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ORIGINAL ARTICLE
Year : 2014  |  Volume : 27  |  Issue : 3  |  Page : 538-543

Study of serum prolactin in primary immune thrombocytopenic patients


1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission17-Feb-2013
Date of Acceptance13-Apr-2014
Date of Web Publication26-Nov-2014

Correspondence Address:
Heba Y Elkholy
Hematology Unit, Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.145508

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  Abstract 

Objectives
The aims of this work were to study serum prolactin (PRL) levels in patients with primary immune thrombocytopenia (ITP) and to investigate its possible correlation with disease activity and manifestations.
Background
ITP is a disorder characterized by immune-mediated accelerated platelet destruction and suppressed platelet production. Hyperprolactinemia (HPRL) has been described in many autoimmune diseases such as systemic lupus erythematosus.
Patients and methods
The study was carried out on 40 cases of primary ITP patients (group I) and 50 healthy controls (group II). PRL was measured directly in the serum samples by VIDAS PRL kits using the ELFA technique for all patients and controls.
Results
Moderate HPRL (serum PRL 30-200 ng/ml) was present in eight (20%) of primary ITP patients, but was not present in any of the 50 controls. Among 22 patients with platelet count below 30 000/μl, eight (36.4%) patients had HPRL and 14 (63.6%) patients had normal PRL levels. HPRL was associated with lower platelet counts.
Conclusion
This study shows that HPRL is present in 20% of patients with primary ITP. Also, patients with HPRL have a lower platelet count than patients with normal PRL levels.

Keywords: autoimmunity, immune thrombocytopenia, platelets, prolactin hormone


How to cite this article:
Gazareen SS, Glal AZ, Shoeib SA, Sonbol AA, El Hafez M, Elkholy HY. Study of serum prolactin in primary immune thrombocytopenic patients. Menoufia Med J 2014;27:538-43

How to cite this URL:
Gazareen SS, Glal AZ, Shoeib SA, Sonbol AA, El Hafez M, Elkholy HY. Study of serum prolactin in primary immune thrombocytopenic patients. Menoufia Med J [serial online] 2014 [cited 2020 Jul 10];27:538-43. Available from: http://www.mmj.eg.net/text.asp?2014/27/3/538/145508


  Introduction Top


Immune thrombocytopenia (ITP) is an autoimmune disease characterized by isolated thrombocytopenia (platelet count<100 000/μl) resulting from accelerated clearance and destruction of antibody-coated platelets by tissue macrophages, predominantly in the spleen. Antiplatelet antibodies also target antigens on megakaryocytes and proplatelets, variably suppressing platelet production [1] .

The long-held dogma of platelet-bound antibodies leading to Fcγ receptor-mediated clearance of platelets by phagocytes residing in the spleen (and liver) continues to be a central theme in our current understanding of ITP. In addition to this, the evidence supports a wide array of immune shifts involving all components of the immune system, resulting in both shortened platelet survival and inhibition of the production of platelets. The initial pathogenic mechanisms underlying primary ITP have not yet been identified. Approximately 80% of patients present with primary ITP and 20% can be identified as secondary ITP [2] .

In primary ITP, adult chronic primary ITP patients have high Th1/Th2 ('helper' CD4+ cells) ratios and high Tc1/Tc2 ('cytotoxic' CD8+ cells) ratios. Furthermore, the Th1/Th2 ratio imbalance is correlated inversely with disease severity. ITP patients also have decreased numbers of CD4+ CD25+ T-regulatory cells (Tregs), which function to downregulate T-cell responses. Not surprisingly, the degree of decrease in the numbers of Tregs is associated with more severe disease in ITP. In addition to these type 1/2-specific changes, the total CD4 : CD8 ratio is also observed to be reduced in ITP and improves with disease remission [3] .

Prolactin (PRL) is produced by lymphocytes and some other immune cells. Increased serum PRL levels interfere with B-cell tolerance induction by impairing B-cell receptor-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity [4] .

In addition, hyperprolactinemia (HPRL) affects dendritic cell maturation, skewing dendritic cell function from antigen presentation to a proinflammatory phenotype with increased interferon α production [5] .

