Lecture 14: biosensors and liver function

Liver Disease in Europe

• 29 million people in Europe have chronic liver disease

• Cirrhosis = 170,000 deaths/year

• 1.8% of all deaths

• Large variation across Europe

• more prevalent in males

• alcohol is a main cause of liver cirrhosis

• liver disease is increasing

Liver disease causes premature death

•Liver disease is the 3rd biggest cause of premature (≤65 years) death in the UK

•Results in 62,000 years of working life lost each year

Natural history of chronic liver disease

• Histology: large nuclei = metabolically active

• Fibrosis is a commonality of liver injury

• Fibrotic tissue is non functional and contracts to prevent blood supply to hepatocytes

• HCC = hepatocellular carcinoma: primary tumour in liver

Progression of Cirrhosis

Compensated Cirrhosis:

• The liver is severely scarred

• but enough healthy cells in your liver to perform its functions adequately

• Ascites: fluid build up in abdomen

Decompensated Cirrhosis:

• liver is not capable of performing all of its normal functions, resulting in several complications

• eg: fluid retention, varicies (blood vessels sag) and mental confusion from ammonium ions (encephalopathy)

Decompensated cirrhosis can be unpredictable….

1. patient enter decompensation

2. emergency hospital admission

3. initial treatment (LVP/diuretics/Lax)

4. optimisation (weight/ U&Es/ cognitive function

5. Discharge

6. Community follow up (OPD or GP)

d-LIVER: Why? A clinical perspective

• To use technology to move management of end-stage liver disease (ESLD) patients out of the clinic and into the home or near-home setting

• To improve quality and length of life by dynamic management of complications (daily, not monthly)

• To improve the quality of life for patients and carers by avoiding burdensome clinic visits

• To reduce the cost of management by earlier cheaper intervention

  • NHS paying for taxis for patients

d-LIVER at home

• monitor patient at home

• chest monitor: heart rate, breathing rates

• small box: biochemistry for liver function

• data monitored by patient coordinator

Work Package 1:

• clinical application senarios and validation

• who benefits from this technique

The Challenge of Liver Disease

• 20,000 deaths per year in the UK

• Only one of the 5 “big” killers increasing in impact

• “Perfect storm” of obesity-related liver disease, alcohol and viral hepatitis

• Impaired quality of life from encephalopathy/itch

  • must moniter bile salts

• There is no primary therapy for liver failure

• Current therapy transplant or support to recovery

• 500 transplants in the UK, 20,000 deaths per year

• Acute on chronic liver disease is not an indication

• No effective artificial bridge to transplant

The Concept of Bridging to Recovery

• Liver has the capacity to recover and re-generate

  • take stressors away

  • bioreactor to carry out funtion of liver

• Patient needs to survive long enough to benefit!

• Loss of liver function is compounded by liver necrosis, leading to hypoperfusion, tissue necrosis and a death spiral

• The concept is for early supportive therapy with d-LIVER system to break the spiral

Hasn’t This Been Tried Before?

• yes but previous approaches were in acute liver failure late in the death spiral

  • Dliver aims to intervine before death spiral begins

• Too great a hurdle to cross and failure inevitable

• Current concept is early light touch intervention

D-LIVER Delivered by 4 scenarios

• Chronic liver failure: – compensated liver become decompensated, treat "acute on chronic liver" failure, reduce intermittent encephalopathy, enable long-term therapy in the home environment and reduce the need for transplantation

• Chronic cholestatic itch: – intermittent long-term therapy to reduce cholestatic itch, measure of bile salt lives, to extend the time before transplantation is inevitable, to improve quality of life

• Bridging therapy before liver transplantation: – help liver regenerate itself, therapy for high-risk patients to reduce duration and incidence of hospitalisation and thus waiting-list mortality

• Acute liver failure: – e.g. "small for size" syndrome to support liver function when resected liver proves insufficient, only one smaller functional liver unit is transplanted, so still can’t cope with metabolic demand

Work Package 8

progenitor cells for bioartificial liver

B-13 pancreatic cell

• B-13 cell is the ideal cell type for a cell-based artificial liver support system.

• B-13 – readily genetically manipulable and expandable rat progenitor cell.

• Dexamethasone transforms them into functional hepatocytes (B-13/H) in vitro (unlike embryonic stem cells).

Effect of Dexamethasone

Gene expression

• Inhibits pancreatic amylase expression, induces expression of the hepatocyte marker transferrin, and induces expression of markers typical of ductal cells

Phenotype

• B-13/H cells express a variety of liver-enriched and liver-specific genes, many at levels similar to hepatocytes in vivo

Maintenance

• B-13/H phenotype is maintained for at least several weeks in vitro

why not use stem cells?

their transformation is linear, difficult to make them stay at desired differentiation stage

How can you test B-13 for hepatocyte activity?

