells at 370C, but do
not directly cause agglutination.
• Cross-match:
• i. Label tube with patient/ unit and test identification.
• ii. Add two drops of patient serum to each tube.
• iii. Prepare a 5% cell suspension in saline from each donor
unit segment.
• iv. Add 1 drop of donor’s 5% red cell suspension to the tube.
• v. Incubate tubes at 370C for minimum 15 minutes. (Follow
manufacturer’s directions when using commercial reagents).
• vi. Wash the cells a minimum of 3 times with saline. Decant
completely after last wash (washing can be done manually or
in automated cell washer).
• vii. Add two drops of antihuman globulin reagent to the dry
cell button.
• was capable of reacting and that the negative anti-globulin
test is valid.
• viii. Mix well and centrifuge at 1000 rpm for 1 minute.
• ix. Re-suspend and read for agglutination. Grade and
record test results immediately.
• x. To all negative anti-globulin tests add 1 drop of lgG-
sensitized control cells. Centrifuge, resuspend and read
for agglutination. Grade and record test results. After
the addition
• of lgG-sensitised control cells to a negative test, the
presence of agglutination indicates that the AHG
serum added
• Interpretation:
• xi. Hemolysis or agglutination indicates the presence of a serologically
incompatible cross-match. This result is interpreted as incompatible.
• xii. Absence of agglutination and hemolysis is a negative test result
and indicates a serologically compatible cross-match. This result is
interpreted as Compatible.
• DOCUMENTATION:
• Enter all results on the transfusion record card and OT/Ward
transfusion register. Enter only the results of compatible units in the
blood compatibility form. The Medical Technologist (Lab) who
performed the test and the one who checked the results sign all
records.
 THE ANTI- GLOBULIN TEST
(COOMB’S TEST)
• THE ANTI- GLOBULIN TEST is introduced in to
clinical medicine by Coomb’s in 1945. It is a
sensitive technique in the detection of incomplete
antibodies, anti bodies that can sensitize but
which fail to agglutinate red cells suspended in
saline at room temperature, mainly IgG. These
antibodies are agglutinated by the anti- IgG in
antiglobulin serum through the linking of the IgG
molecules on neighboring red cells
• Red cells can also be agglutinated by a reaction of
complement components on their surface with
anticomplement serum, in the antiglobulin reagent,
since the anti- C3 in the AHG reagent binds to the C3 on
the sensitized red cells and bridge the gap between the
cell- bound human C3 on adjacent red cells. The anti
complement helps in detecting IgM antibodies, which
bind complement but may elute off the red cells with
increase in temperature, leaving complement
components alone on the red blood cells. They also
enhance the reactions seen with complement binding
antibodies.
• The anti globulin reagent is prepared by
immunizing animals, often rabbits with human
gamma globulin (antibody) and, or beta
globulin (components of complement). There
are two types of anti globulin reagents that
can be used in laboratory procedure: broad
spectrum (polyspecific sera) and monospecfic
sera.
• Broad spectrum (polyspecific): prepared by
combining anti- IgG & anti complement. The reagent
may also contain antibodies of other specificities
such as anti- IgM, anti-IgA, anti C-3, or anti C-4.
• Monospecific: contains only a sigle antibody: anti-
IgG or only anticomplement.
• Two kinds of antiglobulin tests are known: the direct
antiglobulin test (DAT) & the indirect antiglobulin test
(IAT).
• The Direct Antiglobulin Test (DAT)
• It is used to demonstrate whether red cells have
been sensitized (coated) with antibody or
complement in vivo, as in case of HDN, Autoimmune
haemolytic anemia, and drug induced haemolytic
anemia, and transfusion reactions.
• Principle: Patients erythrocytes are washed to
remove free plasma proteins and directly mixed with
AHG, and if incomplete antibodies are present,
agglutination occurs.
• Procedure
• 1. Place one drop of 5% saline red cell suspension in a test tube.
• 2. Wash the red cells 3-4 times using normal saline, ensuring
adequate removal of the supernatant after each wash.
• 3. Add one or two drops of antiglobulin reagent to the tube.
• 4. Centrifuge at 3,400 rpm, for 15 sec.
• 5. Gently re suspend the red cells and examine macroscopically
and microscopically for agglutination or hemolysis.
• 6. Add IgG sensitized red cells as a control, centrifuge and read.
If a negative result is obtained, the test result is invalid if mono
anti complement reagents are used complement sensitized red
cells should be for substituted for IgG sensitized red cells.
• The Indirect Antiglobulin Test (IAT)
• It is used for the detection of antibodies that
may cause red cells sensitization in vitro. The
sensitizing antibody or complement acts as the
antigen for the antiglobulin reagent.
• IAT used in cross- matching, to detect
antibodies that might reduce the survival of
transfused red cells and Du technique, in the
detection of Du antigen.