HPRL have been described in many autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, reactive arthritis, Sjφgren's syndrome, systemic sclerosis, psoriasis, Behcet's disease and polymyositis, type I diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, Addison's disease, lymphocytic hypophysitis, celiac disease, multiple sclerosis, peripartum cardiomyopathy, and uveitis [6] .

The aim of the present study is to study serum PRL levels in patients with primary ITP and examine its possible correlation with disease activity and manifestations.


  Patients and methods Top


The present study included 40 patients with primary ITP (group I); their diagnosis was made on the basis of the American Society of Hematology guidelines [7] :

(1) History compatible with the diagnosis of chronic ITP.

(2) Normal physical examination findings, except for signs of thrombocytopenia (petechiae, purpura, or mucosal bleeding), and no adenopathy or splenomegaly.

(3) Complete blood count showing isolated thrombocytopenia with large platelets, but no anemia unless bleeding or immune hemolysis is present.

(4) Bone marrow examination showing normal or increased numbers of megakaryocytes (not required for diagnosis unless unusual manifestation or age >60 year).

(5) No clinical or laboratory evidence for other causes of thrombocytopenia.

Therefore, hepatitis C virus antibody, HIV antibody, and ANA were assessed for all patients.

Group I included 35 women and five men, ranging in age from 18 to 50 years, selected from among the inpatients and outpatients of the Internal Medicine Department in Menoufia University Hospitals, from September 2012 to July 2013. The ITP patients selected were compared with 50 healthy individuals (controls, group II), 37 women and 13 men, ranging in age from 19 to 50 years.

The following patients were excluded from the study:

(1) Patients with hyperprolactinemia [6] :

(a)

Patients with known causes for hyperprolactinemia, such as pregnancy, lactation, prolactinoma, and pituitary and hypothalamic disorders.

(b)

Hypothyroidism, polycystic ovary syndrome, or other endocrinopathies.

(c)

Taking medications known to induce PRL (e.g. metoclopramide).

(d)

Patients with liver cirrhosis, renal failure.

(2) Patients with PRL deficiency [8] :

(a)

Secondary to general anterior pituitary dysfunction.

(b)

Partial isolated PRL deficiency is rare.

(3) Patients with secondary ITP because of [9] :

(a)

Infection with a number of agents, including hepatitis C virus, HIV, and Helicobacter pylori.

(b)

Underlying autoimmune and lymphoproliferative disorders such as SLE, Wiskott-Aldrich syndrome, chronic lymphocytic leukemia, antiphospholipid syndrome, and common variable immunodeficiency.

(c)

Drugs such as quinine and trimethoprim-sulfamethoxazole.

The procedures followed were in accordance with the ethical standards of the responsible institutional committee on human experimentation and the Helsinki Declaration of 1975, as revised in 1983.

Sampling

From all participants, 2-3 ml peripheral blood samples (plasma collected on EDTA was not used) were obtained in the morning and stored as frozen sera at −25 to 6°C.

Methods

The assay principle combines an enzyme immunoassay sandwich method with a final fluorescent detection (ELFA). The solid phase receptacle (SPR) serves as the solid phase as well as the pipetting device for the assay. Reagents for the assay are ready-to-use and predispensed in the sealed reagent strips. All of the assay steps are performed automatically by the instrument. The reaction medium is cycled in and out of the SPR several times.

The sample is taken and transferred into a well containing alkaline-phosphatase-labeled anti-PRL (conjugate). The sample-conjugate mixture is cycled in and out of the SPR several times to increase the reaction speed. The antigen binds to antibodies coated on the SPR and to the conjugate forming a 'sandwich'.

Unbound components are eliminated during the washing steps. During the final detection step, the substrate 4-methyl-umbelliferyl phosphate is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-methylumbelliferone), the fluorescence of which is measured at 450 nm. The intensity of the fluorescence is proportional to the concentration of PRL present in the sample. At the end of the assay, results are automatically calculated by the instrument in relation to the calibration curve stored in memory, and then printed out [10] .

Statistical analysis [11]

The data collected were tabulated and analyzed using the statistical package for the social sciences (SPSS, version 11; SPSS Inc., Chicago, Illinois, USA) software on an IBM compatible computer.

