Therapy of chronic extensor mechanism deficiency after total knee arthroplasty using a monofilament polypropylene mesh.

Introduction: Lesions of the quadriceps or patellar tendon after total knee arthroplasty (TKA) are a rare but serious complication which, if left untreated, can lead to loss of function of the knee joint. While acute and subacute extensor mechanism disruptions may have several causes, chronic deficiencies are often related to multiple prior revision surgeries for joint infection or aseptic TKA failure. Up to date, biological allograft reconstruction showed unsatisfying results. The use of a monofilament polypropylene mesh is a promising approach for this pathological condition. The aim of the present study was to evaluate clinical, functional and patient reported outcomes of this procedure in patients with chronic extensor mechanism deficiency. Materials and Methods: Twenty-eight patients with chronic extensor mechanism deficiency (quadriceps tendon rupture n = 9, patellar tendon rupture n = 19) after TKA were included in this retrospective study. None of the patients were lost to follow-up. Surgical reconstruction was performed at one institution between 2014 and 2020 with a monofilament polypropylene mesh (Marlex Mesh, Bard, Murray Hill, USA). The mean age at the time of surgery was 69 years. Patients presented with a mean BMI of 33 kg/m2. The mean follow-up period was 23 months. Results: The 2-year survivorship free of mesh revision was 89% [95% confidence interval (CI): 75% to 100%]. Three patients (11%) had to undergo revision because of mechanical mesh failure and received another polypropylene mesh. No further revisions were performed thereafter. Flexion was 87° (range, 30-120°) on average. The majority of patients (75%, 21/28) had a full active extension. The mean active extension lag after surgery was 4 degrees (range, 0-30°). Discussion: We observed a substantial improvement of extensor mechanism function. The majority of patients had full extension and showed good clinical results. A failure rate of over 50% has been published for alternative procedures. Thus, the use of the described augmentation technique represents a reasonable treatment option for chronic extensor mechanism disruptions of the patellar tendon as well as the quadriceps tendon after total knee arthroplasty. However, there might be a potentially higher risk for infection persistence in periprosthetic joint infection cases due to the presence of a foreign material.

[Challenges to endoprosthetic reconstruction after tumor resection around the knee : Management of intra- and postoperative complications]. / Herausforderungen in der Tumorendoprothetik des Kniegelenks : Management von intra- und postoperativen Komplikationen.

BACKGROUND: The good functional outcomes of endoprosthetic knee reconstructions combined with modern oncosurgical concepts have resulted in a decreased rate of primary amputations following tumor resection in the region of the knee, thereby improving the quality of life of affected patients. OBJECTIVE: This study aimed to reappraise complications which are more common than in conventional arthroplasties performed for arthritis or trauma due to the complexity of the surgical procedures, the size of the endoprosthetic reconstructions, and neo-/adjuvant therapies. MATERIAL AND METHODS: Possible intra- and postoperative complications and treatment options are presented. RESULTS: Typical intraoperative complications include malalignment (axis, length, rotation) in implant positioning, unplanned soft tissue defects, impaired implant fixation due to poor bone stock, periprosthetic fractures, vessel/nerve injuries, and lesions/defects of the extensor mechanism. The relevant postoperative complication profile is characterized by local recurrences, periprosthetic infections, thromboembolic events, and mechanical failures (loosening, failure of the constraining mechanism). CONCLUSION: Depending on anatomic conditions and the systemic oncological status of the patient, these complications represent tremendous challenges to reconstructive management. Knowledge of the potential problems enables them to be avoided. This requires profound experience in oncosurgery and tumor endoprosthesis revisions.

[Quadriceps tendon insufficiency and rupture : Treatment options in total knee arthroplasty]. / Quadrizepssehneninsuffizienzen und -rupturen : Verfahren zur Behandlung in der Knieendoprothetik.

