The degree of sensitization is expressed as panel-reactive antibody (PRA). PRA testing evaluates who is most at risk of hyperacute or humoral rejection (*Cecka 2010*). A PRA of 80% reflects that the patient is crossmatch incompatible with 80% of donors. In general, patients with a PRA of more than 10% or more than 80% are considered sensitized or highly sensitized, respectively. However, different centres can use markedly different PRA cut-offs for determining sensitized and highly sensitized patients (*Cecka 2010*). On a typical waiting list for cadaveric renal transplantation, more than 50% of patients have antibodies to more than half of the reference panel, and 20% have antibodies to 90-100% (*Carpenter 1990*).

There are three types of assays used to determine PRA. The oldest is the Complement Dependent Cytotoxicity (CDC) test (*Cecka 2010, Hajeer 2006*). In this test, patient serum is tested against donor lymphocytes (B and T cells). Patients' antibodies will coat antigen expressing lymphocytes and upon administration of complement to the serum, lymphocytes are killed and detected by cell stain. The second type of assay is the Enzyme-Linked Immunoabsorbant Assay (ELISA), a solid phase assay which is more sensitive than the CDC. The third assay is the flow cytometry test. Also, there is the house method where locally acquired whole lymphocytes are used and a microbead method which uses purified HLA antigen coated microbeads. Commerical kits include the Flow PRA and Luminex tests. The CDC is thought to be inferior to the HLA Class I and II ELISA and microbead flow cytometry tests which are similar to each other (*Cecka 2010, Hajeer 2006, Worthington 2001*). Though the superiority of one approach over another is debatable, it is important since PRA may be altered in response to stimuli. PRA response may be altered by the use of medications (rituximab, immune globulin, statins, cyclophosphamide/predisolone with plasmapheresis) or certain Angiotensin Converting Enzyme genotypes (*Vieira 2004, Vo 2008,*

As previously stated, patients with antibodies to a given donor are not suitable for that transplant. Thus, blood transfusions provide a process of negative selection such that transplants destined to early failure are avoided. However, immunosuppressive agents such as cyclosporine, anti-lymphocyte globulins (ALG), etc, may suppress the responses previously subject to negative selection by transfusion, and thus lead to a decline in the transfusion effect. Other factors, such as prompt diagnosis and treatment of early rejections and HLA matching, may also play a role in this decline observed in the transfusion effect.

cannot be predicted from an individual's HLA phenotype.

responders were found to convert after about six months.

*Muhmoud 2007, Nurhan-Ozdemir 2004, Akcay 2004*).

40% (*Opelz 1981b*).

*1990*).

sclerotherapy, transjugular intrahepatic portosystemic shunt placements to relieve the effects of portal hypertension in order to have less gastrointestinal bleeding and to avoid transfusion (*Calcutti 2002*).

However, observations on patients having rejection episodes indicate that a beneficial blood transfusion effect still exists. Recent evidence suggests that the blood transfusion effect remains in certain circumstances, when one considers effects of HLA antigens, rejection episodes, and possibly the prospects of tolerance induction. In a single centre study of a notransfusion policy, the non-transfused group had more early rejection episodes (*Lundgren 1986*). Another study on the relation of rejection activity to previous blood transfusions showed that 63% of the 231 non-transfused recipients had rejection episodes during the first 60 days after transplantation, while 48% of the transfused patients had rejection (*Toyotome 1987*). Additionally, it was found that if a rejection episode occurred, the one-year survival was 49% in those with no transfusions, and 70% in the transfused patients. Hence, the original deficit of 20% poorer survival in the absence of prior transfusions may still be discerned in those patients destined to reject.

Furthermore, though the change in the transfusion effect during the early 1980s (before the introduction of cyclosporine) is most marked by a disappearance of the graded response to increasing numbers of blood units, the deleterious effect of receiving no transfusions remains, with a 10% lower one-year survival rate (*Terasaki 1986*).

Unfortunately, there are no reliable predictive tests to know who would need to have transfusions prior to transplantation.

#### **4. Possible mechanism of beneficial effects of pre-transplant transfusion**

The exact mechanism by which blood transfusion enhances transplant survival remains unknown. However, several possible mechanisms have been postulated. These are as follows:

4.1 Pre-transplant blood transfusions may cause early immunization of some recipients to selected HLA antigens. This enables the pre-transplantation crossmatch to detect those cases where rejection of donor organ would be most likely to occur. Preformed HLA antibodies (presenting as incompatible crossmatch) are a major contraindication to transplantation. About 30% of cases who receive pre-transplant transfusions become highly immunized to HLA. This response may be beneficial by preventing an unsuccessful transplant.

4.2 The beneficial effect of transfusion may also be related to the immunosuppression induced by transfusion. This may occur through enhancement of suppressor T cell activity or induction of immune tolerance by some unknown mechanism.

#### **5. Responses to allogenic blood transfusion and HLA senitization**

There are several individual differences in the effects of allogenic blood exposure. Some of these are discussed here:

5.1 Most patients do not develop anti-HLA antibodies following transfusion. Overall, 30% of transfused individuals develop antibodies (with the rate being higher in previously pregnant females and lower in males) (*Opelz 1981b*). In non transfused multiparous females,

sclerotherapy, transjugular intrahepatic portosystemic shunt placements to relieve the effects of portal hypertension in order to have less gastrointestinal bleeding and to avoid

However, observations on patients having rejection episodes indicate that a beneficial blood transfusion effect still exists. Recent evidence suggests that the blood transfusion effect remains in certain circumstances, when one considers effects of HLA antigens, rejection episodes, and possibly the prospects of tolerance induction. In a single centre study of a notransfusion policy, the non-transfused group had more early rejection episodes (*Lundgren 1986*). Another study on the relation of rejection activity to previous blood transfusions showed that 63% of the 231 non-transfused recipients had rejection episodes during the first 60 days after transplantation, while 48% of the transfused patients had rejection (*Toyotome 1987*). Additionally, it was found that if a rejection episode occurred, the one-year survival was 49% in those with no transfusions, and 70% in the transfused patients. Hence, the original deficit of 20% poorer survival in the absence of prior transfusions may still be

Furthermore, though the change in the transfusion effect during the early 1980s (before the introduction of cyclosporine) is most marked by a disappearance of the graded response to increasing numbers of blood units, the deleterious effect of receiving no transfusions

Unfortunately, there are no reliable predictive tests to know who would need to have

The exact mechanism by which blood transfusion enhances transplant survival remains unknown. However, several possible mechanisms have been postulated. These are as

4.1 Pre-transplant blood transfusions may cause early immunization of some recipients to selected HLA antigens. This enables the pre-transplantation crossmatch to detect those cases where rejection of donor organ would be most likely to occur. Preformed HLA antibodies (presenting as incompatible crossmatch) are a major contraindication to transplantation. About 30% of cases who receive pre-transplant transfusions become highly immunized to

4.2 The beneficial effect of transfusion may also be related to the immunosuppression induced by transfusion. This may occur through enhancement of suppressor T cell activity

There are several individual differences in the effects of allogenic blood exposure. Some of

5.1 Most patients do not develop anti-HLA antibodies following transfusion. Overall, 30% of transfused individuals develop antibodies (with the rate being higher in previously pregnant females and lower in males) (*Opelz 1981b*). In non transfused multiparous females,

HLA. This response may be beneficial by preventing an unsuccessful transplant.

**5. Responses to allogenic blood transfusion and HLA senitization**

or induction of immune tolerance by some unknown mechanism.

**4. Possible mechanism of beneficial effects of pre-transplant transfusion**

transfusion (*Calcutti 2002*).

discerned in those patients destined to reject.

transfusions prior to transplantation.

follows:

these are discussed here:

remains, with a 10% lower one-year survival rate (*Terasaki 1986*).

about 10% develop such antibodies (the response being transient usually). Multiparous women challenged with blood transfusions show an increase in the responder rate to 30- 40% (*Opelz 1981b*).

5.2 Some responders have a highly selective immune response directed to one to four HLA antigens, while others show sensitivity to better than 95% of a reference panel (*Carpenter 1990*).

Thus, even though a genetic control over responsiveness is evident, the responder status cannot be predicted from an individual's HLA phenotype.

The responder status to blood transfusions becomes evident very early after the initiation of blood transfusions. In the earlier days of frequent transfusions, many patients remained negative on antibody screens after more than 50 blood transfusions, and very few nonresponders were found to convert after about six months.

The degree of sensitization is expressed as panel-reactive antibody (PRA). PRA testing evaluates who is most at risk of hyperacute or humoral rejection (*Cecka 2010*). A PRA of 80% reflects that the patient is crossmatch incompatible with 80% of donors. In general, patients with a PRA of more than 10% or more than 80% are considered sensitized or highly sensitized, respectively. However, different centres can use markedly different PRA cut-offs for determining sensitized and highly sensitized patients (*Cecka 2010*). On a typical waiting list for cadaveric renal transplantation, more than 50% of patients have antibodies to more than half of the reference panel, and 20% have antibodies to 90-100% (*Carpenter 1990*).

There are three types of assays used to determine PRA. The oldest is the Complement Dependent Cytotoxicity (CDC) test (*Cecka 2010, Hajeer 2006*). In this test, patient serum is tested against donor lymphocytes (B and T cells). Patients' antibodies will coat antigen expressing lymphocytes and upon administration of complement to the serum, lymphocytes are killed and detected by cell stain. The second type of assay is the Enzyme-Linked Immunoabsorbant Assay (ELISA), a solid phase assay which is more sensitive than the CDC. The third assay is the flow cytometry test. Also, there is the house method where locally acquired whole lymphocytes are used and a microbead method which uses purified HLA antigen coated microbeads. Commerical kits include the Flow PRA and Luminex tests. The CDC is thought to be inferior to the HLA Class I and II ELISA and microbead flow cytometry tests which are similar to each other (*Cecka 2010, Hajeer 2006, Worthington 2001*).

