Det är den tyske f.d doktoranden Matthias Ugele, numera med doktorshatt och en doktorsexamen, som gjort ett hästarbete med att gå till grunden och visa hur väl lämpad Digital Holografisk Mikroskopi (DHM) är för blodsjukdomar (Hematologi).
High-throughput hematology analysis with digital holographic microscopy
är titeln på hans avhandling. Den laddas ner som pdf via länken ovan.
Matthias har gått igenom ett antal DHM-tekniker,däribland PHI´s HoloMonitor, och läst på inom vilka områden dessa har blivit framgångsrika.
I hans avhandling begränsar han sig till 2 blodsjukdomar där DHM-tekniken skulle kunna användas framgångsrikt, Malaria och Leukemi.
Han beskriver hur dagens diagnostiserande ser ut och vilka nackdelar de har mot att man istället skulle använda sig av den digitala holografiska mikroskopin med några modifieringar.
Valda klipp från avhandlingen (fetningar är bloggens egna) :
Abstract
For more than 100 years, the microscopic analysis of Giemsa-stained peripheral blood smears has been the gold standard for the routine diagnosis of hematological disorders.
To accurately interpret peripheral blood smears, well-stained samples and time consuming microscopic analyses are required, which still show high inter-observer variation.
Automated hematology analyzers provide a complete blood count and leukocyte differential, but only flag atypical results as “abnormal”, which provides no clear classification due to method limitations. To overcome the analytical limitations of today’s methods, the analysis of native blood cells in suspension would be highly attractive for routine clinical diagnosis.
Digital holographic microscopy (DHM) was suggested as method of choice for label-free cell imaging, because the phase contrast provides rich intracellular information due to subtle refractive index changes at internal structures.
In this thesis, a flow cytometry-based method for the label-free analysis of unprocessed blood cells by DHM, using highly controlled two-dimensional focusing conditions, is reported.
The combination of a 50 x 500 µm PMMA microfluidics channel for precise microfluidics focusing of the cells with a customized differential DHM, allowed high-throughput imaging without the need for time-consuming autofocusing procedures.
Hereby, a label-free nine-part differential of untouched leukocytes was achieved by principal component analysis of morphological characteristics of the reconstructed images.
In addition, a classification strategy for the differentiation of acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and various myeloproliferative neoplasms was established.
The results suggest the possibility for monitoring of leukemia progression by demonstrating the disappearance of acute myeloid leukemia cells in remission.
To exclude confounding effects, the classification strategy was tested by the analysis of 20 blinded clinical samples.
Here, 70 % of the specimens were correctly classified with further 20 % classifications close to a correct diagnosis, which verified the discrimination strategy.
Apart from a detection and classification of hematological disorders, the developed label-free method was also used for the diagnosis of malaria-infected samples.
Effective malaria treatment requires rapid and accurate diagnosis of infecting species and actual parasitemia.
Despite the recent success of rapid tests, the analysis of thick and thin blood smears remains the gold standard for routine malaria diagnosis in endemic areas.
For non-endemic regions, sample preparation and analysis of blood smears are an issue due to low microscopy expertise, and few cases of imported malaria.
For that reason, automation of microscopy results with minimal sample preparation could be beneficial to quickly confirm suspected infections in such conditions.
In this study, a label-free detection of P. falciparum infection in sphered erythrocytes, with a parasitemia detection limit of 0.01%, was achieved with the high-throughput DHM based approach. Moreover, the differentiation of P. falciparum ring-, trophozoite- and schizont life cycle stages in synchronized cultures demonstrated the potential for future discrimination of even malaria species and hence, an application in routine malaria diagnostics.
Taken together, the findings indicate a broad clinical applicability of the presented imaging cytometry method for automated and reagent‐free diagnosis of hematological disorders and parasitic infections.
To overcome the analytical limitations of today’s methods and significantly reduce sample preparation effort,label-free methods for the analysis of blood cells close to in vivo conditions would be highly attractive.
However, a label-free system for CBC and LDC has to compete with the robustness, accuracy, and throughput of established hematology analyzers, which has not been achieved so far.
For example, label-free methods like tomographic phase microscopy or quantitative phase spectroscopy, offer high resolution, but are too complex and time-consuming to be applicable in a routine workflow.
In contrast, the potential of digital holographic microscopy (DHM) for fast and accurate analysis of blood cells has been shown by various studies, but clinically relevant information has not been shown so far.
