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Association of Heat Stable Placental Alkaline Phosphatase with Severe Preeclampsia Among Pregnant Women at The Federal Teaching Hospital Abakaliki, Southeast, Nigeria.

 

Johnbosco Mamah1, Chichetaram Otu1, Darlington-Peter Chibuzor Ugoji1*, 

Christian Mgbafulu1, Robinson Onoh1, Odidika Ugochukwu Joannes UMEORA1, 

Ortuanya Kelvin Emeka2

 

1Department of Obstetrics and Gynaecology, Alex Ekwueme Federal University Teaching Hospital Abakaliki, Ebonyi State, Nigeria

2Department of Obstetrics and Gynaecology, Enugu State University Teaching Hospital, Parklane, Enugu State, Nigeria

 

Abstract

Background:    So far, there is no accurate diagnostic or predictive investigation for pre-eclempsia. Placental alkaline phosphatase (PLAP) in preeclamptic pregnancies appears to show a trend which may be useful in prognostication.The aim of this study was to evaluate the correlation of placental alkaline phosphatase with severe preeclampsia and pregnancy outcome. Methodology: This was a case-controlled study conducted at the Federal Teaching Hospital Abakaliki. A total of 214 pregnant women consisting of 107 women in the study group and 107 women in the control group. Each patient with preeclampsia was matched with a healthy for gestational age and parity. Data was analyzed using SPSS. Findings were presented in tables using descriptive statistics. Relationships were assessed using Pearson’s correlation and a p-value < 0.05 was statistically significant. Results: The mean values of serum PLAP for the study and control groups were respectively 126.5IU/L and 51.1IU/l, and this was statistically significant (p-value < 0.01). The association between serum PLAP and neonatal birth weight was not statistically significant. The performance of PLAP showed a sensitivity and specificity of 80.0% and 82.0% respectively.

Conclusion: There result showed that Serum PLAP maybe a potential use as an early marker of disease onset and prognostication.

 

Keywords: Adverse pregnancy outcome, correlation, Placental alkaline phosphatase, Severe preeclampsia


*Corresponding Author

 darlingtonpeter2012@gmail.com

Phone number: +2348068748644

 

 

Introduction 

 

Pregnancy is a physiological state associated with significant alteration in virtually all the systems in the body1. In an uncomplicated pregnancy, the parturient tolerates these changes after which there is reversal to the pre-pregnancy state in most cases within six weeks of delivery2,3. Preeclampsia complicates 5-10% of all pregnancies4,5. Hospital based studies in Nigeria report an incidence of between 1.2-16%6-8. Preeclampsia is reported to be the second most common cause of iatrogenic prematurity and perinatal deaths. They account for about 50,000-75,000 of maternal deaths per annum, but this figure might be an underestimation of the actual disease burden because of widespread underreporting in developing countries9. The high burden of preeclampsia and eclampsia in developing countries is thought to be due to limited use of magnesium sulphate, lack of well-defined protocols for case management, poverty, and poor health seeking behaviour10

Understanding the clinical characteristics and maternal biomarkers associated with normal and abnormal pregnancy remains a major goal of obstetrics. It is impossible to manage pregnancy and undertake appropriate interventions without such knowledge. In preeclamptic women, the serum placenta (heat stable) alkaline phosphatase (PLAP) has been reported to constitute about 55% of total alkaline phosphatase11. Numerous studies have reported that variations in serum levels of PLAP may be associated with disease progression in preeclampsia, but these findings are inconsistent11-17. In evaluating a patient for pre-eclampsia, several tests have been extensively studied to screen for and possibly predict onset, but no single candidate test has shown unequivocal accuracy in this regard2,3. Since the placenta is a rich source of enzymes and is at the centre of the disease pathogenesis, assessment of placental function by evaluating placental specific enzymes may play a role in detection and management of the disease.