Though the superiority of one approach over another is debatable, it is important since PRA may be altered in response to stimuli. PRA response may be altered by the use of medications (rituximab, immune globulin, statins, cyclophosphamide/predisolone with plasmapheresis) or certain Angiotensin Converting Enzyme genotypes (*Vieira 2004, Vo 2008, Muhmoud 2007, Nurhan-Ozdemir 2004, Akcay 2004*).

As previously stated, patients with antibodies to a given donor are not suitable for that transplant. Thus, blood transfusions provide a process of negative selection such that transplants destined to early failure are avoided. However, immunosuppressive agents such as cyclosporine, anti-lymphocyte globulins (ALG), etc, may suppress the responses previously subject to negative selection by transfusion, and thus lead to a decline in the transfusion effect. Other factors, such as prompt diagnosis and treatment of early rejections and HLA matching, may also play a role in this decline observed in the transfusion effect.

Effect of Blood Transfusion on Subsequent Organ Transplantation 251

Many people behave as if they were clonally deleted for the HLA antigens of their mothers which they did not inherit. This was first observed in an analysis of end-stage renal disease patients having very high PRA, but consistently having no antibodies against a small number of HLA antigens (*Claas 1988*). This unresponsiveness to antibody response in some sensitized patients was found to be due to a failure to respond to non-inherited maternal HLA antigens. These findings are important as this may be applicable to selection of donors

There have been studies reporting selection of single blood donors from an unrelated population matched for one DR antigen only and not for a whole HLA haplotype (*Lagaaij 1989*). In recipients, anti-HLA antibodies were less frequent as result of one DR matched versus no DR matched transfusions. Such transfusions may induce production of antiidiotypic antibodies which can prevent the response of T cells specific to the immunizing

Studies of renal and heart transplant recipients have shown a reduced rejection frequency and better graft survival when the only blood received prior to transplantation was 1-3 units from donors matched for one DR antigen with the recipient (*Lagaaij 1989*). Anti-HLA antibody production was also diminished in the one DR transfused group. This immunization effect could be due to suppression via some antigen-specific

An alternative possibility could be that provision of self DR on transfused cells induces a different sort of systemic response similar to the autologous mixed lymphocyte response (AMLR) (*Sakane 1979*). However, additional confirmatory studies are needed, along with careful study of possibly different effects when alloantigens are presented in the context of

Additional adequately powered multi-institutional studies should be conducted, because individual centre practices are variable. These studies should have adequate reporting of demographics and either use statistical means to account for confounders or use randomization. Patients receiving or being randomized to no transfusions should be screened to assure that this not only includes transfusions within the dialysis or transplant

The impact of different immunosuppressive regimens on outcomes in patients receiving transfusions should be studied to identify those regimens which can suppress the advantageous or detrimental effects of transfusion on outcomes. This should be specifically evaluated to determine whether transplants need to be encouraged, avoided, or matched

Unlike the prior reports, where pre-transplant transfusions seemed to worsen renal allograft outcomes, transfusions generally have a beneficial to neutral effect on transplant outcomes.

**8. Effect of in-utero (feto-maternal) transfusion in adult renal transplant**

**9. Conditioning with blood transfusions for tolerance induction**

for transplant recipients.

immunoregulatory pathway.

self versus non-self class II HLA.

**10. Conclusions and future research**

centre, but other transfusions as well.

with certain regimens.

HLA antigens (*Phelan 1989, Kawamura 1989*).

Iwaki et al demonstrated a benefit from transfusions in HLA-DR mismatched cases. This benefit was not observed when there were no mismatches. In cases with no mismatches, one-year survival was reported as 80% in both transfused and non-transfused recipients, while transfusions added an 8-10% benefit in the one- and two-DR mismatched groups (*Iwaki 1990*).

### **6. Nonspecific Immune suppression after transfusion**

Allogeneic blood transfusions produce generalized immunosuppression in the recipient. This is due to a variety of changes in the immunological functions, such as decreased function of natural killer cells, macrophage migration to sites of injury, lymphocyte proliferation, and cutaneous delayed hypersensitivity. Donor leukocytes in allogeneic blood may play a role in suppressing cellular immune function.

Serial measurements of cell-mediated responses in previously non-transfused end-stage renal disease patients showed marked reductions in response to mitogens and recall antigens (e.g. PPD, tetanus, mumps, vaccinia) after a single blood transfusion. This effect lasted for over two weeks (*Fischer 1980*). More profound and lasting depression was seen after a second transfusion given after 4 weeks.

Reports have also suggested that allogeneic blood transfusions increase the incidence of postoperative infection and the tumour recurrence rate (*Schriemer 1988, Wu 1988)*. Such postoperative morbidities have been attributed to the immunomodulatory effects of blood transfusion. However, this association is unproven, and there is currently insufficient evidence to recommend the routine use of leukodepleted blood components for surgical patients to prevent either postoperative infection or tumour recurrence.

#### **7. Antigen specific immune-suppression**

The final objective in transplantation is the induction of specific unresponsiveness, or tolerance, so that patients do not need to take anti-rejection medications indefinitely. This unresponsiveness is specific for donor antigens; i.e. the recipients produce perfectly normal responses to cells bearing other HLA antigens.

Patients, who have received pre-transplant transfusions, have marked reductions in cells capable of killing donor cells (*Herzog 1987*). However, full activation by polyclonal mitogens will restore the expected cytotoxic T cells precursor frequency to the normal level (*Dallman 1989*). Thus, the possibility of clonal deletion is unlikely. Though the full T-cell repertoire is present, the individual is functionally unresponsive in the absence of stimuli which bypass inhibitory immunoregulatory influences.

When living donor kidney graft recipients are prepared by single-donor blood transfusions from the potential kidney donor, this is known as "donor-specific transfusions" (DST). Such blood transfusions (which share an HLA haplotype, or at least one DR antigen with the transfused recipient) do not produce an increase in cytotoxic T cells precursor frequency or cell mediated immunity. If such recipients do not develop a positive cross-match, they are still reported to have superior graft survival, close to that of an HLA identical donor (*Salvatierra 1980*).

Iwaki et al demonstrated a benefit from transfusions in HLA-DR mismatched cases. This benefit was not observed when there were no mismatches. In cases with no mismatches, one-year survival was reported as 80% in both transfused and non-transfused recipients, while transfusions added an 8-10% benefit in the one- and two-DR mismatched groups

Allogeneic blood transfusions produce generalized immunosuppression in the recipient. This is due to a variety of changes in the immunological functions, such as decreased function of natural killer cells, macrophage migration to sites of injury, lymphocyte proliferation, and cutaneous delayed hypersensitivity. Donor leukocytes in allogeneic blood

Serial measurements of cell-mediated responses in previously non-transfused end-stage renal disease patients showed marked reductions in response to mitogens and recall antigens (e.g. PPD, tetanus, mumps, vaccinia) after a single blood transfusion. This effect lasted for over two weeks (*Fischer 1980*). More profound and lasting depression was seen

Reports have also suggested that allogeneic blood transfusions increase the incidence of postoperative infection and the tumour recurrence rate (*Schriemer 1988, Wu 1988)*. Such postoperative morbidities have been attributed to the immunomodulatory effects of blood transfusion. However, this association is unproven, and there is currently insufficient evidence to recommend the routine use of leukodepleted blood components for surgical

The final objective in transplantation is the induction of specific unresponsiveness, or tolerance, so that patients do not need to take anti-rejection medications indefinitely. This unresponsiveness is specific for donor antigens; i.e. the recipients produce perfectly normal

Patients, who have received pre-transplant transfusions, have marked reductions in cells capable of killing donor cells (*Herzog 1987*). However, full activation by polyclonal mitogens will restore the expected cytotoxic T cells precursor frequency to the normal level (*Dallman 1989*). Thus, the possibility of clonal deletion is unlikely. Though the full T-cell repertoire is present, the individual is functionally unresponsive in the absence of stimuli which bypass

When living donor kidney graft recipients are prepared by single-donor blood transfusions from the potential kidney donor, this is known as "donor-specific transfusions" (DST). Such blood transfusions (which share an HLA haplotype, or at least one DR antigen with the transfused recipient) do not produce an increase in cytotoxic T cells precursor frequency or cell mediated immunity. If such recipients do not develop a positive cross-match, they are still reported to have superior graft survival, close to that of an HLA identical donor

patients to prevent either postoperative infection or tumour recurrence.

**6. Nonspecific Immune suppression after transfusion**

may play a role in suppressing cellular immune function.

after a second transfusion given after 4 weeks.

**7. Antigen specific immune-suppression**

responses to cells bearing other HLA antigens.

inhibitory immunoregulatory influences.

(*Salvatierra 1980*).

(*Iwaki 1990*).

#### **8. Effect of in-utero (feto-maternal) transfusion in adult renal transplant**

Many people behave as if they were clonally deleted for the HLA antigens of their mothers which they did not inherit. This was first observed in an analysis of end-stage renal disease patients having very high PRA, but consistently having no antibodies against a small number of HLA antigens (*Claas 1988*). This unresponsiveness to antibody response in some sensitized patients was found to be due to a failure to respond to non-inherited maternal HLA antigens. These findings are important as this may be applicable to selection of donors for transplant recipients.

### **9. Conditioning with blood transfusions for tolerance induction**

There have been studies reporting selection of single blood donors from an unrelated population matched for one DR antigen only and not for a whole HLA haplotype (*Lagaaij 1989*). In recipients, anti-HLA antibodies were less frequent as result of one DR matched versus no DR matched transfusions. Such transfusions may induce production of antiidiotypic antibodies which can prevent the response of T cells specific to the immunizing HLA antigens (*Phelan 1989, Kawamura 1989*).

