Validation of tissue microarray technology in squamous cell carcinoma of the esophagus

Tissue microarray (TMA) technology has been developed to facilitate high-throughput immunohistochemical and in situ hybridization analysis of tissues by inserting small tissue biopsy cores into a single paraffin block. Several studies have revealed novel prognostic biomarkers in esophageal squamous cell carcinoma (ESCC) by means of TMA technology, although this technique has not yet been validated for these tumors. Because representativeness of the donor tissue cores may be a disadvantage compared to full sections, the aim of this study was to assess if TMA technology provides representative immunohistochemical results in ESCC. A TMA was constructed containing triplicate cores of 108 formalin-fixed, paraffin-embedded squamous cell carcinomas of the esophagus. The agreement in the differentiation grade and immunohistochemical staining scores of CK5/6, CK14, E-cadherin, Ki-67, and p53 between TMA cores and a subset of 64 randomly selected donor paraffin blocks was determined using kappa statistics. The concurrence between TMA cores and donor blocks was moderate for Ki-67 (κ = 0.42) and E-cadherin (κ = 0.47), substantial for differentiation grade (κ = 0.65) and CK14 (κ = 0.71), and almost perfect for p53 (κ = 0.86) and CK5/6 (κ = 0.93). TMA technology appears to be a valid method for immunohistochemical analysis of molecular markers in ESCC provided that the staining pattern in the tumor is homogeneous.
Introduction
Esophageal carcinoma is the eighth most common type of cancer in the world [13]. Although the recent rise in incidence of esophageal cancer has predominantly been caused by an increase in adenocarcinomas, the majority of esophageal cancer cases globally are squamous cell carcinomas [13]. For both histological types, radical en bloc esophagectomy with an extensive lymph node dissection offers the best chance for cure, leading to an overall 5-year survival rate of around 30% [1, 20].
Well-known histopathological factors for prognostication of esophageal cancer include the TNM stage, the number of positive lymph nodes, and the presence of extracapsular lymph node involvement [16, 24, 26, 32]. Recently, there has been a growing interest in the prognostic value of molecular markers in (esophageal) cancer [21]. The expression of such markers is often studied by immunohistochemistry on formalin-fixed, paraffin-embedded tumor slides. Tissue microarray (TMA) technology has been developed to enable high-throughput immunohistochemical analyses [14]. By inserting small (diameter e.g. 0.6 mm) donor tissue core biopsies into a single recipient paraffin block, this technique allows for rapid analysis of large numbers of tissues under standardized laboratory and evaluation conditions without significantly damaging the patient’s tissue. In addition, TMA technology leads to a significant reduction of the amount of consumables used and time needed for interpretation, increasing cost-effectiveness.
A potential disadvantage compared to full tissue sections is that the donor cores may not be representative for the whole tumor, particularly in case of heterogeneous tumors and heterogeneously expressed molecular markers. Hence, some validation studies have been performed in various cancers using different kinds of antibodies [2, 4, 6, 7, 9, 12, 23, 35]. Although several studies have revealed novel prognostic biomarkers in esophageal squamous cell cancer (ESCC) by means of TMA technology [38, 39, 41], this technique has not yet been validated for these tumors.
The aim of the present study was, therefore, to validate TMA technology in ESCC by assessing the concurrence of immunohistochemical staining scores of established molecular markers with various expression patterns between triplicate 0.6 mm core biopsies of the TMA and their whole tissue section counterparts.
Materials and methods
TMA construction
Formalin-fixed, paraffin-embedded tissues from thoracic ESCCs of consecutive patients having undergone esophagolymphadenectomy at the authors’ institute between 1989 and 2006 were retrieved from the archives of the Department of Pathology. Patients who received neoadjuvant therapy were excluded from this study. The study was carried out in accordance with the ethical guidelines of our institution concerning informed consent about the use of patient’s materials after surgical procedures.
By an experienced pathologist (FtK), three representative tumor regions were marked on one selected hematoxylin and eosin (H&E)-stained section of each tumor, avoiding areas of necrosis. From these three tumor regions, a tissue cylinder with a diameter of 0.6 mm was punched out of the corresponding paraffin block (‘donor block’) and placed into the TMA paraffin block using a manual tissue arrayer (MTA-I, Beecher Instruments, Sun Prairie, USA), which was guided by the MTABooster® (Alphelys, Plaisir, France). The distribution and position of the cores was determined in advance with the TMA-designer Software (Alphelys-TMA Designer®, Version 1.6.8, Plaisir, France). Cores of normal esophageal mucosa, lymph node, kidney, liver, spleen, and prostate were incorporated in the tissue array block as internal controls.
Immunohistochemistry
For each marker, a 4-μm slide of the TMA and one of every selected donor paraffin block were immunohistochemically stained. Table 1 shows the details of all antibodies, dilutions, incubation times and antigen retrieval methods applied in this study.
Table 1Specification of antibodies used and details of tissue processingPrimary antibodyStaining patternSourceaClone and codeAntigen retrievalDilutionIncubation time (min/room temperature)DetectionbPositive controlProcedureCK5/6CytoplasmicChemiconD5/16 B4EDTA pH 9.01:3,00060Strept ABCBreastAutostainerCK14CytoplasmicNeomarkersLL002EDTA pH 9.01:40060PowervisionBreastAutostainerE-cadherinMembranousZymed4A2C7Citrate autoclave pH 6.01:20060PowervisionBreastAutostainerMIB-1 (Ki-67)NuclearDakoM7240Citrate pH 6.01:10060Strept ABCTonsilAutostainerp53NuclearBiogenexBP53-12Citrate pH 6.01:20060Strept ABCSerous adenocarcinoma of the endometriumAutostaineraBiogenex, San Ramon, CA, USA; Chemicon, Chemicon International, Temecula, CA, USA; Dako, DakoCytomation, Glostrup, Denmark; Neomarkers, Fremont, USA; Zymed, Zymed Laboratories, San Francisco, CA, USA.bStrept ABC is biotinylated horse–antimouse Vector BA-2000, diluted 1:500 in PBS, followed by streptavidin–biotin complex, diluted 1:1,000. Powervision ready to use (Poly-HRP-antiMs/Rb/RtIgG biotin-free; ImmunoVision Technologies, Norwell, CA, USA).
