Is flow cytometry better in counting malaria pigment-containing leukocytes compared to microscopy?

<p>Abstract</p> <p>Background</p> <p>Detection of malaria pigment (or haemozoin; Hz)-containing leukocytes may have prognostic relevance in malaria; however, studies reported conflicting results, with microscopic counts suggestive of being inaccurate and imprecise.</p> <p>Methods</p> <p>Numbers of Hz-containing leukocytes from a malaria patient obtained with a flow cytometer counting 50.000 gated events were compared with thin film microscopy as applied under field conditions.</p> <p>Results</p> <p>Flow cytometry identified 5.8% Hz-containing monocytes and 1.8% Hz-containing neutrophils. The microscopic examination yielded 10% and 13% of Hz-containing monocytes, as well as 0% and 0.5% of Hz-containing neutrophils for observers one and two, respectively.</p> <p>Conclusion</p> <p>Novel, robust and affordable cytometric methods should be evaluated in the field as they may assist in utilizing Hz-containing cells as clinically useful parameter.</p

Full blood count and haemozoin-containing leukocytes in children with malaria: diagnostic value and association with disease severity

<p>Abstract</p> <p>Background</p> <p>Diligent and correct laboratory diagnosis and up-front identification of risk factors for progression to severe disease are the basis for optimal management of malaria.</p> <p>Methods</p> <p>Febrile children presenting to the Medical Research Unit at the Albert Schweitzer Hospital (HAS) in Lambaréné, Gabon, were assessed for malaria. Giemsa-stained thick films for qualitative and quantitative diagnosis and enumeration of malaria pigment, or haemozoin (Hz)-containing leukocytes (PCL) were performed, and full blood counts (FBC) were generated with a Cell Dyn 3000^®instrument.</p> <p>Results</p> <p>Compared to standard light microscopy of Giemsa-stained thick films, diagnosis by platelet count only, by malaria pigment-containing monocytes (PCM) only, or by pigment-containing granulocytes (PCN) only yielded sensitivities/specificities of 92%/93%; 96%/96%; and 85%/96%, respectively. The platelet count was significantly lower in children with malaria compared to those without (p < 0.001), and values showed little overlap between groups. Compared to microscopy, scatter flow cytometry as applied in the Cell-Dyn 3000^®instrument detected significantly more patients with PCL (p < 0.01). Both PCM and PCN numbers were higher in severe versus non-severe malaria yet reached statistical significance only for PCN (p < 0.0001; PCM: p = 0.14). Of note was the presence of another, so far ill-defined pigment-containing group of phagocytic cells, identified by laser-flow cytometry as lymphocyte-like gated events, and predominantly found in children with malaria-associated anaemia.</p> <p>Conclusion</p> <p>In the age group examined in the Lambaréné area, platelets are an excellent adjuvant tool to diagnose malaria. Pigment-containing leukocytes (PCL) are more readily detected by automated scatter flow cytometry than by microscopy. Automated Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria. However, this is possibly because of the instrument detection algorithm was not geared towards this task, and data lost during processing; and thus adjusting the instrument's algorithm may allow to establish a meaningful cut-off value.</p

Evidence for Microchimerism in Baboon Recipients of Pig Hearts

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials

Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

Xenotransplantation is a promising approach to reduce organ shortage, while genetic modification of donor pigs has significantly decreased the immunogenic burden of xenotransplants, organ rejection is still a hurdle. Genetically modified pig organs are used in xenotransplantation research, and the first clinical pig-to-human heart transplantation was performed in 2022. However, the impact of genetic modification has not been investigated on a cellular level yet. Endothelial cells (EC) and their sugar-rich surface known as the glycocalyx are the first barrier encountering the recipient's immune system, making them a target for rejection. We have previously shown that wild type venous but not arterial EC were protected against heparan sulfate (HS) shedding after activation with human serum or human tumor necrosis factor alpha (TN

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNF alpha were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs

Hemodynamics in pig‐to‐baboon heterotopic thoracic cardiac xenotransplantation: Recovery from perioperative cardiac xenograft dysfunction and impairment by cardiac overgrowth

