Cell analysis in cerebrospinal fluid (CSF) using Sysmex® hematology analyzers XT-4000i and XE-5000: evaluation with CSF controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine (DGKL).

In cerebrospinal fluid (CSF) analysis, hematology analyzers (HAs) Sysmex® XT-4000i and XE-5000, equipped with flow cytometry (FCM), were used to count cells and differentiate leukocytes into mononuclear and polymorphonuclear cells (MNCs, PMCs) applying body fluid mode. FCM was evaluated with 20 DGKL CSF controls containing viable human leukocytes and erythrocytes. HA values were compared with reference values by Passing/Bablok regression analysis to reveal conformity. Conformity of white blood cells (WBCs) was obtained with native leukocytes, counted in calibrated Fuchs-Rosenthal chamber as reference; red blood cell counts proved inaccurate. CV <40% with WBC counts <20 per µL impairs accuracy. Reference WBC differentiation was assayed using FACS Canto II™ and FC-500 SN with anti-CD45, anti-CD14, anti-CD16, anti-CD16/56 [Becton Dickinson (BD); Beckman Coulter (BC)]. BD FACS lysing solution®-no-wash-procedure was applied. BC pretreatment with Versalyse lysing solution was not recommended. MNCs (lymphocytes + monocytes) were significantly lower (∼14%) on both HAs; PMCs (granulocytes or sum of neutrophils + eosinophils + basophils: range 1-86 M/L) were significantly higher (∼2.2-fold). WBC HA differentiation is not reliable because MNC/PMC differentiation yielded lower and higher values than FACS-FCM references, respectively. This is attributed to incorrect discrimination of leukocytes with rounded/nonrounded nuclei; adding leukocytes with nonrounded nuclei to too low HA MNCs (about 40% not-activated) yielded P/B conformity; subtraction of leukocytes with nonrounded nuclei from elevated HA PMCs showed conformity (about 85% activated). Nucleus/activation state of leukocytes was assessed using microhistology. Sysmex XT-4000i and XE-5000 HAs systems are inappropriate for complete CSF cell analysis.

Evaluation of cell counting and leukocyte differentiation in cerebrospinal fluid controls using hematology analyzers by the German Society for Clinical Chemistry and Laboratory Medicine.

BACKGROUND: Manual cell counting in cerebrospinal fluid (CSF) is technique-dependent, time-consuming, and thus costly and prone to inter-operator variability and low precision. Flow cytometry (FCM) with fast hematology analyzers (HAs) appears to improve accuracy and precision of CSF cell analysis; rapid CSF cell analysis is especially needed in emergency laboratories. Ten external trials of the German Society for Clinical Chemistry and Laboratory Medicine evaluated FCM with Coulter (LH750, 755), Abbott CD3200, CD3500, CD3700, CD4000, Sapphire, ADVIA120 CSF assay, and Sysmex XE-2100 single platform analyzers. METHODS: CSF controls were produced using native blood leukocytes and erythrocytes, resembling CSF and thus rendering the trials feasible and allowing comparison with native manual counting in a Fuchs-Rosenthal chamber and FACScan-CD45-CD14 dual platform analysis, which was used as the reference method. Statistical evaluation was performed using Passing/Bablok regression analysis. RESULTS: Our evaluation revealed significant differences with respect to target values in leukocyte and erythrocyte counts, as well as leukocyte differentiation. These differences were attributed to inaccuracies produced by the HAs, due to blank correction in connection with impedance analysis, leukocyte loss, especially through monocyte injury due to the erythrocyte lysing agent, incomplete erythrocyte lysis, ADVIA cell sphering, cell differentiation using algorithms and peroxidase activity. Erythrocyte counting in the CSF controls was inaccurate with the Coulter and ADVIA analyzers. CONCLUSIONS: Evaluation of HAs by means of the CSF controls revealed inaccuracies in cell counting and leukocyte differentiation. Analyzer techniques, used for CSF cell assays, therefore need to be improved.

Modifications of haematology analyzers to improve cell counting and leukocyte differentiating in cerebrospinal fluid controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine.