For all stainings, sections were deparaffinized in xylene for 10 min followed by dehydration through graded alcohols. Endogenous peroxidase activity was blocked for 15 min in a buffer solution of pH 5.8 (containing 8.32 g citric acid, 21.52 g disodium hydrogen phosphate, 2 g sodium azide in 1 l of water) with hydrogen peroxide (0.3%). After antigen retrieval for 20 min, a cooling down period of 30 min was followed by incubation with the primary antibody. Depending on the antibody used, slides were incubated with the secondary antibody followed by the streptavidin–biotin complex or slides were directly incubated with Powervision (details of both products shown in the legend of Table 1). Then, the peroxidase reactivity was developed by 3,3′-diaminobenzidine for 10 min and slides were counterstained with Mayer’s hematoxylin. In between steps, slides were washed with phosphate-buffered saline (pH 7.4).
Immunohistochemical scoring
By two observers (FtK and JB) conjointly, the degree of differentiation and the percentage of immunohistochemically stained tumor cells were determined in all TMA cores and in the full sections of the selected donor blocks. Histologic grade was scored as well-differentiated (G1), moderately differentiated (G2), or poorly differentiated (G3) [37]. Staining of p53 and Ki-67 were marked as negative (<10% of tumor nuclei stained), weakly positive (10–50%), or strongly positive (≥50%) [10, 40]. Cytokeratin (CK)5/6 and CK14 staining were scored as negative (<10% of tumor cell cytoplasms stained), weakly positive (10–80%), or strongly positive (≥80%). E-cadherin expression was regarded negative when <50% of tumor cell membranes stained and positive when ≥50% stained [29, 30].
Cores were considered lost if <10% of cells contained tumor (‘sampling error’) or when <10% of tissue was present (‘absent core’). Cases were excluded if two out of three cores were lost. When the scores between the cores of a particular case differed, the most frequent score determined the overall score. In case of three different scores in one case, the middle score was chosen. When only two cores were available with both a different score, the case was excluded from further analysis [11].
Statistical analysis
Statistical analyses were performed using the SPSS software for Windows (Version 12.0, SPSS, Chicago, IL, USA). Sixty-four donor blocks (60% of the tumors incorporated in the TMA) were randomly chosen by means of a random selection function of SPSS.
To determine the chance-corrected agreement between the immunohistochemical staining scores of TMA cores and large sections, the Cohen’s weighted kappa statistic was calculated. Chance-corrected agreement was considered poor if κ < 0.00, slight if 0 < κ < 0.20, fair if 0.21 < κ < 0.40, moderate if 0.41 < κ < 0.60, substantial if 0.61 < κ < 0.80, and almost perfect if 0.81 < κ < 1.00 [17]. The overall agreement was defined as the percentage of correct agreement between the TMA and the donor blocks from the total number of cases [15].
Results
Of the 324 (3 × 108) tumor tissue cores that were transferred into the TMA paraffin block, a median of 295 (91%) was available for immunohistochemical scoring on the 6 TMA slides used in this study (Table 2). Of the 64 randomly selected cases, a median of 176 (92%) of 192 cores (3 × 64) was evaluable on the TMA slides.
Table 2Overview of the amount of cores that were evaluable, absent or contained too little tumor in all 108 cases and in the 64 randomly selected cases on the TMA slides H&ECK5/6CK14E-cadherinKi-67p53MedianTotal TMA cases (n = 108) No. of evaluable cores293309294306293295295 Percentage90959194909191 No. of absent cores207229222221 Percentage6273776 No. of cores without tumor11889979 Percentage4333323Randomly selected TMA cases (n = 64) No. of evaluable cores176187176185176176176 Percentage92979296929292 No. of absent cores133134131313 Percentage7272777 No. of cores without tumor3233333 Percentage2122222H&E: hematoxylin and eosin
On the H&E-stained TMA slide, 49 (76%) of the 64 randomly chosen cases were represented by 3 cores; 14 (22%) by 2 cores. One (1.6%) case was excluded from further analysis because only a single core was available. The agreement in the scores for the grade of differentiation between the TMA cores and the full sections is shown in Table 3. The weighted kappa score was 0.65.
Table 3Agreement in the degree of differentiation between TMA cores and full sections Full sectionG1G2G3TotalκTMAG12305 G2221225 G3072633 Total43128630.65G1: well-differentiated, G2: moderately differentiated, G3: poorly differentiated
Fifty-nine (92%) of the 64 randomly selected cases stained for CK5/6 were represented by 3 cores (Fig. 1); the 5 remaining cases by 2 cores. The immunohistochemical scores of the TMA and the donor blocks are shown in Table 4. Overall agreement in CK5/6 scores between the TMA and the donor blocks was 98% with a kappa of 0.93.