Placental (heat-stable) alkaline phosphatase (PLAP) is an isoenzyme of serum total alkaline phosphatase which is specific to the placenta18. Total Serum placental alkaline phosphatase begins to rise at about the 16th week of pregnancy and peaks at about 38 week’s gestation and is completely cleared during the puerperium7,12,13. In some pregnancies complicated by preeclampsia, there is a rise in serum PLAP (yes, there is a rise in all pregnancies but higher when compared, if there is tissue disruption like in placenta diseases e.g., pre-eclampsia and hepatic obstruction) which is thought to be due to the compensatory activities of syncytiotrophoblast cells of the placenta following ischaemic damage to placental tissue that occurs in preeclampsia. This attribute of the placenta may offer a window into understanding the pattern of disease progression using PLAP as a biomarker in disease monitoring and prognostication13,14 (justified by above statement). Some studies carried out outside Nigeria have reported an association between PLAP and preeclampsia. Only one study was identified in Northern Nigeria but none in the Southeast. Determination of this association using a cheap and effective biomarker like PLAP will help in the initiation of policies that will incorporate assessment of serum PLAP as a routine investigation in preeclampsia case management. In Abakaliki, the application of this parameter would be invaluable due to paucity of resources for the more advanced diagnostic tools, especially given that serum PLAP is easy to determine, affordable and tissue specific.

 

Methodology

 

Study design

This was a case-controlled study. The study group consisted of women diagnosed with preeclampsia while the control group consisted of healthy pregnant women matched for parity and gestational age. Deliveries included in the study were from 28weeks, which is the age of viability in our environment. Pregnant women diagnosed with preeclampsia were first evaluated at the antenatal clinic or the Emergency unit of the hospital from where they were admitted into the antenatal ward for stabilization and delivery depending on the disease severity and/or gestational age of the parturient. Patient recruitment followed a systematic sampling method where every 3rd patient was recruited.

The blood pressure was gotten using the Acuson® brand mercury sphygmomanometer with appropriate cuff size covering at least 2/3rd of the length of the patients left arm. Blood pressure was measured with the patient in reclined position. Before taking measurements, the patient is allowed 30 minutes rest. In taking the measurement, the cuff was inflated while simultaneously palpating the radial pulse. Inflation was continued for a further 20mmHg beyond the point where the radial pulse becomes impalpable. Pressure was slowly released at a pace of 2mmHg until the radial pulse becomes palpable again and this indicates the systolic blood pressure. The cuff is then re-inflated, and a stethoscope applied to the cubital fossa and the pressure slowly released as in the previous fashion. The phase 1 Korotkoff sound is recorded as the systolic blood pressure (SBP) while the phase 5 Korotkoff sound was recorded as the diastolic blood pressure (DBP) measurement. Severe hypertension was diagnosed if the patients’ blood pressure was recorded as systolic or diastolic blood pressure of equal to or more than 160mmgHg or 110mmHg respectively. Urinalysis was done by dip stick testing of clean catch midstream urine. The participants were given a wide bore clean universal bottle and asked to collect a clean catch midstream specimen of urine. Prior to taking the sample, the patients were asked to wash their perineum using clean water provided in the toilet. They were educated to stand astride, open the sample bottle, and collect a mid-stream specimen of urine. After specimen collection, urinalysis was performed to test for proteinuria. A proteinuria of 2+ or more was considered significant. Severe hypertension with significant proteinuria confirms a diagnosis of severe preeclampsia. 

After blood pressure measurement, a flexible tourniquet was applied about 10 cm above the ante-cubital fossa of either upper limb to make the veins distend with blood. The skin over the site for venipuncture was cleaned with a cotton wool soaked with 70% Alcohol and allowed to dry. A sterile 21G size hypodermic disposable needle was used to draw 5 ml of blood.  The blood collected was gently emptied into a sterile plain specimen bottle containing no anticoagulant and immediately sent to the chemical pathology laboratory for processing.

 

Ethical consideration

Ethical approval for this study was obtained from the Research and Ethics Committee of the Federal Teaching Hospital Abakaliki. 

