Hemolytic disease of the newborn (HDN) or erythroblastosis fetalis
results from excessive destruction of fetal red cells by maternal antibodies.
This condition in the fetus or newborn infant is clinically characterized by
anemia and jaundice. If the hemoglobin breakdown product, bilirubin, which
produces jaundice, reaches excessive levels in the newborn infant’s
circulation, it accumulates in lipid rich nervous tissue. This deposition of
bilirubin (kernicterus) can cause mental retardation or death.
Mechanism of HDN
In HDN, the erythrocytes of the fetus become coated with maternal
antibodies that correspond to specific fetal antigens. This hemolytic process
reduces the normal 45 to 70 days life span of fetal erythrocytes.
The fetal hematopoietic tissues (the liver, spleen and bone
marrow) respond to hemolysis by increasing production of erythrocytes.
Increased erythrocyte production outside the bone marrow, extramedullary
hematopoiesis can result in enlargement of liver and spleen. If increased erythropoiesis
cannot compensate for erythrocyte destruction, a progressively severe anemia
develops. This severe anemia may cause the fetus to develop cardiac failure
with generalized edema (hydrops fetalis), resulting in death in utero. In
newborn infants, severe anemia can produce heart failure shortly after birth.
Less severely affected infants continue after birth to experience
erythrocyte destruction, which generates large quantities of unconjugated
bilirubin. Before the birth, unconjugated bilirubin is normally bound to
albumin and forms conjugated bilirubin in the mother’s liver through the action
of the enzyme glucuronidase. The newborn infant’s immature liver
does not efficiently synthesize glucuronidase during the first few days of
life. This result in low amounts of the enzyme in the newborn infant’s
circulation. Additionally, the albumin, which is necessary to form conjugated
bilirubin, is limited. This combination of factors in the newborn, which must
excrete large quantities of bilirubin resulting from excessive hemolysis, can
produce the threat of accumulation of free bilirubin in lipid rich tissue of
the central nervous system. Total plasma bilirubin levels approaching 20 mg/dl
can cause mental retardation or death.
Mechanism of antibody transfer from mother to
fetus
The transfer of antibodies from the maternal circulation to the
fetal circulation occurs through the placenta. The only immunoglobulin that is
selectively transported to the fetus is IgG. Although the fetus produces some
IgG, IgG in cord serum is derived almost entirely from the maternal
circulation. Among the five classes of immunoglobulin in humans, only IgG is
found in cord blood in a concentration found in maternal blood. The other
immunoglobulin classes, such as IgM, are either present in much lower
concentrations in the newborn than in the mother or entirely absent.
In the first 12 weeks of gestation, only small amounts of IgG are
synthesized. This concentration of IgG continues to rise until birth. Evidence
suggests that the levels of the IgG subclasses, IgG1 rise at an earlier stage
of gestation than IgG3 subclass levels. At birth, IgG1 levels are higher than
those of IgG3 compared to maternal concentrations. The fetal concentrations of
IgG2 are distinctly lower than those of the other IgG subclass. The structure
of IgG responsible for transplacental passage resides in the gamma chain.
Factors in the antibody production in HDN
1. Genetic make–up of an individual
For antibody formation to take place
the mother must genetically lack the trait and the fetus must genetically
possess the trait, which has been inherited from the father. This genetic
inheritance expresses itself in the mother as her being negative for an
antigen, whereas the fetus possesses the antigen.
2. Antigenicity of a specific
antigen
Antigens vary in terms of the
number of receptor sites on the erythrocyte membrane and their immunogenic
strength. Certain antigens, such as D, c and Kell are known to be very potent
in stimulating the immune system.
3. Actual amount of antigen
introduced into maternal circulation
Transplacental hemorrhage (TPH)
can occur at any stage of pregnancy. Immunization resulting from TPH can result
from negligible does during the first 6 months in utero; however, significant
immunizing hemorrhage usually occurs during the third trimester or at delivery.
