@asherum_ x @dustinealturas x @pattysespina | BSMT-3B 2021 : IMMUNOHEMATOLOGY 13.

(hemolytic disease of the fetus and newborn (HDFN) + RELATIONSHIP/parentage TESTING)

LESSON 13
Date of Lecture : 10-14 May 2021. Dr. Eunice L. Estella
References : Harmening, Denise M. (2012). Modern Blood Banking & Transfusion Practices, Chapters 19 (HDFN), 21-22 (Parentage). (6th ed.). F.A Davis Co.
NOTE : Anything in dark blue mini-boxes (save ink) are not on the slide but mentioned in the audio (side notes)

hemolytic disease of the fetus and newborn
• also known as Erythroblastosis fetalis
• destruction of the red blood cells (RBCs) of a fetus and neonate by antibodies produces by
the mother.
• disorder of the fetus or newborn in which fetal red cells are destroyed by maternal IgG Ab’s.
• Mother can be stimulated to form the antibodies by previous pregnancy or transfusion and
sometimes during the 2nd and 3rd trimester of pregnancy.
• These antibodies directed against fetal antigens cross the placenta, sensitize fetal red cells,
and – therefore – shorten red cell survival of the fetal RBCs.
• Premature red cell destruction varies from mild anemia to death in utero [inside the womb].
1 hydrops fetalis.
overview of the hemolytic disease of the fetus and newborn
Although the clinical findings in the fetus and newborn were noted as early as the 17th century,
it was not until 1939 that Levine and Stetson reported a transfusion reaction from transfusing a
husband’s blood to a postpartum woman. They postulated that the mother had been immunized to
the father’s antigen through fetomaternal hemorrhage. The antigen was later identified as Rh(D).
• Three Important Factors must be present for HDFN to occur:
1. Red cell antibody produced by the mother must be of the IgG Class.
o Only antibodies of the (IgG) class are actively transported across the placenta;
other immunoglobulin classes, such as IgA and IgM, are not.
o Most IgG antibodies are directed against bacterial, fungal, and viral antigens, so
the transfer of IgG from the mother to the fetus is beneficial.
o However, in HDFN, the Ab’s are directed against those Ag’s on the fetal RBCs that
were inherited from the father.
§ Ab from mother, Ag from fetus
o IgG is the only immunoglobulin capable of crossing the placental barrier.
o IgM antibodies such as Anti-Le (a,b), Anti-M, Anti-N and anti-P1 have not been
implicated in HDFN.
2. The fetus must possess an antigen that is lacking in the mother.
o The gene for the antigen is inherited from the father.
3. The antigen must be well developed at birth.
○ Destruction of fetal RBCs and the resulting anemia stimulate the fetal bone marrow to
produce RBCs at an accelerated rate [BM compensates for the anemia in the PB by ↑ its
production of RBCs}, even to the point that immature RBCs (erythroblasts) are released
into the circulation.
○ The term erythroblastosis fetalis was used to describe this finding.
○ When the bone marrow fails to produce enough RBCs to keep up with the rate of RBC
destruction in the periphery, erythropoiesis outside the bone marrow is ↑ in the
hematopoietic tissues of the spleen and liver. These organs become enlarged
(hepatosplenomegaly), resulting in portal hypertension and hepatocellular damage.
• Results in: Severe anemia and hypoproteinemia (caused by ↓ hepatic production of plasma
proteins) lead to the development of ① high-output cardiac failure with generalized edema,
② effusions [presence of fluids in the interstitial spaces], and ③ ascites, a condition known as
○ In severe cases, hydrops fetalis can develop by 18-20 weeks’ gestation.
○ In the past, hydrops fetalis was almost uniformly fatal; today, most fetuses with this
condition can be treated successfully.
• ‼The process of RBC destruction continues even after such an infant is delivered alive‼—in
fact, it continues as long as maternal antibody persists in the newborn infant’s circulation.
• The rate of RBC destruction after birth decreases because no additional maternal antibody is
entering the infant’s circulation through the placenta.
○ However, IgG is distributed both extra- and intravascularly and has a half-life of 25 days,
so Ab-binding and hemolysis of RBCs continue for several days to weeks after delivery.
BILIRUBIN
• RBC destruction releases Hgb, which is further metabolized to bilirubin (Bb).
○ called indirect because indirect methods are required to measure the Bb in the lab.
1. Indirect Bb is transported across the placenta and conjugated in maternal liver to “direct” Bb.
2. The conjugated bilirubin is then excreted by the mother. Although levels of total Bb in the fetal
circulation and amniotic fluid may be elevated, they do not cause clinical disease in the fetus.
○ However, after birth, accumulation of metabolic by-products of RBC destruction can be-
come a severe problem for the newborn infant.
3. The newborn liver is unable to conjugate bilirubin efficiently, especially in premature infants.
4. With moderate to severe hemolysis, the unconjugated, or indirect, Bb can reach levels toxic to
the infant’s brain (generally, > 18-20 mg/dL), and if left untreated, can cause kernicterus or
permanent damage to parts of the brain.

