Immunological and Molecular Assessment of

Umbilical Cord Blood and Maternal Blood

Admixture in a Group of Cameroonian Neonates
Putatively Primed in utero to P.falciparum
Antigens.
Thesis presented in partial fulfillment for the award of the
Medicinae Doctorae Degree by:

BESONG MICHEAL EBANGHA

Director:
Pr. Rose G.F. LEKE

Co-Directors:
Pr. Judith TORIMIRO
Dr. Samuel TASSI YUNGA
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Plan
Introduction
Objectives
Methods
Results and Discussion
Conclusions
Recommendations
Summary in French

INTRODUCTION (1)

Malaria: parasitic disease, Plasmodium sp.

198 million cases worldwide. 80% of cases and 90% of deaths

in Sub-Saharan Africa*
More vulnerable: Children < 5yrs and pregnant women.
MIP accounts for 10.000 maternal and 200.000 foetal deaths.*
Malaria in Pregnancy: sequestration of Pf infected erythrocytes

in placenta.

*WHO 2014

INTRODUCTION (2)

Placental malaria: enhance the in utero exposure of fetus to malaria

antigens.
Past: fetus considered immunologically hypo responsive to in utero

aggression
Recent findings show the contrary, that fetuses can be primed in utero;

cytokines and Ig produced by CB T and B lymphocytes respectively in

response to Pf exposure.
Are these immune responses detected in CB truly of fetal origin or are

from MB lymphocytes that admix with CB?

CB admixture; infusion of MB into CB. It can occur before, during or

immediately after delivery.

4

INTRODUCTION (3)

Methods

used to assess CB
immunological and molecular.

admixture;

enzymatic,

Previous studies have used only single methods but not a

combination of methods which could improve sensitivity.

Molecular analysis have been done only on CB samples

without comparing findings found in CB with MB.

Since assessing CB admixure will comfirm whether fetuses are

really primed in utero,

OBJECTIVES

General objective: Rule out CB admixture with MB in a

group of neonates putatively primed to P.falciparum in utero

using a combination of immunological and molecular
methods.
Specific objectives
To rule out CB admixture with MB using Pf IgM assay

(immunologic method)
To rule out CB admixture with MB using the D1S80

microsatellite genotyping (molecular method).

To rule out CB admixture with MB using a combination of

both Pf IgM assay and D1S80 genotyping methods.

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METHODS (1)

Study type: Cross-sectional

Study duration: 12 months (from July 2014 to June 2015).

Setting: sample collected in another study

Yaounde

(Central maternity, Nkolbisson HD);

Maroua (Regional hospital).

Study population: pregnant women and their neonates at delivery.

Sample size: 43 participants

Sampling: Consecutive

Ethics: Approval by the IRB of the BTC UY1.

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METHODS (2)

Inclusion criteria
Mother-neonate pairs from pregnant women who had
given their consent for another study.
Neonates putatively primed in utero to Pf antigens.
(putative in utero priming was defined as the presence of
Pf specific IgG to at least 1 Pf antigen in in vitro CB
mononuclear cell culture supernatant).
Exclusion criteria.
Maternal samples not available for comparison
Sample pairs with missing baseline data

METHODS (3)

Procedure
Selection of neonates
positive for anti-Pf
IgG in another study

LA
BO
RA
TO
RY

Stored Neonatal CB
and maternal IVS
blood samples sorted
out and thawn

Plasma sample
Packed blood
cells

Plasma
samples
Immunological testing for admixture using the
Pf IgM multiplex assay
Packed blood
cells
DNA extraction
Molecular testing for admixture using PCRbased microsatellite profiling
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METHODS (4)

1. MAP assay for anti-Pf antibodies in CB and MB plasma

PE

PE

PE

Detection
antibody

MagPix Luminex
Results as Median
Fluorescence Intensity (MFI)

Sample
AMA

EBA

MSP1

Luminex plate well

Beads
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METHODS (5)

2. DNA Extraction, Amplification

and Gel Electrophoresis

Thermal
cycler

amplify

Packed
RBC
DNA

D1S80 primers
Separatio
n

Electrophoresis
+
visualization
Amplicon
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METHODS (6)

Data Management
Data entry: MS Excel 2010; Analysis: Graph Pad prism 5.0
Central tendency: means (normal), median (skewed).
Frequencies and proportions used to characterize our study

population.
P values were calculated using the Fishers exact test.
p values <0.05 were considered statistically significant.