Quadriceps tendon injuries and insufficiencies in total knee arthroplasty are rare, but are followed by a devastating complication that left untreated leads to a complete loss of function of the knee. This review article summarizes the functional anatomy, risk factors, and the prevalence and diagnosis of quadriceps tendon injuries, in addition to the possible management options for partial and complete ruptures. The treatment options are adapted according to the extent of the loss of function (partial, complete) and the duration of the injury (acute vs chronic). Furthermore, the choice of treatment should take into account the quality and availability of primary tissue, the patient's general health, along with their likely functional requirements. Conservative treatment is often justified in partial ruptures with good results. Complete ruptures require surgical intervention and multiple operative techniques are described. Treatment options for acute ruptures include direct primary repair with autogenous or synthetic tissue augmentation. In the case of chronic insufficiency and a lack of soft-tissue surroundings, reconstruction with the aid of a muscle flap or allograft tissue can be considered. All surgical intervention techniques used so far have been fraught with complications and rarely lead to satisfactory results. A new surgical approach to the reconstruction and augmentation of the extensor mechanism consists of the use of a synthetic mesh. The technique is described here in detail.

[The Berlin diagnostic algorithm for painful knee TKA]. / Berliner diagnostischer Algorithmus der schmerzhaften Knie-TEP.

BACKGROUND: Approximately 20% of patients are unsatisfied with their postoperative results after total knee arthroplasty (TKA). Main causes for revision surgery are periprosthetic infection, aseptic loosing, instability and malalignment. In rare cases secondary progression of osteoarthritis of the patella, periprosthetic fractures, extensor mechanism insufficiency, polyethylene wear and arthrofibrosis can cause the necessity for a reintervention. Identifying the reason for a painful knee arthroplasty can be very difficult, but is a prerequisite for a successful therapy. AIM: The aim of this article is to provide an efficient analysis of the painful TKA by using a reproducible algorithm. DISCUSSION: Basic building blocks are the medical history with the core issues of pain character and the time curve of pain concerning surgery. This is followed by the basic diagnostics, including clinical, radiological, and infectiological investigations. Unique failures like periprosthetic infection or aseptic loosening can thereby be diagnosed in the majority of cases. If the cause of pain is not clearly attributable using the basic diagnostics tool, further infectiological investigation or diagnostic imaging are necessary. If the findings are inconsistent, uncommon causes of symptoms, such as extra-articular pathologies, causalgia or arthrofibrosis, have to be considered. In cases of ongoing unexplained pain, a revision is not indicated. These patients should be re-evaluated after a period of time.

[Intraoperative Evaluation of Total Knee Arthroplasty: Anatomic and Kinematic Assessment with Trial Components]. / Intraoperative Analyse der Anatomie und Kinematik in der Knieendoprothetik.

The intraoperative use of trial components in total knee arthroplasty (TKA) is of paramount importance to prevent inadequate ligament balance and to achieve optimal position of the definitive components. This review demonstrates an 8-step algorithm to assess the anatomy of the femoral, tibial and patellar component as well as the kinematics of the tibiofemoral and patellofemoral joints. Trial components allow an easy assessment of the anatomic fit of the final implants. Upon the trials insertion, bone coverage and the component overhang should be evaluated. The femoral rotation should be assessed using the transepicondylar axis and for the tibial component rotation assessment, the tibial tuberosity would be the most reliable bony landmark. Addressing the patella, sizing and bone coverage should be thoroughly evaluated. In order to restore physiological kinematics the remnants of the meniscus rim can be used to determine the correct reconstruction of the joint line. A tight extension gap results in limited extension, whereas a tight or unbalanced flexion gap leads to "booking" or "spin-out" of the inlay. The POLO test (pull-out, lift- off) allows an easy assessment of the posterior cruciate ligament tension and the size of the flexion gap as well. To prevent postoperative dislocation and overstuffing, specific tests for correct patellar positioning and tracking support should be performed. The anatomy and kinematics of total knee arthroplasty have to be evaluated by trial components on a routine basis before inserting the final implants in order to identify implant positioning errors and inadequate ligament balance.

[Management of Flexion Contracture in Primary Total Knee Arthroplasty]. / Umgang mit der Beugekontraktur in der primären Knieendoprothetik.

