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J Thorac Cardiovasc Surg 1998;115:1041-1045
© 1998 Mosby, Inc.

SURGERY FOR CONGENITAL HEART DISEASE

Neonatal thymectomy: Does it affect immune function?

Read at the Seventy-seventh Annual Meeting of The American Associationfor Thoracic Surgery, Washington, D.C., May 4-7, 1997.

Received for publication May 6, 1997. Revisions requested June 30, 1997. Revisions received Dec. 29, 1997. Accepted for publication Dec. 29, 1997. Address for reprints: Winfield J. Wells, MD, Associate Professor ofSurgery, Division of Cardiothoracic Surgery, Childrens Hospital Los Angeles,4650 Sunset Blvd., MS 66, Los Angeles, CA 90027.

	Abstract

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
Objective: The purpose of this study wasto determine whether thymectomy in the newborn has a negative effect on immunefunction.
Methods: Twenty-five neonates (<30days) who had thymectomy at congenital heart repair were prospectively studiedto determine immune function. The percentage of T-cell subtypes including CD3(all T cells), CD4 (helper T cells), and CD8 (suppressor T cells) wasdetermined. In six patients, further testing of CD4 cells was done to determinewhether they were newly formed, recent thymic emigrants (CD4, CD45, and RA), orolder educated lymphocytes (CD4, CD45, and RO). Response to the mitogenphytohemagglutinin and to tetanus toxoid were determined, as were antibodytiters to tetanus. Samples were drawn before the thymectomy, at approximately 3months after immunization and at 1 year. Ten age-matched control patients weretested. At follow-up, parents were asked about infections.
Results: Prethymectomy T-cell subsets were all normal andcomparable to controls. At 12 months, the percent of CD3 was significantly lessthan in the control group (48% ± 3% versus 64%± 2% [mean ± standard error of the mean];p  < 0.01) as was CD4 (31%± 2% versus 46% + 2% [mean ± standarderror of the mean]; p = < 0.01). CD8 didnot drop. Surprisingly, the percent of CD4 that were recent thymic emigrants didnot decrease significantly (50% ± 8% versus 60%± 6% [mean ± standard error of the mean];p = not significant). Lymphocyteblastogenesis to phytohemagglutinin and tetanus toxoid and antibody to tetanuswere all normal at 12 months. No patient required readmission for infection, andthere were the expected number of minor infectious events (median 3; 95%confidence interval 1,4).
Conclusion:Thymectomy in neonates results in a modest but significant decrease inT-lymphocyte levels, but there is no compromise in immune function. (J Thorac Cardiavasc Surg 1998;115:1041-6)

	Introduction

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
In the newborn, thymectomy facilitates cannulation for cardiopulmonarybypass and exposure for mobilization or reconstructing of the aortic arch.Although thymectomy in children who are younger than 6 months of age and adultshas been shown to be tolerated without compromise of the immune system, ^1,2there is very little information on the impact of neonatal thymectomy. The onlypublished report specifically addressing this issue suggests avoiding thymectomyin infants younger than 3 months of age because of evidence of impaired immunityas tested in later childhood. ³To further investigate this issue, we have prospectively studied a group ofneonates who underwent thymectomy at the time of cardiopulmonary bypass in thefirst month of life.

	Patients and methods

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
Demographics.
Twenty-five neonates (<30 days of age) who had undergone thymectomy atan operation for congenital heart disease were prospectively entered into thestudy. The anatomic diagnosis of heart disease present in the study patients isdocumented in Table I. Control values for tests of immune function were determined in 10age-matched (3 to 12 months of age) patients who had not undergone thymectomy.No study or control patient carried the diagnosis of DiGeorge syndrome orasplenia.

Immune system testing.
Studies performed to evaluate the immune system are summarized in TableII. T-cell immunophenotyping was performed by staining withfluorescein-labeled monoclonal antibodies to lymphocyte surface antigens CD2,CD3, CD4, and CD8. In addition, in some patients further testing of CD4 cellswas performed to determine whether they were newly formed, naive lymphocytes ofrecent thymic origin (CD4, CD45, and RA) or older, educated memory lymphocytes(CD4, CD45, and RO). CD45 is a glycoprotein surface antigen having the twoisoforms, RA and RO.