1. western blot of protein expression

   • increase conc of dexamethasone = increase in protein expression

2. monitor nitrocatechol production

   • longer incubation with DEX= increase in conc

3D Perfused Bioreactor

• core = capillaries

• outside of capillaries are B13 liver cells

• blood from patient passes into bioreactor> cells remove waste

• reduces metabolic load on liver, so liver can regenerate

work package 4:

blood biochemistry cartrige

parameters to measure in blood chemistry

• amperometric (current) sensor: bilirubin (cholestatic itch)

• impedimetric (resistance) sensor: albumin

• potentiometric (ionic conc.) sensor: creatine, sodium, potassium

• optical sensor: blood clotting time

microfluidic cartridge design

• fingerprick of blood

• clotting assay

• blood is processed to serum

• pump pressurises whole system, forces round to sensor cartriges

microfluidic cartridge requirements

Key Requirements

• Low sample volume (20µl)

• Integrating 6 sensors

• Cheap and simple design

• Sequential flow through

• Microfluidic workflow

Additional Features

• Serum extraction

• Serum dilution

Advantages of microfluidics

• Low fluid volume consumption

• Faster analysis and response times due to short diffusion distances

• System is compact

• Parallelisation allows high-throughput analysis

• Lower fabrication costs

Challenges of microfluidics

• Detection principles may not always scale down

  • electrode SA

• The absolute geometric accuracies and precision in microfabrication are high

work package 3

sensors development

Objective of WP3

• The development and characterisation of physical and biochemical sensors for monitoring:

  • patient at home

  • bio-artificial liver support unit

• 4 physiological parameters:

  • Heart rate

  • Skin body temperature

  • Activity/Posture (alert if fallen over)

  • Blood pressure

• 6 biochemical parameters:

  • Electrolytes (sodium and potassium)

  • Small molecules (albumin, creatinine and bile acids)

  • Clotting time

• Physiological sensors = worn by the patient = wearable sensors

• Biochemical sensors= incorporated in microfluidic cartridge (WP4) and in the bio-artificial liver support unit (WP5)

• Software and signal processing requirements = WP6

why do we need a d-LIVER Wearable Device

• Develop a device for continuous, ambulatory measurements of vital signs for early detection of patient exacerbations.

• Main usage scenarios: Intermittent measurements during shorter time sequences (days, weeks, months), such as assessing medical intervention effects.

1. Establish a baseline at the start of a new treatment programme

2. Lifestyle change, outcome assessment and patient motivational tool (lifestyle changes such as exercise, substance abuse, diet, etc.).

3. Assessment of the effect of medication adjustments.

4. Monitoring requested by medical personnel for patient status assessment.

Target measurement parameters

• • Heart rate

• • Heart rate variability

• • Skin temperature

• • Activity level

• • Step Counter

• • Upper body posture

• • Pulse transit time

Blood Pressure Derived from Pulse Transit Time

• Pulse Transit Time (PTT) = The time is taken from the heart valve’ until the blood pressure wave reaches the periphery.

• PTT depends on blood pressure: higher pressure = shorter PTT.

• Sensors used:

  • Electro Cardiography (ECG): detects heartbeat start impulse

  • Impedance Cardiography (ICG): detects opening of the aortic valves

  • Photo Plethysmo Graphy (PPG): detects peripheral pressure wave

Communication Configuration

• data sent to Liver Paitient Management System (LPMS)

• clinician given data in bar graphs, easy to interpret

• can set alarms to paramaters

Blood clotting analysis

• Complementary metal-oxide semiconductor (CMOS) sensor where the fluidic component is put

• The sample fills the channel by capillarity

• capillary contains clotting agent: thromboplastin, to initiate clotting cascade

• A laser source illuminates the blood sample and RBC diffuse light.

• A moving diffraction picture is formed on the CMOS sensor called speckle diffraction.

• When the blood clots, the movement stops

• clottiing only occurs if all the factors produced by the liver are present

• Clotting is then detected.

Blood clotting data analysis

• A picture generates a correlation factor that is 0 where the cells are moving (before coagulation) and 1 when the cells stop (after coagulation).

• Pictures movements changes are illustrated by a colour change.

• Sample goes from black to white when coagulation occurs.

• Clotting time is detected by the sudden changes in the slope value of the correlation factor curve

Hepatic Encephalopathy (HE)

• A neurological disorder caused by liver dysfunction, esp liver disease or liver failure.

• liver is unable to adequately filter toxins (ammonia) from bloodstream.

• toxins then accumulate and affect brain function, leading to a range of mental and physical symptoms.

Electrolyte and ammonia monitoring

• The printed circuit board (PCB) is composed of 3 working electrodes (for Na+, K+ and NH4+) and one common reference electrode

• Specific PVC chambers are assembled on the PCB

• A membrane is then added to the top of the chamber.

• Each membrane is specific for an ion.

• The specificity is due to an ionophore embedded in the membrane.