Influence of residual platelet count on routine coagulation, factor VIII, and factor IX testing in postfreeze-thaw samples.

The use of frozen-thawed samples rather than fresh samples for specialized coagulation testing is becoming commonplace, thereby creating novel risks that may jeopardize the quality of hemostasis testing. Residual platelets (PLTs) in frozen plasma are most critical as freezing-induced activation and injury may impair routine and specialized testing after thawing. The aim of this study was to verify the impact of postcentrifugation PLT count in postfreeze-thawed samples on activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, factor VIII (FVIII) activity testing, and factor IX (FIX) activity testing. These parameters were herein assessed in postfreeze-thaw paired plasma samples collected from 15 healthy volunteers and subjected to 4 different centrifugation forces (i.e., 3,000, 1,500, 1,000, and 500g), using data obtained with centrifugation force of 1,500g as the gold standard, in agreement with current recommendations. Compared with reference samples, PLT counts in fresh aliquots were indistinguishable in specimens centrifuged at 1,000g, significantly lower in those centrifuged at 3,000g and significantly higher in those centrifuged at 500g. In all cases except samples centrifuged at 3,000g, the PLT count was significantly decreased in postfreeze-thaw compared with paired fresh specimens. In postfreeze-thaw plasma, APTT was not influenced by residual PLT count. The results of PT and fibrinogen were consistently altered in samples centrifuged at 1,000 and 500g, though the correlation with the reference measures remained clinically acceptable. Data obtained for FVIII and FIX activities revealed a positive bias in all postfreeze-thaw plasmas, achieving statistical significance in samples centrifuged at 3,000g. We conclude that alteration of centrifuge speeds away from the recommended 1,500g may influence the level of residual PLTs in sample centrifuged at lower speeds such as 500g, and therefore may make these specimens unsuitable for hemostasis testing in postfreeze-thawed plasma samples. In addition, although the changes seen in FVIII and FIX in samples centrifuged at 3,000g may reflect non-PLT-related effects, such changes should also be considered in this setting.

Sample collection and platelet function testing: influence of vacuum or aspiration principle on PFA-100 test results.

As for other tests of hemostasis, the investigation of platelet function is highly vulnerable to a broad series of preanalytical variables, which span from patient preparation to the final analysis of the specimen and issuance of test results. In particular, there remains much controversy about the influence of manual or vacuum aspiration of blood into primary collection tubes on platelet function testing. Accordingly, we investigated this for the PFA-100. In 12 healthy volunteers, a sample labeled as 'BD-V' was drawn into a 2.7 ml BD Vacutainer tube, whereas two additional samples were collected from the opposite arm into 5.0 ml Sarstedt S-Monovette tubes by vacuum (SD-V) or manual aspiration (SD-A). All sample were tested on PFA-100 with collagen and ADP (CADP) or collagen and epinephrine (CEPI). The values of both CEPI and CADP obtained in SD-A samples were significantly lower than those obtained in SD-V and BD-V tubes, whereas those of the two evacuated tubes did not significantly differ. On average, CEPI values were prolonged by 11% in SD-V and 13% in BD-V, whereas those of CADP were prolonged by 14% in SD-V and 10% in BD-V, respectively. These findings suggests that the lower shear stress generated by the manual aspiration of blood into the primary collection tube would prevent spurious hyper-activation of platelets, thus, preserving the integrity of their function for subsequent testing on PFA-100. This study underscores the need to define or validate local reference ranges for the PFA-100 based on the collection tube used. Different reference ranges of both CEPI and CADP may also be advisable when venous blood samples are collected with manual aspiration or vacuum principle.

Reliability of EGFR and KRAS mutation analysis on fine-needle aspiration washing in non-small cell lung cancer.

INTRODUCTION: Molecular profiling of advanced non-small cell lung cancer (NSCLC) has become essential for predicting customized medical treatment decision. In light of recent advances in non-invasive diagnostic procedures in NSCLC, we aimed to demonstrate the reliability of assessing molecular tests for epidermal growth factor receptor (EGFR) and KRAS genes on cytological samples by comparing the molecular profile obtained on cells from scraped smears with that on paired needle washing in a series of NSCLC cases. METHODS: Thirty-two cytological specimens obtained by fine-needle aspiration biopsy procedures from primary or metastatic lesions of NSCLCs were Giemsa stained for a rapid on-site evaluation and, in case of an adequate sampling, the cellular material obtained from needle washing was collected into a saline solution. Scraped smears and needle washings were tested for EGFR and KRAS by polymerase chain reaction followed by direct sequencing. RESULTS: The concordance between EGFR and KRAS mutational status in 29 paired scraped smears and needle washing was 100%, with 7 paired samples showing the same EGFR mutation (4 L858R mutation, 2 E746_A750 deletion and 1 A767_V769 duplication) and 8 paired samples showing the same KRAS mutations (4 G12D, 1 G12A, 1 G12V and 2 G12C). Three scraped smears, uninformative for poor DNA quality, resulted EGFR mutated on paired needle washings. CONCLUSIONS: Needle washing obtained in the course of NSCLC non-invasive fine needle diagnostic procedures allows reliable mutation testing and can be regarded as an additional important source of biological material for molecular profiling of advanced NSCLC.