Antigenic-specific in vitro responses were tested by stimulatinglymphocytes for 6 days in the presence of tetanus toxoid (MassachusettsBiological Laboratory, Boston, Mass.). Tetanus toxoid has been shown to have avery flat dose-response curve so that, in our laboratory, multiple concentrationtesting is not performed. Our standard concentration of tetanus toxoid is1:1000. Tetanus was pulsed on day 6, and cell harvesting took place on day 7.Normal values were considered to be greater than 3 x 103 cpm(background subtracted). This lower limit and the lower limit for antibody titerto tetanus toxoid have been determined by testing the response of a large numberof control individuals in our laboratory at Childrens Hospital Los Angeles.

To measure T-cell to B-cell cooperation, antibody titer to tetanus toxoidwas measured. A value of greater than 0.1 IU was considered normal. Antibodylevels were determined by use of an enzyme-linked immunosorbent assay andstandardized by use of a reference antiserum with a known concentration ofantibody quantified in international units.

Follow-up.
Study patients were followed for at least 1 year. Hospital charts werereviewed to determine whether there had been an admission for infection orwhether infections had been noted at the time of outpatient visits. At the 3-and 12-month blood testing interval, families were questioned about history ofinfection. The incidence of infection was compared with measures of immunefunction to determine whether there was any correlation.

Statistical analysis.
The significance of differences between means in laboratory indices wasdetermined by use of the Student t test whenthe data were normally distributed. Student Equal Variance ttest and Aspen Welch Unequal Variance test were used when appropriate. When thedata were skewed, Wilcoxon rank sum tests or Mann-Whitney U (for ties) wasapplied. The Spearman rank correlation coefficient was used as a measure ofassociation when one or both variables were skewed. Skewness and kurtosis wasbased on the omnibus normality test statistic.

	Results

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
T-cell subsets.
After thymectomy, patients had a lower proportion of lymphocytes with thesurface antigens CD3 (all T cells), CD4 (helper T cells), and CD8(suppressor/cytotoxic T cells). These changes were most marked at 12 months(Table III).

It is of interest that T-cell subset decreases were more marked for CD4than for CD8 T cells. To test whether this reduction in the CD4 subpopulationwas due to decreased production of new T cells, six patients had determinationof the percentage of CD4, CD45, RA and CD4, CD45, RO cells. As shown in TableIII, the percentage of CD4 lymphocytesthat were recent thymic emigrants although slightly less than control was notsignificantly decreased (p = 0.356). Thusthere was no significant decrease in the production of new CD4 T cells, in spiteof a surgically "complete" thymectomy.

Response to PHA.
Lymphocytes from patients who had undergone thymectomy showed a normalresponse to the mitogen PHA. These results (Table IV) are expressed as countsper minute x 10³.

View larger version (38K): [in this window] [in a new window]   Fig. 1. This graph ofblastogenesis to tetanus toxiod (cumulative percent versus frequencydistribution) shows no significant difference in cumulative percentiles offrequencies of counts per minute in thymectomy and control patients at 3 and 12months.

View larger version (28K): [in this window] [in a new window]   Fig. 2. This graph of antibodytiter to tetanus toxoid (cumulative persent versus frequency distribution) showsno significant difference in cumulative percentiles in antibody response totetanus toxoid in patients after thymectomy at the 3- and 12-month follow-up.

There was no correlation between the number of infectious incidents andthe degree of reduction in T cells (total), T-cell subsets, or response to PHAand tetanus.

	Discussion

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
There is a naturally occurring model of thymic deficiency that occurs inthe DiGeorge syndrome. ^4-6 This is due to developmentalanomalies of the third and fourth pharyngeal pouches and results in hypoplasiaor aplasia of the thymus and parathyroid glands. Because the amount of thymictissue that develops in the DiGeorge syndrome is variable, there is a spectrumof clinical immunologic manifestations. The degree of immunopathy correlateswith the percentage of T cells. When the T-cell count drops to 10% to 15%of lymphocytes, there is usually an increase in infections, particularlycandidiasis; if T cells reach 5% or less, life-threatening infections canoccur. It should be noted that although there was some decrease in T cells inour neonates who had undergone thymectomy, none of them approached thetheoretically critically low level of 20%.

