Research Article (Open access) |
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SSR Inst. Int. J. Life Sci., 7(2):
2774-2780,
March 2021
Cord Blood
Direct Antiglobulin Test (DAT) Positivity in RhD Incompatible Neonates-A Two
Years Study
S. S. S. Quadri1,
Srilatha Bollipogu2*, S. Srujana3, Naval Kishore4
1Associate Professor,
Department of Pathology, Government Medical College, Telangana, India
2Assistant
Professor, Department of Pathology, Government Medical College, Telangana,
India
3Associate
Professor, Department of Pathology, Government Medical College, Telangana,
India
4Professor &
HOD, Department of Pathology, Government Medical College, Telangana, India
*Address for Correspondence: Dr. Srilatha Bollipogu, Assistant Professor, Department of Pathology,
Government Medical College, Mahabubnagar, Telangana, India
E-mail: drsrilathajkp@yahoo.com
ABSTRACT- Background:
Rh D negative blood in mothers carrying Rh D positive fetus is usually
associated with the hemolytic disease of the newborn. Because affected neonates
may develop severe hyperbilirubinemia, early identification of at-risk neonates
is critically important. We hypothesized that the uses of the direct antibody
test (DAT) help us in identifying those neonates who meet the treatment
criteria for hyperbilirubinemia. This study aimed to review the incidence of
positive DAT in cord blood samples at Government General Hospital,
Mahabubnagar, Telangana, India.
Methods:
We
retrospectively reviewed all results of Direct Anti Globulin (DAT) for two
years from January 2019 to December 2020, which was routinely performed in cord
blood samples. Direct Anti Globulin (DAT) test was performed on 854 cord blood
samples using standard procedures.
Results:
29/854(3.39%) of the cord blood samples were DAT positive. In this DAT positive
was seen in 13/29 (44.82%) of O positive blood group; 7/29(24.13%) of A
positive blood group; 9/29(31.03%) of B positive blood group and we did not
encounter any DAT positive cases in AB positive blood group.
Conclusion:
Due
to DAT positivity in cord blood, for the benefit and protection of the neonates
and mother in India, there is a need to establish intervention programmes in
terms of neonatal screening and immunoprophylaxis.
Key
Words: Cord blood, Direct antiglobulin test, Hemolytic
disease of the newborn, Hyperbilirubinemia, Rh-negative mothers
INTRODUCTION- The direct antiglobulin test (DAT), also known as the
Direct Coombs' test is a screen for non-agglutinating antibodies present on an
individual's red cells. The principle of the test was described by Moreschi in
1908, and it was rediscovered by Coombs et
al. [1] 1945. When
a pregnant woman's serum contains a clinically significant immunoglobulin G (IgG)
antibody directed against an antigen present on the red cells of her unborn
child, placental transfer of the antibody into the fetal circulation may
destroy fetal red cells resulting in Hemolytic Disease of the Newborn (HDN). At
its worst, the anaemia may result in hydrops fetalis-gross oedema and heart
failure leading to fetal death.
Neonatal hyperbilirubinemia places the infant at risk
of neurological damage. Phototherapy converts bilirubin to an excretable
photoproduct. [1,2] The indications for phototherapy and exchange
transfusion are determined by the serum bilirubin, age/gestation and
bodyweight.
The Rhesus negative group refers to the
genotype in which the three major genes (CDE) are absent. Thus, the genotype of
the Rhesus negative individual is ‘CDE’ [2]. Sensitization of
Rh-negative mother occurs during childbirth when the mother's bloodstream
penetrates a certain amount of Rh-positive child erythrocytes. Erythrocytes, as
a foreign substance to the mother, encourage her body to begin to produce Rh
antibodies. Therefore, the second and other pregnancy can have complications
related to maternal Rh antibodies to Rh-positive fetal red blood cells. Their
appearance in the circulation is also possible after amniocentesis, spontaneous
or induced abortion, cordocentesis, chorionic villus sampling, ruptured ectopic
pregnancy and blunt trauma to the abdomen [3,4]. An Rh-negative
mother after sensitized produces anti-Rh IgG antibodies that cross the placenta
and the risk factors for antibody production has been reported to include
second and later pregnancies, feto-maternal incompatibility in the ABO system,
paternal zygosity, maternal toxaemia and antigen load.[5] Direct antiglobulin test (DAT) determination
allows us to identify the presence of anti erythrocyte antibodies of IgG
isotope, originating in maternal serum on the surface of the erythrocytes of
the fetus or newborn. [6-8]
Recommendations
by The British Committee for Standardization in Haematology (BCSH) says that
infant born to Rh D negative mothers should have umbilical cord blood ABO and
Rh tested [9,10]. If the baby is Rh D positive, maternal samples
should then be taken for assessment of feto-maternal haemorrhage so that
adequate anti-RhD immunoprophylaxis can be given to the mother. The American
Academy of Pediatrics (AAP) revised its recommendations regarding the
identification and management of neonatal jaundice in 2004. In those
guidelines, the AAP recommends "all pregnant women should be tested for
ABO and Rh-D blood types and have a serum screen for unusual isoimmune
antibodies." The
definition of meeting the treatment criteria for hyperbilirubinemia was
attained if any of the three scenarios occurred during the 1st week of life:
(1) Neonate met the criteria for phototherapy based on AAP guidelines by having
any Total Serum Bilirubin (TsB) level above threshold for phototherapy based on
age since birth, gestational age, and presence of neurotoxicity risk factors;
(2) Received intravenous immunoglobulin (IVIG) for what was deemed severe HDN
by the attending physician; (3) Received a double volume exchange transfusion.