• Principle:
• The serum containing antibodies is
incubated with erythrocytes containing
antigens that adsorb the incomplete
antibodies. After washing to dilute the excess
antibody in the serum, the addition anti
globulin serum produces agglutination in the
presence of incomplete antibodies.
• Procedure (IAT)
• 1. Put 2-4 drops of serum in a test tube
• 2. Add a drop of 5% red cell suspension
• 3. Mix & incubate at 370C for 15-30 minute
• 4. Centrifuge at 3400 rpm, for 15 sec and examine for
agglutination or haemolysis.
• 5. Wash 3-4 times decanting the supernatant.
• 6. Add 1 or 2 drops of antiglobulin reagent.
• 7. Mix and centrifuge at 3400rpm, for 15 sec.
• 8. Examine for agglutination or haemolysis
 HAEMOLYTIC DISEASES
• Autoimmune hemolytic anemia (AIHA)
• AIHA is brought about through the interaction
of red cells and autoantibodies. It is classified
into four groups namely: warm- reactive
autoantibodies, cold – reactive auto
antibodies paroxysmal cold hemoglobinuria
and drug induced hemolysis
• The diagnosis of AIHA depends on the
demonstration of autoantibodies on the patient’s
red cells using the DAT. The autoimmune antibodies
will be either of the ‘warm antibody‘ types or the
‘cold antibody’ type. The warm types have
antibodies active at 370C but no abnormal cold
antibodies. In the cold type the patient’s serum
contains high titer cold agglutinins, optimally active
at 20C but with a temperature range which may go
as high as 320C.
• A positive DAT will be found in both types; in
the cold type it is complement which
sensitizes the cells and give rise to the positive
DAT rather than specifically bound antibody,
whereas in the warm type it is more usually
the attached antibody, although occasionally it
may be due to complement.
• Hemolytic Disease of the New Born (HDN)
• HDN, originally known as erythroblastosis fetalis,
results from blood group incompatibility in which
maternal antibodies destruct fetal red cells. An infant
having inherited an antigen from the father, which is
absent in the mother, causes her to form the
corresponding antibodies.
• These antibodies pass through the placenta by active
transport mechanism, coat the fetal erythrocytes
and cause damage to them.
• Every blood group antibody that can occur as IgG can
cause HDN.
• It is only IgG immunoglobulin that is capable of
passing the placental barrier and which is found in
cord blood in a concentration equivalent to that
found in maternal blood.
• IgM agglutinin though produced in response to fetal
red cells in utero, plays no part in the cause of HDN,
and are either present in much lower concentration
in the new born than the mother or entirely absent.
• Fetal hematopoietic tissue (liver, spleen and
bone marrow) respond to hemolysis by
increased production of RBCs , predominantly
NRBCs . Increased destruction of red cells leads
the fetus to develop anemia and jaundice from
the hemoglobin breakdown product, bilirubin.
• If this blirubin reaches excessive levels in the
newborn or infants circulation it causes mental
retardation or death.
• HDN Due to Rh Blood Group Incompatibility
• HDN due to anti- Rh(D) occurs when mother and infant are
always incompatible with respect to the Rh factor: The
mother Rh(D) negative, and the infant Rh (D) positive
(inherited the D factor from the father).
• ABO incompatibility between the mother and fetus
reduces the chance of maternal immunization to the D Ag.
• This is probably because the fetal cells, which are
incompatible with the maternal ABO antibodies are
destroyed by existing ABO antibodies before they have a
chance to act as an antigenic stimulus.
• The first Rh- incompatible infant is usually
unaffected because the number of fetal cells
that cross the placenta during pregnancy
(after 24 weeks gestation) is small and
insufficient to cause IgG anti D production,
unless a prior transfusion of D positive blood
has been given.
• During transplacental hemorrhage, the amount of fetal
blood that enters the maternal circulation increases, and
in 6 months time after delivery only 10% of these Rh
negative women could produce detectable antibodies.
• The actual production of anti-D antibodies depends on
the dosage and antigenecity of the D antigen, and the
mother’s ability to respond to these foreign antigens.
About one third of mothers are nonresponders, they fail
to form anti- D despite intentional repeated injections of
Rh (D) positive erythrocytes.
• During a second pregnancy with a Rh positive fetus,
small number of fetal cells cross the placenta (20
doses of antigen) stimulating the antibody to high
concentration, mainly Ig G anti- D that passes in to
the fetal circulation destroying fetal red cells.
• The severity of the disease increases with each Rh
positive pregnancy. IgG anti-D is found
predominantly in sub classes IgG1 & IgG3 and these
subclasses possess properties that play and effective
role in erythrocytolysis in vivo.
• HDN due to ABO Blood Group Incompatibility
• HDN due to ABO blood grouping usually
occurs when the mother is invariably group O
(posses Ig G anti- A&B), the infant: group A or
B (usually group A) and when the mother &
infant are Rh compatible.