Flow cytometry (FCM) is used with haematology analyzers (HAs) to count cells and differentiate leukocytes in cerebrospinal fluid (CSF). To evaluate the FCM techniques of HAs, 10 external DGKL trials with CSF controls were carried out in 2004 to 2008. Eight single platform HAs with and without CSF equipment were evaluated with living blood leukocytes and erythrocytes in CSF like DGKL controls: Coulter (LH750,755), Abbott CD3200, CD3500, CD3700, CD4000, Sapphire, ADVIA 120(R) CSF assay, and Sysmex XE-2100(R). Results were compared with visual counting of native cells in Fuchs-Rosenthal chamber, unstained, and absolute values of leukocyte differentiation, assayed by dual platform analysis with immune-FCM (FACSCalibur, CD45, CD14) and the chamber counts. Reference values X were compared with HA values Y by statistical evaluation with Passing/Bablock (P/B) linear regression analysis to reveal conformity of both methods. The HAs, studied, produced no valid results with DGKL CSF controls, because P/B regression revealed no conformity with the reference values due to:-blank problems with impedance analysis,-leukocyte loss with preanalytical erythrocyte lysis procedures, especially of monocytes,-inaccurate results with ADVIA cell sphering and cell differentiation with algorithms and enzyme activities (e.g., peroxidase). HA techniques have to be improved, e.g., using no erythrocyte lysis and CSF adequate techniques, to examine CSF samples precise and accurate.

Deferasirox in MDS patients with transfusion-caused iron overload--a phase-II study.

Blood transfusions represent a main component of supportive care in myelodysplastic syndromes (MDS). To avoid organ damage caused by transfusion-dependent iron overload, an adequate iron chelation therapy is required. Recently, a new oral iron chelator deferasirox (ICL670, Exjade) has become available. A study was conducted to demonstrate the efficacy and tolerability of deferasirox in transfusion-dependent iron-overloaded patients with MDS. The efficacy of deferasirox was monitored by changes in serum ferritin, bone marrow iron, and liver iron concentration (LIC), as determined by T2*-weighted magnetic resonance imaging. Twelve patients with MDS of different subtypes (median age 76 years, range 53-91) were enrolled. Deferasirox administered in a once-daily dose of 20-30 mg/kg for 12 months was effective in reducing median ferritin concentration from 1,515 microg/L (range 665-6,900) to 413 microg/L (range 105-3,052). Within the first 4 weeks of treatment before the continuous decline of ferritin levels, the values markedly rose in eight of 12 patients. The median LIC declined from 315 to 230 micromol/g (p=0.02) at the end of study, accompanied by a reduction of bone marrow siderosis. The most common adverse events were mild and transient gastrointestinal disturbances, skin rash, nonprogressive transient increases in serum creatinine and urine beta2-microglobulin, and a temporary reduction of the creatinine clearance. The renal parameters normalized after end of treatment. No hematologic toxicities were observed. Deferasirox proved to be effective in transfusion-dependent iron overload in MDS by mobilizing iron deposits in liver and at least stabilizing iron stores in bone marrow.

The soluble transferrin receptor (TfR)-F-Index is not applicable as a test for iron status in patients with chronic lymphocytic leukemia.

BACKGROUND: The soluble transferrin receptor (sTfR) is established as a test for iron deficiency (ID). In chronic lymphocytic leukemia (CLL), sTfR is not reliable for screening for ID as the latter is strongly dependent on tumor burden. METHODS: We investigated whether the influence of the tumor load can be excluded or minimized using the sTfR/log ferritin ratio (TfR-F-Index) and the C-reactive protein (CRP)-adjusted TfR-F-Index in 87 patients with CLL. sTfR was measured nephelometrically (normal: 0.81-1.75 mg/L). A cut-off value of 1.5 for the TfR-F-Index and 0.8 for the CRP-adjusted TfR-F-Index, in patients with a CRP >5 mg/L, was used. RESULTS: All Binet A patients had normal sTfR values (1.34+/-0.2 mg/L), TfR-F-Index (0.67+/-0.2) and a CRP-adjusted TfR-F-Index. In Binet B and C, sTfR and the TfR-F-Index were significantly increased compared to Binet A patients (p<0.0001). The differences between Binet B and C were not significant. sTfR was increased in 85%, TfR-F-Index in 46% and the CRP-adjusted TfR-F-Index in 54% of the Binet B patients, in Binet C patients, 80%, 50% and 60% showed increases, respectively. sTfR and the TfR-F-Index decreased or even normalized following successful treatment. CONCLUSIONS: Similar to sTfR, the TfR-F-Index is strongly associated with tumor burden in patients with CLL. Thus, these parameters do not allow for a reliable diagnosis of ID in this patient group.