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RESULTS/DISCUSSION(1)

A total of 43 neonate-mother sample pairs were used.

Most of the samples; 62.8% were collected at the Maroua

regional hospital.
100% of placental cotyledons of the placentas of these

neonates were intact.

53.5% had at least 1 reported episode of malaria during the

pregancy and 64.7% had malaria at delivery confirmed after by

RDT and/or microscopy.
Cord blood microscopy; 0%
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RESULTS/DISCUSSION (2)

Immunological Testing For Cord Blood and Maternal blood

Admixture
Levels of IgM to five Plasmodium falciparum blood-stage antigens in
plasma from mothers and their new-born babies

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RESULTS/DISCUSSION (3)

Interpretation of the plasma IgM status for ruling out maternal

blood admixture with cord blood.
Neonate Mother pairs plasma IgM status and interpretation

Outcome

Interpretation

Neonatal plasma
IgM positive &
Maternal plasma
IgM negative

Neonatal plasma Neonatal

& Neonatal
&
IgM negative & maternal plasma maternal plasma
Maternal plasma IgM positive
IgM negative
IgM positive

Informative :
Admixture ruled out

Not informative:
Admixture Not ruled out

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RESULTS/DISCUSSION (5)

Neonates ruled out for cord blood admixture with maternal blood.
Mother-Neonates
plasma IgM result
Neonate positive &
Mother negative

Admixture ruled
out?
Yes

Number of
sample pairs
1

Percentage of
total
2.3%

79%
Neonate negative
& Mother positive

Yes

33

76.7%

Neonate & Mother

positive

Not informative/
Inconclusive

4.7%

Neonate & Mother

Negative

Not Informative
/inconclusive

21%

16.3%

Each percentage was calculated using the denominator of 43 which is the

total number of mother-newborn blood sample pairs analyzed

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RESULTS/DISCUSSION (6)

Comparison of IgM levels of IgM positive neonates with levels in

their corresponding mothers

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RESULTS/DISCUSSION (7)

Molecular Testing For Cord blood and Maternal Blood Admixture:

D1S80 Microsatellite Genotyping
Interpretation of D1S80 gels for identification of maternal blood
DNA admixture with cord blood.
Newborn

Mothe
r

Informative:
Absence of admixture

Newborn

Mother

Informative:
Absence of admixture

Newborn

Mother

Informative:
Absence of admixture

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RESULTS/DISCUSSION (8)

D1S80-based analysis of cord blood samples for detection of admixture

with maternal blood
Cord-mother sample pairs
All D1S80-genotyped sample
pairs
Not informative pairs
Informative pairs
Only informative sample pairs
Absence of maternal DNA in
cord blood
Presence of maternal DNA in
cord blood

Number of
sample pairs
43

Percentage

27
16

62.8% of all sample pairs

37.2% of all sample pairs

16
16
0

100% of informative
pairs
0% of informative pairs

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RESULTS/DISCUSSION (9)

Ruling out cord blood admixture with maternal blood using a

combination of immunological and molecular approaches
Method used Admixture ruled out?
Yes
P. falciparum
IgM in
Not conclusive
plasma
D1S80
alleles in
blood
mononuclea
r cell DNA

Description
Mother positive but newborn
negative or vice versa
Both mother and newborn are
negative or both positive with
similar IgM titers

Number of
sample pairs
34

Percentage
of total
79.1%

20.9%

Yes

Newborn has two D1S80

bands, one of which is not
present in a heterozygous
mother

16

37.2%

Not conclusive

Both newborn and mother

have the exact same bands
Admixture ruled out by
plasma IgM and/or D1S80
genotyping
Admixture could not be
confirmed or ruled out by
either methods

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62.8%

36

83.7%

16.3%

Yes
Combined
Not conclusive

Each percentage was calculated using the denominator of 43 which is the total number of mother-newborn blood sample pairs
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analyzed

CONCLUSIONS (1)

CB and MB admixture does not occur in a majority

of cases amongst a population of neonates putatively

primed in utero to Pf antigens and thus immune
responses detected in CB were actually produced by
the fetus.

Molecular testing to assess CB admixture is not

very informative when only a single microsatellite

marker is used

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CONCLUSIONS (2)

Plasma IgM assay can be used to assess CB

admixture and the results suggests it is more sensitive

in ruling out admixture than the molecular method.

A combination of 2 or more different methods to test

for CB admixture of given sample pairs has a higher

sensitivity in ruling out CB admixture that using a
single method.