Flexion contracture is a common deformity of the arthritic knee. The present publication describes causes, clinical relevance and surgical technique in the presence of flexion contractures in total knee arthroplasty. Flexion contracture can be attributed to different causes. Basically it is a mismatch between flexion and extension gaps. Moderate and severe deformities have to be corrected by additional surgical interventions. In most cases soft tissue techniques with release of contracted structures, the removal of osteophytes and additional distal femoral bone resection are necessary. The goal of these interventions is to achieve full extension of the knee. During rehabilitation attention has to be paid to maintain it with intensive physical therapy. A remaining flexion contracture is associated with inferior functional outcome and persistent pain.

[A novel technique for a stable intramedullary fixation of static spacers in two-stage septic revision TKA with severe bone defects]. / Technik der stabilen intramedullären Verankerung eines statischen PMMA-Spacers beim zweizeitigen septischen Knie-TEP-Wechsel mit ausgeprägten Knochendefekten.

BACKGROUND: Articulating as well as static PMMA-spacers are routinely used in two-staged septic total knee arthroplasty (TKA) revision surgery. However, if bone defects are excessive and there is substantial instability, conventional spacers without additional stabilisation are inappropriate. Due to the increasing number of multiple revisions and larger bone defects, an absolutely stable spacer construct with appropriate fixation in the remaining bone is required. METHODS: After TKA removal and debridement, AO external fixator steel rods were inserted "press-fit" into the femoral and tibial medullary canal in this technique. Both rods were sealed with bone cement (PMMA) and connected at the joint level in 5° of flexion by an AO external fixator "tube-to-tube" connector. Comparable to a conventional static spacer the joint space is filled with individual antibiotic loaded PMMA. The restoration of the leg axis as well as the distraction of the joint is crucial to preserve the joint space for the second stage TKA reimplantation. Postoperative mobilisation can be conducted under full weight-bearing conditions without external orthosis. CONCLUSION: This technique allows a safe intramedullary fixation of a modified static spacer in septic TKA revision with large bone defects and substantial instability with avoidance of common spacer complications and excellent patient mobilisation.

[Defect Reconstruction in Total Knee Arthroplasty with wedges and blocks]. [Corrected]. / Defektrekonstruktion in der Knieendoprothetik mit Wedges und Blöcken.

OBJECTIVE: Surgical technique for primary and revision total knee arthroplasty to reconstruct bone defects with metal augments and reproducible positioning of the implant at the right joint line. INDICATIONS: Primary and revision total knee arthroplasty with bone defects. CONTRAINDICATIONS: Complete destruction of the metaphysis. SURGICAL TECHNIQUE: Implantation of revision components performed in three consecutive steps: first, positioning of the tibia component at correct height and rotation; second, determination of the posterior joint line in flexion through the size and correct rotation of the femoral implant; third, determination of the distal joint line by use of positioning of the femoral component. These steps are performed independently from bone defects, which are subsequently reconstructed with metal augments. POSTOPERATIVE MANAGEMENT: Mobilization with weight bearing and range of motion as tolerated, depending on osseous and soft tissue condition at primary or revision surgery. RESULTS: In a prospective study, 132 consecutive knee revisions in 76 women and 56 men with an average age of 72.4 years (range 49-93 years) were followed up clinically and radiologically preoperatively and at a mean follow-up of 74 months (range 38-105 months). Clinical results were based on the American Knee Society score. The score was 46.3 (range 31-65) preoperatively and 82.5 (range 61-96) at follow-up. Radiologically 12.1 % of the knees showed lysis around the augment with no clinical signs of loosening. No revisions were performed due to aseptic loosening. The joint line was correctly reconstructed in 84.8 %.

[Does the ideal total knee arthroplasty for younger patients exist?]. / Gibt es die ideale Knietotalendoprothese für jüngere Patienten?