Fetal erythrocytes can also enter the maternal circulation as the result of
physical trauma due to an injury, abortion, ectopic pregnancy, amniocentesis or
normal delivery. Abruptio placentae, cesarean section and manual removal of the
placenta are often associated with a considerable increase in TPH. A significant
fetal–maternal hemorrhage is considered to be 30 ml or greater, evidence exists
to support the theory that a minimal dose for inducing a primary immunization
is probably less than 0.1 ml for some of the more immunogenic antigens.
****** GENERAL ASSESSMENT PROCEDURE FOR HDN ******
1. Prenatal Testing
a. ABO Typing
b. Rh testing for D and Du.
c. Alloantibody screening; if negative, it
should be repeated again at 34 weeks gestation.
d. Alloantibody identification, if the antibody
screening test is positive.
e. Amniocentesis
Amniocentesis is an analysis of
fluid from the amniotic cavity obtained by the transabdominal insertion of a
needle. This gives information of fetal development, cord hemoglobin
concentration, genetic disorders and chromosomal abnormalities
Clinical criteria used in
deciding to perform amniocentesis
a. If a previous infant had HDN, amniocentesis
may be performed as early as 28 weeks of gestation, even if the antibody titer
remains constant. If a previous child was severely afflicted, an amniocentesis
may be performed as early the 22nd week of gestation.
b. When the maternal antibody level accelerates
before the 34th week of gestation.
Spectrophotometric interpretation
of amniotic fluid analysis
a. An optical density (O.D.) of amniotic fluid
at 450 to 460 nm in normal pregnancy is about 0.02 at term.
b. Fluid from an infant with severe HDN has a
greatly increased OD with a peak at 450 to 460 nm, then so called bilirubin
bulge.
c. At 450 nm absorbance, the difference between
the infant’s OD and a baseline value is determined. This value is plotted on a
Lily’s graph and gives a status of severity of HDN:
(1) Zone 1 – represents an unaffected state
in which he fetus is considered not be in danger.
(2) Zone 2 – indicates that the fetus needs
monitoring and the amniotic fluid should be re–tested.
(3) Zone 3 – indicates that the fetus is in
danger
d.
At 650 nm absorbance, it is useful as a
screen or as plat of battery of lung maturity estimates.
2. Post–partum Testing
a. Hemoglobin and hematocrit determination of
cord or infant blood
14.5 – 22. 5 g/dl – normal value
of infant’s cord hemoglobin
10.5 – 14.5 g / dl – cord
hemoglobin value of moderately afflicted infant with HDN.
3.4 – 10.4 g / dl – cord
hemoglobin value of severely affected infants with HDN.
b. Serum bilirubin of cord or infant blood
0.7 – 3.5 mg / dl – normal value
of infant’s cord bilirubin
4.0
mg / dl – indication of exchange transfusion
6.0
mg / dl – cord bilirubin value of infant with severe HDN.
c. ABO and Rh testing of cord or infant blood
d. Direct Antiglobulin Testing
e. Antibody elution and identification, if the
DAT is positive
f. Peripheral blood smear
Hypochromic erythrocytes and
spherocytes are commonly seen in HDN. The number of immature nucleated
erythrocytes is helpful in assessing the erythropoeitic response to hemolysis.
g. Du Rosette Test
This was developed and reported
by Sebring and Polesky in 1982. This test will detect Fetal–maternal hemorrhage
of approximately 10ml in the maternal circulation.
The principle of the test used D+
indicator erythrocytes to form identifiable rosettes around individuals D+
fetal cells that may be in maternal circulation. The indicator erythrocytes
bind to the free second antigenic determinant of the anti–D antibody, thus
forming rosettes. Positive results must be followed by a quantitative procedure
such as Kleihauer–Betke test.
h. Kleihauer–Betke Test
This test can detect a
fetal–maternal hemorrhage as small as 7.5 ml of packed erythrocytes or 15 ml of
whole blood. This procedure test for fetal hemoglobin rather than the presence
of erythrocytes. Fetal erythrocytes contain 53 to 95% hemoglobin F.
The principle of this procedure
is that adult hemoglobin (Hemoglobin A) is soluble in acid, while fetal hemoglobin
(Hemoglobin F) is insoluble at this low pH and not eluted. The disadvantages of
this procedure include false positive results in case of Rh negative mothers
with high levels of fetal hemoglobin and the subjective nature of test
interpretation.