Nevertheless, by its optimization, routine CBC analysis, as well as the detection and classification of hematological disorders seems achievable.
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Malaria
With over 200 million new cases and more than 400,000 deaths per year (World Health Organization, 2017), the treatment and elimination of malaria is still one of the biggest global challenges of today.
Apart from conventional methods, alternative label-free approaches for malaria detection and characterization have been explored with promising results.
Anyhow, the DHM-based approach could contribute to overcome the limitations of conventional diagnosis tools. Blood smear analysis of thin films is cheap and sensitive, but requires time-consuming and error-prone sample preparation and highly trained experts for accurate species and parasitemia determination (Lema et al., 1999). This is an issue in non-endemic regions with low microscopy expertise due to few cases of imported malaria. RDTs are sensitive and easy to use but mainly do not provide sufficient species differentiation, lack parasitemia determination and are relatively expensive compared to blood smear analysis (Lema et al., 1999; Moody, 2002; Wongsrichanalai et al., 2007). PCR analysis is ultra-sensitive and high-throughput capable (Imwong et al., 2014), but requires relatively complex sample preparation and time-consuming DNA amplification. In comparison, with the reduced sample preparation and evaluation effort provided by label-free DHM-based malaria diagnosis, a reliable, fast and user-independent workflow for parasitemia and species determination with price-competitive costs per test may be within reach. Last, apart from detection of malaria-infected cells, our label-free approach offers the potential to assist in the evaluation of potential therapeutics for anti-malarial treatment or monitoring of treatment success (Bettenworth et al., 2014), as well as blood transfusion quality control (Abdullah and Karunamoorthi, 2016; Kitchen and Chiodini, 2006) without the need for time-consuming sample preparation procedures.
Leukemi
källa till ovanstående är wikipedia.
Men forts till doktorsavhandlingen:
Leukemia is responsible for more than 67,000 deaths per year in the EU and USA (Malvezzi et al., 2017; Siegel et al., 2017), whereby leukemia is defined as the uncontrolled proliferation or expansion of hematopoietic cells that do not retain the capacity to differentiate normally to mature blood cells (Sawyers et al., 1991).
The four main subtypes are acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL).
With about 80 %, AML is the most common acute leukemia in adults and mostly appears de novo in previously healthy individuals.
Genetic mutations occur in more than 97 % of all cases, and the two-hit model can be used to describe and classify the mutations associated with AML. According to this model, class I mutations, which activate pro-proliferative pathways must occur in conjunction with class II mutations, which impair normal hematopoietic differentiation. Examples for class I mutations are FTL3, K/NRAS, TP53 and c-KIT, typical class II mutations include NPM1 and CEBPA. Recently, a third class of mutations affecting genes involved in epigenetic regulation, which affect both cellular differentiation and proliferation, has been identified. In more than 40 % of AML cases, class III mutations in the DNA-methylation related genes DNMT3A, TET2, and IDH-1/2 were found. Apart from this, large chromosomal rearrangements can also trigger AML. Clinical manifestations of AML are leukocytosis, bone marrow failure (anemia, thrombocytopenia), fatigue, anorexia and weight loss, which reflect the accumulation of malignant, poorly differentiated myeloid cells within the bone marrow, peripheral blood and other organs.
If untreated, AML patients die within months due to secondary infections or bleeding.
Although advances in supportive care and prognostic risk stratification have optimized established therapies, the overall long-term survival remains poor, as the genetic heterogeneity of AML exacerbates effective treatment (De Kouchkovsky and Abdul-Hay, 2016).
The routine diagnosis of these described leukemias and MPNs requires stained blood smear analysis, which is the gold standard. Modern automated hematology analyzers usually provide a five-part differential separating neutrophils, basophils, eosinophils, monocytes and lymphocytes, but only flag atypical leukocytes as “abnormal“, which provides no clear classification due to method limitations (Barcia, 2009). Additional methods, like flow cytometry, rely on sometimes tedious sample preparation and labeling for the classification of benign and malignant leukocytes, which is expensive and timeconsuming (Filby, 2016).
To overcome the analytical limitations of today’s methods, the analysis of native blood cells in suspension would be highly attractive for routine clinical diagnosis.
DHM was suggested as method of choice for label-free cell imaging, because the phase contrast provides rich intracellular information due to subtle refractive index changes at internal structures.