Studies of renal and heart transplant recipients have shown a reduced rejection frequency and better graft survival when the only blood received prior to transplantation was 1-3 units from donors matched for one DR antigen with the recipient (*Lagaaij 1989*). Anti-HLA antibody production was also diminished in the one DR transfused group. This immunization effect could be due to suppression via some antigen-specific immunoregulatory pathway.

An alternative possibility could be that provision of self DR on transfused cells induces a different sort of systemic response similar to the autologous mixed lymphocyte response (AMLR) (*Sakane 1979*). However, additional confirmatory studies are needed, along with careful study of possibly different effects when alloantigens are presented in the context of self versus non-self class II HLA.

#### **10. Conclusions and future research**

Additional adequately powered multi-institutional studies should be conducted, because individual centre practices are variable. These studies should have adequate reporting of demographics and either use statistical means to account for confounders or use randomization. Patients receiving or being randomized to no transfusions should be screened to assure that this not only includes transfusions within the dialysis or transplant centre, but other transfusions as well.

The impact of different immunosuppressive regimens on outcomes in patients receiving transfusions should be studied to identify those regimens which can suppress the advantageous or detrimental effects of transfusion on outcomes. This should be specifically evaluated to determine whether transplants need to be encouraged, avoided, or matched with certain regimens.

Unlike the prior reports, where pre-transplant transfusions seemed to worsen renal allograft outcomes, transfusions generally have a beneficial to neutral effect on transplant outcomes.

Effect of Blood Transfusion on Subsequent Organ Transplantation 253

Herzog W, Zanker B, Irschick E, Huber C, Franz HE, Wagner H, Kabelitz D. Selective

Iwaki Y, Cecka M, Terasaki PI. The transfusion effect in cadaver kidney transplants-yes or

Kawamura T, Sakagami K, Haisa M, Morisaki F, Takasu S, Inagaki M, Oiwa T, Toshihiko 0,

Lagaaij EL, Hennemann PH, Ruigrok M, deHaan MW, Persijn GG, Termijtelen A, Hendriks

Light J A, Metz S, Oddenino K, et al. Fresh versus stored blood in donor specific transfusion.

Lundgren G, Groth CG, Albrechtsen D, et al. HLA matching and pretransplant blood

Matas AJ, Simmons RL, Buselmeier TJ, Najarian JS, Kjellstrand CM. Lethal complications of

Medawar PB. Immunity to Homologous Grafted Skin. II. Relationship Between Antigens of

Miller WV, Schmidt R, Luke RG, et al. Effect of Cytotoxicity Antibodies in Potential Transplant Recipients of Leukocyte-poor Blood Transfusions. Lancet 1975;1:893. Muhmoud KM, Sobh MA, el Shenawy F, et al. Management of sensitized patients awaiting

and simvastatin offer a solution. Eur J Cancer Clin Oncol 2007;56:202-5. Nurhan-Ozdemir F, Akcay A, Sezer S, et al. Effect of simvastatin in the treatment of highly

Opelz G, Sengar DPS, Mickey MR, Terasaki PI. Effect of blood transfusions on subsequent

Opelz G and Terasaki PI. Poor Kidney-transplant Survival in Recipients with Frozen-blood

Opelz G and Terasaki PI. Prolongation Effect of Blood Transfusions on Kidney Graft

Opelz G, Terasaki PI. Improvement of kidney graft survival with increased numbers of

Opelz G, Terasaki PI. Importance of preoperative (not perioperative) transfusions for

Opelz G, Graver B, Mickey MR, Terasaki PI. Lymphocytotoxic antibody responses to

Opelz G. The Collaborative Transplant Study: Correlation of HLA matching with kidney

transfusion in potential kidney transplant recipients. Transplantation

graft survival in patients with or without cyclosporine treatment. Transplantation

cadaveric kidney transplants. Transplantation 1981a;31:106-198.

well-functioning kidney allograft. Transplantation 1987;43:384-389.

no. Transplantation 1990;49:56-59.

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Transplant Proc 1982;14: 296-301.

cyclosporine. Lancet 1986;i:66-69.

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outcomes. Transplant immunology 2004;13:39-42.

kidney transplants. Transplant Proc 1973;5:253-259.

Transfusions or no Transfusions. Lancet 1974;2:696.

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reduction of donor-specific cytotoxic T lymphocyte precursors in patients with a

Orita K. Induction of antiidiotypic antibodies by donor-specific blood transfusions.

GFJ, Weimar W, Claas F, van Rood JJ. Effect of one HLA-DR antigen-matched and completely HLA-DR mismatched blood transfusions on survival of heart and

transfusions in cadaveric renal transplantation-a changing picture with

bilateral nephrectomy and splenectomy in hemodialyzed patients. Am J Surg

renal transplantation: does sequential therapy with intravenous immunoglobulin

sensitized dialysis patients: the pre and post-renal transplantation follow-up

There is not much support for the belief that transfusions increase the risk of graft rejection. There is evidence that patients receiving pre-transplant transfusions have increased levels of sensitization as assessed by PRA. With regard to rejection, the data are more ambiguous with some analyses showing benefit, some showing a neutral effect, and other analyses showing harm, although the number of studies evaluating more recent time periods is quite limited.

Thus, future application of deliberate HLA antigen exposure in conjunction with novel immunological manipulations may provide a more effective avenue to tolerance induction. However, the literature base is weak and future research is needed to assess the impact of transfusions on allograft and patient survival outcomes in renal transplant recipients.

#### **11. References**

There is not much support for the belief that transfusions increase the risk of graft rejection. There is evidence that patients receiving pre-transplant transfusions have increased levels of sensitization as assessed by PRA. With regard to rejection, the data are more ambiguous with some analyses showing benefit, some showing a neutral effect, and other analyses showing harm, although the number of studies evaluating more recent time periods is quite limited.

Thus, future application of deliberate HLA antigen exposure in conjunction with novel immunological manipulations may provide a more effective avenue to tolerance induction. However, the literature base is weak and future research is needed to assess the impact of transfusions on allograft and patient survival outcomes in renal transplant recipients.

Akcay A, Ozdemir FN, Atac FB, et al. Angiotensin-Converting Enzyme genotype is a

Calcutti RA, Shah OJ, Khan NA, Dar MA, Farooq A. Role of Transfusion Services in Organ

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Claas FHJ, Gijbels Y, van der Velden-deMunck J, van Rood JJ. Induction of B cell

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**16**

Changku Jia

*China*

**Posthepatic Manipulative**

*Department of Hepatobiliary Surgery,*

**Blood Extraction with Blood Transfusion Alleviates Liver**

**Transplant Ischemia/Reperfusion**

**Injury and Its Induced Lung Injury**

*Affiliated Hospital of Hainan Medical college, Haikou, Hainan Province,*

Liver ischemia-reperfusion (I/R) occurs in a number of clinical syndromes, including liver transplantation (Huguet et al., 1994). Liver I/R is a major obstacle to liver transplantation (Rosen et al., 1998; Banga et al., 2005).It causes liver graft primary nonfunction, up to 10% of early liver transplant failures and higher incidence of acute and chronic rejection (Banga et al., 2005; Henderson et al., 1999; Farmer et al., 2008). Despite many experimental improvements and efforts have been made to attenuate liver I/R injury heretofore, clinical liver I/R was not effectively prevented (Olthoff., 2001; Lehmann et al., 2000; Clarke et al., 2009). And apart from the liver injury, extrahepatic factors like pulmonary injury also play an important role in the outcome of liver transplantation recipients (Sykes et al., 2007; Pereboom et al., 2009; Shimizu et al., 2005; Liu et al., 1996). The incidence rate of acute lung injury is about 40% and greatly threatens the lives of recipients and their quality of life after transplantion (Aduen et al., 2003). So it is meaningful to take strategy to protect the lung

Multiple mechanisms are involved in the pathogenesis of liver transplant I/R in which reactive oxygen species (ROS) have been shown to play a central role in aggravating tissue

In previous study, using rat warm I/R injury model, we demonstrated that posthepatic ROS concentration elevated gradually with time after reperfusion. And in the early period of reperfusion posthepatic ROS concentration is higher than that in infrahepatic vena cava (IH-VC). Furthermore, we provided that 2% of body weight SH-VC manipulative blood extraction with blood transfusion at 10 min after reperfusion significantly alleviated liver warm I/R injury (Jia et al., 2008). In this study we evaluated the potential effects of posthepatic manipulative blood extraction and blood transfusion on the liver transplant cold

**1. Introduction**

against liver transplantation induced injury.

injury of the transplant (Zhang et al., 2007).

I/R injury and the lung injury induced by liver cold I/R in rat.