Fig. 1Example of strong CK5/6 staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification ×20. b Enlargement of the middle TMA core depicted in a; magnification ×100. c Part of the slide of the donor block of the same tumor; magnification ×100Table 4Agreement in immunohistochemical scores between TMA cores and full slides stained for CK5/6 and CK14 Full sections<10%10–80%≥80%TotalκTMAsCK5/6 <10%1102 10–80%0404 ≥80%005858 Total1558640.93CK14 <10%5218 10–80%011213 ≥80%043438 Total51737590.71
For CK14, two cases were excluded because only one tumor core was left and three cases were also excluded because the two available cores had discrepant immunohistochemical scores. Fifty (85%) of 59 cases had complete agreement (Table 4). Four cases were scored one class higher on TMA when compared with the full sections. Conversely, five other cases were classified lower on TMA with one case two classes lower. Kappa score was 0.71.
Regarding E-cadherin staining, three assessable cores were present in 89% of the cases; two cores in 11%. Overall agreement in E-cadherin staining scores was accomplished in 72% of cases (Table 5). In one case, a higher score was found on the TMA compared to the full section. In 17 cases, the expression of E-cadherin was scored lower on TMA than on the full sections. The observed kappa was 0.47.
Table 5Agreement in immunohistochemical scores between TMA cores and full slides stained for E-cadherin  Full sectionsE-cadherin<50%≥50%TotalκTMA <50%221739 ≥50%12425 Total2341640.47
Three-core analysis of Ki-67 staining could be performed in 78% of selected cases and two-core analysis in 19%. Two cases were represented by a single tumor core and were, therefore, excluded from further analysis. Ki-67 staining was scored as “moderate” on both TMA and full sections in 42 (69%) of 61 selected cases. In 79% of cases, the Ki-67 scores of the TMA were similar to that of the full sections. Thirteen cases were discordant (Table 6); kappa was 0.42.
Table 6Agreement in immunohistochemical scores between TMA cores and full slides stained for Ki-67 and p53 Full sections<10%10–50%≥50%TotalκTMAsKi-67 <10%2103 10–50%342348 ≥50%06410 Total5497610.42p53 <10%193022 10–50%0123 ≥50%033538Total19737630.86
With regard to p53 staining, two cores were present in 12 (19%) cases and 3 cores were available in 51 (80%) cases. One case was excluded as it was represented by only one TMA core. Complete agreement was achieved in 87% of the selected tumors (Table 6). In the eight nonconcordant cases, the difference was one class, resulting in a kappa of 0.86.
Discussion
After its introduction in 1998, TMA technology has been applied in the immunohistochemical analysis of various malignancies, including squamous cell carcinomas and adenocarcinomas of the esophagus [3, 5, 18, 19, 25, 41]. Although it seems a very attractive method for high-throughput analysis of hundreds of tissues simultaneously, it may have limitations as the evaluation of the marker expression is reduced from full-section analysis to a few tissue cores of only 0.6 mm in diameter, especially for proteins that are heterogeneously expressed or that are cell cycle-dependent [36]. It is, therefore, essential to assess in each type of cancer individually and for every molecular marker whether TMA technology is feasible and valid [8, 33]. To our knowledge, this has not been done in esophageal cancer.
In our TMA containing triplicate cores of 108 ESCCs, a median of 9% of cores was uninformative (6% lost during tissue processing and 3% containing too little tumor), which is comparable to the results reported in other studies [7, 8, 27]. Improper selection of representative tumor areas on the donor block’s H&E slide by the pathologist or incorrect punching of these representative areas out of the donor block can cause tissue cores that contain too little tumor. Possible causes of absent cores are the size and fragility of the tumor tissue used and the aggressiveness of tissue processing applied [31, 35, 42].
Moreover, the number of available cores on the TMA slide depends on the level at which the TMA paraffin block has been sectioned. The slides stained for H&E, CK14, Ki-67, and p53 were one of the first slides that were cut from our TMA block, whereas sections stained for CK5/6 and E-cadherin were taken slightly deeper. On these latter sections, a lower number of absent cores was observed (Table 2), showing that not all cores were placed at the exact same level in the TMA block during TMA construction, mainly due to dissimilar thicknesses of the donor paraffin blocks that were used to construct the TMA [34].
The agreement in immunohistochemical results of the markers between our TMA and the full sections varied from moderate to almost perfect (κ = 0.42 to 0.93), which is consistent with the results reported in other TMA validation studies [4, 7, 8, 31, 35]. The observed variation in agreement could be due to tumor heterogeneity, topographical variation in the expression pattern of the molecular marker, or to the scoring criteria used [31].
Regarding tumor heterogeneity, the optimal amount of tissue cores incorporated in the TMA has been a matter of debate. Several validation studies have shown that three cores are highly representative for the full section [6, 11, 12, 28]. The addition of a fourth core did not add to the percentage of agreement in a colorectal cancer TMA [12]. Moreover, the more cores punched per case, the fewer cases can be placed into the TMA reducing throughput. Adding a fourth core may nevertheless be worthwhile in tissues prone to uninformative cores due to small lesions such as dysplasias or carcinomas in situ [42]. In our TMA, the amount of uninformative cores was low (5–10%), probably because ESCCs have a large diameter, thereby increasing the chance of obtaining a core containing tumor tissue. Taken together, we consider it justified to utilize three biopsy cores in ESCCs. Nonetheless, using such a low amount of cores requires careful selection of the tumor regions by an experienced pathologist to deal with the heterogeneity of the tumor in the TMA [33].
The agreement between TMA and full sections was substantial to almost perfect for CK5/6 and CK14. Because 91% of cases have shown a very strong expression of CK5/6 and only 1 of 64 cases showed negative staining, this molecular marker does not subdivide ESCCs and consequently will not be a prognostic marker for this malignancy. CK14 was more evenly distributed over the three scoring groups, but because one case was scored two classes lower on TMA when compared to the full section, kappa was lower when compared to CK5/6.