Classification of HDN in descending order of
severity
1. Anti–D, either alone or in combination with
anti–c or anti–E
2. Other Rh antibodies, such as anti–c
3. Other blood group system antibodies such as
anti–Kell, anti–Duffy and anti–Kidd.
4. Antibody A and anti–B
************ Rho–HDN
************
The placenta is consists of blood vessels, vascular spaces and
small amounts of supportive tissues. Its prime function is the exchange of
substances between mother and infant.
When hemolytic disease of the newborn due to anti–D occurs, mother
and infant are always incompatible with respect to the Rh factor. The mother is
Rho(D)–negative and the infant has inherited the Rho(D)–positive
factor from the father.
The first Rh–incompatible infant is usually unaffected (but the
second is), while fetal cells do not cross the placenta from the 28th
week of gestation in the first pregnancy; the number of cells is usually small and
insufficient to cause antibody production. In addition to this fact, elevated
steroid levels or other factors associated with pregnancy may suppress the
mother’s primary immune response. At term, however, a relatively large
transfusion of fetal cells into the maternal circulation is common and it is
then that the immune response is initiated.
Anti–Rho(D) is produced in the maternal circulation as
the result of fetal cell stimulation. Subsequent incompatible pregnancies will
be affected by the presence of the antibody, since the gradual crossing of
fetal cells from the 28th week will stimulate the antibody to high
titers. The anti–D formed is IgG and is also capable of crossing the placenta.
It therefore moves into the fetal circulation, combines with fetal Rho(D)–positive
cells and causes their destruction.
Antibodies of the IgG1, IgG2, IgG3 and IgG4 subclasses are all
generally believed to be able to pass through the placental barrier. Because
IgG subclasses have different properties such as their rate of transfer across
placenta and complement fixation, their hemolytic capacity varies.
Approximately 8% of Rh negative women who deliver Rh positive ABO
compatible babies develop detectable anti–Rho within six months if
they are not protected with Rh immune globulin.
Fetal hemolysis is primarily due to antibodies of the IgG1 and
IgG3 subclasses. When IgG1 antibodies are absent, a lower frequency of severe
forms of HDN is observed. Because IgG1 antibodies cross the placenta with
greater ease and earlier (approximately 2 months before IgG3 antibodies), they
are exposed to erythrocyte antigens for a longer period. This makes IgG1
antibodies more competitive in binding to erythrocytic antigens than IgG3
antibodies.
Fc fragment is responsible for hemolytic properties of the anti–D
antibodies because of its placental transfer site and its macrophage binding
site. Severe forms of HDN are significantly higher when the G1m(4) allotype is
present than when it is absent.
Laboratory Diagnosis
1. Prenatal Assessment
a. Antibody titration
b. Non–invasive monocyte monolayer assay
c. Amniocentesis
2. Post–partum Assessment
a. Rh typing with Rho(D) or Du
positive results of the cord or infant’s blood show the mother as Rho(D) and Du
negative
b. Direct antiglobulin test is positive and the
mother demonstrates a positive indirect antiglobulin with anti–D, the
identified antibody.
c. Antibody elution of cord blood cells reveals
the presence of anti–D.
d. Hemoglobin levels of cord blood may be
moderately to severely decrease. Levels ranging from 10.5 to 11.5 g / dl are
seen in moderately afflicted infants, while levels ranging from 3.4 to 10.4
g/dl are seen in severe cases.
e. Bilirubin levels on cord serum range from 3.5
mg / dl to more than 6.0 mg / dl in the most severely afflicted infants.
f. Peripheral blood smears demonstrate the
presence of immature erythrocytes with a range of over 5 to 6 per 100 leukocytes.
Treatment
1. Plasma exchange. An application of this can be
in the treatment of pregnant women in whom a high antibody titer, coupled with
a past history of delivering a stillborn infant due to HDN, indicates a
significant possibility of another fetal loss due to HDN.
Plasma exchange is not accepted
by many clinicians for routine use. Some of the disadvantages include high
cost, discomfort and inconvenience to the patient and the non–selective removal
of all plasma proteins.