A simple, fast, and automated label-free leukemia detection would be a significant advancement to the field to match the unmet need of clinical routine diagnosis.
Amongst other, automated hematology analysis of such complex diseases would potentially reduce manual differentiation of leukocytes efforts in central laboratories, and could improve patient diagnosis at the point of care level.
In conclusion, after optimization, the DHM approach bears the potential to circumvent the interobserver dependence of peripheral blood smear analysis (Bacus, 1972; Pierre, 2002; van der Meer et al., 2005, 2004), and it can be envisioned that leukocyte viability and function close to in vivo conditions (Vuorte et al., 2001) potentially add promising new information to hematology diagnosis, which is not covered by today’s CBCs measured using automated hematology analyzers (Singhal and Agrawal, 2018).
Och i hans sammanfattning och slutsatser klipper jag in:
- Collectively, the suitability of the system for a label-free detection and classification of various hematological disorders could be shown. With an increased number of clinical samples and improved data handling and analysis, the DHM based approach bears the potential to circumvent the limitations of state-of-the-art hematology analyzers and reduce the need of peripheral blood smear analysis for the routine diagnosis of hematological disorders. Thereby, workflow integration for automated, high-throughput analysis in a clinical environment is critical and should be addressed by ongoing research. In conclusion, the results obtained in this thesis provide the basis for the development of a next generation hematology analyzer, which addresses the unmet needs of clinical routine diagnosis by reduced sample preparation together with improved clinical output.
Min kommentar
Det Matthias belyser är 2 blodsjukdomar och hur dessa diagnostiseras idag.
Han lyfter fram kända nackdelar med dagens diagnosteknik och visar på hur man löser denna problematik på ett hyfsat enkelt sätt --> ett teknikskifte.
Matthias har gjort ett gediget grundarbete som jag sällan sett i andra avhandlingar.
Kolla bara in alla referenser han har med på sin lista som han betat sig igenom. Bloggen får det till över 200 forskningsrapporter Matthias förmodligen lusläst och byggt sin forskning vidare på.
Denna doktorsavhandling lär knappast gå de större kommersiella instrumenttillverkande företagen förbi.
Vi pratar alltså om en synnerligen framsynt prognos som kommer ha ( enligt bloggens syn ) påverkan på framtida tekniker som löser / förenklar dagens problematik. Men som vi vet är forskare konservativa och med det innebärande skepsis till ny modern teknik. Med denna epokavgörande doktorsavhandling kommer marknaden med dess användare förr eller snarare applicera en modern teknologi som bättre löser möjlighet att diagnostisera och med det snabbare sätta in adekvat behandling. Bloggen rekommenderar att ladda ner den 125 sidiga doktorsavhandlingen och ta del av en riktigt gedigen forskning.
Bloggen har tidigt lyft fram DHM som ett superbt instrument att kunna använda i otillgängliga miljöer för att snabbt kunna utföra ex malariadetektioner. Men även Zika-viruset och Dengue-feber är andra sjukdomar DHM är användbart för. Använd denna sidas sökfunktion för malaria så hittar ni tidiga inlägg i ämnet.
Med denna enormt grundliga (bloggens subjektiva åsikt) forskningsrapport har Matthias visat för instrumentmarknaden med dess aktörer och ledare hur framtiden kommer att se ut. DHM är lösningen för enbart 2 identifierade blodsjukdomar,fler områden lär komma.
Bloggen har länge väntat på en tungt vägande akademisk rapport som stärker och bekräftar undertecknads syn på att DHM är ett verkningsfullt instrument inom tropiska sjukdomar som ex malaria.
Nu visar Matthias med sin doktorsavhandling att marknaden strax är redo för detta paradigmskifte med ny teknik,DHM.
Bloggen tänker att en större aktör med brett distributionsnät och större forskning och utvecklingsavdelning mycket väl kan bygga vidare på en teknik som ex PHI har. Och med det få ut instrument riktade till blodsjukdomar som ex ovan angivna.Denna marknad tror undertecknad är riktigt stor och då intressant för de större tillverkarna. ThermoFisher är en av dessa och om nu TF är en av friarna till PHI har de förmodligen gjort sin analys och sett att potentialen HoloMonitor erbjuder går förbi immunologi och cancerforskning.
Mvh the99
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