### **Posthepatic Manipulative Blood Extraction with Blood Transfusion Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury**

Changku Jia

*Department of Hepatobiliary Surgery, Affiliated Hospital of Hainan Medical college, Haikou, Hainan Province, China*

#### **1. Introduction**

254 Blood Transfusion in Clinical Practice

Parfrey PS, Forbes RDC, Hutchinson TA, Beaudoin JG, Dauphinee WD, Hollomby DJ,

Phelan DL, Rodey GE, Anderson CB. The development and specificity of antiidiotypic

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Sakane T, Green I. Specificity and suppressor function of human T cells responsive to

Salvatierra 0, Vincenti F, Amend W, Potter D, Iwaki Y, Opelz G, Terasaki PI, Duca R,

Sanfilippo FP, Bollinger RR, MacQueen JM, Brooks BJ, Koepke JA. A randomized study

Schriemer PA, Longnecker DE, Mintz PD. The possible immunosuppresive effect of perioperative blood transfusions in cancer patients. Anesthesiology 1988;68:422-428. Suarez-Ch R and Jonasson O. Isoimmunization of Potential Transplant Recipients: General Frequency and Some Associated Factors. Transplant. Proc. 1972; IV:577. Tang H, Chelamcharla M, Baird BC, et al. Factors affecting kidney-transplant outcome in

Terasaki PI, Himaya NS, Cecka M, Cicciarelli J, Cook DJ, Ito T, Iwaki Y, Mickey MR, Takiff

Vieira CA, Agarwal A., Book BK, et al. Rituximab for reduction of anti-HLA antibodies in

Vo A.A., Lukovsky M, Wang J, et al. Rituximab and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med 2008;359:242-51. Waanders MM, Roelen DL, de Fijter JW, et al. Protocolled blood transfusions in recipients of

Worthington JE, Robson AJ, Sheldon S, et al. A comparison of enzyme-linked

Wu H-S, Little AG. Perioperative blood transfusions and cancer recurrence. J Clin Oncol

H, Tiwari JL, Toyotome A. Overview. In: Clinical Transplants 1986. Edited by PI Terasaki. Los Angeles, CA, UCLA Tissue Typing Laboratory, 1986, pp 367-398. Toyotome A, Terasaki PI, Salvatierra O, et al. Early graft function. In: Clinical Transplants

1987. Edited by PI Terasaki. Los Angeles, CA, UCLA Tissue Typing Laboratory,

patients awaiting renal transplantation: safety, pharmacodynamics, and

a simultaneous pancreas-kidney transplant reduce severe acute graft rejection.

immunoabsorbent assays and flow cytometry techniques for the detection of HLA

cadaver renal allograft recipient. Transfusion 1985; 25(2):116-19.

recipients with lupus nephritis. Clin Transplant 2008;22:263-72.

pharmacokinetics. Transplantation 2004;77:542-8.

specific antibodies. Hum Immunol 2001;62:1178-84.

Transplantation 2008;85:1668-70.

Transfusions on Renal Allograft Survival. Transfusion 1976;16:536.

renal transplant recipients. Transplantation 1984;37:461-466.

autologous non-T cells. J Immunol 1979;123:584-589.

Transplantation 1989;48:57-60.

1980;192:543-552.

1987:435-452.

1988;6:1348-1354.

Guttmann RD. The clinical and pathological course of hepatitis B liver disease in

antibodies in renal transplant recipients receiving single-donor blood transfusions.

deglycerolized red cells on renal allograft survival. Transplantation 1977;24: 449-52.

ed. Progress in hematology. *vol* XVI. Orlandu: Grune and Stratton Inc. 1986:99-122.

Cochrum K, Hanes D, Stoney RJ, Feduska NJ. Deliberate donor-specific blood transfusions prior to living related renal transplantation-a new approach. Ann Surg

comparing leukocyte depleted versus packed red cell transfusion in prospective

Liver ischemia-reperfusion (I/R) occurs in a number of clinical syndromes, including liver transplantation (Huguet et al., 1994). Liver I/R is a major obstacle to liver transplantation (Rosen et al., 1998; Banga et al., 2005).It causes liver graft primary nonfunction, up to 10% of early liver transplant failures and higher incidence of acute and chronic rejection (Banga et al., 2005; Henderson et al., 1999; Farmer et al., 2008). Despite many experimental improvements and efforts have been made to attenuate liver I/R injury heretofore, clinical liver I/R was not effectively prevented (Olthoff., 2001; Lehmann et al., 2000; Clarke et al., 2009). And apart from the liver injury, extrahepatic factors like pulmonary injury also play an important role in the outcome of liver transplantation recipients (Sykes et al., 2007; Pereboom et al., 2009; Shimizu et al., 2005; Liu et al., 1996). The incidence rate of acute lung injury is about 40% and greatly threatens the lives of recipients and their quality of life after transplantion (Aduen et al., 2003). So it is meaningful to take strategy to protect the lung against liver transplantation induced injury.

Multiple mechanisms are involved in the pathogenesis of liver transplant I/R in which reactive oxygen species (ROS) have been shown to play a central role in aggravating tissue injury of the transplant (Zhang et al., 2007).

In previous study, using rat warm I/R injury model, we demonstrated that posthepatic ROS concentration elevated gradually with time after reperfusion. And in the early period of reperfusion posthepatic ROS concentration is higher than that in infrahepatic vena cava (IH-VC). Furthermore, we provided that 2% of body weight SH-VC manipulative blood extraction with blood transfusion at 10 min after reperfusion significantly alleviated liver warm I/R injury (Jia et al., 2008). In this study we evaluated the potential effects of posthepatic manipulative blood extraction and blood transfusion on the liver transplant cold I/R injury and the lung injury induced by liver cold I/R in rat.

Posthepatic Manipulative Blood Extraction with Blood Transfusion

Figure 1.

sites of vascular occlusion.

described in previous study (Jia et al., 2008).

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 257

containing fresh blood was connected to a 5.5# scalp needle and was place on microinfusion pump. Right external jugular vein of the tested rats was exposed and the scalp needle was inserted into it. The pipeline was maintained by small flow of blood transfusion (1 ml/h). To obtain pure and immediate SH-VC blood after 1 h of reperfusion, an indwelling intravenous line (BD Vialon Biomaterial) was cannulated in the IH-VC with the catheter tip positioned between the liver and diaphragm. A slipknot around the indwelling intravenous line was tied using the pre-placed 7# silk suture to separate blood between both sides of the suture. Then SH-VC was occluded at the level of diaphragm and blood transfusion was sped up to 90ml/h at the same time. Then pure SH-VC blood was drawn-out through the catheter with the speed matching that of transfusion. The bleeding volume is 2% of body weight. After the blood extraction SH-VC and IH-VC was orderly unblocked followed by the catheter withdrawn. A schematic representation for blood extraction and transfusion is shown in

Fig. 1. Schema of blood extraction in SH-VC, blood transfusion in external jugular vein and

To obtain pure and immediate SH-VC blood, the vena cava was occluded between the liver and the right atrium and between the liver and right adrenal vein. Pure SH-VC blood was

Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group (IB, n=6): One hour after transplantation, two percent of body weight IH-VC manipulative blood extraction with blood transfusion was performed by the method

drawn-out through the catheter with the speed matching that of transfusion. (From Jia C, et al. Suprahepatic vena cava manipulative bleeding alleviates hepatic

ischemia-reperfusion injury in rats. Dig Liver Dis, 2008, 40(4):285-292).

### **2. Materials and methods**

#### **2.1 Animals and reagents**

Male Sprague-Dawley rats, weighing 200-250 g, were supplied by the Shanghai Experimental Animal Center. Rats were housed under standard environmental conditions with a 12:12-h light-dark cycle. Before use in experiments, rats were fasted overnight with free access to water. All animals received humane care in accordance with the Guidelines of the Animal Care Committee of Zhejiang Medical University.

Reagents:(1) MDA and myeloperoxidase (MPO) assay kit (Jiancheng Bioengineering Co.Ltd, Nanjing, China). (2) Naphthol ASD chloroacetate esterase (Sigma Chemical Co., St. Louis, MO). (3) IL-6 ELISA assay kit (Quantikine,MN, USA). (4) TNF-α ELISA assay kit (Quantikine, MN, USA).

#### **2.2 Part I: Liver transplantation and determination of peak production of posthepatic ROS after transplantation**

#### **2.2.1 Surgical procedures, experimental groups and sample harvesting**

The animals were anesthetized with intraperitoneal injection of 4% chloral hydrate. Orthotopic liver transplantation was performed by Kamada's two-cuff technique (Huguet et al., 1994) (Kamada et al., 1983). Blood was taken at 5 min, 20 min, 30 min, 1 h, 2 h, 6 h after reperfusion respectively (n=7 for each time point). Pure posthepatic blood and IH-VC blood were sampled by the method described in previous study (Jia et al., 2008). Sera were separated for lipid peroxide detection. Median cold ischemia time in all transplantation procedures in this study including in Part I and Part II was 50.22 min.

#### **2.2.2 Lipid peroxide assay**

The amount of oxidative stress was assessed by determining serum levels of malonaldehyde (MDA). Serum MDA was determined by the thiobarbituric acid reaction. Sera were isolated then MDA was detected according to manufacturer's instructions.

#### **2.3 Part II: Posthepatic manipulative blood extraction on liver transplant I/R injury and hepatic cold I/R induced lung injury**

#### **2.3.1 Surgical procedures, experimental groups and sample harvesting**

Rats were randomly divided into four groups and subjected to the following treatments.

IR control group (IR, n=5): The first group involved liver transplantation but without any intervention after transplantation.

Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group (PB, n=7): One hour after transplantation, two percent of body weight posthepatic manipulative blood extraction with blood transfusion was performed by the method described in previous study (Jia et al., 2008). After isolation of IH-VC, a 7# silk suture was placed just above the level of right adrenal vein behind the IH-VC. All the structures in the portal triad were clamped by a Bull-dog clamp. Fresh abdominal aorta blood was collected from other SD rats and was anti-coagulated using heparin. The syringe

Male Sprague-Dawley rats, weighing 200-250 g, were supplied by the Shanghai Experimental Animal Center. Rats were housed under standard environmental conditions with a 12:12-h light-dark cycle. Before use in experiments, rats were fasted overnight with free access to water. All animals received humane care in accordance with the Guidelines of

Reagents:(1) MDA and myeloperoxidase (MPO) assay kit (Jiancheng Bioengineering Co.Ltd, Nanjing, China). (2) Naphthol ASD chloroacetate esterase (Sigma Chemical Co., St. Louis, MO). (3) IL-6 ELISA assay kit (Quantikine,MN, USA). (4) TNF-α ELISA assay kit

**2.2 Part I: Liver transplantation and determination of peak production of posthepatic**

The animals were anesthetized with intraperitoneal injection of 4% chloral hydrate. Orthotopic liver transplantation was performed by Kamada's two-cuff technique (Huguet et al., 1994) (Kamada et al., 1983). Blood was taken at 5 min, 20 min, 30 min, 1 h, 2 h, 6 h after reperfusion respectively (n=7 for each time point). Pure posthepatic blood and IH-VC blood were sampled by the method described in previous study (Jia et al., 2008). Sera were separated for lipid peroxide detection. Median cold ischemia time in all transplantation

The amount of oxidative stress was assessed by determining serum levels of malonaldehyde (MDA). Serum MDA was determined by the thiobarbituric acid reaction. Sera were isolated

**2.3 Part II: Posthepatic manipulative blood extraction on liver transplant I/R injury and**

Rats were randomly divided into four groups and subjected to the following treatments.