Quantitative data were expressed as mean and SD (X+SD); qualitative data were expressed as number and percentage [N (%)] and analyzed using the χ2 -test.

Quantitative data were analyzed using the Student t-test, analysis of variance test (f-test), and the Kruskal-Wallis test.

The correlation coefficient test (Pearson's correlation) (r-test) was used to detect the association between quantitative variables.


  Results Top


The study was carried out on 40 cases of primary ITP patients (group I) and 50 healthy controls (group II). The mean age of the patients (group I) was 30.4 years (range 18-50 years) and that of the controls (group II) was 31.1 years (range 19-50 years). There was no significant difference between cases and controls in age in years (P > 0.05).

In group I, there were 35 (87.5%) women and five (12.5%) men, and in group II, there were 37 (74%) women and 13 (26%) men. There was no significant difference between cases and controls in sex (P > 0.05) ([Table 1]).
Table 1: Demographic characteristic of the studied groups


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There were nine ITP patients who were discovered accidentally to have platelet count more than 30 000/μl. There was a highly statistically significant difference between the accidentally discovered ITP patients in terms of the platelet count (P = 0.001) ([Table 2]).
Table 2: Clinical presentation of immune thrombocytopenia in terms of platelet count


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Sixteen ITP patients presented with petechiae or ecchymosis; of these, eight (50%) patients had platelet count above 30 000/μl and eight (50%) patients had platelet count less than 30 000/μl. There was no statistically significant difference between the patients who presented with petechiae or ecchymosis in terms of the platelet count (P > 0.05) ([Table 2]).

Fifteen ITP patients presented with petechiae or ecchymosis; of these, one (6%) patient had a platelet count above 30 000/μl and 14 (94%) patients had a platelet count lower than 30 000/μl. There was a highly statistically significant difference between the patients who presented with mucosal bleeding in terms of the platelet count (P = 0.001) ([Table 2]).

In group I, eight (20%) patients were found to have HPRL. In group I, the PRL value was between 4.3 and 72 ng/ml (mean 25.4 ± 20.6 ng/ml), whereas in group II, PRL levels were between 4 and 31 ng/ml (mean 18.9 ± 5.7 ng/ml). Thus, all control healthy individuals had normal serum levels of PRL (<25 ng/ml in men and <35 ng/ml in women). There was a highly statistically significant difference between group I and group II in PRL levels (P = 0.001) ([Table 3]).
Table 3: Comparison between immune thrombocytopenia cases and control of prolactin levels


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In group I, eight patients had HPRL and all of them were women. There was no significant difference between patients with normal PRL and patients with HPRL in terms of sex (P > 0.05) ([Table 4]).
Table 4: Comparison between immune thrombocytopenia patients with and without hyperprolactinemia in terms of sex


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In group I, there were nine accidentally discovered patients, all (100%) of whom had normal PRL levels, and 16 patients presented with mild bleeding in the form of petechiae or ecchymosis; of these, 11 (68.8%) patients had normal PRL levels and five (31.2%) patients had hyperprolactinemia and 15 patients presented with mucosal bleeding. Of these, 12 (80%) patients had normal PRL levels and three (20%) patients had HPRL. There was no statistically significant difference in the effect of high PRL levels and the presentation of ITP patients (P > 0.05) ([Table 5]).
Table 5: Comparison between immune thrombocytopenia patients with and without hyperprolactinemia in terms of clinical presentation


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In group I, 22 patients had platelet count below 30 000/μl; of these, eight (36.4%) patients had HPRL and 14 (63.6%) patients had normal PRL levels and 18 patients had platelet count above 30 000/μl. All these patients (100%) had normal PRL levels. There was a highly statistically significant difference between patients with HPRL and those who had normal PRL in terms of the platelet count of ITP patients (P = 0.004) ([Table 6] and [Figure 1] and [Figure 2]).
Table 6: Comparison between immune thrombocytopenia patients with and without hyperprolactinemia in terms of platelet count


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Figure 1: Mean serum prolactin level in immune thrombocytopenia (ITP) cases and controls. There was highly statistically significant difference between cases (group I) and controls (group II) in prolactin levels (P < 0.05).