The relatively moderate concordance in case of Ki-67 may be explained by the fact that almost 80% of cases were situated in one category (staining of 10–50% of tumor cells) with 13 discordant cases deviating from this category. E-cadherin also had a moderate concordance, mainly because the relatively faint staining intensity of this molecular maker made its assessment in our TMA very difficult (Fig. 2).
Fig. 2Representative example of E-cadherin staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification ×20. b Enlargement of the right TMA core depicted in a; magnification ×100. c Part of the slide of the donor block of the same tumor; magnification ×100
TMA technology was also found to be valid for determining the histologic grade of differentiation in ESCC. Complete agreement between TMA and full sections occurred in 78% (49 out of 63; κ = 0.65) of selected cases, which is high when compared to the 40% agreement achieved in a TMA of bladder cancer [22]. Due to its homogeneous staining pattern, p53 showed excellent concordance (κ = 0.86) in our microarray (Fig. 3).
Fig. 3Representative example of p53 staining in TMA cores and the corresponding full section. a Three TMA cores representing one tumor; magnification ×20. b Enlargement of the left TMA core depicted in a; magnification ×100. c Part of the slide of the donor block of the same tumor; magnification ×100
The concurrence between the TMA and the full sections is affected by the cut-off values of the immunohistochemical scoring system of the stainings as well [7, 31]. The application of a two-class scoring system in an endometrial cancer TMA improved κ to 1.0 compared to 0.81 with a three-class system [7]. In our study, the two-class scoring system did not substantially affect the kappa (data not shown). Because the E-cadherin expression had a very low intensity in our ESCCs, we have chosen to apply a two-class system. In addition, the cut-off values indicating a strong immunohistochemical expression were set higher in the cytokeratins (80%) than in the other molecular markers (cut-off value 50%) because otherwise practically all tumors would be designated having a strong expression of cytokeratins.
Now that our esophageal cancer TMA has been validated, it will be used to correlate the expression of various molecular pathways with clinicopathologic data, aiming at detecting markers of prognostic significance and molecular targets for new therapies. Because the agreement between TMA slides and full sections depended on the molecular marker stained for, it should be considered to assess the expression pattern of a marker on a full section first, before staining a TMA slide. When a focal or heterogeneous expression pattern is noticed, it might be more valuable to assess marker expression by means of full sections instead of TMA. On the other hand, when a marker shows a homogeneously diffuse expression pattern, staining a TMA slide does allow for high-throughput screening of tumors. When a prognostic molecular marker has been identified by means of TMA technology, it is recommended to verify the results by full-section analysis.
In conclusion, this study has demonstrated TMA technology to be a valid method for immunohistochemical analysis in ESCC with agreement levels for well-known molecular markers with different staining potential between TMA and full sections ranging from moderate to almost perfect.

squamous cell carcinoma
validation studies
biological markers
esophageal neoplasms
protein microarray analysis


Renal replacement therapy for acute renal failure in children: European Guidelines

Acute renal failure (ARF) is uncommon in childhood and there is little consensus on the appropriate treatment modality when renal replacement therapy is required. Members of the European Pediatric Peritoneal Dialysis Working Group have produced the following guidelines in collaboration with nursing staff. Good practice requires early discussion of patients with ARF with pediatric nephrology staff and transfer for investigation and management in those with rapidly deteriorating renal function. Patients with ARF as part of multi-organ failure will be cared for in pediatric intensive care units where there should be access to pediatric nephrology support and advice. The choice of dialysis therapy will therefore depend upon the clinical circumstances, location of the patient, and expertise available. Peritoneal dialysis has generally been the preferred therapy for isolated failure of the kidney and is universally available. Intermittent hemodialysis is frequently used in renal units where nursing expertise is available and hemofiltration is increasingly employed in the intensive care situation. Practical guidelines for and the complications of each therapy are discussed.
Introduction
Acute renal failure (ARF) is uncommon in childhood, but its incidence may be increasing and modalities of treatment changing with an increasing number of children being treated in the intensive care unit (ICU) with multi-organ failure. Traditionally children with ARF with renal involvement were only treated with peritoneal dialysis, but extracorporeal techniques are being increasingly used in ICUs.
Members of the European Pediatric Dialysis Working Group reviewed all modalities of renal replacement therapy for ARF in children and developed the following guidelines in collaboration with nursing staff during three meetings and extensive e-mail discussion. There are no randomized trials of renal replacement treatment in children with ARF. The guidelines are based upon published reports and consensus opinion to emphasize good practice.
ARF is recognized when renal excretory function declines rapidly. Rising values of plasma urea and creatinine are usually accompanied by oliguria (<1 ml/kg per hour), but occasionally patients may be polyuric. The cause of ARF may be pre-renal, intrinsic, or post-renal (obstructive) problems, and causes differ between neonates and older children [1, 2, 3].
The incidence of ARF in children is hard to define, as often renal insufficiency in the newborn and on ICUs is conservatively managed by ICU staff. Outside the neonatal period, ARF is an uncommon condition accounting for 8 referrals per million population per year to one regional pediatric nephrology unit in the United Kingdom [4].
ARF may occur as isolated failure of the kidneys alone, with other organ systems functioning normally, or in association with multiple organ failure. The mortality of the latter group is considerably higher, especially with the growth in pediatric intensive care. For example, the mortality in neonates and infants is 51% after cardiac surgery for congenital heart defects [4], but only 3%–6% for children with intrinsic renal disease such as hemolytic uremic syndrome (HUS) in developed countries [5, 6].