2 3
pathogenesis HDFN classification
Harmening’s (pp. 429-430) • HDFN is often classified into 3 categories based on antibody specificity:
HEMOLYSIS, ANEMIA, AND D ABO Other antibodies
ERYTHROPOIESIS
• the most antigenic; for this • Of the non-Rh system Ab’s, anti-Kell
• Hemolysis occurs when maternal reason, only Rh(-) blood is is considered the most clinically sig.
IgG [the result of previous transfused to Rh(-) females of in its ability to cause HDFN.
exposure/tran sfusion prior to childbearing potential • Kell antigens are present on
pregnancy] attaches to specific
• Other Ag’s in the Rh system, immature erythroid cells in the bone
antigens of the fetal RBCs.
such as C, E, and c, are also marrow, so severe anemia occurs
• The Ab-coated cells are removed potent immunogens although not only by destruction of circulating
from the circulation by the less immunogenic than D. RBCs but also by precursors.
macrophages of the spleen.
• The rate of RBC destruction depends on ① Ab titer and specificity and ② on the number of
antigenic sites on the fetal RBCs.

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PART III Transfusion Practice | BSMT-3B 2021 : IMMUNOHEMATOLOGY 13.2
Introduction circulation. When D antigen is inherited from the father,
these fetal cells immunize the mother and stimulate the
3.1Hemolytic disease of the fetus and newborn (HDFN) is production of anti-D. Once the mother is immunized to
Rh-HDFN
the destruction of the red blood cells (RBCs) of a fetus D antigen, all subsequent offspring who inherit the D anti-
and neonate by antibodies produced by the mother. The gen will be affected. The maternal anti-D crosses the pla-
(PATHOPHYSIOLOGY)
mother can be stimulated to form the antibodies by previ- centa and binds to the fetal Rh-positive cells (Fig. 19–1).
ous pregnancy or transfusion and sometimes during the The sensitized RBCs are destroyed (hemolyzed) by the fetal
Side notes: second and third trimester of pregnancy. Previously, about monocyte-macrophage system, resulting in anemia.
1) …conceive a 95% childof –thesocases
fetusof HDFN were caused by antibodies in
the mother directed against the Rh antigen D, or Rh(D).
There are several factors that affect immunization and
severity of HDFN, including antigenic exposure, host factors,
is Rh+ (takes after father) of the disease caused by anti-D has steadily
The incidence immunoglobulin class, antibody specificity, and influence of
4) Because thedecreased
womansince is Rh(-
1968 with the introduction of Rh-immune the ABO group.
globulin (RhIG). Rh(D) incompatibility is common, al-
), “woman becomes…”though other RBC incompatibilities have surpassed D in Antigenic Exposure
5) If this is the first pregnancy,
frequency at referral centers.1 Because Rh(D) incompati-
bility washappen;
a major concern for many years, the diagnosis Fetomaternal hemorrhage during pregnancy can cause
nothing will significant increases in maternal antibody titers, leading to
and treatment of HDFN caused by anti-D has been the
however, “in emphasis
the next of muchRh-investigation. These findings can be increasing severity of HDFN. Transplacental hemorrhage
positive...” applied to other clinically significant RBC antibodies that of fetal RBCs into the maternal circulation occurs in up to
4
7% of women during gestation, as determined by the acid-
… resulting intocauseHDFN.HDFN, with the exception of ABO antibodies, which
are described separately.
3.2
In addition to the use of RhIG, many other advances
elution method for detecting fetal hemoglobin.2 In addi-
tion, interventions such as amniocentesis and chorionic management
ABO HEMOLYTIChave been made in the diagnosis and treatment of HDFN.
DISEASE • The and management of HDFN requires close cooperation among the pregnant patient, her
Ultrasonography, Doppler assessment of middle cerebral
OF THE FETUS AND
artery NEWBORN
peak systolic velocity, cordocentesis, allele-specific
Hemorrhage of D-positive fetal RBCs into D-negative mother obstetrician, her spouse or partner, and the personnel of the clinical laboratory performing the
• Moregenecommonly involved
amplification studies newborn
on fetalincells in amniotic red
fluid, Maternal antibody formed against paternally inherited D antigen serologic testing.
fetal DNA analysis in maternal plasma, and intravascular
cellintrauterine
destruction (thanhave
transfusion anti-D)
greatly. increased the success Next D-positive pregnancy • Serologic and clinical tests performed at appropriate times during the pregnancy can
• Most cases are
of accurately subclinical
diagnosing and treating
and adequately do not this accurately determine the level of antibody in the maternal circulation, the potential of the
Placental passage of maternal IgG anti-D
disease.
necessitate treatment. antibody to cause HDFN, and the severity of RBC destruction during gestation