Inclusion criteria

  1. Women who give an informed consent to participate in the study.
  2. Women diagnosed with preeclampsia at a gestational age of 28 weeks or more.
  3. Healthy normotensive pregnant women who met the matching criteria. 

 

Exclusion criteria

  1. Women with chronic medical diseases such as renal, liver or heart diseases
  2. Multiple pregnancies
  3. Gestational trophoblastic diseases.
  4. Urinary tract infection and malaria in pregnancy.
  5. Pregnant women with malignancy.
  6. Women who smoke.
  7. Anaemia in pregnancy.
  8. Over-weight and obese women

 

Blood Sample Analysis and Extraction of PLAP

Reagents were manufactured by Quimica Clinica Applicada (QCA) Laboratories limited, Spain20. It is stable to expiry when stored at +2 to +8oC.It had three components:

  1. Solution A: This is a ready-to-use 25ml solution of 65mM Chromogenic substrate (Phenolphthalein monophosphate in 7.8M of 2-amino-2-methyl-1-propanol at pH of 10.4).
  2. Solution B: Two colour developers (containing 0.1M phosphate, Na3PO4, buffer).
  3. Solution C: Standard. Ready-to-use.

At the laboratory, serum extraction was done by centrifugation at 8000 rpm for 5 minutes. The extracted serum was stored in a regulated freezer at -20oC until a total sample of 24 (12 for each of study and control groups) was reached and then analysed.

 

Technique

The Phenolphthalein Monophosphate Substrate Method (Chromogenic method) of estimation of placental alkaline phosphatase was used for this study. This method is based on the principle that serum alkaline phosphate (ALP) hydrolyzes a colourless substrate of Phenolphthalein Monophosphate giving rise to phosphoric acid and phenolphthalein which at an alkaline PH of 10.4 (using 2-Amino-2-methyl-1-propanol buffer) turns into a pink colored solution whose absorbance can photometrically be determined at 546nm wavelength21. The rate of increase in absorbance is directly proportional to the alkaline phosphatase activity in the serum (concentration).

To determine the level of placental alkaline phosphatase, 0.5ml of serum was put into a thin-walled glass tube and immersed in a thermostatically controlled water bath (Incubator) which was maintained at 65 0C for 30 minutes. PLAP remains stable at this temperature while alkaline phosphatases from other sources are denatured. The serum was cooled in an ice bath for 3 minutes before returning it to room temperature. To estimate the concentration of serum PLAP in the sample, a drop (0.04ml) of the chromogenic substrate was diluted in 1ml of deionized water and incubated at 37oC, and then 0.1ml of the cooled serum was added to the mixture and allowed to stand for 20 minutes. Thereafter, 5ml of the colour developer was added to the mixture and read spectrophotometrically. The value of serum PLAP was documented in the proforma.

 

Quality Control

To ensure precision within batch analysis, control sera were procured from QCA laboratories (Serriscann Normal and Seriscann Anormal)20 which were used to test for accuracy of each batch analysis. Also, each batch was analysed by the same laboratory scientist using reagents constituted from the same reagent pack.

 

Labour And Delivery                                            

For those who had vaginal delivery, their labour

was managed using the departmental protocol for management of preeclampsia. This entailed the use of partographs and Cardiotocographs for monitoring of labour events. Those that were delivered abdominally had the procedure performed by a senior registrar or consultant in the Obstetrics and Gynaecology department. All the deliveries were attended by the neonatology team. 

 

Post Delivery

 

Baby: Following the delivery of the baby, the umbilical cord stump left on all the neonates were reduced to a length of 5cm. This was to avoid over estimation of the neonatal birth weight. All the babies were weighed unclad, using the same weighing scale which was checked daily for zero error. 

 

Placenta: The placenta and membranes were severed from the umbilical cord at the point of attachment. The placenta was examined under clear running water and a bright light source to remove blood clots and examined for completeness. Thereafter, the placenta was placed in a bowl of known weight and weighed using a zero-error electronic weighing scale. The actual placental weight was determined by subtracting the weight of the empty bowl (predetermined) from the final weight of the bowl with placenta. The same weighing scale and bowl were used for all the patients.  

