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 Table of Contents  
Year : 2021  |  Volume : 11  |  Issue : 2  |  Page : 92-96  

Correlation between CD 34 and CD 68 expression in placental malaria with maternal anemia

1 Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
2 Department of Anatomical Pathology, Labuha Hospital, South Halmahera, North Maluku, Indonesia

Date of Submission30-Sep-2020
Date of Decision18-May-2021
Date of Acceptance14-Jul-2021
Date of Web Publication20-Oct-2021

Correspondence Address:
Primariadewi Rustamadji
Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya 6 Jakarta
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tp.TP_108_20

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Background: Malaria is the second most life-threatening infectious disease in Indonesia, causing approximately 1–3 million deaths annually. Histopathologic studies assessing CD 68 and CD 34 protein expression in placental malaria and its association with maternal anemia are essential to determine the prognosis of malaria in pregnancy.
Materials and Methods: This cross-sectional study was carried out in 2017. Thirty biopsy samples of human placental tissue were obtained from Timika and Sumba, and ten normal biopsy samples were taken from the Pathological Anatomy Department of Cipto Mangunkusumo General Hospital as comparisons. CD 34 and CD 68 protein expressions were determined using immunohistochemistry, and the resulting data were analyzed using SPSS.
Results: Average hemoglobin (Hb) level was 9.5 mg/dL, 11.5 mg/dL, and 9.9 mg/dL in acute infection, chronic infection, and latent infection, respectively. A positive correlation was found between CD 68 protein expression and maternal Hb level. No correlation was found between CD34 expression and maternal anemia.
Conclusions: CD 68 expression in placental tissue biopsy from Timika and Sumba residents with placental malaria was shown to be positively correlated with maternal anemia. Immunohistochemical examination of CD 68 may play a role in the early diagnosis of malaria.

Keywords: CD 34, CD 68, maternal anemia, placental malaria

How to cite this article:
Rustamadji P, Takbir M, Wuyung PE, Kusmardi K, Wiyarta E. Correlation between CD 34 and CD 68 expression in placental malaria with maternal anemia. Trop Parasitol 2021;11:92-6

How to cite this URL:
Rustamadji P, Takbir M, Wuyung PE, Kusmardi K, Wiyarta E. Correlation between CD 34 and CD 68 expression in placental malaria with maternal anemia. Trop Parasitol [serial online] 2021 [cited 2023 Feb 9];11:92-6. Available from: https://www.tropicalparasitology.org/text.asp?2021/11/2/92/328689

   Introduction Top

Malaria is the second most life-threatening infectious disease in Indonesia following tuberculosis. Approximately 350–500 million people were infected, and 1–3 million died annually.[1] The World Health Organization reported that globally malaria causes mortality for over 10,000 pregnant women and 200,000 neonates every year.[2] Pregnancy exacerbates malaria, and malaria progression threatens pregnancy and fetal well-being. Pregnant women contracting malaria are at increased risk for anemia, cerebral malaria, lung edema, kidney failure, and death,[3],[4] while the fetus may be subjected to intrauterine growth and developmental abnormality, resulting in abortion, premature birth, low birth weight, and fetal death.[5],[6]

Plasmodium falciparum, the most common cause of malaria infection, causes 95% of malaria mortality.[7] Sequestration of infected red blood cells in the placenta was one of the contributing factors in the progression of malaria in pregnancy. To make things worse, pregnant women are more vulnerable to malaria due to the physiological immunodeficiency during pregnancy.[8],[9] Histologic examination performed on biopsy of placental tissue is the gold standard in diagnosing placental malaria, with higher sensitivity compared to peripheral or placental blood smears.[10] Parasite sequestration induces maternal inflammatory response of monocyte and macrophage migration towards the intervillous space, where the nitrite oxide released by macrophages may further the progression of malaria in the placenta.[11] Previous researchers, such as Ordi et al., Menendez et al., and Rogerson et al., demonstrated that this process increases the risk of low birth weight[12],[13],[14] as well as maternal anemia.[14] Histologically, the placenta infected by P. falciparum demonstrates increased inflammation in the intervillous space hallmarked by the accumulation of monocytes and macrophages, which can be detected using the CD 68 antibody.[14] Immunohistochemistry can be utilized to determine maternal immune response against malaria during pregnancy.

Endometrial development and changes in pregnancy rely on angiogenesis. Immunohistochemistry performed to evaluate CD 34 expression can exhibit placental angiogenesis and vascularization. As not many investigations have been conducted on CD 68 and CD 34 protein expression in malaria, we decided to further explore the correlation between CD 68 and CD 34 expression and maternal anemia by performing histological examinations. We are hopeful that our study may contribute to the earlier diagnosis of malaria during pregnancy.