[11]
Determination of blood type in ABO Rh D
negative pregnant women allows reasonable precautions which limit the risk to
the fetus. Erythroblastosis is a very serious medical condition for about 4000
babies a year. In 15% of cases of babies die before birth. Those who survive
may suffer from jaundice, which leads to deaf-muteness, speech disturbances,
cerebral palsy and mental retardation. The use of the direct antiglobulin test
(DAT) would be of high predictive value in identifying those neonates most
likely to meet treatment criteria for hyperbilirubinemia.[3,4]
MATERIALS
AND METHODS
Data
Collection- Data of women visiting for antenatal
care at Government General Hospital, Mahabubnagar, from January 2019 to
December 2020, for deliveries were collected from records. Blood group details
of the patients visited for antenatal care during each month of the year were
noted down. The number of candidates belonging to the A, B, AB and O groups was
consolidated. Rh factor details of the patients were also consolidated.
Inclusion
Criteria- A total of 895 healthy Rhesus negative pregnant
mothers, who attended the antenatal clinic, were included in the study. We
retrospectively reviewed all results of DAT, which was routinely performed in
cord blood samples of all infants born to Rh-negative mothers between January
2019 to December 2020. During this period, a total of 854 cord blood samples
were analyzed.
Exclusion
Criteria- Rh positive pregnant mothers, who attended the
antenatal clinic between January 2019 to December 2020 were excluded from the
study.
Slide technique- Hemagglutination was used for the
determination of ABO blood grouping. For the direct phase, commercial reagents
were used that contained monoclonal antibodies with anti-A, anti-B and anti-AB
specificity (Tulip Diagnostics private limited).
A drop of blood from each volunteer was placed on a glass slide in three places. A drop of each of the antisera A, B and D was added and mixed with each blood sample, with the aid of glass rods. Monoclonal blood grouping antibodies, the in-vitro diagnostic reagent of Tulip Diagnostics private limited are used. Then, the mixture was rocked gently for 60 seconds to observe for agglutination. The results of agglutination were recorded immediately after mixing. The agglutination in blood drop A was considered as group A and agglutination in blood drop B as group B. The agglutination in both drops was considered as group AB, and if both blood drops were not agglutinated, it was considered as group O. The agglutination in rhesus blood drop was considered as rhesus positive and non-agglutination as rhesus negative.[12]
After delivery of newborn DAT was
performed by using whole blood samples obtained by a puncture of the umbilical
vessel at the end of the placenta immediately. The umbilical cord was clamped
at the end attached to the neonate. Another cord clamp was placed 8 to 10
inches away from the first. The isolated section was cut and the fetal blood
sample was collected into an ethylene diamine tetraacetic acid (EDTA)
container. The bottle was labelled with patient identity including name and
hospital number. Immediately at 4000 rpm for 2-3 minutes the umbilical cord
blood was centrifuged to enable the red cells to sediment. Supernatant plasma
was removed leaving the fetal red cells in the tube.
Direct Antiglobulin Test (DAT) on cord blood was performed by
taking two tubes (12×75 mm) which were labelled
‘test’ and ‘control’ and a drop of a 3% suspension of cord blood added to them.
The cells in the tube were washed three times with saline and the saline was
completely decanted after the last wash. To the test tube labelled ‘test’ was
added 2 drops of Anti Human Globulin (AHG) reagent (Monoclonal anti-IgG Tulip Diagnostics
private limited) while 2 drops of 3% of bovine albumin (Tulip Diagnostics
private limited) was added to the tube labelled ‘control’.