IR control group (IR, n=5): The first group involved liver transplantation but without any

Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group (PB, n=7): One hour after transplantation, two percent of body weight posthepatic manipulative blood extraction with blood transfusion was performed by the method described in previous study (Jia et al., 2008). After isolation of IH-VC, a 7# silk suture was placed just above the level of right adrenal vein behind the IH-VC. All the structures in the portal triad were clamped by a Bull-dog clamp. Fresh abdominal aorta blood was collected from other SD rats and was anti-coagulated using heparin. The syringe

**2.2.1 Surgical procedures, experimental groups and sample harvesting**

procedures in this study including in Part I and Part II was 50.22 min.

then MDA was detected according to manufacturer's instructions.

**2.3.1 Surgical procedures, experimental groups and sample harvesting**

the Animal Care Committee of Zhejiang Medical University.

**2. Materials and methods 2.1 Animals and reagents**

(Quantikine, MN, USA).

**ROS after transplantation**

**2.2.2 Lipid peroxide assay**

**hepatic cold I/R induced lung injury**

intervention after transplantation.

containing fresh blood was connected to a 5.5# scalp needle and was place on microinfusion pump. Right external jugular vein of the tested rats was exposed and the scalp needle was inserted into it. The pipeline was maintained by small flow of blood transfusion (1 ml/h). To obtain pure and immediate SH-VC blood after 1 h of reperfusion, an indwelling intravenous line (BD Vialon Biomaterial) was cannulated in the IH-VC with the catheter tip positioned between the liver and diaphragm. A slipknot around the indwelling intravenous line was tied using the pre-placed 7# silk suture to separate blood between both sides of the suture. Then SH-VC was occluded at the level of diaphragm and blood transfusion was sped up to 90ml/h at the same time. Then pure SH-VC blood was drawn-out through the catheter with the speed matching that of transfusion. The bleeding volume is 2% of body weight. After the blood extraction SH-VC and IH-VC was orderly unblocked followed by the catheter withdrawn. A schematic representation for blood extraction and transfusion is shown in Figure 1.

Fig. 1. Schema of blood extraction in SH-VC, blood transfusion in external jugular vein and sites of vascular occlusion.

To obtain pure and immediate SH-VC blood, the vena cava was occluded between the liver and the right atrium and between the liver and right adrenal vein. Pure SH-VC blood was drawn-out through the catheter with the speed matching that of transfusion.

(From Jia C, et al. Suprahepatic vena cava manipulative bleeding alleviates hepatic ischemia-reperfusion injury in rats. Dig Liver Dis, 2008, 40(4):285-292).

Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group (IB, n=6): One hour after transplantation, two percent of body weight IH-VC manipulative blood extraction with blood transfusion was performed by the method described in previous study (Jia et al., 2008).

Posthepatic Manipulative Blood Extraction with Blood Transfusion

criteria (Suzuki et al., 1993).

**2.11 Statistical analysis**

statistically significant.

**3. Results**

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 259

necrosis of parenchymal cells were evaluated semiquantitatively according to Suzuki's

These data are expressed as mean ± SD. Calculations were made using the SPSS 13.0 for windows computer software (SPSS Inc., Chicago, IL). Statistical comparisons were performed using one-way analysis of variance (ANOVA) and the LSD t-test for blood MDA, IL-6, liver PMNs, TNF-α and enzyme levels. P-values less than 0.05 were considered

Figure 2 represents the kinetic changes of MDA in all time points in experiment Part I. In this study MDA concentration within SH-VC peaked at 1 h after transplantation. MDA concentration at 1 h was significantly higher than that at other time points except for 5 min. Furthermore, MDA concentrations in SH-VC were significantly higher than that in IH-VC at 20 min,30 min,1h (*P*<0.05) and slightly higher at all other time points. MDA

**3.1 Time course and peak level of posthepatic MDA after transplantation**

Fig. 2. Time course and peak level of MDA after liver transplantation in Part I

**3.2 MDA and PMO levels in the sera**

SH-VC represents the group that blood was taken form SH-VC. IH-VC represents the group that blood was taken form IH-VC. a*P* < 0.05 vs. 60 min within SH-VC. b*P* < 0.05 vs. IH-VC.

In experiment Part II, serum MDA level at 6 h after transplantation was slightly lower in PB, IB and PR group than in IR group but no statistically significant. Whereas serum MPO level in PB and PR groups (48.78±9.36 pg/ml and 49.36±18.32 pg/ml, respectively) were significantly lower than that in IR group (71.34±22.14) (*P*=0.022, *P*=0.045, respectively). Serum MPO level in IB group was not significantly lower than that in IR group (Figure 3).

concentration within IH-VC peaked at 2 h after transplantation.

Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group (PR, n=5): Ringer's lactate solution was used for transfusion while posthepatic manipulative blood extraction.

In IR group, a section of the liver was sampled and fixed in 10% formaldehyde, and blood samples were taken from IH-VC at 7 h after transplantation to meet the time course of other groups. In PB, IB and PR group, a section of the liver was sampled and fixed in 10% formaldehyde, and blood samples were taken from IH-VC at 6 h after blood extraction. A section of lung was sampled and fixed in 10% formaldehyde for pathological examination at 6 h after blood extraction. Another section of lung was sampled for the analysis of cytokine and tissue edema.

#### **2.4 Myeloperoxidase and lipid peroxide assays**

Sera were isolated and detected for MPO and MDA, an indicator of oxidative injury. Serum MPO contents were measured to analyze degree of neutrophil sequestration and activation. Serum MDA and MPO were assessed according to manufacturer's instructions.

#### **2.5 Liver function**

To assess hepatocellular injury in liver transplant, ALT and AST levels in Sera were measured using an Olympus AU600 Analyser (Olympus Optical, Tokyo, Japan).

#### **2.6 Measurement of serum IL-6 level**

We use commercially available ELISA kits for the determination of serum IL-6 level according to the manufacturer's instructions.

#### **2.7 Measurement of TNF-α level in lung**

We use commercially available ELISA kits for the determination of lung TNF-α level according to the manufacturer's instructions.

#### **2.8 PMN infiltration in the transplant and in the lung**

PMN infiltration in the transplant and lung was evaluated by staining for naphthol ASD chloroacetate esterase, a neutrophil specific marker. PMNs were identified by positive staining and were counted in 5 high-power fields under a light microscope (×400).

#### **2.9 Lung edema assay**

After resection, lung samples were weighed and then placed in an oven at 60°C until a constant weight was obtained. In this determination, edema is represented by an increase in the wet-to-dry weight ratios(Peralta et al., 1999).

#### **2.10 Histopathological analysis**

Liver and lung tissues were fixed with 10% neutral formalin and were stained with hematoxylin and eosin. The degrees of sinusoidal congestion, cytoplasmic vacuolization and necrosis of parenchymal cells were evaluated semiquantitatively according to Suzuki's criteria (Suzuki et al., 1993).

#### **2.11 Statistical analysis**

These data are expressed as mean ± SD. Calculations were made using the SPSS 13.0 for windows computer software (SPSS Inc., Chicago, IL). Statistical comparisons were performed using one-way analysis of variance (ANOVA) and the LSD t-test for blood MDA, IL-6, liver PMNs, TNF-α and enzyme levels. P-values less than 0.05 were considered statistically significant.

#### **3. Results**

258 Blood Transfusion in Clinical Practice

Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group (PR, n=5): Ringer's lactate solution was used for transfusion

In IR group, a section of the liver was sampled and fixed in 10% formaldehyde, and blood samples were taken from IH-VC at 7 h after transplantation to meet the time course of other groups. In PB, IB and PR group, a section of the liver was sampled and fixed in 10% formaldehyde, and blood samples were taken from IH-VC at 6 h after blood extraction. A section of lung was sampled and fixed in 10% formaldehyde for pathological examination at 6 h after blood extraction. Another section of lung was sampled for the analysis of cytokine

Sera were isolated and detected for MPO and MDA, an indicator of oxidative injury. Serum MPO contents were measured to analyze degree of neutrophil sequestration and activation.

To assess hepatocellular injury in liver transplant, ALT and AST levels in Sera were

We use commercially available ELISA kits for the determination of serum IL-6 level

We use commercially available ELISA kits for the determination of lung TNF-α level

PMN infiltration in the transplant and lung was evaluated by staining for naphthol ASD chloroacetate esterase, a neutrophil specific marker. PMNs were identified by positive

After resection, lung samples were weighed and then placed in an oven at 60°C until a constant weight was obtained. In this determination, edema is represented by an increase in

Liver and lung tissues were fixed with 10% neutral formalin and were stained with hematoxylin and eosin. The degrees of sinusoidal congestion, cytoplasmic vacuolization and

staining and were counted in 5 high-power fields under a light microscope (×400).

Serum MDA and MPO were assessed according to manufacturer's instructions.

measured using an Olympus AU600 Analyser (Olympus Optical, Tokyo, Japan).

while posthepatic manipulative blood extraction.

**2.4 Myeloperoxidase and lipid peroxide assays**

**2.6 Measurement of serum IL-6 level**

according to the manufacturer's instructions.

according to the manufacturer's instructions.

the wet-to-dry weight ratios(Peralta et al., 1999).

