Vincenzo Rocco

Background: Severe high molecular weight kininogen (HK) deficiency is an autosomal recessive defect of the contact system caused by mutations in KNG1. Limited scientific interest in HK deficiency due to the rarity of the seemingly asymptomatic condition may increase, as HK, the precursor of bradykinin, is now discussed as a therapeutic target e.g. in hereditary angioedema. Aims: We provide a comprehensive analysis of the diagnostic, clinical, and genetic features of HK deficiency and estimate its frequency. Methods: We identified a new case of HK deficiency, systematically review the literature, conduct new genetic studies of reported cases, and comprehensively analyze the clinical course and diagnostic criteria. Clotting activity of HK and prekallikrein (PK) (HK:C/PK:C) and antigen (HK:Ag/PK:Ag) were determined and genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. Characteristics deduced from the known HK deficiency-causing variants were used to estimate the frequency of HK deficiency from the KNG1 variants aggregated in GnomAD. Results: 677 studies were identified by systematic review of the literature for HK deficiency. 27 of these contained individual cases of HK deficiency including 6 studies not listed in PubMed. Little-noticed cases from the gray literature account for more than one-third (16/39) of the extracted, unrelated cases. We genotyped one newly diagnosed HK-deficient case and 2 cases described in the literature and additionally evaluated all 10 studies reporting genetic data in HK-deficiency (including one case previously misdiagnosed as having PK deficiency). A total of 10 KNG1 variants causing HK deficiency (one new) were found, the most frequent being c.586C>T, p.Arg196* (4 unrelated families). Interestingly, all HK deficiency-causing variants are truncating, whereas two amino acid substitutions with presumed functional consequence, have been described as the cause of hereditary angioedema. Conservative prevalence estimates based on all known and putative HK deficiency-causing variants extracted from GnomAD (truncating variants in KNG1, including indels, nonsense and canonical splice site mutations located in that part of the gene, where relevant mutations have been described) revealed a frequency of 1 case of HK deficiency among 7,925,172 with slight differences in the analyzed ethnicities (see table). In addition, although not to the same extent as seen in PK deficiency, HK deficiency apparently is more prevalent in Africans. While it is already well known that HK deficiency causes decreased PK levels, our data indicate that factor XI levels are also frequently decreased, albeit to a lesser extent. The number of cases detected so far is too low for a more detailed analysis regarding bleeding, thrombotic, and cardiovascular events or immunological abnormalities. Conclusion: HK-deficiency is probably more frequent than previously thought. Suspected cases of contact phase defects should at least be analyzed for HK activity (besides factor XII, XI and PK activity) to facilitate conclusive evaluation of the clinical significance in the future.

Introduction: 3D‐DIFF scattergram of the Mindray BC‐6800 haematological analyser shows morphological abnormalities and lymphocyte cluster splitting related to the presence of reactive lymphocytes.This study aims to assess whether these cytographic changes are useful in detecting both activated and apoptotic lymphocytes, leading to an improvement in the laboratory diagnostic process of infectious mononucleosis.Methods: Two hundred three samples with modified shape and doubled lymphocyte cluster of DIFF scattergram (study group) were divided into two different subgroups: with and, respectively, without serological evidence of ongoing IM. Activated and apoptotic cells in peripheral blood were counted by light microscopy or gating in the instrumental dot plots. Values of apoptotic cells counted by microscopy were compared with those resulting from gating.Results: Samples with both shape change and doubled lymphocyte cluster had serological profiles according to the diagnosis of ongoing infectious mononucleosis. Blood smears review was positive for reactive lymphocytes in all 112 samples (100%). An underestimation of apoptotic cell count by light microscopy compared with the gating in the instrumental scatterplot was also observed (96 out of 112, 85.7%).Conclusion: The additional lymphocyte cluster was significantly associated with activated and apoptotic lymphocytes in samples with serology suggesting ongoing infectious mononucleosis. Considering the significance of clue for infectious mononucleosis assigned to the apoptotic lymphocytes, a specific flag such as "apoptotic cells?" could be associate with the related cluster. Such a flag could be used for dedicated rules for smears review, thus increasing infectious mononucleosis detection in laboratories that do not usually practise instrumental cytograms observation.

Introduction: Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a life‐threatening coagulopathy. Once it is suspected, ATRA should be started. Appreciation of APL details is critical, but an experienced hematopathologist may not be available. We developed an algorithm, based on the parameters generated by automated blood cell counter ADVIA 2120i Siemens that can aid the diagnosis of APL.Methods: All parameters in the algorithm were selected on the bases of the pathophysiology of the APL and the analyzer's technology. We used c1 cutoff: PLT < 150 × 10³; % Mono 4. Satisfying at least five of six cutoffs, we obtained an “APL criteria”. It was tested on 247.209 raw‐data from routine and emergency samples and on 124 raw‐data from APL. We performed a multiparametric analyses and obtained definitive cutoffs (c2). It was validated on a new group of 51.002 raw‐data from routine and emergency. Finally, it was tested on AML and ALL, MDS, MPN, oncologic samples, and hemolytic and megaloblastic anemia.Results: The algorithm provided a high sensitivity (86.30%) and high specificity (99.83%) in APL with high or normal number of WBC and satisfactory results in APL with cytopenia (80%). The specificity was very high also in groups of hematological and nonhematological diseases. It can be used as an “APL criteria” to alert pathologists of the possible presence of APL. It together with instrumental and classical morphology may allow to reduce the time of diagnosis with further reduction in early death.