BACKGROUND: A total knee arthroplasty (TKA) is still not able to reinstate the physiological kinematics of the knee. This results in a considerable number of unsatisfied patients, especially if they are younger and active. Recently developed TKA systems claim to consider these biomechanical properties. The current study investigates the outcome (range of motion, stability) of the new ATTUNE™ TKA system (DePuy, Warsaw, IN, USA) in in young patients. MATERIAL AND METHODS: This was a prospective study design (55 patients, 55 knees, age < 70 years, 01/2012-07/2012). Patients received an ATTUNE™ TKA and were examined preoperatively and at 6 months postoperatively for range of motion and stability (Knee Society score). RESULTS: Age: 63 ± 8 years; range of motion increased from 112.33 ± 13.6° to 123.60 ± 11,1° (p < 0.001). Coronal stability improved significantly (preoperatively vs. postoperatively, p < 0.001). Sagittal stability was not significantly different (preoperatively vs. postoperatively, p > 0.05). CONCLUSION: The data of this study show improved early functional results in younger patients in comparison to the current literature. However, until now no existing TKA system can be identified as ideal. But taking recent biomechanical knowledge into consideration, modern TKA designs have the potential to improve the functional outcome. Especially for younger patients with superior muscle-status and coordinative abilities this seems beneficial.

[Early results of gender-specific posterior stabilized total knee arthroplasty without patella resurfacing]. / Konventionelle und geschlechtsspezifische kreuzbandersetzende Knietotalendoprothesenimplantation. Vergleich frühfunktioneller Ergebnisse.

INTRODUCTION: To address anatomical gender differences in total knee arthroplasty (TKA) specific total knee prostheses have been developed for women. Potential benefits of these modified prostheses are currently under debate. The present study investigated whether the modified design features bring benefits compared to uni-sex TKA. METHODS: A total of 80 prospectively blinded and randomized patients underwent implantation of unilateral TKAs with NexGen LPS Gender Solutions (Zimmer, Warsaw, USA, group gender-specific GS prosthesis, n = 40) or NexGen LPS Flex (Zimmer, Warsaw, USA, control group standard prosthesis ST, n = 40) The follow-up was carried out 10 days and 6 weeks postoperatively. Clinical data and the subjective assessment of quality of life were evaluated using the Knee Society Clinical Rating System (KSS), the short form 36-item health survey (SF-36) and the Western Ontario and McMaster Universities OA Index (WOMAC). RESULTS: The two groups showed equal values in KSS, SF-36 and WOMAC preoperatively and ten days postoperatively the GS group reached an average KSS knee score of 62.6 ± 16.1 points (ST group 56.9 ± 14.7, p = 0.184) and a functional score of 28.5 ± 12.1 (ST group 24.3 ± 15.3, p = 0.082). In the overall score the GS group reached 91.1 ± 24.1 points (ST group 81.0 ± 27.1, p = 0.104). The GS group reached a knee score of 85.5 ± 14.4 points (ST group 77.8 ± 16.8, p = 0.03) and a functional score of 68.1 ± 20.7 points (ST group 62.3 ± 18.5, p = 0.185) 6 weeks postoperatively. In the overall score the GS group reached 153.7 ± 30.7 points (ST group 139.6 ± 32.4, p = 0.048). The analysis of SF-36 and WOMAC showed no significant differences at all time points. No evidence of loosening or migration was observed in both groups. CONCLUSIONS: Based on the data presented, gender-specific TKA type NexGen LPS Gender Solutions has advantages in terms of early functional outcome. This result is not reflected in the patient satisfaction and is not considered to be clinically relevant.

[Patella height after total knee replacement: influence of the radiological setting]. / Patellahöhenstand nach Kniegelenkendoprothesenimplantation: Einfluss der radiologischen Aufnahmebedingungen.