Monoclonal anti-idiotype antibody 6G6.C4 fused to GM-CSF is capable of breaking tolerance to carcinoembryonic antigen (CEA) in CEA-transgenic mice.

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF). Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P=0.005) and to CEA (P=0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.

Urokinase-type plasminogen activator induces proliferation in breast cancer cells.

Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPA-interacting molecules on proliferation of uPA-suppressed MDA-MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal anti-uPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phospholipase C (PI-PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway.

Evaluation of the automated coagulation analyzer Sysmex CA-7000.

INTRODUCTION: Centralization of laboratory diagnostics and an increasing number of urgent requests and nonstandard samples raise the need for short turn-around times and high-throughput analyzers for coagulation tests. The aim of the present study was to evaluate the analytical and technical performance of the Sysmex CA-7000 coagulation analyzer under routine laboratory conditions. MATERIALS AND METHODS: We evaluated the Sysmex CA-7000 in comparison to the Sysmex CA-6000 analyzer for PT, INR, aPTT, Clauss, and derived fibrinogen. We also compared antithrombin (AT) measured on the Sysmex CA-7000 and Dimension RxL. Imprecision studies were performed, and throughput, online, and STAT functions and the handling of routine samples were evaluated. RESULTS: The Sysmex CA-7000 showed very low intra-assay and interassay variability for all parameters. The method comparison study showed good comparability to the Sysmex CA-6000 and Dimension RxL. The throughput of the Sysmex CA-7000 was 2.5--3 times faster than that of Sysmex CA-6000. No interference was seen for total bilirubin up to 710 micromol/L. Triglyceride levels >11.4 mmol/L resulted in invalid PT measurements and levels >3.42 mmol/L gave invalid results for Clauss fibrinogen on the CA-7000 analyzer. CONCLUSIONS: This is the first published evaluation of the analytical and technical performance of the coagulation analyzer Sysmex CA-7000. We conclude that the CA-7000 is well suited for coagulation laboratories with high sample throughput and a high number of nonstandard samples.

Porcine spermatozoa contain more than one membrane progesterone receptor.

Progesterone has been shown to be a physiologically relevant inducer of the sperm acrosome reaction. A novel protein intrinsic to microsomal membranes, membrane progesterone receptor (mPR, now termed progesterone membrane receptor component 1, PGMRC1) that binds progesterone with high affinity has been cloned from porcine liver previously, and corresponding antibodies mitigate the progesterone induced acrosome reaction. In this study we aimed at the localization of mPR in porcine spermatozoa. Immunostaining suggested the exclusive occurrence of mPR in a hardly accessible place, possibly the inner acrosomal membrane, with digitonin dramatically increasing the number of positively stained cells. Consistent with the structure prediction for mPR, its short N-terminus (NT) but not the large C-terminal part becomes accessible from outside after digitonin treatment as evidenced by the staining pattern of antibodies directed against different regions of the protein. However, digitonin treatment solubilizes a progesterone binding activity of approximately 140 kDa molecular weight, that is different from mPR, which remains in the cell membrane as demonstrated by Western blotting. Ligand binding studies confirm the dissimilarity of mPR and the digitonin-soluble progesterone binding protein. Chemical modification studies also indicate that the digitonin-soluble progesterone binding protein has a binding site that differs from that of mPR. It is concluded that more than one progesterone receptor is present in porcine spermatozoa.

Tumor stroma is the predominant uPA-, uPAR-, PAI-1-expressing tissue in human breast cancer: prognostic impact.