   Materials and Methods Top

Study design and data collection

This cross-sectional study was carried out at the Pathological Anatomy Department, Cipto Mangunkusumo General Hospital, Jakarta, and the Eijkman Institute, Jakarta, throughout February–March 2017. Thirty biopsy samples of human placental tissue which are positive for malaria obtained from Timika and Sumba (ten acute infection, ten chronic infection, and ten latent infection biopsy samples), as well as ten normal biopsy specimens from the Pathological Anatomy Department, were observed in the study. Samples were collected using consecutive sampling. Inclusion criteria include placental tissue biopsy positive for acute, chronic, or latent malaria infection stored in the Eijkman Institute archive. Exclusion criteria include disqualified slides or paraffin block and incomplete data in the Eijkman Institute.

Operational definition

Tissue biopsy was classified as noninfected when no parasite of hemozoin pigment was found in the placenta. Acute infection was defined when a parasite with no or minimal pigment deposit (1–2 focus of pigment under × 400 total magnification) was observed. Chronic infection was defined when parasites and numerous pigment deposits (pigment observed under total magnification of × 100–200) were observed. If pigment deposits without parasites were found in the biopsy sample, the case was defined as latent. Anemia was defined as a hemoglobin (Hb) level of <11 g/dL.

Immunohistochemistry staining and histopathologic observation

Tissue was cut 4 μ thick, deparaffinized with xylol (Merk, Jakarta, Indonesia), and processed with dual endogenous enzyme block, which is H2O2 0.5% (Merk, Jakarta, Indonesia) and ethanol 95% (Merk, Jakarta, Indonesia) followed by heat-induced epitope retrieval. Preparation was then incubated with primary and secondary antibodies (labeled polymer horseradish peroxidase) for CD 34 dan CD68 (Merk, Jakarta, Indonesia). The preparation was further assessed for histological observation.

The main focus of histological observation on the preparation is to assess the focus of inflammation. Inflammation, defined as the number of monocytes or macrophages observed in the intervillous space stained by CD 68 antibody, was categorized as minimal (no intervillous inflammation), moderate (scattered monocytes in the intervillous space), and massive (intervillous space is dense with monocyte). Positive staining appears as brownish decoloration in the monocyte or macrophage cytoplasm. Pigment deposit was separated into three categories: the first category is when ≤10% of pigment deposit was discovered in the intervillous space, the second category 10%–40%, and the third category ≥40%, each observed under 10 high-power fields (HPFs) using both standard (Olympus C × 21) and polarized microscopes. The intensity of CD 68 was categorized into 0 (negative), 1 (weak), 2 (intermediate), and 3 (strong).[15] The amount of monocyte and macrophage in 5 HPF was calculated and recorded as mean and standard deviation. The distribution of CD 34 protein expression, observed as staining of blood vessel endothelium by CD 34 antibody, was grouped into five categories: 0 (negative), 1 (<20%), 2 (20%–50%), 3 (51%–80%), and 4 (>80%). CD 34 was calculated using the Immunoreactivity Scoring System (IRS).[16] The score was further categorized into negative (0), weak (1–5), intermediate (6–9), and strong (>9). The observation was performed by two observers in the region with the highest number of expressions.

Statistical analysis

CD 34 and CD 68 protein expression were recorded as categorized data using Microsoft Excel (Microsoft Corp, Redmond, WA, USA). The Pearson correlation between both CD34 and 68 to Hb concentration was carried out using the Statistical Package for Social Sciences/SPSS version 20 (IBM Corp, Armonk, NY, USA) program for data analysis.

Ethical clearance

This research had obtained ethical clearance from the Health Research Ethics Committee, Faculty of Medicine University of Indonesia–Cipto Mangunkusumo General Hospital on October 30, 2017, numbered ND-710/UN2.F1/ETIK/PPM.00.02/2019. The study was undertaken with the understanding and written consent of each patient, and that the study conforms with the Code of Ethics of the World Medical Association (Declaration of Helsinki).[17]

   Results Top

Acute placental malaria was found in patients 17–33 years old, with an average age of 24 years old [Table 1]. Chronic and latent infection was found in older patients, 29 years old and 26 years old, respectively. Average Hb level among patients was 9.5 mg/dL in acute infection, 11.5 mg/dL in chronic infection, and 9.9 mg/dL in latent infection. CD 34 IRS score average was 7, 9, and 9 for acute, chronic, and latent infection, whereas the average for CD 68 was 1.8, 0.8, and 0.7 for acute, chronic, and latent infection, respectively [Figure 1], [Figure 2], [Figure 3].
Figure 1: Monocyte (red arrow) scattered around the intervillous space of the placenta (black arrow) with minimal inflammation, total magnification ×400 (a) and ×1000 with hematoxylin and eosin staining (b)

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Figure 2: Immunohistochemistry of CD 68 in the intervillous space of the placenta

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Figure 3: Immunohistochemistry of CD 34 in acute infection (a and b), chronic infection (c and d), and latent infection (e and f) of placental malaria

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Table 1: Distribution of age, hemoglobin level, cluster of differentiation 34, and cluster of differentiation 68 in acute, chronic, and latent malaria infection in samples

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Pearson correlation test was performed, and a positive correlation was shown between CD 68 expression and Hb level (P = 0.03, confidence interval [CI] =95). Unfortunately, no correlation between CD 34 IRS score and Hb level was found (P = 0.85, CI = 95).