The case mix in different units treating ARF, and hence mortality and morbidity rates, will therefore vary according to local clinical activity and resources [7, 8]. Many pediatric renal units will be close to pediatric ICUs (PICUs) in hospitals that may offer cardiac surgery, liver transplantation, and specialist treatment for metabolic disorders, oncology patients, etc. [6]. Other renal units may be in hospitals that do not have a PICU on site and conversely there may be hospitals offering pediatric intensive care with no specialist pediatric nephrology service.
Recommendations
All children with ARF require discussion with a pediatric nephrologist. Early transfer for investigation and management is essential in those with rapidly deteriorating renal function or in those with hemodynamic or biochemical disturbances (good practice) [9].
All children with ARF as part of multi-organ failure require transfer to a designated regional pediatric ICU where there should be access to pediatric nephrology advice and support (good practice).
Rationale
Since there are few comprehensive regional pediatric nephrology centers the distances that families may have to travel can be considerable. Children with acute renal impairment may be managed in local hospitals, but it is essential that early referral is made, especially if children have evidence of rapidly deteriorating renal function and require an urgent histological diagnosis to determine if immunosuppressive therapy or other treatment is required. Indications for referral include oligoanuria, especially if associated with fluid overload, hypertension, hyperkalemia, hyponatremia, acidosis, or the need for blood transfusion. Dialysis is often accompanied by early nutritional support and pediatric nephrology units should be equipped to provide the necessary medical and nursing expertise, combined with dietetic and psychosocial support. The latter support is also important if the child is managed conservatively.
Neonates and premature infants with ARF require transfer to a tertiary neonatal unit with pediatric nephrology team expertise. Patients with ARF and multi-organ failure require prompt transfer to a designated regional PICU.
The choice of dialysis therapy for ARF depends upon the clinical circumstances, patient location, and expertise available. Peritoneal dialysis (PD) has generally been considered the preferred therapy if there is isolated failure of the kidneys, such as HUS. It is regarded as a simpler technique that is universally available. However, hemofiltration (HF) and hemodiafiltration (HDF) are increasing in popularity in PICUs where the facilities to perform hemodialysis (HD) may not be available. HD may be the preferred mode of treatment in more-stable patients with adequate vascular access treated on renal units where specialist nurses are available.
Although extracorporeal techniques such as continuous venovenous hemofiltration (CVVH) or continuous venovenous hemodiafiltration (CVVHDF) are used quite frequently in adult ICUs, there is still limited expertise in many PICUs. Such techniques are very dependent on technology and are more costly than PD [10]. They are also dependent upon the availability of appropriate nursing expertise [11]. Such expertise can be developed and maintained in units remote from the pediatric nephrology center by an outreach service using a renal critical care nurse educator [12].
Recommendation
There is no evidence for the optimum level of renal function for starting renal replacement therapy nor for the optimum dialysis modality. Advantages and disadvantages are listed in Table 1. Consideration should be given to establishing national and international databases to collect these data along with patient outcomes [6, 13].Table 1 Advantages and disadvantages of various modalities of renal replacement therapy for acute renal failure (CVVH continuous venovenous hemofiltration, CVVHDF continuous venovenous hemodiafiltration)TypeComplexityUse in hypotensionEfficiencyVolume controlAnticoagulationPeritoneal dialysisLowYesModerateModerateNoIntermittent hemodialysisModerateNoHighModerateYesCVVHModerateYesModerateGoodYesCVVHDFHighYesHighGoodYes
Choice of therapy
Acute PD
The main advantage of PD is that it is continuous therapy that requires neither anticoagulation nor vascular access, and the technique can be used in hemodynamically unstable patients [14]. Acute PD can be performed in units with no HD expertise and is effective in children of all ages, including neonates [15, 16, 17, 18]. PD has been used in treating acute pancreatitis, tumor lysis syndrome, intoxications, metabolic diseases, and other pathological conditions in children [19, 20, 21, 22]. The choice of PD as therapy has always to be individualized, balancing advantages against disadvantages.
Limitations in the use of PD
Inborn areas of metabolism in the newborn period lead to acute accumulation of neurotoxic metabolites that can be better removed using techniques such as CVVHDF [23, 24]. The latter technique requires good vascular access, which can still be a major problem in small children [25]. Newborns with respiratory diseases, even if on ventilatory treatment, can be treated with PD provided that the fill and exchange volumes are adapted to the clinical situation. However, caution is necessary in neonates with necrotizing enterocolitis and older children with suspected bowel perforation [26].
Preparation for PD
Dialysis is only possible if the access provides free flow in and out of the abdomen. The choice is between catheters inserted at the bedside under sedation or the placement of a chronic PD catheter by a pediatric surgeon in the operating theater, or exceptionally at the bedside in the ICU.
The rigid Trocath catheter with a stylet has largely disappeared and surgically placed Tenckhoff catheters are reported to have fewer complications [27, 28, 29]. However, small catheters for percutaneous placement using a Seldinger technique are invaluable in providing acute PD rapidly, especially in the neonatal PICU [13, 30].
Blockage by the omentum is always a risk with PD catheters. If the catheter is to be placed surgically then consideration should be given to partial omentectomy [31].
In patients who are having a PD catheter inserted under general anesthetic a cephalosporin antibiotic (20 mg/kg) should be given as a single intravenous dose up to 1 h prior to implantation of the catheter [32]. Any subsequent accidental contamination should result in the use of prophylactic antibiotics, e.g., cefuroxime 125 mg/l in the dialysate for 48 h.
For catheters that are inserted percutaneously, prophylactic antibiotics, e.g., cefuroxime 125 mg/l, should be added to the dialysis fluid unless the patient is on systemic treatment.