UNLABELLED: Urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor PAI-1, play a key role in tumor invasion and metastasis. uPA and PAI-1 were the first novel tumor biological factors to be validated at the highest level of evidence regarding their clinical utility in breast cancer. Their antigens are determined in tumor tissue extracts by standardized, quality-assured immunometric assays (ELISA). Since the late 1980s, numerous independent studies have demonstrated that patients with low levels of uPA- and PAI-1 in their primary tumor tissue have significantly better survival than patients with high levels of either factor. However, it is unclear whether it is their (relative) levels in the tumor stroma or in the tumor cells themselves that is most relevant to patient outcome. This missing knowledge leads to an uncertainty concerning the management of breast cancer tissue specimens. It is unclear how much tumor stroma is allowed in one tumor tissue specimen for an adequate assessment of the patients' outcome. This is the first study in which tumor cells and stromal tissue of invasive breast carcinomas (n=60) were separated by laser capture microdissection followed by ELISA-based determination of the uPA-, uPAR- and PAI-1-levels. In addition, we have assessed uPA-, uPAR- and PAI-1 distribution in formalin-fixed, paraffin-embedded breast cancer specimens (n=60) by immunohistochemistry. The uPA-, uPAR- and PAI-1 in tumor stroma only, tumor cells only and not separated tumor tissue did not show any significant differences in protein-levels determined by ELISA. Cox regression analysis showed that patients with high uPA-, high uPAR-, and/or high PAI-1-levels, as compared to patients with low levels of either factor, showed a significantly shorter relapse-free survival and overall survival (p=0.000001). These results suggest that a strong expression of uPA, uPAR and PAI-1 in the tumor stroma, as well as in tumor cells, have the same impact on the clinical behaviour of breast cancer. CONCLUSION: When using uPA- and PAI-1 levels as prognostic and predictive factors in breast cancer the quantity of tumor stroma in the tumor tissue specimen is not relevant for the assessment of the patients' outcome.

The soluble transferrin receptor in dysplastic erythropoiesis in myelodysplastic syndrome.

OBJECTIVES: In individuals without iron deficiency, the soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity. This study investigated sTfR concentrations in ineffective, dysplastic erythropoiesis in myelodysplastic syndrome (MDS). METHODS: To exclude influences of other myeloid cells on sTfR, only patients with refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and 5q(-) syndrome were included. sTfR was measured nephelometrically (normal range 0.81-1.75 mg/L). RESULTS: Thirty-four untreated MDS patients (RA = 14, RARS = 10, 5q(-) syndrome = 10) were enrolled and analysed. The mean sTfR value of all MDS patients (1.30 +/- 0.8 mg/L, range 0.2-3.8) did not differ from our control group. In 5q(-) syndrome, the mean sTfR concentration (0.80 +/- 0.5 mg/L) was significantly lower than in RA (1.32 +/- 0.4 mg/L, P = 0.02) and RARS (1.75 +/- 1.1 mg/L, P = 0.03). Subdividing MDS according to their amount of erythroid mass in bone marrow a significant difference of sTfR between patients with decreased (0.70 +/- 0.4 mg/L), normal (1.32 +/- 0.4 mg/L) and increased (2.06 +/- 0.9 mg/L) erythropoiesis was observed. MDS patients with sTfR values below the reference range of 0.81 mg/L required transfusions in 90% of cases and showed higher erythropoietin levels compared to MDS patients with sTfR levels > or =0.81 mg/L (P = 0.01). There was a good agreement between sTfR and the amount of polychromatic erythroblasts observed (r = 0.68, P < 0.001). CONCLUSION: In conclusion, the serum concentration of sTfR reflects erythropoietic activity in MDS, but it is in particular determined by the degree of erythroid maturation and the severity of ineffective erythropoiesis. Low sTfR values in MDS are associated with a reduced, poorly differentiated erythropoiesis and requirement of blood transfusions.

The soluble transferrin receptor reflects tumor load in chronic lymphocytic leukemia.

BACKGROUND: The soluble transferrin receptor (sTfR) is a parameter of erythropoietic activity and iron deficiency. Increased levels have also been described in hematological malignancies, especially in chronic lymphocytic leukemia (CLL). METHODS: We investigated the value of sTfR in the assessment of tumor mass in 61 previously untreated CLL patients. Both hemolysis and iron deficiency were excluded. sTfR was measured nephelometrically (normal 0.81-1.75 mg/L). RESULTS: All Binet A patients had normal sTfR values (1.36+/-0.22 mg/L). In Binet B patients, the sTfR was increased (3.08+/-1.70 mg/L, p<0.0001) compared to Binet A patients. Binet B patients with normal sTfR had a small tumor load and no abdominal involvement. A further increase of sTfR in Binet C (3.75+/- 2.32 mg/L) was not significant compared to Binet B patients. sTfR values decreased or even normalized after successful treatment, whereas relapse or disease progression was associated with another increase of sTfR. CONCLUSIONS: The sTfR concentration directly reflects the tumor burden in CLL. Therefore, sTfR may be of clinical value in monitoring disease activity, response to treatment and disease progression.