Introduction Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. Methods We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. Results Aortic pressures and heart rates of the recipients’ hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. Conclusion The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size

Hemozoin-detection by Laser-Flowcytometry in malaria patients in Lambaréné, Gabun: Sensitivity, specificity and prognostic value

Malaria ist weltweit eine der bedeutendsten Infektionskrankheiten. Jährlich erkranken bis zu 500 Millionen Menschen an einer Malariainfektion, etwa zwei Millionen Menschen versterben an einer durch Plasmodium falciparum hervorgerufenen Malaria tropica. Besonders gefährdet durch Malaria sind Kleinkinder, die noch keine ausreichende Immunität besitzen. Neben umfassender Prävention und adäquater Behandlung der Infektion mit Chemotherapeutika hat vor allem die rechtzeitige Diagnose große Bedeutung im Management der Malaria. In den letzten Jahren wurde der Nachweis von hämozoinhaltigen Leukozyten als Marker für eine akute Malariainfektion wiederentdeckt. Hämozoin, ein Abbauprodukt des Malariaerregers, wird von Monozyten und neutrophilen Granulozyten phagozytiert und ist mikroskopisch an seiner doppelbrechenden optischen Eigenschaft erkennbar. Der Nachweis von pigmenthaltigen Leukozyten wurde nicht nur als diagnostisches Kriterium beschrieben, sondern auch als prognostischer Marker mit der Schwere der Erkrankung in Verbindung gebracht. Laser-Durchflusszytometer vom Typ des Blutanalysegerätes Cell-Dyn 3000® sind in der Lage, hämozoinhaltige Leukozyten anhand der optischen Eigenschaften des Hämozoin-Kristalles als fehlklassifizierte Punktsignale zu identifizieren. In verschiedenen Studien wurden bereits sog. atypische violette Punktsignale zur Malariadiagnostik genutzt. Unklar war bisher die Bedeutung von atypischen grünen Punktsignale geblieben, welche hämozoinhaltige neutrophile Granulozyten repräsentieren. In der vorliegenden Studie wurden die durchflusszytometrischen Daten von Kindern und schwangeren Frauen in Lambaréné, Gabun, untersucht, um Sensitivität, Spezifität und prognostischen Wert des Cell-Dyn 3000®-Gerätes in Bezug auf Malariainfektion zu prüfen. Es stellte sich heraus, dass bei Kindern das Auftreten von atypischen violetten und grünen Punktsignalen hoch sensitiv und spezifisch für eine Malariainfektion (AVPs: 95,4% respektive 95,4%; AGPs: 82,9% respektive 96,3%) ist. Erstaunlicherweise sind Sensitivität und Spezifität bei Erwachsenen deutlich niedriger (AVPs: 81,3% respektive 83,6%; AGPs: 68,8% respektive 95,2%), hohe Raten an falsch positiven Fällen wurden beobachtet. Ursächlich für diese Ergebnisse sind neben der noch nicht vollständig ausgereiften Identifizierungsmethode von atypischen Punktsignalen auch die unterschiedliche Immunitätslage von Kindern und Erwachsenen, insbesondere schwangeren Frauen. In holoendemischen Gebieten wie Lambaréné scheint die Durchflusszytometrie als Screeningmethode durchaus sinnvoll zu sein. Die Ergebnisse in der Gruppe der schwangeren Frauen jedoch müssen durch zukünftige Studien genauer evaluiert werden. Des weiteren wurden bei Kindern atypische Punktsignale auf ihr Potential zur Diagnose der Schwere der Malaria untersucht. Es zeigten sich signifikante Unterschiede in den Mengen der atypischen violetten und grünen Punktsignale zwischen unkomplizierter und komplizierter Malaria (21 AVPs vs. 33 AVPs [p=0,026] respektive sechs AGPs vs. 15 AGPs [p<0,001]). Allerdings scheiterten Versuche, einen Schwellenwert zu finden, welcher dem Kliniker eine Klassifikation in komplizierte und unkomplizierte Malaria allein anhand der Anzahl der Punktsignale ermöglichen sollte. Die Signale sind in beiden Gruppen zu gleichmäßig verteilt, als dass eine klare Unterscheidung möglich ist. Weitere Studien sind notwendig, um die Schwellenwerte der Cell-Dyn 3000® für atypische Signale zu verbessern und genauere Aussagen über die Verlaufsprognose von malariakranken Patienten zu ermöglichen.Malaria is one of the most important infectious diseases worldwide. Every year Plasmodium spp. causes 500 million acute illnesses and an estimated two million deaths predominantly due to Plasmodium falciparum malaria (malaria tropica). The main burden of morbidity and mortality is found in children, as immunity