2. Intraperitoneal and Intrauterine
Transfusion. In this
procedure, blood is usually selected on the basis of compatibility with
maternal serum and the matching of donor and maternal erythrocytes with respect
to the antigen causing the hemolysis. Because of the high mortality in infants
with severe HDN born before the thirtieth week of gestation, premature delivery
may not be an option.
One of the risks of transfusion
is the introduction of other antigens into the circulation. Enhanced antibody
production has been observed after intrauterine transfusion of common
non–Rhesus antigens which may also cause HDN, for example, Kidd (JK) and Duffy
(Fy).
3. Exchange Transfusion. The actual transfusion
procedure takes place by way of umbilical vessels. The immediate effectiveness
of a two volume exchange transfusion is 45 to 50%
Objective of exchange
transfusion:
a. To lower the serum bilirubin concentration in
order to prevent kernicterus.
Kernicterus is the deposition of increased
bilirubin, a red cell breakdown product in lipid–rich nervous tissue such as
the brain, which can produce mental retardation or death in newborn. This
condition can occur when circulating plasma bilirubin levels reach 30 mg / dl
in a full term infant and at lower levels in a premature infant.
b. To remove the baby’s red blood cells which
have been coated with antibody and would be more rapidly destroyed.
c. To provide substitute compatible red blood
cell with adequate oxygen–carrying capacity.
d. To reduce the amount of incompatible antibody
in the baby.
Criteria for an exchange
transfusion
a. If the cord bilirubin is 5 mg / dl at birth,
with the plasma bilirubin rising to above 11.5 mg / dl at 12 hours after birth
and above 16 mg / dl after 24 hours.
b. If the rate of increase in plasma in plasma
bilirubin is more than 0.5 mg / dl / hour or anemia (hemoglobin below 14 g /
dl) exist.
c. Prematurity is also a consideration. In
premature infants, the threshold for bilirubin toxicity occurs at lower levels,
along with decreased albumin binding capacity as well as a higher risk of
acidosis, hypoglycemia, hypothermia, hypoxia and sepsis.
Requirements for donor blood for
exchange transfusion
a. Lack the red cell antigens corresponding to
the maternal antibodies.
b. Be crossmatched with the mother’s serum
c. Be less than five days old.
d. Be free of hemolytic anti–A if newborn is
blood group A or be free of hemolytic anti–B if blood group B.
Rules governing the choice of
blood in exchange transfusion
a. Avoid the antigen responsible for the
antibodies present in the mother’s serum.
b. Administer blood with phenotype specific to
mother as to group, etc.
c. Use group specific cells compatible with
mother and child of group O with lower titer serum antibody.
4. Antenatal Rh Immune Globulin (RhoGAM). All pregnant Rh negative women
should receive Rh IgG even if the Rh status of the fetus is unknown because
fetal D antigen is present in fetal erythrocytes as early as 38 days from
conception. Administration of Rh IgG at 28 weeks gestation (antenatal) has
decreased the incidence of primary immunization in Rho(D) negative
woman.
Calculation of the number of
vials of Rh IgG to be administered:
In this procedure, the volume of
fetal–maternal hemorrhage (Kleihauer–Betke test) can be calculated by
multiplying the percentage of fetal cells by a factor of 50. The volume of
fetal blood is divided by 30 to determine the number of vials of IgG needed.
Example:
a. Kleihauer–Betke reported as 3%
b. 3% x 50* = 150 ml of
fetal blood
c. 150 ml / 30 = 5.0 = 6** doses of Rh IgG
* Factor of 50 = 5000 ml
(estimated maternal blood volume) x 1/1000 (%)
** If the number to right of the
decimal point is less than 5, round down and add one vial. If the number to the
right of the decimal point is 5 or greater, round up to the next number and
then add one vial.
Criteria for
the administration of Rh IgG:
a. For prophylactic treatment of previously
unsensitized Rh (D) negative women within 72 hours of delivery or obstetric
intervention
(1) After
delivery of an Rh (D) positive infant.