Zinc protoporphyrin, a useful parameter to address hyperferritinemia.

Zinc protoporphyrin (ZPP) is produced instead of heme as soon as iron support to erythropoiesis becomes insufficient. In iron deficiency the intra-erythrocytic ZPP concentration is increased. The aim of this study was to investigate whether ZPP is influenced by increased iron levels in hereditary hemochromatosis (HE) and is useful in the clarification of hyperferritinemia. Twenty HE patients and 160 patients with hyperferritinemic caused by anemia of chronic disorders, liver diseases, transfusional iron overload and hematologic or solid malignancies were enrolled. ZPP was measured using the Aviv front-face hematofluorometer (normal

Excessive erythrocytosis does not elevate capillary oxygen delivery in subcutaneous mouse tissue.

OBJECTIVE: Acclimatization to reduced environmental oxygen includes erythropoietin-regulated increase in erythrocytes enhancing the blood's oxygen content. However, increased hematocrit levels result in elevated blood viscosity that might impair microcirculation and tissue oxygenation. To assess this oxygen supply to the skin, the authors used erythropoietin overexpressing transgenic mice (tg6) that develop excessive erythrocytosis in an oxygen-independent manner. These animals have been previously reported to elevate their blood viscosity 4-fold. METHODS: The partial oxygen pressure (pO2) distribution was evaluated in microvessels as well as in subcutaneous interstitial tissue within a dorsal skinfold chamber of resting conscious mice using automated phosphorescence quenching. RESULTS: Compared to wildtype (wt) animals, transgenic blood viscosity increased 4-fold but microvessel diameter was not altered. Despite sharing similar blood pO2 as the wt siblings, tg6 animals nearly doubled their oxygen content. Moreover, tg6 erythrocytes reduced hemoglobin's oxygen affinity by decreased 2,3-DPG levels and an increased Hill number. Transgenic arterioles and venules showed increased pO2 compared to wt controls whereas capillary and tissue pO2 were not altered. CONCLUSIONS: Excessive erythrocytosis does not elevate capillary oxygen delivery.

Improvement of technical and analytical performance in DNA sequencing by external quality assessment-based molecular training.

BACKGROUND: From 2003 to 2005, the European Union supported the EQUAL-initiative to develop methodological external quality assessment (EQA) schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). As a relevant part of the EQUALseq program, a training course was held subsequent to the first EQA Program (EQAP1). The success of this course was reassessed in a 2nd EQUALseq round (EQAP2). METHODS: In September 2005, a 3-day training course took place. We invited 8 laboratories with below-average performance in EQAP1 to improve their methodological and analytical/proficiency skills by lectures and practical work. To compare the results of the pretraining and posttraining EQUALseq rounds, we distributed 2 samples used in the first EQUAL round, but this time we provided different oligonucleotide sets. We evaluated the results by means of a previously described scoring system. RESULTS: In EQAP2, 6 laboratories returned complete data sets, corresponding to an overall 14% of the 43 laboratories that had finished EQAP1. The scoring results for samples A (P=0.0025) and B (P=0.0125) demonstrated a significant improvement in EQAP2. Overall, a substantial improvement of technical and interpretative skills was demonstrated (P=0.0051). In general, the workshop experience was highly rated by the participants. CONCLUSIONS: Methodologic EQAPs in DNA sequencing are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, emphasizing the need for mandatory EQAPs. Training courses, together with 2nd-round reiterations, should be implemented into methodological EQAPs in molecular diagnostics to improve technical performance and proficiency in genetic testing.

Methodologic European external quality assurance for DNA sequencing: the EQUALseq program.

BACKGROUND: DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program. METHODS: The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation. RESULTS: Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P <0.05). Interestingly, more than 30% of participants neither added comments to their data nor made efforts to identify the gene sequences or mutational positions. CONCLUSIONS: Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.

Soluble transferrin receptor and zinc protoporphyrin--competitors or efficient partners?