 

Outcome Measures

 

Primary/main outcome measures

Mean serum level of PLAP in preeclamptic and normotensive pregnant women.

 

Secondary outcome measures

  1. Mean neonatal and placental birth weights.
  2. Number of participants who suffered maternal and neonatal complications.
  3. Gestational age at delivery

 

Cofounders

  1. Maternal age
  2. Gestational age
  3. Parity

 

Data Collection and Analysis

The study lasted for six months between 1st of September, 2017 to 28th February, 2018. Samples were collected at delivery and matched.  A structured proforma was used to collect data. Analyzed was done using International Business Machine-Statistical Package for Social Sciences Version 22 (IBM-SPSS 22, 2015, Atlanta Georgia). Data was presented in tables, means and standard deviation. Means were compared using the Z-test for continuous variables and Chi square test for categorical variable. Associations between variables were compared using Pearson’s correlation and statistical significance was at P< 0.05. Receiver Operating Curve Characteristics (ROC) was plotted to elicit the cut off, sensitivity and specificity of PLAP.

 

Results

 

In Table 1, The mean maternal age of 29.4±6.9years was significantly higher in the study group compared to the mean age of the control group, 27.3±5.5 (p-0.019). The mean systolic and diastolic blood pressures of preeclamptic women was 179.2+17.6mmHg and118.6+13.1mmHg respectively which were significantly higher 

 

Table 1: Comparison of the clinical parameters of the study and control groups

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*Significant

 

Table 2: Correlation Between Blood Pressure, Proteinuria and PLAP in the Study Group

 

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compared to the normotensive controls (118.6+13.1 and 71.5 + 7.5 mmHg), p-value of 0.000. The mean values of serum PLAP for the study and control groups were respectively 126.5IU/L and 51.1IU/L and the p-value was 0.0000. 

In Table 2, there was a positive correlation between the serum PLAP of women in the study group with their blood pressure and degree of proteinuria, p-value 0.000. There was a significant positive correlation between the serum PLAP and gestational age of the study group (Table 3).

 

Table 3: Gestational age at delivery and PLAP

 

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Table 4: Correlation between neonatal birthweight and PLAP

 

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Table 5: Correlation between mean placental weights and PLAP

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Table 6: Pregnancy outcome of the study and control groups

 

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Table 4 shows that there was no statistically significant correlation between the neonatal birth weights of participants in the study and control groups. Even though the mean placental weight was lower for the study group participants (0.63kg versus 0.78kg), there was a significant (p-value 0.000) positive correlation (0.463) between the mean placental weights and serum PLAP of the study group. Correlation was weak (0.085) and not significant for the control group, p-value 0.391 (Table 5).

There were more caesarean sections in women in the study group compared to the control group (64.7% versus 23.1%). The commonest indication for caesarean section in the study group was unfavorable cervix (31.4%) followed by fetal distress, 23.5%. Instrumental deliveries were similar in both arms, 3.8 and 3.9%. Eclampsia was the commonest complication among the study group. There was no maternal death in the control, but it was 2.0% in the study group. Mild and severe birth asphyxia were recorded in 37% of the study group participants but this was observed in only 9.6% of the control group. NICU admission was the 

imageFigure1: Receiver Operating Curve Characteristics (ROC)

 

Table 7: Key        

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commonest adverse neonatal outcome in the study group. Perinatal death among babies born in the study group was almost five-fold that of those born in the control group, 10.8% versus 1.9% (Table 6). The performance of serum PLAP was evaluated using the receiver operating characteristics curve (ROC) which shows that the association of serum PLAP and disease severity had a sensitivity and specificity 80.0% and 82.0% respectively while the optimum cut off value was 54.5 IU/L (Figure 1 and Table 7). They presented with the complication of HELP syndrome and were recruited as such.