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Figure 2: Prolactin and platelet count in immune thrombocytopenia (ITP) patients. There was a highly statistically significant difference between patients with hyperprolactinemia and those who had normal prolactin in terms of the platelet count of ITP patients (P < 0.05).

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  Discussion Top


ITP is an autoimmune disease characterized by isolated thrombocytopenia (platelet count, 100 000/μl) resulting from accelerated clearance and destruction of antibody-coated platelets by tissue macrophages, predominantly in the spleen. Antiplatelet antibodies also target antigens on megakaryocytes and proplatelets, variably suppressing platelet production [12] .

Autoimmune mechanisms are complex and influenced by genetic factors, immune system abnormalities, hormonal factors, and environmental influence. Sex hormones are probably partly responsible for the higher occurrence of autoimmune disorders in women [13] .

Elevated serum PRL levels interfere with B-cell tolerance induction by impairing B-cell receptor-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity [14] .

In our study, we found that 20% of ITP patients had HPRL and none of the controls had HPRL; this is in agreement with other studies that have described HPRL in many autoimmune diseases such as SLE, rheumatoid arthritis, reactive arthritis, Sjφgren's syndrome, systemic sclerosis, psoriasis, Behcet's disease and polymyositis, type I diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, Addison's disease, lymphocytic hypophysitis, Celiac disease, multiple sclerosis, peripartum cardiomyopathy, and uveitis [7] .

In the Gazareen et al.'s [15] study, HPRL was detected in 20% of SLE patients with a mean serum PRL of 17 ng/ml (range 7.9-33 ng/ml) in mild active disease and 23.4 ng/ml (range 11-44.5 ng/ml) in highly active disease (P < 0.05).

In the Shahin's [16] study, HPRL was detected in 30.3% of SLE patients, with a mean serum PRL of 680.7 ± 1021.5 mIU/l (range 198-4500 mIU/l, median 318 mIU/l).

Extra pituitary PRL synthesis is regulated by an alternative promoter, which contains a single-nucleotide polymorphism at the region 21 149 G/T. Higher PRL mRNA expression is associated with the G allele in lymphocytes. A high frequency of the G allele was described in patients with SLE [17] .

A study was carried out on polymorphisms in three genes (HLA class II, MICA, PRL) located within the susceptible locus for SLE on the chromosome 6. Genes in this region show a high level of polymorphism and linkage disequilibrium [17] .

Immune cells from SLE patients produce more PRL than those from healthy individuals. Lymphocyte PRL synthesis is regulated by an alternative, extra pituitary promoter, and the G allele of the −1149 G/T single-nucleotide polymorphism of extra pituitary PRL promoter (rs1341239) leads to higher PRL expression [18] .

In the present study, there was no relationship between PRL level and clinical presentation of ITP.

In the Gazareen et al.'s [15] study, there was statistically significant difference between SLE patients with hyperprolactinemia and SLE patients with normal PRL levels in terms of nephritis and cerebritis and no significant statistical difference between groups in terms of malar rash, mucosal ulcers or arthritis, malar rash, mucosal ulcers, or arthritis.

In the Shahin's [16] study, Pearson's correlation showed no significant correlation between the age of the SLE patients, disease duration, SLEDAI score, and their serum PRL levels. The patients' general signs of illness, particularly elevated temperature, articular manifestations, vasculitis, encephalopathy, APLs, or thrombocytopenia, showed significantly higher mean values of serum PRL compared with patients without these manifestations.

In our study, we found that in 22 patients with platelet count below 30 000/μl, eight (36.4%) patients had HPRL and 14 (63.6%) patients had normal PRL levels and 18 patients had platelet count above 30 000/μl; all these patients (100%) had normal PRL levels.

The Shahin's [16] study indicated that the platelet count was correlated inversely with PRL. Furthermore, thrombocytopenia and leukocytosis were the best independent predictors of elevated serum PRL in the Saudi SLE patients studied. Therefore, bromocriptine, as an anti-PRL, can be used as a relatively safe adjunctive therapy in the treatment of recalcitrant SLE patients with thrombocytopenia and leukocytosis, who are unresponsive to traditional approaches [11] .

In agreement with our study, in the Gazareen et al.'s [15] study, there was a statistically significant difference between patients with HPRL and patients with normal PRL levels in nephritis proteinuria and no significant difference between groups in anemia, leukopenia, thrombocytopenia, or ANA.