OBJECTIVES: Soluble transferrin receptor (sTfR) and zinc protoporphyrin (ZPP) are both parameters of iron deficient erythropoiesis (IDE), the sTfR measurement is commonly regarded to be the more sensitive test. sTfR also reflects erythropoietic activity, it is increased in enhanced erythropoiesis. METHODS: We investigated the diagnostic accuracy of sTfR in assessment of iron deficiency (ID) and compared it with ZPP. The study was performed on 174 subjects, in which ID has been precisely staged. RESULTS: Individuals without ID and patients with storage iron depletion only, had normal sTfR values. Patients classified as IDE and patients with iron deficiency anemia had significantly increased sTfR. There was a good correlation between sTfR and hemoglobin (r = -0.86; P < 0.0001) and between sTfR and ZPP (r = 0.86; P < 0.0001). When diagnosing ID, ZPP was the more sensitive test. In mildly developed IDE associated with ZPP-ratios between 40 and 70 micromol/mol heme, the sTfR concentration was elevated in only 25% of the cases. Reliably elevated sTfR values were observed only in more advanced IDE, associated with ZPP > 70 mumol/mol heme. CONCLUSIONS: ZPP is not inferior to sTfR when diagnosing IDE. Given the good correlation between sTfR and ZPP and because ZPP is uninfluenced by the erythropoietic activity, sTfR and ZPP are not competitors, rather efficient partners in diagnosing anemias. By measuring ZPP and sTfR simultaneously, the diagnostic uncertainty inherent in each of them individually can be eliminated. In particular, the simultaneous determination of ZPP and sTfR enhances the diagnostic power of sTfR in assessment of the erythropoietic activity.

Sickle cell disease--pathophysiology, clinical and diagnostic implications.

We review the current knowledge of the pathophysiology of sickle cell disease (SCD), the clinical complications and the state of the art in SCD diagnostics. Today, a flexible laboratory concept allows the fast and economic clarification of the patients' sickle cell hemoglobin (HbS) status, e.g. additional compound heterozygosities. In contrast to a well-investigated pathophysiology of the disease, factors influencing the severity of symptoms as well as some laboratory findings in SCD still lack a final explanation. In this review, we focus on red cell lysis resistance as an additional diagnostic tool in SCD. There is a need for further studies regarding lysis resistance in blood samples from patients with HbS.

Limitations of the immunocytochemical detection of isolated tumor cells in frozen samples of bone marrow obtained from melanoma patients.

We report on a case of a 70-year-old woman with an ocular melanoma, which was diagnosed and treated 14 years ago. The patient was referred to the hospital with a suspected lymphoma. Cytological examination of bone marrow proved a marked infiltration with melanoma cells. Because detection of isolated tumor cells in the bone marrow of patients with various types of tumors was shown to be of prognostic significance and since current tumor-staging techniques are unable to detect single disseminated tumor cells or small aggregates of tumor cells, which might be the seed for subsequent metastatic relapse, we therefore evaluated the feasibility of immunocytochemical screening of bone marrow aspirates of 36 melanoma patients in different clinical stages using three monoclonal antibodies against melanoma-associated antigens in comparison with 43 non-melanoma control patients. Two of these antibodies (HMB45 and NKI-beteb) are directed against the melanoma antigen gp100/pmel17, whereas the third one (TA99) recognizes gp75/Tyrosinase-related protein 1 (TRP-1). None of the patients demonstrated a macroscopic bone marrow infiltration as was present in our patient with metastatic ocular melanoma. Seven (20.6%) of the 34 eligible melanoma patients presented with cells in the bone marrow positive for one or more of the above-mentioned melanosomal markers. Four of the positive patients were clinically free of tumors by the time of puncture, whereas the remaining 3 patients showed overt metastases in the subcutaneous fat (2 patients) and the brain (1 patient). On the other hand, 20 (66%) of the 29 patients with negative bone marrow findings also presented with clinical advanced disease with overt metastasis in the skin, lymph node, spleen, liver, lung, bone and brain. In conclusion, immunocytochemical screening of bone marrow samples is a feasible procedure that allows the detection of micrometastatic tumor cells in a subset of melanoma patients. Massive invasion of bone marrow with melanoma cells is a rare event even in far-advanced metastatic stages and no clear correlation between tumor load and bone marrow infiltration could be established.