 

Discussion

 

Many biomarkers have been tried in a bid to predict and prognosticate this disease, but findings remain inconclusive13. The role of placental alkaline phosphatase in the aetiopathogenesis of preeclampsia has been a subject of considerable interest but findings are inconsistent22-28. In this study, the mean serum Placental Alkaline Phosphatase (PLAP) level in the study group was higher than that of the control group (126.5IU/L versus 51.1IU/L), although these values are within the normal reference range (24-161IU/L)13reported by Aliyu et al in their study population, the difference was statistically significant in our study. This is likely due to the ethnic variation between the study participants. While the predominant ethnic group in the Aliyu study was Hausa/Fulani, the Ibos make up much of our study population, study group and control alike. PLAP has been reported to have ethno-racial variation11-17.

Interestingly, the blood pressure variation followed a similar trend re-enforcing the fact that serum PLAP rose with disease severity. This agrees with the findings by Aliyu et al13, Rajagambeeram et al5 and Mangal et al22. The rise in serum PLAP among preeclamptics is thought to be due to compensatory increase in syncytiotrophoblast cell activity in response to placental dysfunction occasioned by preeclampsia. PLAP plays an important role in fetal metabolism and wellbeing22,29. Although Sindu et al23 and Nahar et al25 found a significantly lower PLAP level among preeclamptic women in their study, this was attributed to severe widespread placental damage in the face of severe preeclampsia which overwhelms the compensatory response mounted by the remaining functional trophoblastic tissues. 

We found a positive correlation between the serum PLAP and disease severity which was based on patients’ blood pressure and the level of proteinuria. This observation is remarkable for its potential use in disease prognostication as was reported by Rajagambeeram et al12 and Aliyu et al in Zaria13. Since the aetio-pathogenesis of preeclampsia is in the placenta, a significant rise in the serum PLAP reflects the extent of placental dysfunction and should serve as a tool to alert the Obstetrician of possible fetomaternal compromise if timely intervention is not initiated. The association observed in the study group with advancing gestational age can be attributed to not only the presence of placental disease but also due to the natural history of PLAP in pregnancy which typically rises with advancing gestational age even in normal pregnancies24,30. A similar correlation between serum PLAP and gestational age was also reported by Mangal et al22 who found an increased PLAP activity with advancing gestational age in pregnancies complicated by preeclampsia while Sulaiman31 found a significantly reduced PLAP levels in women who had miscarriages, implying that serum PLAP activity was low at early gestational age and failing pregnancies31. 

Overall, the serum PLAP showed a positive but weak correlation with the neonatal birth weight of the study group participants. This correlation was not statistically significant (p-value of 0.512) and was rather negative for the control group. The finding of a negative correlation for the control group is surprising because reduced levels of PLAP in normal pregnancy may suggest intrauterine growth restriction32 which is apparently not the case here and calls for further research. Onyesom et al found a significant positive correlation between serum PLAP level and cord blood glucose, albumin, and neonatal birth weight among healthy pregnant controls26,28. Strongly in support of the fact that the placenta is the source of PLAP, and its serum level is a reflection of syncytiotrophoblast activity in response to pregnancy stressors, we found a positive correlation between the mean placental weight and serum PLAP which was statistically significant. Conversely, the correlation was weak and not significant for the control group. This comparison is a major strength of this study as other scholars did not evaluate it11-13,22.

Caesarean section rate was high for the study group compared to the control group (64.7% versus 23.1%). The commonest indication for caesarean section in the study group was unfavorable cervix (31.4%) followed by fetal distress, 23.5%. This was because a high proportion of the patients did not present in labour, and when they did, intrapartum fetal distress in some cases necessitated emergent delivery to avert adverse fetal outcomes. Even when delivery was timely, prior mismanagement before arrival to the unit in the cases of referred patients, contributed to adverse outcomes reported in this study. For instance, Eclampsia was the commonest complication among the study group. Delayed treatment of preeclampsia may result in Eclampsia6,7. There was no maternal death in the control group but 2% of women in the study group died because of their disease. Birth asphyxia was recorded in 37% of the babies born to the study group participants but this was observed in 9.6% of the control group. An association between serum PLAP and fetomaternal complications was not assessed for which is a weakness of this study.