Both tubes were centrifuged at 3000 rpm for 15 to 20 seconds. Following
centrifugation, the cell pellet was completely re-suspended by gentle tipping
and rolling of the tube. The macroscopical examination was done and
agglutination was scored. In doubtful cases, the presence or absence of
agglutination was confirmed with low power magnification. For control, to all
negative tubes was added 1 drop of control cells weakly sensitized with IgG, it
was mixed, centrifuged and observed for agglutination. A mixed field weakly
positive reaction at this stage indicates that the AHG had been added to the
tube and it was still reactive. All reactive results at this stage were
considered valid. The
rate of alloimmunization in vivo
among Rh D negative mothers has not been determined.
Statistical Analysis- The prevalence in this study was determined from the
proposition of individuals and the total study population and is expressed as a
percentage. Descriptive statistics of variable and other characteristics of the
sampled population were calculated and tabulated are expressed as percentages.
All the statistical analysis was done using the Statistical Package for Social
Sciences (SPSS) software package version 20.
Ethical
Approval- Ethical review and clearance were obtained from the
Hospital and Ethics Committee. Department protocol was via informed written
consent before data collection.
RESULTS- We found that a total of 20863 deliveries
were conducted from January 2019 to December 2020. We evaluated the ABO blood groups
of 895/20863 (4.58%) Rhesus D negative pregnant mothers in two years study and
out of 895 deliveries, 854 (95.41%) were Rh positive babies born to Rh-negative
mothers and 41(4.58%) were Rh negative babies born to Rh-negative mothers as
shown in Fig. 1 and were excluded from the study. DAT positive cases were seen
in 29/854 (3.39%) of cord blood samples and DAT negative cases are seen in
825/854 (96.6%) in Government General Hospital,
Mahabubnagar
as shows in Fig. 2.
Fig.
1: Distribution of total Rh positive and Rh negative babies born in Government
General Hospital, Mahabubnagar, from January 2019 to
December 2020
Fig.
2:
Relative percentage of DAT positive and negative cases at Government General
Hospital, Mahabubnagar
Of 854 Rh-positive babies
303/854 (35.48%) babies are of blood group O, 216/854 (25.29%) group A, 254/854
(29.74%) group B and 81/854 (9.48%) group AB as shown in Fig. 3.
In this DAT positive was seen in 13/29(44.82%)
of O positive blood group; 7/29(24.13%) of A positive blood group;
29(31.03%) of B positive blood group and we did not encounter any DAT positive
cases in AB positive blood group as shown in Fig. 4. In this study, it is shown
that majority of DAT positive cases were seen in the O positive blood group,
which is shown in Table 1.
Fig.
3:
Total distribution of blood groups in positive blood group babies born in
Government General Hospital, Mahabubnagar from January 2019 to December 2020
Fig.
4: Relative distribution of
positive and negative Direct Antiglobulin Test (DAT) in individual blood group
Table 1: Prevalence of Positive DAT among Rh-Positive
Babies Born to Rh Negative mothers in Government General Hospital, Mahabubnagar
Direct Antiglobin Test
Positive In Positive Blood Group |
Frequency |
Percentage (%) |
A
Rh D Positive |
7 |
24.13 |
B
Rh D Positive |
9 |
31.03 |
O
Rh D Positive |
13 |
44.82 |
AB
Rh D Positive |
0 |
0 |
Total |
29 |
100 |
Fig.
5: Distribution of the total
number of direct antiglobulin test positive cases monthly wise in the years
2019 and 2020
Table
2:
Distribution of DAT in Cord blood samples of Rh Positive Babies Born to Rh
Negative mothers in Government General Hospital, Mahabubnagar
Direct antiglobulin test |
Frequency |
Percentage (%) |
Negative |
825 |
96.60 |
Positive |
29 |
3.39 |
Total |
854 |
100 |
DISCUSSION-
The
knowledge of the blood groups and Rhesus factor is important in evolution,
related to diseases and environment, essential in blood transfusion, organ
transplantation, forensic pathology, anthropology and training ancestral relation
of human, and also helps to prevent complications due to Rhesus
incompatibility. [13-15] The prevalence of Rh D negative women in
this study is 4.58%. This is similar to previous studies done at Ibadan and
Abraka in Nigeria. [16,17] This rate shows a low frequency of Rh D
negative blood group system in this environment. This finding is similar to
that previously reported amongst African subjects, West Indians, and blacks in
Great Britain.[18]. The results are, however, different from those
reported from the Eastern highlands of Papua Guinea where the entire population
was reported to be 100% Rh D positive. [19] DAT is a useful tool in
the early prediction of jaundice or hyperbilirubinemia. In our study, 3.39% of
the prevalence rate of positive DAT was seen which is shown in Table 2.