Although there is still uncertainly about exactly what point in timethymectomy might alter immune function, there are a number of studies that haveaddressed the issue. In landmark studies reported in 1961, Miller and associates ^7,8showed that thymectomy in neonatal mice led to immunodeficiency, principallyaffecting cell-mediated immune response. It was subsequently shown thatthymectomy in older children (>6 months of age) and adults has no effect onimmunity. ^1,2 It might be speculated that thedifference between rodent and human immune outcomes after neonatal thymectomyrelate to the relative level of gestational maturity at the time of birth.Because rodents are less mature when born, neonatal thymectomy might be expectedto have a greater impact on immune function. In the only report that has focusedon the effect of neonatal thymectomy in human beings, Brearley and associates ³ concluded that, although theclinical consequences of early thymectomy were unclear, there was evidence ofimpaired immunity as measured in later infancy. In this retrospective study, theauthors documented a decreased number of T cells and T-cell subsets that weresignificantly lower than age-matched controls (as in this study) but did notapproach the critically low value of 20% of lymphocytes as T cells.Although previous work by Hisatomi and associates ⁹ had suggested that cardiopulmonarybypass (in adults) caused a transient decrease in T-cell subsets and PHAresponse lasting approximately 1 week, there was no such evidence in the studyconducted by Brearley and associates ³for control patients who had not undergone thymectomy and who had had heartsurgery when measured at the later interval of 9 to 36 months after theoperation. Brearley and associates also found a marginally diminished responseto PHA at the lowest dilution of 4 µg/ml. In our laboratory, most controlpatients respond maximally to a PHA dilution of 20 µg/ml. Maximalstimulation has not been seen at lower doses such as 4 µg/ml. In normalpatients, the response to PHA at 4 µg/ml has been erratic and notpredictive of immune function. We did, in fact, test some of our study andcontrol patients with the lower concentration of 4 µg/ml and found minimalresponses in both control and study patients. Despite the subtle evidence ofdecreased immune function and no evidence of an increased incidence ofinfections, Brearley and associates ³concluded that thymectomy in pediatric cardiac surgery should be avoided. Theyadvocated splitting the thymus vertically so that it could be held back bypericardial sutures or, if thymectomy was required, they suggested a partialexcision. On the basis of our study, we do not concur. Removal of the thymusprovides significantly better exposure, and splitting or partial resection ofthymic tissue often results in a hemorrhagic gland that may be a source ofcontinued bleeding.

The present study suggests that there must be tremendous redundancy orreserve built into the thymus. Despite what was a surgically "complete"resection of the encapsulated neonatal thymus, including the upper poles thatextend into the cervical area, there was a relatively small effect on T cells.Most surprisingly, in the patients in whom the number of new thymic emigrants(CD4, CD45, and RA) were tested, there was no evidence that thymectomy led to adecrease in the number of newly formed, naive T cells. The continued productionof new T cells suggests that retained portions of the thymus, however small, arecapable of maintaining adequate T-cell production.

The thymus normally undergoes involution, and by the second decade oflife the number of recent thymic emigrants (CD4, CD45, and RA) begins tosignificantly decrease. ^l0The difference in the appearance of the pediatric versus adult thymus gland asseen at sternotomy is consistent with this decrease. The significance of thediminished capacity to produce new thymic T cells is not entirely clear, but itcould be speculated that the increased susceptibility of some elderlyindividuals to new pathogens, such as new strains of influenza, may be relatedto their inability to produce new CD4, CD45, and RA cells. Given thispossibility it remains to be seen whether patients who have had neonatalthymectomy will more rapidly lose their ability to produce new T cells(accelerated thymic involution) and what the impact on their immunity will belater in life.

Limitations of the study
Lack of "true" controls.
Because surgical exposure would be significantly compromised withoutthymectomy, it was not deemed appropriate to prospectively randomize neonatesundergoing cardiac repair into thymectomy and no thymectomy groups. Thepossibility of leaving a portion of the thymus gland was considered. However,this was believed to be undesirable because of bleeding issues but could havebeen a second arm to a future study if "complete" thymectomy hadproved deleterious to immune function.

Lack of ability to measure retained thymus.
Although the entire encapsulated thymus was removed (including the upperpoles extending into the base of the neck), there is no way to determine howmuch thymic tissue remained. On the basis of our ability to visualize themediastinal portion of the thymus at sternotomy, we would speculate that theretained portion was in the neck although there could be small detached thymicrests near the phrenic nerves where extensive dissection is avoided. It is alsopossible that there was significant thymic regeneration from small retained"rests" of tissue. However, our experience with staged reoperation,which was carried out in a small number of the patients, did not reveal ananatomically identifiable thymic structure.