This chapter will discuss the disease process, diagnosis, Maternal antibody attaches to fetal RBCs
e.g. and
o therapy, Type O of
outcomes mother
HDFN. + Type A/B/AB
Hemolysis of fetal RBCs
4.1
father PRENATAL TESTING: ABO, RH AND AB SCREENING:
Etiology
• Occurs in 1 in 150 births. Immune • The recommended obstetric practice is to perform the type and antibody screen at the first
Although the clinical findings in the fetus and newborn were
• These cases can usually be treated with
noted as early as the 17th century, it was not until 1939 that
+ System
prenatal visit, preferably during the 1st trimester.
Phototherapy
Levine and Stetson[orreported
exposure to sunlight]
a transfusion reaction from trans- + • The antibody screening method must be able to detect clinically significant IgG alloantibodies
o fusingMother norblood
a husband’s baby are treated;
to a postpartum woman. Theybabypos- that are reactive at 37°C and in the antiglobulin phase.
tulated that the mother had been immunized to the father’s
usually develops jaundice after birth.
antigen through fetomaternal hemorrhage. The antigen was • At least 2 separate reagent screening cells, covering all common blood group antigens
• Unlike
later the Rh-HDFN,
identified as Rh(D). the firstborn infant may (preferably homozygous), should be used.
HDFN is causedas
be affected by thewell
destructionas of the RBCs of a fetus
subsequent
by antibodies produced by the mother. Only antibodies of
• For tube testing, an antibody-enhancing medium such as polyethylene glycol (PeG) or low ionic
pregnancies in which
the immunoglobulin theclass
G (IgG) mother is group
are actively O
transported strength solution (LISS) can increase sensitivity of the assay.
andacross
the newborn
the placenta; is group
other A, B, or AB.
immunoglobulin classes, such as
4.2
IgA and IgM, are not. Most IgG antibodies are directed HDFN
• Theagainst
IgG bacterial,
antibody, anti-A, B in the mother’s
fungal, and viral antigens, so the transfer RhIG
circulation,
of IgG from the crosses
mother to the the placenta
fetus is and
beneficial. However,
• Active immunization induced by RBC antigen can be prevented by the concurrent
attaches
in HDFN, tothethe ABO-incompatible
antibodies antigens
are directed against those antigens
on the fetal RBCs that were inherited from the father. administration of the corresponding RBC antibody.
of the fetal RBCs.
○ This principle has been used to prevent immunization to D antigen by the use of high-
Rh HDFN 3.3 titered RhIG.
ALLOANTIBODIES
In Rh(D) HDFN, theCAUSING HDFNinfant of an Rh-
Rh-positive firstborn • During pregnancy and delivery, fetal and maternal blood are mixed. If the mother is Rh-
negative
OTHER mother usuallyANTI-D
THAN is unaffected because the mother
has not yet been immunized. During gestation and partic- negative and the fetus is Rh- positive, the mother has up to a 16% chance of being stimulated
Figure 19–1. Pathogenesis of hemolytic disease of the fetus and newborn
• Anyularly
IgGat delivery,
antibody whenisthecapable of causing
placenta separates from the caused by D incompatibility between the fetus and mother. The pathogenesis of to form anti-D.
HDFN,
uterus,ifvariable
the numbers
fetal red cells
of fetal RBCs possess the
enter the maternal other blood group incompatibilities (except ABO) follows the same pattern.
○ As little as 1 mL of fetal RBCs can elicit a response.
antigen and the antigen is well developed at birth. ○ Before delivery, the risk of sensitization is 1.5-1.9% in susceptible women, indicating that
• Anti-K and Anti-c are next most common antibodies to cause HDFN after Anti-D. a significant amount of fetal RBCs can enter the maternal circulation during pregnancy.
ABO HDFN Rh HDFN • RhIG administered to the mother within 72 hours following delivery is used to prevent active
Clinical Jaundice Mild to Moderate Moderate to Severe immunization by the Rh(D) antigen on fetal cells.
Findings Edema No Mild to Severe • RhIG attaches to fetal Rh-positive RBCs in maternal circulation, blocking immunization and
Serologic Mother Type O Type D-negative subsequent production of anti-D.
Results Baby Type A or B Type D-positive 4.3
(-) or “weakly +” Anti-A, Anti- KLEIHAUER-BETKE TEST
DAT Antibody Positive Anti-D
B, Anti-AB • Flow cytometry is used to quantitate the number of fetal Rh-positive cells in the mothers
Hematology Anemia Mild Moderate to Severe circulation as a result of a fetomaternal hemorrhage.
Results Reticulocyte Ct. Mild ↑ Greatly ↑ • Active immunization induced by RBC antigen can be prevented by the concurrent
Morphology Spherocytes Macrocyte, Hypochromia administration of the corresponding RBC antibody.
○ This principle has been used to prevent immunization to D antigen by the use of high-
titered RhIG.
• Massive fetomaternal hemorrhages > 30 mL of whole blood occur in < 1% of deliveries.