1-9% of prevalence rate was reported by
Dinesh [1] and Cianciarullo [18] in White populations.
The incidence of a positive DAT in newborns was 3.39%, a percentage higher than
that (2.3%) reported by Dillon et al.
[24] but lower than that (3.5%) of Hershel et al. [20] Higher the prevalence rate of positive cord
blood DAT places women at higher risk of developing the hemolytic disease of
the newborn. 23% of infants with positive DAT require phototherapy was reported
by one study. [20] In another study of the infants receiving
phototherapy, 15.1% show a DAT positive [21]. Detection of positive
DAT on cord blood enables the newborn at risk of clinically relevant jaundice
to benefit from programs aimed at identifying risk or early detection of
various adverse clinical conditions such as hyperbilirubinemia, encephalopathy,
prolonged hospitalization, intense hemolytic anaemia or others. By inclusion of
DAT in neonatal screening allows the early intervention with prophylactic
phototherapy [22].
In vivo phenomenon associated with a
positive DAT could be due to several factors such as transfusion, drug-induced
and autoimmune hemolytic anaemia [23]. For women with a high
percentage having received a previous blood transfusion and with multiple
pregnancies, it is possible that alloantibodies in the recipient Rh D negative
mothers must have sensitized their red cells to cause in vivo sensitization.
DAT results in our study showed a
promising ability to predict the treatment criteria for hyperbilirubinemia in
high-risk neonates. Positive DAT result close to birth may alert the clinician
to apply more aggressive management techniques (e.g., frequent laboratory
draws, prophylactic phototherapy, etc.), while a negative result may allow
clinicians to adopt a more conservative approach and spare the neonate from
overaggressive laboratory investigations or other invasive modalities. Early
risk-stratification using DAT results obtained early, can be one of the many
tools available to clinicians in identifying and efficiently managing patients
at risk for severe hyperbilirubinemia.
CONCLUSIONS- DAT being a useful tool in the early
prediction of hyperbilirubinemia and if included as a neonatal screening test
will help in identifying at-risk neonates. Maternal
screening tests and a careful look at the history of drug administration during
pregnancy could identify other important but rare causes of DAT positivity. Inclusion of DAT in neonatal screening
allows early and immediate intervention with prophylactic phototherapy of
at-risk neonates.
The use of
universal DAT testing along with maternal isoimmunization will lead to further
investigation and discussion to determine its efficacy and cost-effectiveness
in predicting severe hyperbilirubinemia.
CONTRIBUTION OF AUTHORS
Research
concept- Dr. Srilatha Bollipogu
Research
design- Dr. S.S.S.Quadri
Supervision- Dr.
S.S.S.Quadri
Materials- Dr. Srilatha
Bollipogu
Data
collection- Dr. Srilatha Bollipogu
Data
analysis and Interpretation- Dr. Srilatha Bollipogu
Literature
search- Dr. S. Srujana
Writing
article- Dr. S. Srujana
Critical
review- Dr.S. S.S.Quadri
Article
editing- Dr.S. S.S.Quadri
Final approval- Dr. Naval Kishore
REFERENCES
1. Dorothy D. Review of positive direct antiglobulin
tests found cord blood sampling. J Paediatr Child Health, 2005; 41(9-10):
504-07.
2. Mollison
PL, Engelfriet CP, Contreras M. Blood Transfusion in Clinical Medicine.
(10thedn), Oxford, UK: Blackwell Sci., 1993; 543–91.
3.
Sebija I. Occurrence of ABO and RhD
Incompatibility with Rh Negative Mothers, Mater Sociomed. 2013; 25(4): 255-58. doi: 10.5455/msm.2013.25.255-258.
4.
Phibbs, Roderic H. Hemolytic Disease of
the Newborn (Erythroblastosis Fetalis). In: Rudolph’s Pedriatic, ed. Abraham,
M. Rudolph, et al. Stamford: Appleton & Lange, 1996.
5.
James RM, McGuire W, Smith DP. The
investigation of infants with RhD negative mothers, can we safely omit the
umbilical cord blood direct antiglobulin test. Arch Dis Child Fetal Neonatal
Ed., 2011; 96: F301-304.
6. Dittmar
K, Procter JL, Cipolone K, Njoroge JM, Miller J, et al. Comparison of DATs
using traditional tube agglutination to gel column and affinity column
procedures. Transfusion, 2001: 41: 1258-62.
7.
Novaretti MC, Jens E, Pagliarini T,
Bonifacio SL, Dorlhiac-Llacer PE, et al. Comparison of conventional tube test
technique and gel microcolumn assay for direct antiglobulin test: a large
study. J Clin Lab Anal., 2004; 18: 255-58. doi:
10.1002/jcla.20033.
