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Effect of selected osteopathic lymphatic techniques on immune system in healthy subjects: A randomized control trial

Ahmed Samir Ibrahim Abdelfattah1, Neveen Abdel Latif Abdel Raoof1, Samy Abdul-Sammad Nasef1, Rania Reffat Ali1, Rania Shafeek Swailm2 1Basic Science Department, Faculty of Physical Therapy, Cairo University, 2Clinical Pathology Department, Faculty of Medicine, Alexandria University, Egypt Immune system Effect of selected osteopathic lymphatic techniques on immune system in healthy subjects: A randomized control trial


Introduction
Osteopathic Manipulation Techniques are therapeutic manual applications used to improve the physiological system as well as restore the fluid base of the body that can be changed by structural problems [1]. It was suggested that fighting disease is naturally stimulating the body's immune system [2] . The immune system is the body's defense against infectious organisms and other invaders [3]. The immune system is divided into two parts, called the innate immune system and acquired immune system (adaptive immune), while each of these plays a role in defending the body [4]. Spleen, lymphatic system, gums, bone marrow, white blood cells, antibody, hormones are the basic components of the immune system [5]. T-and B-cells are types of lymphocytes which represent the adaptive immune system [6]. T-helper lymphocytes regulate both the innate and adaptive immune responses [7]. Thoracic lymphatic pump technique (TLPT) is a method of manipulation used to enhance the rate of lymphatic flow to overcome infection; these methods increase the flow of lymph through the thoracic duct [8]. Splenic pump technique (SPT) is a technique involving manipulation of the spleen that has an important role in lymphatic and macrophage systems by eliminating the damaged RBCs and in producing antibodies [9]. Sternum pump and recoil techniques are working on vegetative system which in turn enhances the lymphatic flow and drainage [10]. This study is unique as regarding to this literature; there is a gap in knowledge addressing these techniques especially those in the first group (sternal techniques), also there is a relationship between these techniques and lymphatic cells, researches need to be done to make it more evidence-based. Thus, this study was designed to investigate and compare the efficacy of selected osteopathic lymphatic techniques on the immune system in healthy subjects.

Material and Method
Forty-five healthy subjects (33male and 12 female) aged 20 to 50 years participated in this study. Subjects were randomly assigned into 3 equal groups using the permuted blocks randomization in selection. The first group received sternal pump and sternal recoil techniques, the second group received thoracic lymphatic pump and splenic pump techniques and the third was control group and didn't receive any manipulative techniques. This study was conducted at the outpatient clinic of the Faculty of Physical Therapy, Cairo University, Alexandria Main University Hospital "Clinical Pathology Lab" and Outpatient Clinic in Alexandria. The signed consent form for participation in the study was obtained from the subjects. The eligibility criteria for the study were healthy subjects; all subjects who had thoracic trauma, inflammatory or systemic diseases, malignancy, osteoporosis, and autoimmune diseases were excluded. All subjects had signed the consent form. This study was approved by the ethical committee of Cairo University No: P.T.REC/012/00945 All subjects in this study were subjected to the following:

1-Peripheral blood sampling:
Peripheral blood samples (4 ml) were collected under complete aseptic conditions; these were divided into: a-2 ml on K2 EDTA for complete blood count (CBC) morphological studies. b-2ml on K2 EDTA immune-phone type for CD4 detection

2-CBC and morphological studies:
CBCs were performed on ADVIA 2120 automated cell analyzer (Siemens healthcare diagnostics, Forchheim, Germany) films were stained with Leishman stain, and were used for morphological identification of the differential count and calculation of the absolute CD4 count [11].

3-Detection of CD4:
In this study, the direct immunofluorescence technique was employed using labeled Ab according to the manufacturer instructions for CD4 detection. Immunofluorescence on the viable peripheral blood cells in suspension was analyzed using Becton Dickinson, FACS caliber flow cytometer equipped with Cell Quest Software (USA) [12].

Treatment techniques: Group A: Sternal pump and recoil techniques:
1-The subject was in the supine position. The practitioner stood at the head of the table. 2-Both hands were on top of each other on the subject's sternum, with the thenar eminence on the sternal angle. 3-The subject was asked to inhale deeply. During the deep exhalation, pressure was applied with both hands caudally and posteriorly. The pressure was released on the next inhalation. 4-This process was repeated five to six times. The Practitioner made recoil technique at the beginning of next inhalation, then left his hands rapidly so this made sternum to recoil [10].  6-The subject was asked to breathe during pressure which was decreased slightly gradually [13].

Splenic pump technique:
1-The subject was in the supine position, legs bent. The practitioner stood on the subject's right side.
2-The fingers of practitioner's right hand with the side of the little finger were placed below the left costal arch. 3-The left hand was placed on top of the right hand. With both hands, oscillation was in the direction of the spleen, i.e. pressing lightly and intermittently in a cranial-lateral direction with a frequency of 150-180/min and continuing these oscillations for about 2 min [10].

Statistical Analysis
All statistical measures were performed using the Statistical Package for Social science (SPSS) program version 23 for windows. The current test involved two independent variables. The first one was the (tested group) between-subjects factor which had three levels (Group A, Group B, and Group C). The second one was the (measuring periods) within-subjects factor which had two levels (pretreatment and post-treatment). In addition, this test involved one tested dependent variable (CD4). Preliminary assumption checking revealed that data was normally There were no outliers, as assessed by examination of studentized residuals for values greater than ±3. There was the homogeneity of variances (p > 0.05) and covariances (p > 0.05), as assessed by Levene's test of homogeneity of variances and Box's M test, respectively. All these findings allowed the researchers to conduct a parametric analysis. So, 3×2 mixed design ANOVA was used to compare the tested variables of interest at different tested groups and measuring periods. The alpha level was set at 0.05.