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@asherum_ x @dustinealturas x @pattysespina | BSMT-3B 2021 : IMMUNOHEMATOLOGY 13.3

○ These massive hemorrhages 2

can lead to immunization if
adequate RhIG is not
history
administered. ● 1920’s: ABO blood group system was used to establish non-paternity issues in Vienna (three-
• A maternal sample should be allele theory)
obtained within 1 hour of delivery and ○ The processes currently used to resolve cases of questioned parentage can be traced
screened—using a test such as the back to the statistical analysis published by Bernstein in 1924 demonstrating that the ABO
rosette technique—for massive blood group frequency distribution in Austria was most compatible with a three-allele
fetomaternal hemorrhage. theory.
○ If positive, quantitation of the ○ ABO blood group test results were first used to establish non-paternity in Vienna in 1926.
hemorrhage must be done by ○ As other blood groups were described and their inheritance established, they were used
Kleihauer-Betke or by flow with the ABO system for paternity studies.
cytometry. ● 1970’s: Extreme polymorphism of the human leukocyte antigen (HLA) system was
• In the Kleihauer-Betke test, a demonstrated at the Evian workshop organized by Dausset. (1972)
maternal blood smear is treated with acid and then stained with counterstain. Fetal cells ○ This next step occurred in 1972
contain fetal Hgb (Hgb F), which is resistant to acid and will remain pink. ○ All these genetic systems were expressed on different elements of the blood (RBCs,
• The maternal cells will appear as ghosts [lysed by the acid in the stain]. After 2,000 cells are proteins, and leukocytes), but they had one important aspect in common: the detection
counted, the percentage of fetal cells is determined, and the volume of fetal hemorrhage is of the polymorphisms was all dependent on a complex biochemical expression of the
calculated using this formula: original genes.
■ This group of genetic systems is often termed the classical systems – makes use
𝑛𝑜. 𝑜𝑓 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 × 𝑀𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙. of the ABO and HLA systems
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑓𝑒𝑡𝑜𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 ℎ𝑒𝑚𝑜𝑟𝑟ℎ𝑎𝑔𝑒 =
𝑛𝑜. 𝑜𝑓 𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑐𝑒𝑙𝑙𝑠
3
• The calculated volume of fetomaternal hemorrhage is then divided by 30 to determine the
HLA complex
number of required vials of RhIG. ● Represents the most polymorphic genetic system in the human genome
• A simpler way to calculate the dose is to multiply the fetal cell percentage by 50, which gives ● Only antigens expressed by the A and B loci are considered for relationship testing with non-
the volume of fetomaternal hemorrhage in milliliters. DNA-based typing.
• If needed, additional vials of RhIG should be administered within 72 hours of delivery or ASAP. ● Historical Perspective
○ Evidence for human leukocyte blood groups was first advanced in 1954 by Jean
Dausset,with the observation that patients whose sera contained leukoagglutinins had