**2.8 PMN infiltration in the transplant and in the lung**

**2.7 Measurement of TNF-α level in lung**

and tissue edema.

**2.5 Liver function**

**2.9 Lung edema assay**

**2.10 Histopathological analysis**

#### **3.1 Time course and peak level of posthepatic MDA after transplantation**

Figure 2 represents the kinetic changes of MDA in all time points in experiment Part I. In this study MDA concentration within SH-VC peaked at 1 h after transplantation. MDA concentration at 1 h was significantly higher than that at other time points except for 5 min. Furthermore, MDA concentrations in SH-VC were significantly higher than that in IH-VC at 20 min,30 min,1h (*P*<0.05) and slightly higher at all other time points. MDA concentration within IH-VC peaked at 2 h after transplantation.

Fig. 2. Time course and peak level of MDA after liver transplantation in Part I SH-VC represents the group that blood was taken form SH-VC. IH-VC represents the group that blood was taken form IH-VC. a*P* < 0.05 vs. 60 min within SH-VC. b*P* < 0.05 vs. IH-VC.

#### **3.2 MDA and PMO levels in the sera**

In experiment Part II, serum MDA level at 6 h after transplantation was slightly lower in PB, IB and PR group than in IR group but no statistically significant. Whereas serum MPO level in PB and PR groups (48.78±9.36 pg/ml and 49.36±18.32 pg/ml, respectively) were significantly lower than that in IR group (71.34±22.14) (*P*=0.022, *P*=0.045, respectively). Serum MPO level in IB group was not significantly lower than that in IR group (Figure 3).

Posthepatic Manipulative Blood Extraction with Blood Transfusion

were not significantly lower than in IR group.

Fig. 5. ALT and AST levels in all groups in Part II

PR group was not significantly lower than that in IR group.

Fig. 6. Lung TNF-α level in all groups in Part II

group. a*P* < 0.05 vs. IR.

**3.5 TNF-α level in lung**

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 261

significantly lower in PB and IB group (460.00±126.65UI/L and 766.00±165.02 UI/L, respectively) than in IR group (1494.00±1015.05UI/L and 1976.00±1262.79 UI/L, respectively) (*P*=0.043, *P*=0.043, respectively). ALT and AST levels at 6 h in IB and PR group

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion

As shown in Figure 6, lung TNF-α level at 6 h in Part II were significantly lower in PB group (21.01±7.87 pg/ml) than in IR group (47±19.62 pg/ml) (*P*=0.038). Lung TNF-α level in IB and

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood

extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

Fig. 3. Serum MPO level in all groups in Part II

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

#### **3.3 Serum IL-6 level**

As shown in Figure 4, IL-6 level at 6 h in Part II were significantly lower in PB and IB group (470.88±101.36 pg/ml and 392.71±259.80 pg/ml, respectively) than in IR group (1067.42±547.91) (*P*=0.017, *P*=0.008, respectively). IL-6 level at 6 h in PR group was not significantly lower than in IR group.

Fig. 4. Serum IL-6 level in all groups in Part II

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

#### **3.4 Liver function**

Figure 5 showed posthepatic manipulative blood extraction significantly attenuated hepatocellular injury, as compared with I/R control group. ALT and AST levels at 6 h were significantly lower in PB and IB group (460.00±126.65UI/L and 766.00±165.02 UI/L, respectively) than in IR group (1494.00±1015.05UI/L and 1976.00±1262.79 UI/L, respectively) (*P*=0.043, *P*=0.043, respectively). ALT and AST levels at 6 h in IB and PR group were not significantly lower than in IR group.

Fig. 5. ALT and AST levels in all groups in Part II IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

#### **3.5 TNF-α level in lung**

260 Blood Transfusion in Clinical Practice

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion

As shown in Figure 4, IL-6 level at 6 h in Part II were significantly lower in PB and IB group (470.88±101.36 pg/ml and 392.71±259.80 pg/ml, respectively) than in IR group (1067.42±547.91) (*P*=0.017, *P*=0.008, respectively). IL-6 level at 6 h in PR group was not

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood

extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

Figure 5 showed posthepatic manipulative blood extraction significantly attenuated hepatocellular injury, as compared with I/R control group. ALT and AST levels at 6 h were

Fig. 3. Serum MPO level in all groups in Part II

group. a*P* < 0.05 vs. IR.

**3.3 Serum IL-6 level**

**3.4 Liver function**

significantly lower than in IR group.

Fig. 4. Serum IL-6 level in all groups in Part II

As shown in Figure 6, lung TNF-α level at 6 h in Part II were significantly lower in PB group (21.01±7.87 pg/ml) than in IR group (47±19.62 pg/ml) (*P*=0.038). Lung TNF-α level in IB and PR group was not significantly lower than that in IR group.

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

Posthepatic Manipulative Blood Extraction with Blood Transfusion

lactate solution transfusion group. aP<0.05 versus IR.

A B

C D

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 263

Fig. 8. PMN infiltrations in the transplants at 6 h after manipulation in Part II

PMN kinetics in the liver after reperfusion. IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's

#### **3.6 Lung edema assay**

Wet-to-dry weight ratio of the lung tissue in group PB was 4.25±0.79, which was significnagly lower than that in IR group (7.0±4.03) (*P*=0.039). Wet-to-dry weight ratio of the lung tissue in IB and PR group were not significantly lower than that in IR group (Figure 7).

Fig. 7. Wet-to-dry weight ratio of the lung tissue in all groups in Part II IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

#### **3.7 PMN infiltration in the transplant and in the lung**

To determine whether posthepatic manipulative blood extraction affected local leukocyte infiltration in transplant and lung, we assessed PMNs infiltration using naphthol ASD chloroacetate esterase staining. In this study, PMN counts in liver transplant were significantly lower in PB, IB and PR group (11.75±5.02 counts/HPF, 8.53±1.98 counts/HPF and 13.6±4.07 counts/HPF, respectively) than that in IR group (24.46±13.87 counts/HPF) (*P*=0.006, *P*=0.002, *P*=0.031, respectively) (Figure 8). PMN counts in lung were significantly lower in PB and IB group (44.13±19.29 counts/HPF and 56.57±9.10 counts/HPF, respectively) than that in IR group (82.8±25.01 counts/HPF) (*P*=0.001 and *P*=0.030, respectively). PMN infiltration in PR group was not significantly lower than that in IR group (Figure 9).

Wet-to-dry weight ratio of the lung tissue in group PB was 4.25±0.79, which was significnagly lower than that in IR group (7.0±4.03) (*P*=0.039). Wet-to-dry weight ratio of the lung tissue in IB and PR group were not significantly lower than that in IR group (Figure 7).

Fig. 7. Wet-to-dry weight ratio of the lung tissue in all groups in Part II

**3.7 PMN infiltration in the transplant and in the lung**

IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion

To determine whether posthepatic manipulative blood extraction affected local leukocyte infiltration in transplant and lung, we assessed PMNs infiltration using naphthol ASD chloroacetate esterase staining. In this study, PMN counts in liver transplant were significantly lower in PB, IB and PR group (11.75±5.02 counts/HPF, 8.53±1.98 counts/HPF and 13.6±4.07 counts/HPF, respectively) than that in IR group (24.46±13.87 counts/HPF) (*P*=0.006, *P*=0.002, *P*=0.031, respectively) (Figure 8). PMN counts in lung were significantly lower in PB and IB group (44.13±19.29 counts/HPF and 56.57±9.10 counts/HPF, respectively) than that in IR group (82.8±25.01 counts/HPF) (*P*=0.001 and *P*=0.030, respectively). PMN infiltration in PR group was not significantly lower than that in IR group

**3.6 Lung edema assay**

group. a*P* < 0.05 vs. IR.

(Figure 9).

Fig. 8. PMN infiltrations in the transplants at 6 h after manipulation in Part II PMN kinetics in the liver after reperfusion. IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. aP<0.05 versus IR.

Posthepatic Manipulative Blood Extraction with Blood Transfusion

C D

E F

G H

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 265

At 6 h after transplantation, conspicuous necrosis and sinusoidal congestion were observed

Fig. 10. Representative histologic findings in transplant at 6 h after manipulation.

in IR and PR group (A:IR group, original magnification ×100; B:IR group, original magnification ×400; G:PR group, original magnification ×100; H:PR group, original magnification ×400). Moderate severity of necrosis and congestion were found in IB group (E:IB group, original magnification ×100; F:IB group, original magnification ×400). Only ballooning of parenchymal cells and minimal necrosis were seen in PB group (C: original

magnification ×100; D: original magnification ×400).

Fig. 9. PMN infiltration in the lung in all groups in Part II PMNs were seen as deep-brown dots (original magnification ×400). A:IR group; B: PB group; C:IB group; D: PR group. E: the comparison of PMN infiltration in the lung between groups. IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

#### **3.8 Histopathological findings in Part II**

Histological changes of the transplant were in keeping with the aforementioned biochemical observations. Liver sections of IR and PR rats at 6 h after manipulation showed sinusoidal congestion, cytoplasmic vacuolization and conspicuously focal necrosis. Moderate severity of necrosis and congestion were found in IB group. In marked contrast, liver sections in PB rats showed significant preservation of the lobular architecture with ballooning of parenchymal cells and minimal signs of hepatocyte necrosis (Figure 10). Four out of seven rats in PB group showed + hepatic injury, 2 showed ++ and 1 showed +++, whereas rats in other groups showed ++~+++ hepatic injury according to Suzuki's criteria. The histological study of the lungs revealed a diffuse interstitial thickening with marked edema and polymorphonuclear infiltration 6 h after liver transplant in IR group. In contrast, mild lesions were seen in PB group with discrete thickening of the alveolar septa and the presence of scattered inflammatory cells (Figure 11).