Results
As indicated by the one-way ANOVA, there were no statistically significant differences (P>0.05) between subjects in three groups concerning age, body mass, height and BMI (Table 1). Statistical analysis using mixed design ANOVA analyzed 45 patients assigned into three equal groups. It revealed that there were significant within-subject effect (F =14.53, p = 0.0001) and between-subject effect (F= 11.921, p = 0.0001*). Additionally, there was significant treatment-time effect (F = 23.534, p = 0.0001).

Discussion
Osteopathic lymphatic manipulative techniques are used in practice to enhance the immune and defense of the body to fight the organisms. This study was designed to investigate and compare the efficacy of selected osteopathic lymphatic techniques on the absolute CD4+ count in healthy subjects. The multiple pairwise comparison tests revealed that there was a significant increase in the CD4 post-treatment compared with pretreatment in Group B only. Regarding between-subject effects, multiple pairwise comparisons revealed that there was a significant increase in favor to Group B compared to Group A and C at post-treatment. Regarding the effects of the first group (sternal pump and recoil techniques) on immune system, according to the analysis in the current study, the results of the Group A (sternal pump and recoil techniques) revealed that there was a non-significant effect within and between groups in the values of absolute CD4 pre-treatment Mean/SD values and post-treatment, this was due to the application of these techniques on sternum so they can affect the mobility of manubrium, thorax and or lungs, however the neuro-vegetative effect of these techniques and the pressure changes in thorax after applying these techniques, but they didn't make any change to blood counting. Firsova [14] studied the efficacy of recoil technique on the cardiovascular and the respiratory systems. The result was significant on systolic blood pressure; the study concluded that there was a change in blood pressure after application of recoil technique. There are no studies related to blood counting or lymphocytes according to sternal techniques however they have a vegetative effect on the circulatory system. Regarding the effects of the second group (Thoracic lymphatic pump technique and splenic pump technique) on immune system, according to analysis in the current study, the results of the group B (TLPT&SPT) revealed that there was a significant improvement within and between groups in the values of absolute CD4 pre-treatment Mean/SD values and increased significantly post-treatment, this was due to the forces exerted on the lymph vessels, increase force would also compress lymph vessels and increase lymph flow and manual techniques on spleen so it released the WBCs into bloodstream. Breithaupt et al [15] examined the impact of LPT on the immune reaction incorporates few randomized controlled examinations researching the impact of LPT on immune reaction to vaccination, and the study concluded that application of LPT with flu immunization enhanced general immunity. Mesina [16] studied an application of LPT on seven normal subjects and the study showed an increase in the Basophils, WBCs counts. In a study by Hodge et al. [17], TLPT has shown an increase in blood leukocyte numbers of macrophages, neutrophils and CD4‫‬ T-cells in both thoracic and mesenteric duct lymph. Leukocytes released from mesenteric lymph nodes by the effect of TLPT into thoracic duct lymph and stimulate flux of leukocytes in mesenteric duct lymph and thoracic duct lymph. Measel and Kafity [18] studied the pump techniques and showed an increase of WBCs count, B-cells and T-cells, this explains the important role of the spleen in the im-  [20] studied TLPT and showed that TLPT enhanced thoracic duct lymph and leukocyte concentrations in dogs and rats. In the study by Rockson [21], TLPT and intestinal motility exerted during SPT increase lymphatic transmural pressure due to compressing lymph vessels so the lymph flow increased consequently. Noll et al. [22] examined the importance of osteopathic treatment protocol including the thoracic lymphatic pump on elderly resident patients in hospitals who suffered from pneumonia, the results showed a significant difference in the rate of change in WBCs counts between days 1 and 3, also he dedicated it to the spleen that acts as a reservoir of antibodies and leukocytes that expelled into circulation by contraction of the spleen. This augments importance of the application of SPT to enhance the adaptive immunity in infectious diseases. This study was limited by the lack of studies regarding the use of lymphatic techniques. According to the results of this study, the following further researches are highly recommended: 1-Evaluation of T-helper lymphocyte cells number of normal subjects on a larger sample size. 2-The other study could be done on a wide range of young age group (infant) or different age groups. 3-Other studies could be done to determine the effect of osteopathic manual therapy program on other immune system components (innate and adaptive). 4-Studying the effect of osteopathic manual therapy program in the T-helper lymphocyte cells percentage and cytotoxicity. 5-Studying the effect of thymus gland removal (thymectomy) in myasthenia gravis patients and its effect on muscle efficiency. 6-Studying the effect of osteopathic manual programs on the level of Vit D.

Conclusion
From the finding of the current study, it was concluded that the osteopathic techniques (TLPT & SPT) had a significant effect on the absolute CD4 count than the other techniques (sternal pump & recoil techniques) on enhancing the immune system in healthy subjects.

Scientific Responsibility Statement
The authors declare that they are responsible for the article's scientific content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientific review of the contents and approval of the final version of the article.
Animal and human rights statement All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.

Funding: None
Conflict of interest None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.