Relationship testing (parentage testing) received a larger number of blood transfusions than other patients.
○ He determined that these agglutinins were not autoantibodies as had been thought
• The field of parentage testing, or more appropriately known now as “relationship testing,” has
previously but rather were alloantibodies produced by the infusion of cells bearing
expanded over the past 20 years. This has now included criminal investigations, immigration,
alloantigens not present in the recipient.
and identification of human remains from disasters, war, or genocide.
○ Dausset determined that patients with alloantibodies segregated into two groups, based
• Genetic disease, transplantation, and even transfusion of specialized blood products may call
upon their reactivity; antibodies from group 1 specifically recognized antigens on group
for basic knowledge and ability in predicting the passage of genetic traits from one generation
2. Through this method, Dausset identified HLA-A2, the first known HLA molecule.
to the next.
● HLA genetic region
1 ○ A series of closely linked genes that determine major histocompatibility factors—that is,
Relationship or Parentage Testing surface antigens or receptors responsible for the recognition and elimination of foreign
tissues.
● Refers to the testing of genetic markers that are inherited to confirm or refute a biological
○ The region is also referred to as the major histocompatibility complex (MHC).
relationship.
○ The HLA complex contains an estimated 35-40 genes physically grouped into three
○ Most common example is determining whether a male is the biological father of the child
regions located on the short arm of chromosome 6:
○ It can also apply to determining maternity or other kinships
CLASS I REGION CLASS II REGION CLASS III REGION
● The ultimate goal of relationship (parentage testing) is to confirm or refute a specific biological
relationship between two or more individuals whose relationship is in question, usually a ● encodes genes for ● encodes genes ● encodes structurally and
father or a mother or sometimes a sibling. the classic for the functionally diverse
○ When applied to the usual trio of mother, child, and alleged father, the goals are to transplantation molecules molecules, including C2,
exclude all falsely accused men, and to provide compelling inclusionary evidence if a molecules HLA-A, HLA-DR, HLA- C4, Bf (the C’ factors), 21-
man is not excluded. HLA-B, and HLA-C. DP, and HLA- hydroxylase, and TNF.
● The blood group systems used most often in paternity testing are ABO, Rh, MNSs, Kell, Duffy, ● It also encodes for DQ composed ● Two other genes,
additional of both α and β glyoxalase-1 (GLO) and
and Kidd
○ These systems have been in use for a very long time and their potential pitfalls in nonclassic genes, chains. phosphoglucomutase-3
interpretation are well-known from the vast worldwide experience accumulated. including HLA-E, (PGM-3) are linked with
○ Phenotype determination is based on standard RBC agglutination techniques with HLA-F, and HLA-G. the HLA complex.
regulated reagents and, with some extra built-in quality control steps, is indistinguishable
from testing performed daily in most blood banks or transfusion services.