BACKGROUND: Acquired patella baja is a possible cause of painful total knee arthroplasty (TKA) and a poor functional outcome. Standardized lateral radiographs - often missing in the daily routine - are required for evaluating the patella height. It is unknown which patella index is insensitive to the radiological setting so that it can be used efficiently in the clinical routine. PATIENTS AND METHODS: This prospective study included 25 patients after TKA. Their average age was 69.5+/-8.8 (51-80) years. In two cases (8%), resurfacing of the patella was performed. The modified Insall-Salvati index (mIS) and the Caton-Deschamps index (CD) were determined and compared on lateral radiographs with the patient's knee extended while lying down, extended while standing, and 30 degrees flexed while lying down. RESULTS: The average mIS in extension was 1.63+/-0.31 with the patient lying and 1.68+/-0.31 with the patient standing. In 30 degrees flexion and lying, it was 1.7+/-0.3. The mIS on the recommended radiographs (30 degrees flexion) correlated well with the mIS in extension while lying (R=0.48, p<0.001) and standing (R=0.99, p<0.001). The CD in extension was 0.78+/-0.21 while lying and 0.49+/-0.12 while standing; it was 0.48+/-0.15 in 30 degrees flexion. The CD on the recommended radiographs (30 degrees flexion) correlated satisfactorily with the CD in extension while standing (R=0.93, p<0.001) but not in extension while lying (not significant). CONCLUSION: The mIS is less dependent on the radiological setting than the CD is. The mIS can be used efficiently for evaluating an acquired patella baja in radiographs performed in different, not standardized, conditions.

[C-reactive protein. An independent risk factor for the development of infection after primary arthroplasty]. / Das C-reaktive Protein. Ein unabhängiger Risikofaktor für die Entwicklung eines Infekts nach Primärendoprothetik.

BACKGROUND: Infection is a severe complication after primary arthroplasty of the hip (THA) or knee joint (TKA). Based on its high sensitivity, the C-reactive protein (CRP) concentration has become a valuable tool in the diagnosis of infection, although it has only moderate specificity. Because of this, it remains unclear whether a preoperative increased CRP without clinical symptoms is a risk factor for infection after primary arthroplasty. MATERIAL AND METHODS: In a retrospective analysis, we investigated individuals with infection after primary THA or TKA and matched them with patients without infection after similar operations. Matching criteria were age, gender, and present diseases. The average age of the 50 included individuals was 67.4 (range 48-81) years, with eight men and 17 women in each group. In addition to preoperative CRP, specific patient and surgery data and microbiological and histopathologic findings were obtained. RESULTS: The average preoperative CRP concentration in the infected patient group was 1.3+/-2.5 mg/dl, in contrast to 0.4+/-0.7 mg/dl in the noninfected group. A threshold of 0.5 mg/dl was appropriate for discriminating between the two groups [13/25 (52%) in the infection group vs. 3/25 (12%) in the control group, p=0.003]. Independent from the patient group, CRP concentrations were significantly increased in individuals with diabetes mellitus (1.2+/-1.5 vs. 0.7+/-2.0 mg/dl, p=0.03). CONCLUSION: An increased preoperative CRP concentration without clinical findings of infection is a risk factor for prosthetic infection after primary THA or TKA with a threshold concentration of 0.5 mg/dl. Latent local or systemic infections or aseptic inflammation with subsequent local immune suppression seem to be responsible. We recommend evaluating CRP before every THA and TKA. For values beyond 0.5 mg/dl, an exploration for infection should be done. Otherwise, the patient should be informed about the increased risk of infection.

Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines.

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.

Quantitative molecular monitoring of residual tumor cells in chronic lymphocytic leukemia.

New therapeutic approaches for the treatment of B-cell chronic lymphocytic leukemia (B-CLL) can induce remarkable responses. Molecular remissions have been observed occasionally after high-dose chemotherapy. Thus, new improved techniques to monitor residual tumor cells on a molecular basis in CLL are warranted. For this purpose, a real-time quantitative allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) for patients with B-CLL was designed. In the present study, the PCR assay was standardized with identical cycling parameters as well as primer, probe, and MgCl(2) concentration for each patient. Ten patients were monitored with DNA samples obtained at 52 time points (median: 5.2 per patient). The median follow-up per patient was 11.4 months. Nine of ten patients had PCR-detectable residual tumor cells in the peripheral blood after therapy. One patient became PCR negative with a combination of fludarabine and rituximab after the end of treatment. The MRD levels in patients with detectable disease ranged from 0.002% to 10.1% after therapy. We conclude that real-time quantitative ASO-PCR can be utilized for quantitative molecular monitoring of minimal residual disease (MRD) in B-CLL patients in complete remission (CR), that new effective treatment approaches such as combined chemo/immunotherapy can render CLL patients PCR negative, and that different MRD levels in PCR-positive patients were observed warranting further investigation into possible correlation with clinical outcome.