   Discussion Top

Studies on the expression of CD 34 and CD 68 in malaria are scant, despite histopathological examination being the gold standard in malaria diagnosis, including placental malaria. Our study was the first to observe CD 34 and CD 68 expression in placental malaria occurring in malaria-endemic Timika and Sumba, eastern regions of Indonesia. Malaria infection during pregnancy brings about numerous morbidity and mortality to the pregnant woman and the unborn child. Severe anemia was associated with ethnic groups, low economic status, and HIV. We found that maternal Hb level was correlated to the CD 68 expression, although no correlation was found with CD 34 expression.

Quintero et al., in 2011, conducted US-based research, which demonstrated that malaria-related anemia was less common compared to Indonesia.[18] Less incidence of malaria cases and better health services are possible contributing factors to this finding. Ordi et al. carried out in Tanzania, another malaria-endemic area, showed that CD 68 expression was expressed in a higher amount in chronic malaria infection (10.6 ± 9.0) compared to other types of infection.[12] The researcher explained that only minimal inflammation occurs in acute infection, whereas a stronger, more visible inflammatory reaction was observed in the intervillous space in the chronic inflammation. In our research, a more aggressive inflammation was found during the acute infection, hence the more pronounced CD 68 expression in acute infection.

Another study by Shulman et al. in Kenya revealed that active malaria infection was associated with anemia, while chronic infection contributed to low birth weight.[19] Similarly, we found that placental malaria was correlated with maternal anemia. Research by Rogerson et al. was able to show that malaria infection was related to maternal Hb level.[14] Anemia was more common in chronic malaria infection (53%) compared to acute infection (7.7%) and latent infection (20.9%). Diagnosis of anemia was often made during antenatal care. The prevalence of severe anemia at the gestation age of 34 weeks was 17.5% for primigravida and 17.3% for multigravida pregnant women, thus necessitating intermittent antenatal antimalarial treatment in malaria-endemic areas. Our results differ with this study as we found that maternal Hb was lowest during acute infection, which may be due to a smaller number of samples and more robust inflammation occurring during the acute stage.

We did not find any correlation between CD 34 expression and maternal Hb. This finding is dissimilar with the results of a study by Panichakul et al. in 2012, which demonstrated that Plasmodium vivax infection might directly inhibit growth and differentiation of erythroid progenitor, leading to anemia.[20] Another recent research by Moeller et al. compared placental vascularization between malaria-infected and healthy Tanzanian women.[21] This study involved 138 patients and exhibited that the placenta infected with malaria before the gestational age of 15 weeks experienced decreased villi volume but the increased area of blood vessel diffusion. In women infected with malaria in the later term, increased vascularization was found, predictably due to a compensatory mechanism.

   conclusion Top

As it is quite challenging to locate patients with placental malaria, our study involved a small number of patients, which is a study limitation that might affect our results. Therefore, we suggest that larger studies assessing the correlation between placental malaria and maternal anemia, birth weight, and parity are carried out in future.

Financial support and sponsorship

This study was financially supported by Anatomical Pathology Department, Universitas Indonesia.

Conflicts of interest

There are no conflicts of interest.

   References Top

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Uneke CJ. Impact of placental Plasmodium falciparum malaria on pregnancy and perinatal outcome in sub-Saharan Africa: I: Introduction to placental malaria. Yale J Biol Med 2007;80:39-50.  Back to cited text no. 4
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Rogerson SJ, Pollina E, Getachew A, Tadesse E, Lema VM, Molyneux ME. Placental monocyte infiltrates in response to Plasmodium falciparum malaria infection and their association with adverse pregnancy outcomes. Am J Trop Med Hyg 2003;68:115-9.  Back to cited text no. 14
Rustamadji P, Wiyarta E, Bethania KA, Kusmardi K. Potential of AKT2 expression as a predictor of lymph-node metastasis in invasive breast carcinoma of no special type. J Pathol Transl Med 2021;55:271-8.  Back to cited text no. 15
Meyerholz DK, Beck AP. Principles and approaches for reproducible scoring of tissue stains in research. Lab Invest 2018;98:844-55.  Back to cited text no. 16
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Quintero JP, Siqueira AM, Tobón A, Blair S, Moreno A, Arévalo-Herrera M, et al. Malaria-related anaemia: A Latin American perspective. Mem Inst Oswaldo Cruz 2011;106 Suppl 1:91-104.  Back to cited text no. 18
Shulman CE, Marshall T, Dorman EK, Bulmer JN, Cutts F, Peshu N, et al. Malaria in pregnancy: Adverse effects on haemoglobin levels and birthweight in primigravidae and multigravidae. Trop Med Int Health 2001;6:770-8.  Back to cited text no. 19
Panichakul T, Payuhakrit W, Panburana P, Wongborisuth C, Hongeng S, Udomsangpetch R. Suppression of erythroid development in vitro by Plasmodium vivax. Malar J 2012;11:173.  Back to cited text no. 20
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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