(2) After
amniocentesis, abortion, miscarriage or ectopic pregnancy
(3) Before
delivery in selected cases (antenatal)
b.
Laboratory criteria
(1) The mother
is D and Du negative.
(2) The
screening test for allantibodies is negative for anti–D antibody.
(3) The infant
is D or Du positive. In obstetric cases where the Rh cannot be determined, it
must be assumed that this criterion has been met.
(4) The direct
antiglobulin test on cord cells or infant’s cells, if available, is negative.
If a positive DAT test result is obtained, an elution technique should be used
to establish anti–D is not the coating antibody.
Types of responses to Rh
immunization
a. Nonresponders
About one third of all Rho(D)
negative persons are classified here. They fail to form anti–D despite
intentional repeated injections of Rh(D) positive erythrocytes.
b. Responders
They form anti–G, a component of
anti–D.
c. Hyperresponders
They produce extremely high
titers of both IgM and IgG types of anti–D. Some of them may additionally
produce antibodies to antigens to which they were never exposed. This
hyperresponsive condition is referred to as augmentation. The Rh –augmented
immune response is responsible for very rare cases of primary Rh immunization.
************ ABO – HDN
***********
The hemolytic disease of the newborn resulting from ABO
incompatibility (ABO – HDN), the IgG anti–A, anti–B and anti–A,B are present in
the mother’s plasma without the requirement for prior immunization by foreign
red blood cells. Thus, any pregnancy including the first may be involved.
A group O mother with IgG anti–A becomes pregnant with a fetus
group A. The A cells from the fetus cross the placenta from the 28th
week of gestation, stimulating the anti–A to a higher titer. The IgG anti–A
crosses the placenta into the fetal circulation, where it attaches to the fetal
cells, resulting in their destruction.
Severe forms of the disease are rare, however, when they do occur,
some infants are stillborn or kernicteric or require exchange transfusion. The ABO–HDN
does not usually develop until a day or so post partum. Direct antiglobulin
test results are often unreliable and may appear negative in ABO hemolytic
disease.
The generally mild form of HDN may be due to several factors: (1) fewer A and B antigen sites on the fetal/newborn
erythrocytes (2) weaker antigen strength fetal/newborn A and B antigens and (3)
competition for anti–A and anti–B between tissues as well as erythrocytes.
Laboratory diagnosis:
1. ABO grouping of mother and baby
2. Direct antiglobulin test
3. Antibody elution testing
4. Hemoglobin assay
15–20 mg /dl – normal value of
cord and venous hemoglobin concentration
5. Bilirubin assay
1–3 mg/dl – normal value of cord
serum bilirubin
12 mg/dl – peak bilirubin value
that is clinically significant.
6. Peripheral blood smear morphology
This may reveal an anemia with
erythrocyte abnormalities such as hypochromia, microspherocytosis and
reticulocytosis. Immature nucleated erythrocyte may be seen in small numbers.
Treatment
Phototherapy is the usual treatment. Phototherapy uses ultraviolet
light that reacts with bilirubin near the surface of the skin. This process
slowly decomposes/converts bilirubin into a non–toxic isomer, photobilirubin,
which is transported in the liver to the liver. There, the molecules are
rapidly excreted in the form of bile without being conjugated.
************ THE Du ROSETTE TEST ************
Principle
This screening procedure detects fetal D positive cells in the
circulation of a D negative postpartum woman. The test uses D positive red
cells as the indicator to demonstrate antibody coating. These indicator cells
combine with the anti–D present on the coated red cells to form easily visible
rosettes of several cells clustered around each antibody–coated D positive red
blood cells present in the mixture. Because this procedure is considered only
as a screening test, specimens producing a positive result should be tested
with a quantitative method, such as Kleihauer–Betke acid elution, to quantitate
the fetal cells present.
Specimen
5 to 7 ml of
blood (red top or lavender top)
Must be retained
under refrigeration for 7 days
Procedure
1. Prepare a 3 to 4% saline suspension of washed
red blood cells from the patient’s postpartum blood sample.
2. Add one drop anti–D antisera
3. Add one drop of maternal red blood cell.
4. Incubate at 37oC for 15 to 30
minutes.