Heparin, 500 units/l, should be prescribed to prevent catheter blockage with fibrin. This is generally maintained for the first 48 h, and longer if the PD fluid remains slightly bloodstained [33, 34].
PD prescription
This needs to be individualized according to patient size and condition. Automated PD machines are the preferred method for delivering the individualized dialysis prescription and accurately measuring ultrafiltration [30]. Such machines are now available that can deliver dialysis volumes accurately down to 60 ml with 10-ml increments. Although such machines now have improved accuracy of ultrafiltration measurements, the dead space of the tubing can reduce dialysis efficiency. A manual PD set can be used, using burettes that can accurately measure inflow and outflow, with the PD fluid warmed appropriately [35]. With manual sets, attempts should be made to maintain a closed drainage system, which can help reduce the frequency of peritoneal contamination [36]. Such manual PD sets are commercially available for neonatal patients.
Choice of dialysis solution
The choice of dialysis solution will depend upon the weight, blood pressure, and hydration status of the child, bearing in mind the need to create nutritional space as part of the management strategy [37].
The general principle is to commence with the lowest concentration of glucose solution possible (1.36%), with stepwise increments. Care is needed if 3.86% glucose solution is required as (1) rapid ultrafiltration can occur (especially in infants) and (2) hyperglycemia may develop (especially in septic and multi-organ failure patients) leading to hyperosmolarity and loss of effective ultrafiltration.
Icodextrine solutions need a longer dwell time to obtain significant ultrafiltration and so are rarely indicated in ARF. Lactate-containing dialysis solutions are likely to be replaced by bicarbonate solutions, which are being evaluated in chronic PD. The routine use of bicarbonate solutions should be considered in neonates or in patients with reduced lactate metabolism or with lactic acidosis [38, 39].
Practical points
Patients should be connected and automated PD or manual cycles started immediately after catheter implantation. Heparin (500 units/l) should be added to the dialysis fluid to prevent fibrin deposition and to improve peritoneal solute permeability [33, 34], but it can be absorbed and care is needed in patients with coagulation disorders.
Dialysis fill volumes of 10–20 ml/kg (300–600 ml/m2) should be used initially, depending on the body size and cycle in and out, until the dialysate becomes clear.
A PD program with 1-h dwells should be used during the first 24 h. Shorter cycles can be considered initially if hyperkalemia needs urgent treatment.
The program should be adjusted with increasing dwell times and cycle fill volume (if no leakage problems) until the desired fill volume (800–1,200 ml/m2) is achieved, with adequate ultrafiltration and biochemical control [40].
High intraperitoneal pressure (IPP) can be a problem in the first 2–3 days after surgical catheter insertion. The measurement of IPP may limit the risk of leakage when the fill volumes are being increased and allow optimized pain management, but is not yet in routine use [41].
Inflow/outflow pain on PD usually diminishes with time. Tidal dialysis is an alternative [42] and bicarbonate dialysis should be considered [43].
The amount of ultrafiltration that is prescribed will partly depend upon the volume of oral, nasogastric, or total parenteral nutrition that is required, combined with fluid for drugs. Ultrafiltration may not be enough without the use of 2.27% or 3.86% glucose solutions.
The clinical, biochemical, and nutritional status of the patient should be assessed regularly in conjunction with an experienced renal dietitian [44]. Optimal nutrition is necessary to avoid a catabolic state and associated production of blood urea nitrogen and uremic products.
Rationale
Patients with ARF need constant assessment while on PD, and adequacy should be judged in terms of clinical status, ultrafiltration achieved, and biochemical parameters, particularly urea, creatinine, and bicarbonate levels [40]. Although a link between the dialysis dose and the outcome of adult patients in ARF has been established [45], there are no guidelines as to what constitutes adequate PD in a child with ARF. The aim is to deliver maximum clearance to compensate for the catabolic stress.
Complications of acute PD
Leakages can be a difficult problem and are mostly due to a leakage around the catheter. The incidence can be reduced by proper surgical technique when using a Tenkhoff catheter [46] or resuturing around a percutaneous catheter. Fibrin glue injected into the catheter tunnel is a technique under evaluation [47].
Poor drainage due to mechanical blockage or catheter migration is all too common. Flushing the catheter and preventing fibrin accumulation by increasing the heparin dosage and/or urokinase is suggested initially [48]. A plain abdominal X-ray is rarely justified, as repeated poor drainage will require catheter relocation. If available, a laparoscopic technique may be used to correct poor drainage or replace the malfunctioning catheter [49].
Hernias can be a problem in neonates and infants, particularly males. They do not usually require interruption of PD and can be repaired electively by laparoscopic or direct measures when the child’s clinical condition has improved or stabilized.
Peritonitis remains a constant threat, especially if there has been a lot of manipulation of the catheter. The standard features of cloudy PD fluid require urgent attention [50].
Continuous extracorporeal techniques
Continuous arteriovenous hemofiltration (CAVH) has largely been replaced by pumped CVVH and CVVHDF, particularly in ICUs [51]. Such continuous renal replacement therapies (CRRT) have expanded the possible role of blood purification in the management of critically ill patients. However, there is a lack of randomized trials in patients with sepsis, and a recent analysis failed to show a benefit for hemofiltration [52]. Studies in adult ICU patients have shown a lower mortality in patients treated with CRRT compared with intermittent HD. However, a recent meta-analysis of studies before 1996 concluded that the evidence was insufficient to draw strong conclusions regarding the mode of renal replacement therapy for ARF in the critically ill [53]. A recent randomized trial in adult ICU patients showed a significant survival advantage when the intensity of ultrafiltration was increased [54].