8.
Meberg A, Johansen KB. Screening for
neonatal hyperbilirubinaemia and ABO alloimmunization at the time of testing
for phenylketonuria and congenital hypothyreosis. Acta Paediatr., 1998; 87:
1269-74.
9.
Jeremiah ZA, Pwana FE, Mgbere O.
Positive Cord Blood Direct Anti Globulin Test (DAT) is Strongly Associated with
Parity and Maternal Age among Rh Negative Mothers in Maiduguri, Nigeria. J
Blood Disord Transfus., 2013; S10: 002. doi: 10.4172/2155-9864.S10-002.
10. Gooch
A, Parker J, Wray J, Qureshi H. British Committee for Standards in Haematology
Blood Transfusion Task Force, Guideline for blood grouping and antibody testing
in pregnancy. Transfus Med., 2007; 17: 252-62. doi:
10.1111/j.1365-3148.2007.00767.x.
11. American
Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of
hyperbilirubinemia in the newborn infant 35 or more weeks of gestation.
Pediatr., 2004; 114(1): 297–316.
12. Sultana
R, Yousuf R, Rahman Z, Helali AM, Mustafa S, et al. Study of ABO and RH-D Blood
Groups among the common people of capital city of Bangladesh. Int J Pharm Pharm
Sci., 2013; 5(3): 814-16.
13. Khurshid
B, Naz M, Hassan M, Mabood SF. Frequency of ABO and Rh (D) blood groups in
district Swabi, NWFP, Pakistan. J. Sci. Tech. Univ. Peshawar, 1992; 16: 5-6.
14. Bamidele
O, Arokoyo DS, Akinbola AO. Distribution of ABO and rhesus blood groups among
medical students in Bowen University, Iwo, Nigeria. Ann Biol Res., 2013; 4
(11): 1-6.
15. Zaman et al. Study of ABO and Rh-D blood group
among the common people of Chittagong city corporation area of Bangladesh. J
Public Health Epidemiol., 2015; 7(9): 305-10.
16. Onwukeme
KE. Blood group distribution in blood donors in Nigeria population. Niger J
Physiol Sci., 1990; 6: 67- 70. doi: 10.23937/2377-9004/1410034.
17. Kotila
TR, Odukogbe AA, Okunola MA, Olayemi O, Obisesan KA. The pregnant Rhesus
negative Nigerian women. Nigerian J Clinical Practice, 2005; 15: 4.
18. Cianciarullo
MA, Ceccon ME, Vaz Fv. Prevalence of immunohematologic tests at birth and the
incidence of hemolytic disease in the newborn. Rev Assoc Med Bras., 2003; 49(1): pp. 45-53. doi: 10.1590/S0104-42302003000100033.
19. Prevalence
of rhesus negativity among pregnant women, International Journal of Research in
Medical Sciences Nagamuthu EA et al. Int J Res Med Sci., 2016; 4(8): 3305-09. doi:
http://dx.doi.org/10.18203/2320-6012.ijrms20162284.
20. Herschel
M, Karrison T, Wen M, Caldarelli L, Baron B. Isoimmunization is unlikely to be
the cause of hemolysis in ABO-incompatible but direct antiglobulin
test-negativeneonates. . doi:
21. Kaplan
M, Kaplan E, Hammerman C, Algur N, Bromiker R, et al. Post-phototherapy
neonatal bilirubin rebound: a potential cause of significant
hyperbilirubinaemia. Arch Dis Child, 2006; 91: 31-34. doi: 10.1136/adc.2005.081224.
22.
Adeniji AA, Fuller I, Dale T, Lindow SW.
Should we continue screening rhesus D positive women for the development of
atypical antibodies in late pregnancy? J Matern Fetal Neonatal Med., 2007; 20:
Pages 59-61. doi:
10.1080/14767050601123317.
23. Baptista-Gonzaliz
H, Hemandez-Martinez A, Galindo-Delgado P, SantamariaHernandez C,
Rosenfield-Mann F. Usefulness of direct antiglobulin test in neonatal
screening. Bol Med Hosp Infant Max., 2009; 66: 502-10.
24. Dillon
A, Chaudhari T, Crispin P, Shadbolt B, Kent A. Has anti-D prophylaxis increased
the rate of positive direct antiglobulin test results and can the direct
antiglobulin test predict need for phototherapy in Rh/ABO incompatibility? J
Paediatr Child Health, 2011; 47(1-2): 40-43. doi: 10.1111/j.1440-1754.2010.01888.x.