5. Washed the cell suspension at least four
times. Decant the saline completely after the last wash.
6. To the dry cell buttons, add one drop of
indicator red blood cells to each tube and mix thoroughly to resuspend.
7. Centrifuge the tubes for 15 seconds at 3,400
rpm.
8. Resuspend the cell button and examine the
cell suspension microscopically at 100x magnification
9. Examine at least 10 fields and count the
number of cell rosettes in each field.
Interpretation
The absence of rosettes is a negative result. However, if enzyme–treated
indicator red blood cells were used; a maximum of 1 rosette per 3 fields is
considered a negative result. Use of other enhancement medium, a maximum of 6
rosettes per 5 fields constitutes a negative result.
************ ELUTION TECHNIQUES ************
Uses of elution
1. Verify the diagnosis of hemolytic disease of
the newborn due to ABO incompatibility.
2. Identify the antibody causing hemolytic
disease of the newborn from the infant’s cells, if the maternal serum is
unavailable.
3. Aid in the identification of the antibody
coating transfused red cells that are responsible for a transfusion reaction
4. Identify specific antibodies and non–specific
autoantibodies from the red cells of patients with autoimmune hemolytic
disease.
5. Identify mixtures of antibodies by absorption
techniques
6. Removal of antibody from red cell surface to
enable cells to be typed.
Methods of elution
1.
Heat elution
2.
Ether elution
3.
Cold alcohol precipitation
4.
Acid elution
5.
Freeze–thaw elution
6.
Xylene elution
LANDSTEINER AND MILLER ELUTION TECHNIQUE
1. Wash antibody coated red cells 4x with normal
saline.
2. To one volume of packed red cells, add one
half the volume of normal saline.
3. Agitate this suspension continuously in a
waterbath kept at 56oC for 10 minutes.
4. Centrifuge for 3 minutes at 3000 rpm. Preheat
centrifuge cups are recommended.
5. Remove the supernatant containing the eluted
antibody as quickly as possible. Test the eluate by appropriate technique.
ETHER ELUATE
Caution: Ether
is highly volatile and explosive. Be careful to avoid sparks of flame.
1. Wash approximately 1 ml of packed red cells
six times with saline.
2. To the washed, packed cell volume, add ½
volume of saline.
3. Add a volume of reagent grade diethyl ether,
equal to the total volume of saline plus packed red cells.
4. Close with stopper and mix by inversion for
about 1 minute, removing stopper occasionally to release volatile ether.
5. Centrifuge for 1 minute at 3600 rpm.
6. There will be three distinct layers: upper
– containing clear ether, middle – containing denatured red cells stroma
and bottom – containing hemoglobin stained eluate. Remove the ether
layer by aspiration with suction. Carefully insert pipette tip through the
stroma layer and remove the eluate (bottom layer) to another tube.
7. Incubate in unstoppered tube at 37oC
for 30 to 45 minutes, in an open waterbath to drive off any residual ether.
8. Centrifuge for 1 minute at 3600 rpm to
separate any remaining particles.
· This method is best for removal of IgG
antibodies.
LUI EASY FREEZE ELUTION TECHNIQUE
1. To six or eight drops of washed, packed red
cells, add one or two drops 0.9% NaCl, mix and stopper.
2. Coat sides of tube by rotation. Place the
slanted tube at –6 to –30oC for 10 minutes.
3. Thaw rapidly (under running water).
4. Centrifuge the hemolyzed cells.
5. Test clear hemolysate against group A, B and
O cells by routine technique.
Preparation of eluate:
a. Break up the clotted specimen of cord blood
with applicator stick or obtain 1 or 2 ml of free cells from an EDTA specimen. Wash
the cells by hand at least three times with large quantities of saline. If less
than 1 ml of free cells is used, there will be insufficient eluate for testing.
b. Pack the cell mass and remove all of the last
wash completely.
c. To one volume of packed cord cells, add one
volume of 6% bovine albumin and resuspend.
d. Constantly agitate the cell–albumin mixture
in a 56oC waterbath for 7 minutes.
e. Preheat centrifuge holders by placing them in
a 56oC watebath.
f. Immediately remove the supernatant (eluate)
and use it for testing. Do not remove any cells with the eluate because they
will reabsorb the antibody. This eluate is used in the same manner as serum.