to Plasmodium spp. is not yet sufficiently developed. Besides extensive prevention and adequate treatment with chemotherapeutics opportune diagnosis of malaria infection is of utmost importance in disease management. In the last years detection of hemozoin containing leukocytes as a marker for acute plasmodium falciparum infection has been rediscovered. Hemozoin, a side product of parasite metabolism, is phagocytosed by monocytes and neutrophile granolocytes and can by detected microscopically by its birefringent nature. Detection of hemozoin containing leukocytes was not only described as a diagnostic tool but was also found to be a prognostic marker for disease severity. Laser-Flowcytometry as used by the cell-counter Cell-Dyn 3000® displays hemozoin containing leukocytes as misclassified signals in the machine’s scatter blots due to the optical properties of the hemozoin crystal. Various studies already used atypically distributed purple dots (representing hemozoin containing monocytes) for diagnosis of malaria infection. Yet the relevance of atypical distributed green dots which seem to represent hemozoin containing neutrophile granolocytes remained unclear. The present study evaluates flowcytometric data of children and pregnant women in Lambaréné, Gabun, to assess sensitivity, specificity and prognostic value of the Cell-Dyn 3000® in patients with malaria infection. In children, detection of atypically distributed purple (AVPs) and green dots (AGPs) was found to be highly sensitive and specific for malaria infection (AVPs: 95.4% and 95.4%, respectively; AGPs: 82.9% and 96.3%, respectively), whereas in adults results were lower (AVPs: 81.3% and 83.6%, respectively; AGPs: 68.8% and 95.2%, respectively) and high rates of false positive cases were observed. These findings could be explained as follows: (i) the identification process of atypically distributed signals is still experimental and not yet developed to its full potential and (ii) the immune status is different in children and adults, in particular in pregnant women. In holoendemic regions like Lambaréné flowcytometry seems to be helpful as a screening method. Nevertheless, the results concerning pregnant women should be reassessed in further studies. In children atypically distributed signals were investigated to determine their diagnostic potential regarding the severity of disease. In uncomplicated and complicated malaria the amount of atypical distributed purple and green dots differed significantly (21 AVPs vs. 33 AVPs [p=0,026] and six AGPs vs. 15 AGPs [p<0,001]). Although, attempts to create a threshold allowing a classification of complicated and uncomplicated malaria according to signal counts failed. Signals within both groups were almost evenly distributed making a clear differentiation impossible. Further studies are therefore necessary to improve threshold values for atypically distributed signals of the Cell-Dyn 3000®, thus providing a more accurate prognosis in the course of malaria infection

Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation

The porcine cytomegalovirus (PCMV) is a herpesvirus that may pose a risk for xenotransplantation using pig cells, tissues, or organs. Here, three orthotopic pig heart transplantations into baboons were studied. To detect PCMV, a real-time PCR and a Western blot assay based on four PCMV protein sequences, including two tegument proteins, were used. The transmission of PCMV from the donor pig to the recipient baboon was found in two cases, despite PCMV not being detected in the blood of the donor pigs by real-time PCR. Although it was not in the blood, PCMV was detected in different organs of the donor pigs, and in sibling animals. Immunohistochemistry using an antiserum that is specific for PCMV detected virus protein-expressing cells in all of the organs of the recipient baboon, most likely representing disseminated pig cells. Therefore, for the first time, the distribution of PCMV in organs of the donor pigs and the recipient baboons was described. In addition, baboon cytomegalovirus (BaCMV) was found activated in the recipient, and a screening for hepatitis E virus (HEV) and porcine lymphotropic herpesviruses (PLHV) was performed. For the first time, a cross-reactivity between antibodies directed against PCMV and BaCMV was found