The infectious mononucleosis (IM) includes clue elements, apoptotic and atypical lymphocytes. In IM, the evaluation of dot plot provided by Sysmex XN-1000 analyzer revealed a stretched lymphocytic cluster, white cell differential channel (WDF), on cytogram. In this study, we analyzed 698 samples that include 39 IM, 76 chronic lymphoproliferative disorders, 25 nonclonal lymphocytosis, and 40 healthy donors. Five hundred eighteen samples with other diseases or interference were evaluated. The algorithm was validated on 40,000 files that were received from internal database of Sysmex-Dasit. The analysis of flow cytometry standard (FCS) files in WDF channel and presumed apoptotic lymphocytes counts on side scatter/forward scatter (SSC/FSC) and SSC/SFL (where SFL is side fluorescence) dot plot revealed excellent correlation among apoptotic cells on peripheral blood smear (R(2) = 0.79 and 0.75). There was a variation of positional parameters in lymphocyte clusters WX, WY, and WZ. If WX-SSC > 500 and WY-SFL > 1,000 and WZ-FSC > 700, specificity equals to 99% and sensitivity equals to 100%. If nucleated red blood cell (NRBC) <0.03 × 10(3) /μl, specificity equals to 100%. In received files, positives were 1% adding the simultaneous presence of a percentage of events in the two gates relating to the apoptotic lymphocytes of 1.2% of WBC. On Sysmex XN-1000, dot-plot observation allowed immediate detection of IM. Meanwhile, an algorithm based on the parameters on these plots can be calculated with excellent performance.

I pitfalls sono trappole in grado di alterare i dati numerici citometrici e che bisogna conoscere bene al fine di evitarle. Il riconoscimento dei pitfalls è essenziale per il miglioramento dell’accuratezza e tutto il personale operante in Laboratorio che esegue e referta i test in urgenza deve essere in grado di riconoscerli. Il citofluorimetro Dasit “SYSMEX UF-1000i”, tramite il dimensionamento (FSC), la complessità cellulare (SSC) e la fluorescenza alta e bassa (FLH e FLL), è in grado di fornire citogrammi e distinguere le varie popolazioni cellulari urinarie, con notevole vantaggio nell’individuazione delle micro-ematurie.

Imerslund-Gräsbeck syndrome is a rare autosomal recessive disorder, characterized by vitamin B12 deficiency due to selective malabsorption of the vitamin and usually results in megaloblastic anemia appearing in childhood. It is responsive to parenteral vitamin B12 therapy.

Nel corso di uno screening preoperatorio viene individuato un paziente con notevole allungamento del tempo di tromboplastina parziale attivato (aPTT), normale tempo di protrombina (PT) e anamnesi negativa per precedenti episodi di sanguinamento. Vengono eseguiti l’aPTT sulla miscela 1:1 (plasma paziente:pool) e successivamente il dosaggio dei fattori di pertinenza della via intrinseca. La normalizzazione dell’aPTT nella miscela 1:1 (plasma paziente:pool) orienta per deficit di uno o più fattori della “via intrinseca”, tuttavia la valutazione dei fattori XII, XI, IX e VIII non evidenzia carenze. Viene pertanto ipotizzato un deficit di uno degli altri fattori della fase di contatto (pre-callicreina, chininogeno ad alto peso molecolare). La mancata normalizzazione dell’aPTT nella miscela 1:100 (pool:plasma paziente) consente di escludere la carenza di pre-callicreina (PK), rendendo più consistente l’ipotesi di un deficit di chininogeno ad alto peso molecolare (HMWK). In assenza del saggio immunologico, viene eseguito l’aPTT sulla miscela costituita dal plasma del paziente e dal 12,5% di plasma normale. La correzione dell’aPTT consente di confermare il sospetto di deficit di HMWK. Il paziente viene pertanto regolarmente sottoposto a intervento chirurgico senza complicanze emorragiche. Anche in assenza di test specialistici e costosi è possibile giungere a diagnosi di patologie piuttosto rare, utilizzando metodologie diagnostiche economiche e disponibili in tutti i Laboratori, quali l’attenta analisi dell’anamnesi in combinazione con la valutazione dei risultati dell’aPTT e del test di miscela. A patient with significant prolongation of activated partial thromboplastin time (aPTT), normal prothrombin time (PT) and with negative clinical history for previous bleeding was identified during a preoperative screening. An aPTT was carried out on the mixture 1:1 (plasma patient:pool) and, at a later stage, a dosage of the factors relevant to the intrinsic way was also obtained. The normalization of aPTT in mixture 1:1 (plasma patient:pool), points out to a deficit of one or more “intrinsic way” factors; nevertheless, the evaluation of factors XII, XI, IX and VIII, does not highlight any shortages; therefore, it is possible that there might be a deficiency in one of the other factors in the contact phase (pre-kallikrein, high molecular weight kininogen). Missing normalization in aPTT mixture 1:100 (pool:plasma patient), allows us to exclude a pre-kallikrein shortage (PK), so as to consolidate the hypothesis of a high molecular weight kininogen shortage (HMWK). If there is no immunological sample, the aPTT is carried out on the mixture made of patient plasma and 12.5% normal plasma. The correction in the aPTT allows us to confirm our suspicion of an HMWK deficit, therefore, the patient can undergo surgery without incurring in complications due to possible hemorrhages. Even without carrying out expensive and specialized tests, it is possible to diagnose rare diseases using inexpensive diagnostic methods which are available in all laboratories, such as a careful anamnesis analysis together with an evaluation of aPTT results and mixture tests.