Note: If antibodies other than anti–A or anti–B are
suspended, identify the antibody using a reagent red blood cell panel with the
eluate.
Testing procedure
1. Label (4) 10 x 75 mm test tube: A, B, I, II
2. Add 2 drops of eluate to each tube.
3. To each of the labelled tubes, add one drop
of 3% suspension of adult A1 cells, B cells and I,II.
4. Mix the cells thoroughly and incubate at 37oC
for 30 minutes.
5. Wash the contents of each tube three times
with normal saline. Decant completely after the last wash.
6. Add 2 drops of antiglobulin serum to each
tube.
7. Mix all tube and centrifuge at 3400 rpm for
15 seconds.
8. Gently resuspend the cells and examine each
tube macroscopically and microscopically for agglutination. Record the results.
9. To each tube showing no agglutination, add 1
drop of Coomb’s control check cells.
10. Mix the
tubes and centrifuge for 15 seconds at 3400 rpm.
11. Gently
resuspend the cells and examine each tube macroscopically for agglutination. If
any of the tubes show no agglutination, the entire procedure is invalid and
must be repeated.
Interpretation:
Agglutination occurring when the eluate is tested against RBC’s of
appropriate phenotypes, indicates that an antibody has been recovered from the
original cells.
KLEIHAUER–BETKE TEST: HEMOGLOBIN F
DETEMINATION BY ACID ELUTION
(Modified by Shepard, Weatherall and Conley)
Principle
After blood smears are fixed with ethyl alcohol, a citric acid
phosphate buffer solution removes (elutes) hemoglobin other than hemoglobin F
from erythrocytes. The hemoglobin F (fetal hemoglobin) – containing
erythrocytes are visibly identifiable upon microscopic examination when
appropriately stained. Shortly after birth, the amount of hemoglobin F in
humans decreases to low levels. Increased amounts of hemoglobin F are found in
various hemoglobinopathies such as hereditary persistence of fetal hemoglobin,
sickle cell anemia and thalassemias.
Specimen
Capillary blood
or EDTA anticoagulated blood
Procedure
1. Make four thin blood smears from each
specimen: patient, normal control, neonatal control, label.
2. Allow these smears to air dry for 10 to 60
minutes.
3. Prepare the working citric acid phosphate
buffer solution. Transfer to a staining jar and cover. Incubate at 37oC
for 30 minutes.
4. The solutions needed for steps 5 to 9 should
be prepared and filtered (if needed) and dispensed into labeled containers
before proceeding with the next step.
5. Place the dry slides into 80% ethyl alcohol
for about 5 minutes. At the end of this time, gently rinse or dip the smears in
distilled water and allow to air–dry.
6. After the smears are completely dry, place
the slides in the pre–warmed citric acid– phosphate buffer solution for 5
minutes. At the end of 1 minute, dip the slides up and down. Repeat again at 3
minutes.
7. After 5 minutes, remove the slides from the
citric acid–phosphate buffer solution and rinse with distilled water, air–dry.
8. After the smears are completely dry, stains
in Mayer’s hematoxylin for 3 minutes. Rinse with distilled water.
9. Place the slides in Erythrosin B for 4
minutes. Rinse with distilled water and allow to dry.
10. Examine with
(100x) OIO for Hemoglobin F. Cells containing Hemoglobin F stain a dark–red–orange
color depending upon the concentration of Hemoglobin F. Normal adult
erythrocytes appear as ghost cells. The neonatal blood sample should have many
dense–appearing erythrocytes per field.
Calculations
The percentage of Hemoglobin F containing cells can be determined
by counting the number of dense–staining cells and the number of ghost cells
per field. Using the high dry (43–44x) objective, count 500 ghost cells and
record the number of dense hemoglobin F– containing cells seen during the
count.
Normal values
Normal adults –
less than 1%
Newborn infants –
70 – 90%
No comments:
Post a Comment