PMNs were seen as deep-brown dots (original magnification ×400). A:IR group; B: PB group; C:IB group; D: PR group. E: the comparison of PMN infiltration in the lung between groups. IR: IR control group. PB: Two percent of body weight posthepatic manipulative blood

extraction with blood transfusion group. IB: Two percent of body weight IH-VC manipulative blood extraction with blood transfusion group. PR: Two percent of body weight posthepatic manipulative blood extraction with Ringer's lactate solution transfusion group. a*P* < 0.05 vs. IR.

Histological changes of the transplant were in keeping with the aforementioned biochemical observations. Liver sections of IR and PR rats at 6 h after manipulation showed sinusoidal congestion, cytoplasmic vacuolization and conspicuously focal necrosis. Moderate severity of necrosis and congestion were found in IB group. In marked contrast, liver sections in PB rats showed significant preservation of the lobular architecture with ballooning of parenchymal cells and minimal signs of hepatocyte necrosis (Figure 10). Four out of seven rats in PB group showed + hepatic injury, 2 showed ++ and 1 showed +++, whereas rats in other groups showed ++~+++ hepatic injury according to Suzuki's criteria. The histological study of the lungs revealed a diffuse interstitial thickening with marked edema and polymorphonuclear infiltration 6 h after liver transplant in IR group. In contrast, mild lesions were seen in PB group with discrete thickening of the alveolar septa and the

Fig. 9. PMN infiltration in the lung in all groups in Part II

**3.8 Histopathological findings in Part II**

(E)

presence of scattered inflammatory cells (Figure 11).

A B

Fig. 10. Representative histologic findings in transplant at 6 h after manipulation. At 6 h after transplantation, conspicuous necrosis and sinusoidal congestion were observed in IR and PR group (A:IR group, original magnification ×100; B:IR group, original magnification ×400; G:PR group, original magnification ×100; H:PR group, original magnification ×400). Moderate severity of necrosis and congestion were found in IB group (E:IB group, original magnification ×100; F:IB group, original magnification ×400). Only ballooning of parenchymal cells and minimal necrosis were seen in PB group (C: original magnification ×100; D: original magnification ×400).

Posthepatic Manipulative Blood Extraction with Blood Transfusion

G H

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 267

magnification ×100; H:PR group, original magnification ×400). In contrast, mild lesions were seen in PB group with discrete thickening of the alveolar septa and the presence of scattered

Our results provided evidence for the role of ROS in the pathogenesis of liver transplant I/R. In Part I of this study, pure SH-VC and IH-VC blood were taken at multiple time points to determine the peak production of posthepatic ROS after transplantation. We found that serum MDA concentration in SH-VC increased and peaked at 1 h after transplantation. And the MDA concentrations in SH-VC were significantly higher than that in IH-VC at 20 min,30 min,1h (*P*<0.05). The phenomenon that MDA concentration increased slowly in the early stage after transplantation is due to its incomplete reflow and injured microcirculation including endothelial cell swelling (Vollmar et al., 1994), vasoconstriction (Marzi et al., 1994), leucocyte entrapment (Fondevila et al., 2003; Yadav et al., 1998) and possibly intravascular haemoconcentration (Menger et al., 1988). This process prolongs the period of hypoxia, with areas of the liver remaining ischemic after early period of transplantation. In the later stage, neutrophils infiltrate into the liver increased with time, so

In Part II of this study, we evaluated the potential effects of posthepatic manipulative blood extraction and blood transfusion at 1 h after reperfusion on the liver transplant cold I/R injury and liver I/R induced lung injury. We found that 2% of body weight posthepatic manipulative blood extraction with blood transfusion (PB) significantly reversed the elevations in the serum MPO, IL-6, TNF-α, liver enzyme levels and dramatically decreased PMNs in the liver and lung. Oxidative and other environmental stress lead to an upregulation of proinflammatory mediators (Otterbein et al., 2000; Otterbein et al., 2003). Increased production of IL-6 is thought to be involved in the pathogenesis of cold hepatic I/R injury (Tomiyama et al., 2008; Uchida et al., 2009). IL-6 is considered a marker for liver

The histological study of the lungs revealed a diffuse interstitial thickening with marked edema and polymorphonuclear infiltration in I/R, IB and PR group (A:IR group, original

Fig. 11. Representative histologic findings in lung at 6 h after transplantation.

magnification ×100; B:IR group, original magnification ×400; E:IB group, original magnification ×100; F:IB group, original magnification ×400; G: PR group, original

inflammatory cells (C: original magnification ×100; D: original magnification ×400).

**4. Discussion**

companied by increased ROS.

Fig. 11. Representative histologic findings in lung at 6 h after transplantation. The histological study of the lungs revealed a diffuse interstitial thickening with marked edema and polymorphonuclear infiltration in I/R, IB and PR group (A:IR group, original magnification ×100; B:IR group, original magnification ×400; E:IB group, original magnification ×100; F:IB group, original magnification ×400; G: PR group, original magnification ×100; H:PR group, original magnification ×400). In contrast, mild lesions were seen in PB group with discrete thickening of the alveolar septa and the presence of scattered inflammatory cells (C: original magnification ×100; D: original magnification ×400).

#### **4. Discussion**

266 Blood Transfusion in Clinical Practice

A B

C D

E F

Our results provided evidence for the role of ROS in the pathogenesis of liver transplant I/R. In Part I of this study, pure SH-VC and IH-VC blood were taken at multiple time points to determine the peak production of posthepatic ROS after transplantation. We found that serum MDA concentration in SH-VC increased and peaked at 1 h after transplantation. And the MDA concentrations in SH-VC were significantly higher than that in IH-VC at 20 min,30 min,1h (*P*<0.05). The phenomenon that MDA concentration increased slowly in the early stage after transplantation is due to its incomplete reflow and injured microcirculation including endothelial cell swelling (Vollmar et al., 1994), vasoconstriction (Marzi et al., 1994), leucocyte entrapment (Fondevila et al., 2003; Yadav et al., 1998) and possibly intravascular haemoconcentration (Menger et al., 1988). This process prolongs the period of hypoxia, with areas of the liver remaining ischemic after early period of transplantation. In the later stage, neutrophils infiltrate into the liver increased with time, so companied by increased ROS.

In Part II of this study, we evaluated the potential effects of posthepatic manipulative blood extraction and blood transfusion at 1 h after reperfusion on the liver transplant cold I/R injury and liver I/R induced lung injury. We found that 2% of body weight posthepatic manipulative blood extraction with blood transfusion (PB) significantly reversed the elevations in the serum MPO, IL-6, TNF-α, liver enzyme levels and dramatically decreased PMNs in the liver and lung. Oxidative and other environmental stress lead to an upregulation of proinflammatory mediators (Otterbein et al., 2000; Otterbein et al., 2003). Increased production of IL-6 is thought to be involved in the pathogenesis of cold hepatic I/R injury (Tomiyama et al., 2008; Uchida et al., 2009). IL-6 is considered a marker for liver

Posthepatic Manipulative Blood Extraction with Blood Transfusion

source for TNF-α release during liver I/R (Vollmar et al., 1994).

elevations in the some parameters, it was not statistically significant.

liver transplantation. Liver Transpl, 2003; 9: 764-771

should be further investigated.

Br J Surg 2005; 92:528–538.

**5. References**

Alleviates Liver Transplant Ischemia/Reperfusion Injury and Its Induced Lung Injury 269

after hepatic I/R (Suzuki et al., 1993). In this study, as decreased in serum MPO, TNF-α, a pleiotropic pro-inflammatory cytokine which can induce liver and lung injuries, was also decreased via posthepatic blood diacharge in that the liver by itself is also an important

So in this study, the protective effect of posthepatic manipulative blood extraction was, in part, due to the inhibition of neutrophil infiltration and activation via its inhibition of tissue TNF-a production, which was further demonstrated by its suppression of PMNs accumulation in lung. In addition, we found that water content of the lung significantly increases after reperfusion. TNF-a and activated neutrophils contribute to the damage through the release of ROS. Increased accumulation of pulmonary TNF-a and neutrophil results in increased lung capillary permeability in that it can damage both vascular endothelium cells and lung epithelial cells. And the toxic lipid peroxidation reaction products mediated by ROS will furthermore cause cell injury and death (Marzi et al., 1994). The protective mechanisms involved in PB were the direct decreasing in circulating MPO and pulmonary TNF-α via manipulative blood extraction. And the lung pathological findings parallel with biochemistry parameters mentioned above. So it demonstrated that posthepatic manipulative blood extraction alleviates lung injury induced by liver ischemiareperfusion. Although other groups, compared with IR group, slightly reversed the

In conclusion, we provided the first evidence that posthepatic ROS concentration elevated with time and peaked at 1 h after transplantation. Posthepatic manipulative blood extraction and blood transfusion alleviates liver transplant cold IR injury and lung injury induced by liver cold IR injury through its marked decrease in serum PMO, IL-6, inhibition neutrophil infiltration in transplant, so significantly attenuating hepatocellular injury. Our data strongly support that targeting posthepatic manipulative blood extraction with blood transfusion represents a useful approach to prevent IR injury in rats and maybe a potential way in human. It is possible to use this strategy clinically due to its facility and practicability. In human liver transplantation, for example, the posthepatic manipulatively extracted blood can be re-transfused after re-oxygenation and removal of ROS and proinflammatory cytokines. It might improve the overall success of liver transplantation and

Aduen JF, Stapelfeldt WH, Johnson MM, Jolles HI, Grinton SF, Divertie GD, Burger CD.

Banga NR, Homer-Vanniasinkam S, Graham A, Al-Mukhtar A, White SA, Prasad

Clarke CN, Kuboki S, Tevar A, Lentsch AB, Edwards M.CXC chemokines play a critical role in liver injury, recovery, and regeneration. Am J Surg 2009; 198:415-419.