  Conclusion Top


This study shows that moderate HPRL (serum PRL 30-200 ng/ml) is present in 20% of patients with primary ITP and that patients with HPRL have a lower platelet count than patients with normal PRL levels.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.Cines DB, McMillan R. Pathogenesis of chronic immune thrombocytopenic purpura. Curr Opin Hematol 2007; 14 :511-514.  Back to cited text no. 1
    
2. Cines DB, Liebman H, Stasi R. Pathobiology of secondary immune thrombocytopenia. Semin Hematol 2009; 46 :S2-S14.  Back to cited text no. 2
    
3. Hu Y, Ma Dx, Shan Nn, et al. Increased number of Tc17 and correlation with Th17 cells in patients with immune thrombocytopenia. PloS One 2011; 6 :e26522.  Back to cited text no. 3
    
4. Saha S, Tieng A, Pepeljugoski KP, et al. Systemic lupus erythematosus, and autoreactive B cells: lessons learnt from murine models. Clin Rev Allergy Immunol 2011; 40 :8-15.  Back to cited text no. 4
    
5. Jara LJ, Benitez G, Medina G. Prolactin, dendritic cells, and systemic lupus erythematosus. Autoimmun Rev 2008; 7 :251-255.  Back to cited text no. 5
    
6. Orbach H, Shoenfeld Y. Hyperprolactinemia and autoimmune diseases. Autoimmun Rev 2007; 6 :537-542.  Back to cited text no. 6
    
7. Neunert C, Lim W, Crowther M, et al. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 2011; 117 :4190-4207.  Back to cited text no. 7
    
8. Toledano Y, Lubetsky A, Shimon I. Acquired prolactin deficiency in patients with disorders of the hypothalamic-pituitary axis. J Endocrinol Invest 2007; 30 :268-273.  Back to cited text no. 8
    
9. Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009; 113 :2386-2393.  Back to cited text no. 9
    
10.Morton RF, Hebel JR, Mccarter RJ. Medical statistics. In: A study guide to epidemiology and biostatistics. 5th ed. Gaithersburg, Maryland: Aspen Publication, 2001. 71-74.  Back to cited text no. 10
    
11.Sapin R, Simon C. False hyperprolactinemia corrected by the use of heterophilic antibody-blocking agent. Clin Chem 2001; 47 :2184-2185.  Back to cited text no. 11
    
12.Ghanima. W, Godeau B, Cines DB, Bussel JB. How I treat immune thrombocytopenia: the choice between splenectomy or a medical therapy as a second-line treatment. Blood 2012; 120 :960-969.  Back to cited text no. 12
    
13.Sadovnick AD. Genetic background of multiple sclerosis. Autoimmun Rev 2011; 9 :1058-1079.  Back to cited text no. 13
    
14.Saha S, Tieng A, Pepeljugoski KP, Zandamn-Goddard G, Peeva E. Prolactin, systemic lupus erythematosus, and autoreactive B cells: lessons learnt from murine models. Clin Rev Allergy Immunol 2011; 40 :8-15.  Back to cited text no. 14
    
15.Gazareen S, El-Najjar M, Lassien Y, Dawood A, El Shebinie A, Soliman SG, El-Saied JK. Prolactin hormone: a possible marker of the activity of systemic lupus erythematosus. Menoufiya Med J 2009; 22 :67-80.  Back to cited text no. 15
    
16.Shahin D. Thrombocytopenia and leukocytosis are independent predictors of hyperprolactinemia in systemic lupus erythematosus patients. Egypt Rheumatol 2011; 33 :77-83.  Back to cited text no. 16
    
17.Fojtíková M, Èerná M, Èejkova P, et al. Extrapituitary prolactin promoter polymorphism in Czech patients with systemic lupus erythematosus and rheumatoid arthritis. Ann Rheum Dis 2007; 66 :706-707.  Back to cited text no. 17
    
18.Fojtíkov M, Novota P, Èejková P, et al. HLA class II, MICA and PRL gene polymorphisms: the common contribution to the systemic lupus erythematosus development in Czech population. Rheumatol Int 2011; 31 :1195-1201.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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