The Receiver operating characteristics curve (ROC) of serum PLAP showed a serum cut-off value of 54.5IU/L with a sensitivity of 80.0% and specificity 82.0% and a significant area under the curve (AUC-0.823) for the study group. At this cut-off value, patients with severe preeclampsia should come under close surveillance and stable patients should be delivered to avert potential fetomaternal compromise should the pregnancy be allowed to continue.

In conclusion, this study has shown that heat stable alkaline phosphatase may have a role in the monitoring of women diagnosed with severe preeclampsia and would come handy in delivery planning. It is cheap and easy to of process which makes it suitable for developing economies.

 

Source of funding: Cost of the research was burn by the researchers.

 

Conflict of interest: There was no conflict of interest

 

References

 

  1. Ghandi M, Chavda R, Saini HB. Comparative study of serum lactate dehydrogenase and uric acid in hypertensive versus normotensive pregnant women. Int J Biomewd Res. 2015;6:25-28.
  2. Cunningham F, Lenevo K, Bloom S, Hauth J, Gilstrap L, Wenstrom K (eds). Maternal physiology. In: Williams Obstetrics, 23rd ed. New York. Lange; 2011: Pp107-131.
  3. Backes CH, Markham K, Moorehead P, Cordero L, Nankervis CA, Giannone PJ. Maternal preeclampsia and neonatal outcomes. Journal of Pregnancy. 2011, 2011. doi:10.1155/2011/214365.
  4. Tejal P, Astha D. Relationship of serum uric acid level to maternal and perinatal outcome in patients with hypertensive disorders of pregnancy. Gujarat Med J. 2014;69:45-47.
  5. Chaiworapongsa T, Romero R, Kusanovic, Mittal P, Kim SK, Gotsch F et al. Plasma soluble endoglin concentration in preeclampsia is associated with an increased impedance to flow in the maternal and fetal circulations. Ultrasound Obstet Gynecol.2010;35:155-162.
  6. Jido TA, Yakasai IA. Pre-eclampsia: A review of evidence. Ann Afr Med. 2013;12:75-85
  7. Swati S, Ekele BA, Shehu CE, Nwobodo EI. Hypertensive disorders in pregnancy among pregnant women in a Nigerian hospital. Niger Med J. 2014; 55: 384-388
  8. Ugwu EO, Dim CC, Okonkwo CD, Nwankwo TO. Maternal and perinatal outcome of severe pre-eclampsia in Enugu, Nigeria After introduction of Magnesium sulphate. Niger J Clin Pract. 2011;14:418-21.
  9. WHO, 2004. Bethesda, MD. Global Burden of Disease for the Year 2001 by World Bank Region, for Use in Disease Control Priorities in Developing Countries, National Institutes of Health: WHO. Make every mother and child count. World Health Report, 2005, Geneva:World Health Organization, 2005. 2nd ed. 
  10. Langer AV, Tell K, Villar  J, Kim T, Kennedy S. Reducing eclampsia related deaths  – a call to action.  Lancet. 2008; 371: 705 – 706.
  11. Carty DM, Delles C, Dominicezak AF. Novel Biomarkers of Predicting Preeclampsia. Trend Cardiovasc Med. 2008;18:186-194
  12. Rajagambeeram R, Raghavan SA, Ghosh S, Basu S, Ramasamy R, Murugaiyan SB. Diagnostic utility of heat stable alkaline phosphatase in hypertensive disorders of pregnancy. J Clin Diag Res.2014;8:10-13.
  13. Aliyu IS, Isah HS, Afonja OA. Relationship between heat-stable alkaline phosphatase activity and blood pressure in patients with preeclampsia and eclampsia. Ann Afr Med. 2006;5:38-41.