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4 Indirect Exclusion
● Also referred to as: Apparent Opposite Homozygosity
New Systems ● Exists when the child demonstrates only ONE marker and the alleged father demonstrates a
DNA Polymorphism different single marker (e.g. when the child appears to be homozygous for that marker)
● 1985: Jeffrey – The molecular genetics technology developed over the next decade laid the ● As this situation requires assumption for homozygosity, it is considered as an indirect evidence
foundation for a revolution in parentage testing, heralded by the description by Jeffreys in 1985 ● For example, in a paternity testing, an indirect exclusion occurs when a child does not express
of a new type of polymorphism in human DNA – Hypervariable minisatellites: referred to as an antigen that he should have expressed based on the fathers presumed genotype.
variable number tandem repeats (VNTR) ● For example, the child has (Fya – b +) phenotype, the mother has (Fya – b +)phenotype, while
○ New genetic systems the father has a phenotype of (Fya +, b –), the father’s phenotype is opposite to that of the
○ Tested by technologies that directly assess the variability of DNA, have now become child; so the child does not express the antigen that he/she should have expressed based on
the norm. These are called DNA polymorphisms. the father’s presumed genotype
● VNTR systems were tested by treating the extracted DNA with restriction enzymes followed
by Southern blotting and hybridization to VNTR probe(s). Table 1. The
● This technology is known as restriction fragment length polymorphism (RFLP) testing. Genetic System is
○ This dominated the field for several years D2S1338: the
DNA polymorphisms by RFLP Biological Mother
● The sample required can be any tissue or body fluid from which sufficient DNA can be has markers 8 &
extracted. 10, while the
● Peripheral blood, buccal smears, amniotic fluid, chorionic villi, and tissue biopsies can be Alleged Father
analyzed. has markers 10 &
● Once extracted, DNA can be kept frozen for an indefinite period of time 12, while the child
● The technique is time consuming and labor-intensive, and turnaround times are long. manifested a
marker for the
Polymerase Chain Reaction: PCR allele as 10 and 12. It can clearly be said here that the 12 comes from the Alleged Father while the 10
● In PCR, the DNA fragment of interest is amplified in amount thousands of times, and the comes from the Biological Mother.
resulting product is normally identified directly through fluorescence resulting from
fluorescently labelled primers incorporated in the PCR reaction. Next, we have DS21358: the Biological Mother has markers 14 & 17, while the Alleged Father has markers
● RFLP testing ceded precedence to polymerase chain reaction (PCR) technology. 8 & 11, while the child has markers 11 & 14 – the 11 came from the Alleged Father, while 14 came from the
● Originally, PCR was often used to detect polymorphisms caused by point mutations or Biological Mother.
sequence-specific polymorphisms but soon was used to analyze VNTR-type loci in which the
repeated unit was small (i.e., 4 to 5 nucleotides). Such loci are termed short tandem repeat or Mismatch
small tandem repeat (STR) loci. ● When there is an inconsistency in the Inheritance pattern, which is detected in the DNA system,
● the term exclusion is not used; instead, we use the term mismatch or inconsistency. They are
5 both used to indicate that the alleles (or bands) between the child and the alleged father do
not match.
terminology and the interpretation of results ○ The term exclusion is reserved for the final interpretation of the entire set of genetic
● In interpreting the testing results on a classic trio (mother, child, and alleged father), it is always systems tested.
assumed that the mother is the biological mother. This assumption sets the stage for the ● A minimum of 2 mismatches are required before an opinion of nonpaternity or nonmaternity
analysis of results in that it is expected that the mother and child will share at least one allele. is rendered. This could also be changed depending on the laboratory because some
● Therefore, the remaining allele in the child’s profile is of paternal origin and can be compared laboratories require 3 mismatches to conclusively exclude an alleged parent especially if all 3
with the profile of the alleged father (AF). mismatches involve single repeat changes.
○ If the AF lacks the paternally inherited allele in the child, he is excluded as the biological
father. Table 2. We have the genetic
○ If he is not excluded, a statistical analysis is performed to determine his relative system D2S1338 as an example:
probability of being the biological father of the child as compared with a random man the alleged father has markers
unrelated to him or to the mother. 12 & 13, while the child has
● There are two terms that ultimately define the overall result of the testing and those are markers 8 & 9; so, either of those
exclusion and inclusion. markers (8 or 9) did not come
5.1 from the alleged father à the
DIRECT EXCLUSION Parentage Index is 0 (zero).
● Occurs when a marker is detected in the child and is absent in the mother and the alleged
father (child is K+ and both mother and father is K –); or Let us go to D10S1008 (4th row):
● when the alleged father’s phenotype demonstrates two markers and the child has neither one the alleged father has markers
of them (AB alleged father and an O child) 7 & 12, while the child has markers 15 & 18. The markers of the child (15 & 18) did not come from the
False Direct Exclusions Alleged Father so the Parentage Index is zero.
● Can occur as a result of mutations that are significant enough to alter the final product. It could
be a lack of precursor substance, suppressor activity at a locus unlinked to the one tested, or
chimeric state of one of the tested individuals.