Practical guidelines for prescription
Since the concentration of solutes in the filtrate is the same as in the plasma, biochemistry is controlled by removing large volumes of filtrate and replacing it with electrolyte-containing fluid (HF replacement fluid). As most solutes are distributed within the extracellular and intracellular fluid compartments (total body water), the exchange volume of filtration necessary to control biochemistry relates to total body water. Clinical experience has shown that a turnover of approximately 50% of body weight in 24 h is usually adequate for CVVH.
The extracorporeal circuit requires good central venous access, usually via a dual-lumen catheter, to allow the high blood flows necessary to prevent clotting in the hemofilter. Suggested catheter sizes in French gauge (FG) are:Patient size (kg)Vascular access2.5–106.5-FG dual-lumen (10 cm)10–208-FG dual-lumen (15 cm)>2010.8-FG or larger dual-lumen (20 cm)
For neonates a 5-FG dual-lumen catheter may be adequate, and access can be obtained via the umbilical vein [55]. A single-lumen catheter using a “single needle” for CVVHD in very low birth weight infants has also been described [56], but this method may be compromised by high recirculation rates with most available systems. However, the smaller the access the greater the problems [57]. It is possible to consider placing two small single-lumen catheters in different central veins.
A low blood flow rate, high hematocrit, and high plasma protein concentration will limit the rate at which filtration can occur and solutes (particularly of higher molecular weight) are removed. For a given blood flow rate, pre-dilution results in higher clearance of solutes than does post-dilution [58], but at the expense of greater use of replacement fluid (approximately 20%–50% more). Pre-dilution has the potential for extending filter life.
As with HD, the blood volume in the extracorporeal circuit should be less than 10% of the patient’s circulatory volume. Blood flows of 6–9 ml/kg per min or 8% of circulating blood volume prevents excessive hemoconcentration in the filter. Automated machines with appropriate accuracy for children are recommended for delivering the CRRT prescription safely [59], and have replaced pump-assisted hemofiltration using volumetric pumps [60].
To achieve a 50% exchange of total body water in 24 h, an appropriate filter should be selected with a surface area of no more than the surface area of the patient. Suggested maximum filtration rates are:Patient size (kg)Maximum filtration rate (ml/h)<8.52508.5–20500>202,000Under post-dilution conditions, the filtration rate should never exceed one-third of the blood flow.
Several filter materials are now available. Synthetic membranes have replaced cellulose acetate, as they are more biocompatible, causing less complement reaction and anticoagulation needs. The synthetic polysuphone membranes are also thought to aid convective clearance of solutes through solute drag [61].
A variety of replacement fluids are available such as lactate, bicarbonate, and buffer-free solutions. Bicarbonate or buffer-free solutions should be used in young infants and those intolerant of lactate. If a commercially available bicarbonate solution were freely available, then this would be the solution of choice. Careful monitoring of electrolytes, glucose, and phosphate is essential, as the constituents vary between the solutions.
Anticoagulation
The goals of anticoagulation are to prevent clotting of the circuit and maintain adequate clearances with minimal risk to the patient. Heparin is the standard anticoagulant in Europe, but the choice of dosage will depend upon the patient’s coagulation status, adequacy of blood flow, and blood viscosity. In most patients, heparin should be administered as an initial bolus (maximum 50 units/kg) at the time of connection to the extracorporeal circuit, followed by a continuous infusion of 0–30 units/kg per hour. The activated clotting time (ACT) or whole blood activated partial thromboplastin time (aPPT) are usually used to monitor treatment. The optimal ACT during hemofiltration is 120–180 s. The aPPT should be between 1.2 and 1.5 times the respective baseline value. Some patients can be treated without heparin in the circuit [6].
In those patients who are severely thrombocytopenic or where there is suspected heparin-induced thrombocytopenia, alternative treatment with prostaglandin infusions or recombinant hirudin [62], a direct thrombin inhibitor, can be considered [63].
Regional anticoagulation with citrate has been favored by some centers [64, 65]. Sodium citrate chelates ionized calcium necessary for the coagulation cascade and systemic anticoagulation is avoided by infusing calcium through a separate central line. The disadvantages include the possibility of various acidbase and electrolyte disturbances, including hypernatremia, hypocalcemia, and metabolic alkalosis.
Adjustment of the prescription
Any formula for the prescription of HF is at best an approximation or starting point, as the needs will be determined by many unmeasured variables, such as the rate of solute production, nutritional intake, and the actual volumes of the extracellular fluid and intracellular fluid compartments.
If only fluid removal is required, then relatively low rates of filtration are needed, often referred to as slow continuous ultrafiltration (SCUF). There will be negligible solute removal under these circumstances.
Correction of “uremia” and electrolyte disturbance requires the turnover of large volumes per kilogram of fluid, typically of the order of 50% of body weight per day for post-dilution and 75% for pre-dilution (approximately 20–30 ml/kg per hour).
In catabolic patients, the clearances achieved with standard CVVH may not be sufficient. Solute removal may be increased by attempting “high-volume exchange,” but this may be limited by the practical problems of pediatric patients with limitations of vascular access and hemoconcentration in the filter. In these cases, small solute clearances can be maximized by establishing diffusive mass transport via a dialysis circuit. This can be performed with CVVHDF or without an additional major ultrafiltration component (CVVHD). CVVHDF latter technique requires an additional pump to achieve separate control of the dialysate in- and outflow and of the replacement fluid flow. CVVH substitution fluid bags can be used as dialysis fluid. Dialysis fluid flow should be 2–3 times the blood flow if maximal efficacy is desired. This setting requires frequent manual bag exchanges and continuous supervision of the system. For practical purposes, the HD component can be added for several hours per day to a CVVH regimen.