Length of follow-up.
We have demonstrated no significant defects in immune function in thepresent study. A longer follow-up would provide information on the persistenceof the trend toward lower T-cell counts and the natural history of thymicinvolution in these patients. It is our intent to continue the follow-up.

	Appendix: Discussion

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 
Dr. Steven R. Gundry (Loma Linda, Calif.). I congratulate Dr. Wells and his coauthors on a very nicely designed and presented study about a question that has worried most congenital heart surgeons since the onset of congenital heart surgery; that is, what to do with a thymus in your way and does it have any consequences. As most of us surgeons know, when something is in our way we usually throw it away and then worry about the consequences later. It is a delight to find some real science placed on this quite puzzling question.

In infant pediatric cardiac transplantation, we have believed that the elimination of the thymus and perhaps its T cells may have an effect on the early T-cell–mediated rejection of the new allograft. However, we have been disappointed in our large series at Loma Linda to find that T-cell–mediated rejection, despite a total thymectomy in all of our patients, is the predominant form of rejection. T-cell-mediated rejection is only slightly less vigorous than in a corresponding adult cardiac transplantation population. So we too have been unable to prove that thymectomy is beneficial for us in transplantation or, for that matter, negatively affects immune function.

Also, we have been surprised, often times greatly, by the regrowth of thymic tissue that we have found during those few occasions when retransplantation is necessary. During your few reoperations on patients, have you, in fact, rediscovered thymic tissue at the time of the operation?

I have only two questions. First, was the total thymectomy done by pulling both lobes of the thymus out of the neck or were the actual superior aspects of these lobes visualized directly?

Second, you point out in your article that the control patients did not have cardiac surgery. Perhaps the author could speculate as to the effect of cardiopulmonary bypass, in and of itself, on immune function and T-cell population apart from removal of the thymus.

Dr. Wells. Your first question concerns regrowth of the thymus seen at the time of a reoperation. In the moderate number of patients who have undergone staged procedures, we have not seen a significant amount of thymic tissue at the time of resternotomy.

Your second question involved the extent of the thymectomy and particularly the superior aspect of the thymic lobes as they extend into the cervical area. We have found that it is easiest to remove the neonatal thymus if you take all of the encapsulated tissue including its cervical extensions. By pulling the superior poles downward, you can usually identify and ligate the small vessels that enter the gland from the neck. The fact that we have made an effort to remove all of the encapsulated tissue may account for the lack of thymus as seen at reoperation, yet we speculate that there must be residual small rests of thymus to account for the presence of the CD4, CD45, and RA (recent thymic emigrants) identified in our patients after thymectomy. These residual thymic sites would most likely be high in the neck or laterally along the area of the phrenic nerves, where our dissection tends to be less aggressive.

Finally, there was a question on the effect of cardiopulmonary bypass on T-cell populations and immune function. We have not done these studies; and although I doubt that there would be an effect, particularly months after the procedure, it would not be difficult to get these controls.

	Acknowledgments

 
We thank Earl Leonard (MS, Biostatistics) for his help withstatistical analysis of the data.

	References

Top Abstract Introduction Patients and methods Results Discussion Appendix: Discussion References

 

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4. Lischner HW, Dacou C, DiGeorge AM. Normallymphocyte transfer (NLT) test: negative response to a patient with congenitalabsence of the thymus. Transplantation 1967;5:555-7.[Medline]
   
5. Lischner HW, Punnett HH, DiGeorge AM.Lymphocytes of extrathymic origin from an infant with congenital absence of thethymus: behavior in vitro. Nature (Lond) 1967;214:580-3.
   
6. DiGeorge AM. Immunologic deficiency diseasesin man. In: Bergsma D, editor. Birth Defects Orig Artic Ser 1968;4:116-21.
   
7. Miller JF. Immunological functions of thethymus. Lancet 1961;2:748-9.[Medline]
   
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9. Hisatomi K, Isomura T, Kawara T, YamashitaM, Hirano A, Yoshida H, et al. Changes in lymphocyte subsets, mitogenresponsiveness, and interleukin-2 production after cardiac operations. J ThoracCardiovasc Surg 1989;98:580-91.[Abstract]
   
10. Mackal CL, Fleisher TA, Brown MR, AndrichMP, Chen CC, Feurersten IM, et al. Age, thymopoiesis, Arj CD4+ T-lymphocyteregeneration after intensive chemotherapy. N Engl J Med 1995;332:143-9. [Abstract/Free Full Text]
    

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