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@asherum_ x @dustinealturas x @pattysespina | BSMT-3B 2021 : IMMUNOHEMATOLOGY 13.5

5.2
INCLUSIONARY CALCULATIONS This slide shows the usage of relationship testing in our common
Inclusionary Terms setting. We can have Parentage/Paternity Testing where there is
● There are 3 types of statistical evaluations that can be calculated when the Alleged Father is the Alleged Father; what is known are the mother and the child.
not excluded. These are:
1. Parentage Index (PI) 2. Probability of Paternity (PP) 3. Probability of Exclusion (PE) For Reverse Parentage Testing, this is used for Missing Persons
Investigation. The one in question is the person that is missing and
○ The 3 calculations producing the values defined by these terms are based on the we have the phenotype & genotype of the Alleged father and the
assumption that the random man with whom the tested man is compared is biologically Alleged mother.
unrelated to him or to the mother.
■ It is unacceptable if the random man could be a brother, father, uncle, or other
Rules of Inheritance
close biological relative of the tested man.
1. Child has two alleles for each autosomal marker (one from
○ Another important aspect of the calculations is the need to have accurate allele
mother and one from biological father)
frequencies for each genetic system tested, estimated from databases drawn from an
2. Child will have mother’s mitochondrial DNA haplotype
adequate sample population of the appropriate ethnic group.
(barring mutation)
■ e.g. in our setting, we need calculations that are in the Philippine/Filipino setting.
3. Child, if a son, will have father’s Y-chromosome haplotype
○ When referring to a sample size for a database, the term adequate will depend largely (barring mutation)
upon the polymorphism exhibited by the genetic marker in question.
■ In general, the larger the number of alleles exhibited by the marker, the larger the
population sampling needed to create a valid allele frequency database.

5.2.a
Paternity Index (PI) SHARAWAT
● The paternity inde x (PI) compares two mutually exclusive hypotheses that are expressed as a
Shanaenae and Momo – I honestly don’t know where I’d be if it were not for you two, but
likelihood ratio.
● The numerator generally states the mathematical probability of producing a child genetically we’re down to the final two weeks. I have nothing but endless gratitude.
identical to the tested child, through a mating of the known parent with the alleged parent. Za, An, Ella, Sir Lui, Queen P, Steff, and Sir Kobs – I have loathed every waking day for the
past four months, but you have made it much more bearable. You are the only thing I will miss this
● The denominator mathematically states sem.
the probability of producing the child Choline and Pael – thanks for taking the time to correct my notes. Vv helpful hema kweens.
through a mating of the known parent with Gly, Abi, Mardsz, and Teya – I am never not laughing with you guys. If f2f pa ni, di ra ko
a randomly selected, unrelated individual
maguol bc kamo akong kuyog. Tenks for the friendship. See u in July 🤞
in the same ethnic population.
● The PI is calculated one genetic system at Romelle – KDJHJSJLKA IMY thanx for keeping me sane at 2:30 in the morning with a random
a time and is sometimes referred to as the system index (SI). Tiktok. Stream “One Whole Day”. #RVComebackWhere ???
● If all the genetic systems tested are independent from one another, the overall final paternity Balaycee, Martits, and Elek – you are my rock, needless to say. Never would’ve come this far
index is the product of all the system indices calculated. if it weren’t for you.
● When the PI is greater than 100, the evidence for paternity is considered very strong. Dindin and Paté – my day 1’s. Through every high and low, we’ve seen each other through.
Here we are, finally. Would never trade you two for the world.
5.2.b Elli and Bry – I miss you every day. You both have my whole heart, but you know that fully
Probability of Paternity (PP) / Probability of Parentage
well. See you soonest.
● a restatement of the weight of the evidence for relatedness, expressed in percentage form
● To calculate the PP, the PI previously discussed is incorporated into a statistical theory. Apeiron - ‼! One last push 🥲 I pray that finals be the last time you ever have to pick up notes
(PI)(prior for) before reviewing for boards. Let’s go MTI ‼
𝑷𝑷 =
(PI)(prior for) + prior against
● Currently, laboratories target a PP of 99% or greater as the threshold of proof of parentage for
an alleged parent

5.2.c
Probability of Exclusion (PE) Looking for a summer getaway? Check in at www.southpalmsresort.com
● the probability of excluding a falsely accused man, given the phenotypes of the M&C
● entirely dependent on the mother-and-child phenotype combination and does not require
knowledge of the alleged father’s phenotype.
● It is evaluated by first calculating the proportion of men who would not be excluded – termed
as Random Men Not Excluded (RMNE).
○ In the Layman's point of view, the probability of exclusion represents the proportion of
untested men who would have been excluded by the extent of testing performed

“Failure teaches us something success never does.” Miss Velez 2019 (idc idc)