  14. Cunningham FG, Leveno KJ, Bloom SL, Spong CY, Dashe JS, Hoffman BL, Casey BM, Sheffield JS. Hypertensive Disorders. Williams Obstetrics. 24th Edition. New York. McGraw-Hill; 2014: 728—779.
  15. National Institute for Health and Care Excellence (NICE). Hypertension in Pregnancy: The Management of Hypertensive Disorders during Pregnancy. Clinical Guideline 107. London. 2010.
  16. Waugh JS, Smith MC. Hypertensive disorders. In: DK. Edmonds (ed). Dewhurst’s textbook of Obstetrics and Gynaecology. 8th edition. London. Wiley-Blackwell; 2012:101-110.
  17. Ghadei R, Mohanty GS. Placental laterality as a predictor of preeclampsia. J Evolution Med Dent Sci. 2017;6:2885-2887.
  18.   Kuc S, Wortboer E, Van Rijn BB, Franx A, Visser of GH, Schielen PC. Evaluation of 7 Serum Biomarkers and Uterine Artery Doppler for First- Trimester Prediction of Preeclampsia: A Systemic review ObstetGynecolSurv.. 2011; 66: 225-239.
  19. Ebonyi State Government Strategic Health Development Plan (2010-2015). Ebonyi State Ministry of Health, March 2010.
  20. QuimicaClinicaAplicada (QCA). IFCC Method for invitro estimation of serum alkaline phosphatase. PRO4­REG9_FALLIQ_5A. revised 01/2017.
  21. Massod MF, Werner KR, McGuire BA. Kinetic determination of alkaline phosphatase activity. Available from www.academic.oup.com/ajcp/article-abstract/54/1/110/1764881   Accessed 4/4/2018.
  22. Dixon CL, Urrabaz-Garza R, Trivedi J, Menon R. Placental alkaline phosphatase; is it placenta specific? Am J Obstet Gynecol. Suppl. 2018;S361.
  23. Sindu PC, Ramakrishnan VR. Role of heat stable fraction of Alkaline Phosphatase, Lipid peroxidation and Uric acid in the early detection of pre-eclampsia. J Dental Med Sci. 2016;15:28-32.
  24. Aliyu IS, Afonja OA, Isah HS. Reference interval of serum heat-stable alkaline phosphatase activity in pregnant women in Zaria. Niger Postgraduate Med J. 2006;13:1-4.
  25. Nahar NS, Monjur-E-Elahi M, Islam MS, Amin MZ, Rahman T, Das SC et al. Alkaline Phosphatase activity in Preeclampsia. Dinajpur Med Col J. 2017;10:244-249.
  26. Onyesom I, Opajobi OA, Uzuegbu UE, Oriero D, Mordi J, Awhin PE et al. Relationship between placental alkaline phosphatase activity and cord blood glucose, albumin and neonatal birthweight at term. Invest Clin. 2009;50:491-495.
  27. Onyesom I, Ebeigbe PN. The level of heat-stable alkaline phosphatase in serum of some Nigerian pregnant women. Ethiop J Biol Sci. 2008;7:85-93.
  28. Onyesom I, Opajobi AO, Uzuegbu  UE, Ebeigbe PN, Anyanwu BE, Suru SM et al. Relationship between placental alkaline phosphatase activity and some biochemical indices of foetal nutrition among the ethnic group in the Western Niger Region of Nigeria. World J Med Sci. 2008;3:39-42.
  29. ShevadeSp, Arole V, Paranjape VM, Bharambe VK. Histochemistry of placental alkaline phosphatase in pre-eclampsia. Int J Biomed Adv Res. 2016;7:323-328.
  30. Onwumeze IC, Onwubere BJ, Ezeoke AC. Serum heat-stable alkaline phosphatase activity in normal pregnancy. East Afr Med J. 1999;76:341-3.
  31. Sulaiman RB. Impact of serum placental alkaline phosphatase level on abortion. Int J Res Pharm Chem. 2016;6:156-161.
  32. Miranda J, Pauels C, Kinhal V, Lai A, Palma C, Gratacos E. Placental derived exosomes in pregnancies complicated with fetal intrauterine growth restriction at term. J Ultrasound ObstetGynaecol. 2017;50:48-15