Myocardial fibrosis in transforming growth factor-beta(1) (TGF-beta(1)) transgenic mice is associated with inhibition of interstitial collagenase.

BACKGROUND: TGF-beta(1) mediates effects on fibroblast proliferation and collagen synthesis in the myocardium. The extracellular matrix remodeling depends on the fibrillar collagen degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The in vivo effects of TGF-beta(1) on the MMP/TIMP system in TGF-beta(1) overexpressing transgenic mice were studied. METHODS: Male Alb/TGF-beta(1)(cys(223,225)ser) transgenic mice (TG) and nontransgenic controls (C; 8 weeks) were examined. Protein expression of collagen type I, -III, interstitial collagenase (Int Coll), MMP-2, -9, TIMP-1, -2, -4 and TGF-beta(1) as well as enzyme activity (MMP-2, -9) were measured (Western blots, zymographic assays). mRNA expression of the interstitial collagenase and MMP-9 was studied with the Light-Cycler based real-time PCR. RESULTS: Overexpression of TGF-beta(1) resulted in a 10-fold increase in plasma and a seven-fold increase in myocardial TGF-beta(1) concentrations. Relative heart weights increased (mg g(-1): 7.8 +/- 0.4 vs. 4.8 +/- 0.6, n = 6; P < 0.01) in TG compared to C. Collagen type I and III increased in TG (1.9-fold and 1.7-fold) compared to controls. Interstitial collagenase protein activity (- 91%) and mRNA expression (-75%) in TG were reduced (P < 0.05-P < 0.001). Gelatinase (MMP-2, MMP-9) expression and activity were not significantly alterated. MMP-inhibitors were increased 2.5-fold (TIMP-1, -4) and 6-fold (TIMP-2) in TG. CONCLUSIONS: TGF-beta(1) produces myocardial fibrosis in vivo. This effect is not only produced by a stimulation of matrix protein formation: a complex regulation of MMP and TIMP interaction, namely decrease of expression and activity of interstitial collagenase and an enhanced inhibition by increased levels of TIMPs, are involved. These mechanisms are optional targets for therapeutic interventions in myocardial diseases.

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

A real-time PCR assay for the quantification of residual malignant cells in B cell chronic lymphatic leukemia.

Several new therapeutic approaches for the treatment of monoclonal B cell lymphomas are currently being investigated. In parallel with new therapeutic modalities, more sensitive diagnostic methods are needed. These methods should be highly sensitive in detecting very low amounts of malignant cells and should be specific for the malignant clone. In addition, these methods should allow the quantification of residual tumor cells. In this study a new real-time polymerase chain reaction (LightCycler) was evaluated to quantify residual tumor cells in monoclonal B cell malignancies. This technology combines the advantages of rapid cycling PCR with the online detection of PCR products using fluorescent dyes. Our assay is based on immunoglobulin heavy chain (IgVH)-specific PCR with allele-specific primers complementary to hypervariable CDRII and CDRIII regions. A set of framework region III (FRIII)-specific hybridization probes was used for detection of the specific amplification product, and IgVH copy number was quantified with the cloned IgVH sequence as an external standard. The approach was evaluated with the Hodgkin lymphoma cell line L428 in order to quantify L428 dilutions. L428 cells mixed with peripheral blood mononuclear cells (PBMNCs) were detected and quantified with a sensitivity of one cell within 1 x 10(5) PBMNCs. Sample DNA from the peripheral blood and from the bone marrow of two patients with B-CLL was analyzed in the new set up at different time points before and after therapy. Statistically significant changes in IgVH copy numbers were documented in both patients. We conclude that this technology offers an additional opportunity to detect and quantify residual tumor cells in B-CLL over several log steps with a high sensitivity. The kinetics of residual tumor cell counts in B-CLL can be analyzed by this method.