CVVHD has recently been recommended as the method of choice for the treatment of inborn errors of metabolism, since it supplies maximal clearance of ammonium and other neurotoxic metabolites. When CVVHD is unavailable, large volume turnover of body water with CVVH will provide the next best therapy. Rates of up to 100 ml/kg per hour have been reported [66]. If possible, the blood pump speed also needs to be increased.
When high turnover and blood flow rates are in use, patients should be carefully monitored for hypothermia, hypokalemia, and circulatory failure. Hypothermia may need to be treated with an external warming blanket and hypokalemia will require replacement. Blood flow should not be increased if the patient develops cardiovascular instability.
CVVH and extracorporeal membrane oxygenation
In the authors′ experience, the best results are achieved when pre-diluted fully automated CVVH is used, attached to the venous (outflow from patient) side of the extracorporeal membrane oxygenation (ECMO) circuit. This appears to reduce problems of shunting blood around the oxygenator and overcomes the problems of the increased hematocrit that may be associated with ECMO. It also reduces the complications of excessive fluid and solute clearances, with a free flow when systemic hemofilters are used in line with the ECMO circuit. When using CVVH in the suggested configuration, the “pigtails” provide access with very little resistance, causing the arterial and venous pressure alarms to activate and shut down the circuit. Therefore, three-way taps are used to create more resistance to flow into and out of the CVVH circuit. When treating neonatal patients, the ECMO circuit increases the extracorporeal blood volume very significantly. Therefore, the blood pump speed should be calculated taking into account the patient’s blood volume and the priming volume of the ECMO circuit.
Complications of continuous extracorporeal techniques
Complications of continuous extracorporeal techniques are described in reference [67].
Hypotension
Hemofiltration is most commonly used in sick septic children, many of whom will be on pressor therapy. Indeed, the need for pressor agents gives a poorer prognosis [6]. Care should have been taken to minimize the amount of blood in the extracorporeal circuit and blood priming of the HF circuit may be necessary at the outset. Fluid removal is obviously adjusted according to the patient’s clinical state during the treatment.
Clotting of the filter and lines
This is one of the commonest complications and again is related to the patient’s changing clinical status and problems with anticoagulation. This complication occurred in 24% of 89 patients treated with CVVH in a 2-year local audit (B. Harvey, unpublished observations).
Other potential complications of bleeding, anticoagulation toxicity, and infections appear to be minimal. Air embolism is a rare but preventable complication of extracorporeal circuits, and is greatly reduced with the proper use of automated machinery.
Intermittent HD
The advantages and limitations of intermittent HD are described in reference [68].
Advantages
The main advantage of HD is the relatively rapid removal of uremic toxins and ultrafiltration of fluid. This makes the technique well suited for acute situations.
Limitations
HD is not a continuous therapy and it requires good vascular access as with HF. A purified water supply is also required, as well as anticoagulation, which should always be minimized. The technique might not be applicable for hemodynamically unstable patients. Often the major limiting factor is the availability of expert nursing staff [69], especially in the ICU [70].
Practical guidelines for prescription
HD is only possible with good vascular access provided either by a double-lumen HD catheter or a single-lumen catheter of sufficient diameter to achieve flows for single-needle dialysis. Catheter lengths vary from 5 cm for neonates to 20 cm for large adolescents.
Bloodline choice depends on the priming (extracorporeal) volume, which traditionally has not exceeded 10% of the blood volume (approximately 80 ml/kg).
Dialyzer choice depends on the priming volume and maximum flow rate, with a surface area that should not exceed the child’s surface area and with a urea clearance between 3 and 5 ml/kg per min. There is no evidence for dialyzer choice in pediatric practice, but meta-analysis in adult patients with ARF suggested synthetic membranes conferred a significant survival advantage over cellulose-based membranes, but with no similar benefit for recovery of renal function [71].
Bloodline priming is usually performed with isotonic saline. Small babies, anemic patients, and those in an unstable cardiocirculatory condition, require priming with albumin or blood.
HD catheter care
After the session the catheter should be flushed with isotonic saline and filled with undiluted heparin (1,000 IU/ml), with volumes according to manufacturer’s recommendations (usually marked on the catheter itself).
HD prescription
The first session should not exceed 2–3 h, but the standard time is usually 4 h. Longer sessions are advisable to avoid too-rapid ultrafiltration and disequilibrium syndrome.
All children should be dialyzed using volume-controlled machines and with bicarbonate dialysate.
The blood pump rate is usually 6–8 ml/kg per min, but depends upon the catheter and patient size [69].
The ultrafiltration target should not exceed 0.2 ml/kg per min for acute patients who should be carefully monitored for hypovolemia and hypotension. Sodium profiling is rarely used in pediatric HD practice. Anticoagulation is usually with heparin (50–100 IU/kg per session including initial bolus). Reinfusion is usually performed with isotonic saline.
Complications occurring during acute HD
For hypotension, the ultrafiltration should be switched off and isotonic saline infused into the venous line until the blood pressure normalizes; additional 20% albumin 5 ml/kg might be helpful.
Hypertension is treated according to standard hypertension protocols available elsewhere [72].
Disequilibrium syndrome is now a rare event with adequate control of ultrafiltration and stepwise reduction of uremic toxins.
Hypoglycemia should not occur with the use of glucose-containing dialysis fluid.
In cases of anemia transfusions are avoided unless patient symptomatic. Erythropoietin may be given intravenously at the end of dialysis (50–200 IU/kg) to maintain hemoglobin levels.
Medications
The clearance of drugs on HD or during CRRT needs to be considered. Reference should be made to standard texts [73, 74].

acute renal failure
guidelines
peritoneal dialysis
hemodialysis
hemofiltration