Epidermal growth factor receptor and Kras gene expression: reliability of mutational analysis on cytological samples.

Epidermal growth factor receptor (EGFR) and Kras gene mutations are crucial for discriminating patients responsive to anti-EGFR drugs in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), respectively. The majority of NSCLCs come to clinical attention at an advanced stage when surgery is no longer recommended and a considerable number of them are diagnosed by cytology only. A large number of metastatic CRCs are also diagnosed by imaging and minimally invasive techniques such as fine-needle aspiration biopsy. Here, we report our experience in the mutation analysis of EGFR and Kras on cytological material obtained from superficial and deep lesions of NSCLC and CRC. Our series included 63 cytological specimens from primary or metastatic lesions of 42 NSCLCs and 21 CRCs. The cytological material was adequate for the mutation analysis in 39/42 (93%) NSCLCs and in 20/21(95%) CRCs. EGFR and Kras mutations were found in 9 (23%) and 9 (23%) NSCLC cases, respectively. Kras mutations were found in 9/20 (45%) CRC specimens. Histological samples from the primary tumors were available in 9/42 NSCLCs and in 17/21 CRCs. The agreement of EGFR and Kras mutational status in cytological vs. histological samples was 100% for NSCLC and 88% for CRC. Our results suggest that standard cytology provides adequate material for the assessment of EGFR and Kras mutational status in NSCLC and CRC patients and could be specifically indicated in patients not eligible for surgery but candidate to anti-EGFR therapy.

Influence of mechanical hemolysis of blood on two D-dimer immunoassays.

Although there is broad information about the influence of spurious hemolysis on several laboratory tests, less is known on the bias produced on D-dimer testing. Four different pools were obtained from primary blood tubes, and each of them was divided into four aliquots. The first nonhemolyzed was centrifuged, the plasma was separated and then tested for hemolysis index and D-dimer. The second (hemolyzed aliquot A), third (hemolyzed aliquot B) and fourth (hemolyzed aliquot C) aliquots were mechanically hemolyzed by aspirating whole blood one, two and three times through a fine needle. The plasma was then separated and tested for hemolysis index and D-dimer. D-dimer was quantified by HemosIL AcuStar D-dimer and HemosIL D-dimer HS for ACL TOP. Undetectable hemolysis was present in aliquot nonhemolyzed (<0.5 g/l), whereas the concentration of cell-free hemoglobin significantly increased from hemolyzed aliquot A (5.5-7.0 g/l hemoglobin) to hemolyzed aliquot B (11.5 and 15.0 g/l hemoglobin) and hemolyzed aliquot C (20-22 g/l hemoglobin). The plasma concentration of D-dimer decreased from aliquots nonhemolyzed to hemolyzed aliquot C, achieving clinical significance fromhemolyzed aliquot A and hemolyzed aliquot B when measured with D-dimer HS for ACL TOP and AcuStar D-dimer, respectively. The decrease with AcuStar D-dimer was -5 ± 3% in hemolyzed aliquot A, -7 ± 3% in hemolyzed aliquot B, and -9 ± 3% in hemolyzed aliquot C, whereas the decrease with D-dimer HS for ACL TOP was -5 ± 3% in hemolyzed aliquot A, -8 ± 3% in hemolyzed aliquot B and -9 ± 3% in hemolyzed aliquot C. The similar trend towards decreasing values observed when measuring D-dimer with chemiluminescent and turbidimetric immmunoassays on four heterogeneous plasma pools suggest that the hemolysis interference is more likely to be biological than analytical. The modest bias observed in samples with frank hemolysis (i.e. cell-free hemoglobin of 11.5 g/l) confirms that both methods are robust against this type of interference, so that test results might be released in the majority of mildly hemolyzed samples.