Clinical relevance of time of onset,duration, and type of pulmonary edema after

KR.Ischaemic preconditioning in transplantation and major resection of the liver.

injury severity (Jin et al., 2006) and it has been demonstrated that IL-6 overexpression leads to detrimental effects in some studies (Monbaliu et al., 2007; Wustefeld et al., 2000). Here, prompt inflammatory response was evident in our model of cold hepatic I/R injury by the elevation of serum IL-6 level at 6 h of reperfusion. Then we have shown that posthepatic manipulative blood extraction reversed this elevation.

In Part II of this study, although serum MDA level was no statistically significant between groups, serum MPO levels in PB was significantly lower than that in IR group. Posthepatic manipulative blood extraction significantly reduced serum MPO level, indicating suppression of neutrophil accumulation and activation, which play important roles in I/R-induced ROS production (Parks et al., 1988; Zimmerman et al., 1990). This result shows the imbalance of oxidation/antioxidation and increase of lipid peroxide in vivo. So the protective effect of posthepatic manipulative blood extraction with blood transfusion was, in part, due to the inhibition of neutrophil infiltration and activation, which was further demonstrated by its suppression of PMNs accumulation in liver transplant and lung. Experimental evidence shows that there are two distinct phases of liver reperfusion injury (Jaeschke et al., 1990). The early phase covers the first 2 hours after reperfusion (Tamaki et al., 1996). So in the view of time course, we conclude that posthepatic manipulative blood extraction mainly alleviates liver IR injury in late phase in that it significantly decrease serum MPO, IL-6, liver enzyme levels and PMNs in the liver at 6 h after reperfusion.

Although IB group significantly reversed the elevations in the serum IL-6, liver enzymes and even PMN infiltrations in transplant, it did not exert significant protective effects in terms of serum MPO and did not improve transplant pathological injury. So we thought that the posthepatic manipulative blood extraction takes advantages over IH-VC manipulative blood extraction on attenuating hepatic cold I/R injury. Both blood/ROS extraction and fresh blood transfusion are indispensable in alleviating liver transplant I/R injury in our model. And the reason why PR group significantly decrease serum MPO and PMN infiltrations in the liver remains unclear. Blood dilution maybe at least one of the causes for this phenomenon.

The liver contains great concentration of Kupffer cells (Kamada et al., 1983). During the initial stage of hepatic ischemia, Kupffer cells are activated and these activated Kupffer cells released a lot of pro-inflammatory cytokines, and proteolytic enzymes. After reperfusion, activation products generated in the ischemic liver, including proinflammatory cytokines such as TNF-α, are released into the systemic circulation. Yoshidome et al demonstrated, in a rat hepatic I/R model, that hepatic-derived TNF-α plays a central role in the induction of lung neutrophil recruitment and tissue injury (Peralta et al., 1999). It initiates an inflammatory response both in liver and remote organs such as the lungs and kidneys (Rosen et al., 1998). These factors, after the reperfusion, can cause hepatocellular injury and hepatic microcirculation disorder. On the other hand, these active factors can reach to the pulmonary vascularity through systemic circulation to activate lung capillary endothelial cells, neutrophils and lead to the inflammatory reaction of the lung tissues, causing acute lung injury and increase the lung capillary permeability. This is because the pulmonary endothelium is sensitive to these cytokines, and because the pulmonary circulation represents the first vascular bed to which these mediators are delivered (Peralta et al., 1999). Furthermore, elevated levels of TNF-α are associated with neutrophil-dependent lung injury after hepatic I/R (Suzuki et al., 1993). In this study, as decreased in serum MPO, TNF-α, a pleiotropic pro-inflammatory cytokine which can induce liver and lung injuries, was also decreased via posthepatic blood diacharge in that the liver by itself is also an important source for TNF-α release during liver I/R (Vollmar et al., 1994).

So in this study, the protective effect of posthepatic manipulative blood extraction was, in part, due to the inhibition of neutrophil infiltration and activation via its inhibition of tissue TNF-a production, which was further demonstrated by its suppression of PMNs accumulation in lung. In addition, we found that water content of the lung significantly increases after reperfusion. TNF-a and activated neutrophils contribute to the damage through the release of ROS. Increased accumulation of pulmonary TNF-a and neutrophil results in increased lung capillary permeability in that it can damage both vascular endothelium cells and lung epithelial cells. And the toxic lipid peroxidation reaction products mediated by ROS will furthermore cause cell injury and death (Marzi et al., 1994).

The protective mechanisms involved in PB were the direct decreasing in circulating MPO and pulmonary TNF-α via manipulative blood extraction. And the lung pathological findings parallel with biochemistry parameters mentioned above. So it demonstrated that posthepatic manipulative blood extraction alleviates lung injury induced by liver ischemiareperfusion. Although other groups, compared with IR group, slightly reversed the elevations in the some parameters, it was not statistically significant.

In conclusion, we provided the first evidence that posthepatic ROS concentration elevated with time and peaked at 1 h after transplantation. Posthepatic manipulative blood extraction and blood transfusion alleviates liver transplant cold IR injury and lung injury induced by liver cold IR injury through its marked decrease in serum PMO, IL-6, inhibition neutrophil infiltration in transplant, so significantly attenuating hepatocellular injury. Our data strongly support that targeting posthepatic manipulative blood extraction with blood transfusion represents a useful approach to prevent IR injury in rats and maybe a potential way in human. It is possible to use this strategy clinically due to its facility and practicability. In human liver transplantation, for example, the posthepatic manipulatively extracted blood can be re-transfused after re-oxygenation and removal of ROS and proinflammatory cytokines. It might improve the overall success of liver transplantation and should be further investigated.

#### **5. References**

268 Blood Transfusion in Clinical Practice

injury severity (Jin et al., 2006) and it has been demonstrated that IL-6 overexpression leads to detrimental effects in some studies (Monbaliu et al., 2007; Wustefeld et al., 2000). Here, prompt inflammatory response was evident in our model of cold hepatic I/R injury by the elevation of serum IL-6 level at 6 h of reperfusion. Then we have shown that posthepatic

In Part II of this study, although serum MDA level was no statistically significant between groups, serum MPO levels in PB was significantly lower than that in IR group. Posthepatic manipulative blood extraction significantly reduced serum MPO level, indicating suppression of neutrophil accumulation and activation, which play important roles in I/R-induced ROS production (Parks et al., 1988; Zimmerman et al., 1990). This result shows the imbalance of oxidation/antioxidation and increase of lipid peroxide in vivo. So the protective effect of posthepatic manipulative blood extraction with blood transfusion was, in part, due to the inhibition of neutrophil infiltration and activation, which was further demonstrated by its suppression of PMNs accumulation in liver transplant and lung. Experimental evidence shows that there are two distinct phases of liver reperfusion injury (Jaeschke et al., 1990). The early phase covers the first 2 hours after reperfusion (Tamaki et al., 1996). So in the view of time course, we conclude that posthepatic manipulative blood extraction mainly alleviates liver IR injury in late phase in that it significantly decrease serum MPO, IL-6, liver enzyme levels and PMNs in the liver

Although IB group significantly reversed the elevations in the serum IL-6, liver enzymes and even PMN infiltrations in transplant, it did not exert significant protective effects in terms of serum MPO and did not improve transplant pathological injury. So we thought that the posthepatic manipulative blood extraction takes advantages over IH-VC manipulative blood extraction on attenuating hepatic cold I/R injury. Both blood/ROS extraction and fresh blood transfusion are indispensable in alleviating liver transplant I/R injury in our model. And the reason why PR group significantly decrease serum MPO and PMN infiltrations in the liver remains unclear. Blood dilution maybe at least one of the

The liver contains great concentration of Kupffer cells (Kamada et al., 1983). During the initial stage of hepatic ischemia, Kupffer cells are activated and these activated Kupffer cells released a lot of pro-inflammatory cytokines, and proteolytic enzymes. After reperfusion, activation products generated in the ischemic liver, including proinflammatory cytokines such as TNF-α, are released into the systemic circulation. Yoshidome et al demonstrated, in a rat hepatic I/R model, that hepatic-derived TNF-α plays a central role in the induction of lung neutrophil recruitment and tissue injury (Peralta et al., 1999). It initiates an inflammatory response both in liver and remote organs such as the lungs and kidneys (Rosen et al., 1998). These factors, after the reperfusion, can cause hepatocellular injury and hepatic microcirculation disorder. On the other hand, these active factors can reach to the pulmonary vascularity through systemic circulation to activate lung capillary endothelial cells, neutrophils and lead to the inflammatory reaction of the lung tissues, causing acute lung injury and increase the lung capillary permeability. This is because the pulmonary endothelium is sensitive to these cytokines, and because the pulmonary circulation represents the first vascular bed to which these mediators are delivered (Peralta et al., 1999). Furthermore, elevated levels of TNF-α are associated with neutrophil-dependent lung injury

manipulative blood extraction reversed this elevation.

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Species in Mediating Hepatic Ischemia-Reperfusion Injury and Its Therapeutic

### *Edited by Puneet Kaur Kochhar*

Blood Transfusion in Clinical Practice focuses on the application of blood transfusion in different clinical settings. The text has been divided into five sections. The first section includes a chapter describing the basic principles of ABO blood group system in blood transfusion. The second section discusses the use of transfusion in various clinical settings including orthopedics, obstetrics, cardiac surgery, etc. The third section covers transfusion transmitted infections, while section four describes alternative strategies to allogenic blood transfusion. The last section speculates over immunomodulatory effects of blood transfusion.

Photo by PongzaA / iStock

Blood Transfusion in Clinical Practice

Blood Transfusion in

Clinical Practice

*Edited by Puneet Kaur Kochhar*