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RECOMMENDATIONS

Further analyse the neonate-mother sample pairs that

were not informative using other immunological

markers such as PLAP, IgE or molecular markers
such as D1S11, D17S and many more.

Further evaluate CB and MB admixture in this

population of neonates using

A larger sample size
More than two different molecular markers
Using a higher resolution electrophoresis technique which

can distinguish alleles with very close molecular weight

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Rsum

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Dans les rgions o le paludisme est endmique, les ftus

RESUME

peuvent tre exposs in utero aux produits de parasites du

paludisme de leurs mres infectes.
le

ftus
ait
longtemps
t
considr
comme
immunologiquement hypo-sensible aux agressions in utero

Des tudes rcentes ont montr le contraire que les ftus

peuvent tre sensibiliser in utero.

Mais la question qui se pose encore est celle de savoir si ces

rponses immunitaires sont vraiment d'origine ftale, ou alors

si elles viennent des lymphocytes du sang maternel qui se sont
mlang avec le sang du CO.
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Etudes: valuer le mlange de sang du CO ont utilis soit une

RESUME

mthode enzymatique, immunologique ou molculaire mais

pas une combinaison de plusieurs mthodes.
Une combinaison de plusieurs mthodes augmente la

sensibilit de dtecter le mlange de sang du CO.

Dautres auteurs: utilis des mthodes molculaires mais

sans comparer les rsultats obtenus dans le sang du CO avec

le sang maternel correspondant
Sachant que lvaluation de mlange de sang du cordon avec

le sang maternel confirmera si les ftus sont vraiment

sensibiliss in utero
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RESUME

Objectif gnrale: Ecarter le mlange de sang du CO avec le

sang maternel chez certains nouveau-ns camerounais qui

sont prsums avoir t sensibilis in utero au Pf utilisant une
combinaison des mthode immunologique et molculaire.
Objectifs spcifiques:
Ecarter le mlange de sang du CO avec le sang maternel

utilisant une mthode immunologique

Ecarter le mlange de sang du CO avec le sang maternel
utilisant une mthode molculaire
Ecarter le mlange de sang du CO avec le sang maternel
utilisant une combinaison des deux mthodes
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RESUME

Type dtude: transversal

Sur les echantillons des Paturientes et nouveau-ns

prelever dans les maternit de lhopital central de

Yaound, du district de sant de Nkolbisson, et de
lhopitale regionale de Maroua.
Le sang du cordon ombilicale et le sang de lespace

inter-villeux du placenta stocke etait utiliser.

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La technologie MAP quantification des IgM dirigs contre les

RESUME

antignes palustre
LADN tait extrait des GR concentrs , gnotypage par la PCR

du microsatellite D1S80.
Donnes: microsoft excel 2010 GraphPad Prism 5.0
Test de fisher. P<0.05 statistiquement significatif

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RESUME

43 paires dchantillons
Mthodes de dosage IgM plasmatique: 79% exclu pour le

mlange de sang du CO.

Mthodes

de gnotypage D1S80: 62.8% ntait pas

concluant. 37.2% instructif; pas dADN maternel dans le sang
du CO.

Combinaison des deux mthodes; 83.7% des nouveau ns

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En conclusion:
le mlange ne sest pas produit dans la majorit des nouveau-

RESUME

nes dans notre population dtude

Le mthode molculaire est peu concluant quand une seul

marquer microsatellite est utiliss

La mthode immunologique est plus sensible a carter le

mlange que la mthode molculaire

Nos rsultats montrent aussi que utiliser une combinaison de la

mthode immunologique et molculaire augmente la sensibilit

dcarter le mlange de sang du CO.
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Nous recommandons:

RESUME

d'analyser davantage les paires dchantillons qui ntaient

pas concluants en utilisant d'autres marqueurs molculaires

tels que D1S111 et D17S
Analyser le mlange dans cette population utilisant:
une plus grande taille dchantillons
Dautre marqueur immunologique et molculaires
dutiliser une technique d'lectrophorse

haute
rsolution qui permet de distinguer les allles ayant un
poids molculaire trs proche.

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MERCI POUR VOTRE

AIMABLE ATTENTION

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Special Thanks to!!!

All participants in the study
Directors
Staff of the Immunology Laboratory, BTC UY1, for the

technical support
FOGARTY and IMPM for financial support
University of Hawaii
Staff and teachers of FMBS
Parents and friends

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