• The fetal red cells cross the placenta in to the maternal
circulation stimulating the existing anti A& B to high titers;
the “immune” anti A& B stimulated is largely IgG.
• ABO HDN occurs in the first pregnancy because anti A & anti-
B are always present (naturally occurring) and therefore
readily stimulated.
• Although ABO incompatibilities between mother and baby
occur frequently and represent a common form of HDN, the
clinical course of ABO HDN is relatively mild, probably
because of the antigenic development of the fetal red cells
and may also be due to the presence of A&B substances in
the fetal tissues and fluids that will neutralize the anti A &
anti – B antibodies before they can attack the fetal red cells.
• Assessment of HDN
• Prenatal
• Some investigations are carried out on blood of
the mother to identify women at risk of having a
child affected with HDN.
• It is recommended that all pregnant women at
their first attendance at a clinic need to have ABO
grouping, Rh typing for D & Du, alloantibody
screening test and amniocentesis.
• Postnatal
• determining the presence and assessing the severity of
HDN.
• 1. ABO & Rh Typing: the ABO group of the infant is based
on forward (cell) grouping as anti- A and anti B
agglutinins do not develop until a few months after birth.
• - ABO grouping most commonly reveals the mother to be
group O and the baby to be group A or possible group B.
• - Rh typing shows the baby to be D or DU positive and
the mother D or DU negative
• 2. DAT on cord or infants blood:
• - In ABO HDN, DAT is usually negative or
weakly positive , as weak Ag-Ab interaction
which cause the antigen to be removed during
the washing phase of the DAT. Also, due to too
low antibody titer to be detected.
• - In Rh HDN, DAT gives positive result.
• 3. Antibody elution test of cord blood: done if
DAT is positive, may reveal the presence of
immune anti- A or anti-B in ABO HDN and anti-
D in Rh HDN.
• 4. Hgb level of cord blood: may be slightly to
severely decreased
• 5. Serum bilirubin level on cord serum: may
exceed the normal values of cord total serum
bilirubin of 1 to 3 mg/ml.
• 6. Peripheral blood smears on cord blood: blood smear
evaluation shows anemia with RBC morphology
abnormalities: hypochromia, microspherocytosis with the
demonstration of reticulocytes and immature nucleated
RBCs.
• 7. Kleihauer- Betke acid elution test: is a test to be
performed for quantitating the extent of fetal maternal
hemorrhage (number of fetal cells in the maternal
circulation). It is an indicator for treatment of the mother
with anti-D immunoglobulin, more importantly used to
determine the size of dose to be given.
• Reagents: 80% ethanol
• Solution A: 0.75 haematoxylin in 96% ethanol
• Solution B : 2.4 g FeCl3 & 2 ml of 25% HCl in
100 ml distilled water
• Elution solution: 2 parts of A mixed with 1 part
of B & 9 part of 80% ethanol
• Counter stain: 0.1% erythrosin or 0.5%
aqueous eosin.
• Method: Mother’s whole blood is diluted with an equal
quantity of saline and used for making films. The slides are
fixed in 80% ethanol for 5 minutes at RT.
• They are thoroughly dried. They are then placed for 20
seconds in the elution solution, rinsed in distilled water and
counterstained for 2 minutes. At least 50 fields are
examined using a magnification x 400.
• The white cells, including lymphocytes, stain gray, the adult
red cells, which have soluble hemoglobin appear as ‘ghost’
cells, while the fetal cells stain bright red. Report the
percent & fetal cells after counting 2000 cells in the acid
elution smear.
• Prevention of HDN
• Fetal red cells in the maternal circulation might be
destroyed by administration of suitable quantity of IgG
anti- D to prevent Rh immunization of the mother, given
to Rh-negative women within 72 hours of delivery. This
dramatically decreases the incidence of anti-D HDN.
Combined prenatal-postnatal treatment is more effective
than postnatal treatment alone in suppressing Rh
immunization. All pregnant Rh- women should receive Rh
IG even if the Rh status of the fetus is unknown because
fetal D antigen is present in fetal erythrocytes as early as
38 days of conception.
• Dose:
• The usual recommended dose (contained in one vial)
is about 300 μg which is believed to offer protection
against a fetomaternal hemorrhage of 30 ml (15 ml
packed cells) or less.
• If a massive fetomaternal hemorrhage has occurred,
the volume of the hemorrhege must be determined to
calculate the number of vials of Rh (D) immune
globulin to administer.
• Calculated as follows:
• Volume of fetomaternal Percentage of fetal cells
• hemorrhage = (seen in the acid elution stain)
x*50 No of vials of Rh IG = volume of
fetomaternal hemorrhagex*2 30
• NB: Factor of *50 = 5000 ml (estimated maternal
volume) x 1/100%
• *2 is a common factor, because the actual
fetomaternal hemorrhage may be twice the
estimate.