Application of modern laboratory techniques and self-developed measurement methods

Photo credit: Nicolas LoranThe research group collaborates with various units at the Vrinnevi Hospital in Norrköping, Sweden (e.g., psychiatry and medical clinic). For several years the research group has also collaborated with the Vikbolandet healthcare centre. Another collaboration partner in Alzheimer’s research includes Karolinska Institutet, Huddinge (Professor B. Winblad). Additionally, there is an academic collaboration with the Pain Clinic in Linköping (professor B. Gerlde).

At Vikbolandet, the research group follows people with hypertension over a long period (10 years). They examine the function of thrombocytes and how the inflammatory response predicts long-term survival.

At Vrinnervi Hospital, they are studying individuals with bipolar disorder, which is viewed as being a risk factor for arteriosclerosis. Research is being conducted on thrombocytes and inflammation predicting the number of relapses in the disorder, the response to the administered medication and long-term survival.

The Medical Clinic in Norrköping examines individuals who have had strokes. Physical exercise is studied to see if it improves the functional capacity amongst individuals who have had severe strokes. There is also a study examining whether exercise affects thrombocytes, inflammation and the risk for the development of blood clots.

In several articles from the Vrinnevi Hospital, the research group has published a number of potential bio-markers that can differentiate Alzheimer’s from different forms of dementia (10, 11, 13, 14). Studies of the new bio-markers continue at Karolinska Institutet. They want to see if they can forecast how quickly the memory deteriorates and if they can predict which people with “mild cognitive impairment” who go on to develop Alzheimer’s.

Micha Milovanovic studies thrombocytes and the inflammatory response in Fibromyalgia. The preliminary results are promising and the condition is associated with significant changes to thrombocytes. Currently there are no bio-markers for Fibromyalgia. Eventually the research group will examine if the changes to the thrombocytes they have identified in Fibromyalgia can be used for diagnosis.

Thrombocytes 

Thrombocytes play an important part in haemostasis – the body’s ability to stop bleeding. When blood vessels are injured, collagen builds up. The Von Willebrand factor (vWf) then binds to the collagen and via the glycoprotein Ib receptor, thrombocytes bind to vWf. And so the thrombocytes attach to the damaged endothelium. The cell is then activated and the thrombocyte’s glycoprotein IIb/IIIa receptors bind the fibrinogen. Many thrombocytes bind to the same fibrinogen molecule and each thrombocyte binds many fibrinogen molecules and the cells react. As a result, a plug of thrombocytes is formed which primarily stops the bleeding.  

Thrombocyte density (kg/L)

The density of thrombocytes (kg/L) varies considerably. Denser thrombocytes contain more α- and dense-granules. The density of thrombocytes reflects the reactivity of the population – i.e., their tendency to react. To separate the blood’s cells following their density, a specifically developed method is used (1, 2, 8, 10, 11, 14, 15). This involves gradients being created with help from a density medium (Percoll™). They also include substances that block thrombocyte activation in the test tube. Upon centrifugation, the cells are separated by density. Micha is working with the density-separated thrombocytes in the following studies (2, 8, 11, 15). The group has also used this method to examine the sub-populations of red cells and granulocytes in cases of Alzheimer’s (10, 14).

Fibrinogen 

After thrombocyte-bound stimulation (in vitro) with different activators, the thrombocyte’s tendency to react – their reactivity – is reflected. The number of in vivo fibrinogens reflect thrombocyte activity in the body. Micha has studied thrombocyte-bound fibrinogens in several basic scientific studies (1, 2, 4, 10, 11). Furthermore, the markers have been applied as measurements of thrombocyte reactivity in coronary artery spasms (6), diabetes (7), atrial fibrilation (9) and acute stroke (12). The research group has also studied the differences between men and women with cases of angina pectoris (5) and acute stroke (16). The thrombocyte-bound fibrinogens are analysed with flow cytometry techniques.

P-selectin

P-selectin is stored in the thrombocytes’ α-granules. When they are activated, the molecules move to the cell surface. Here, the protein acts as a receptor for neutrophil granulocytes. Then the P-selectin rapidly divides from the thrombocyte membrane and then circulates in the plasma (sP-selectin). The level of intra-cellular P-selectin reflects the level of α-granules (2, 8). After provocation with agonists, the membrane-bound P-selectin is a measure of the thrombocyte’s reactivity. The cell-bound P-selectin without stimulation and sP-selectin are used as activity markers. The thrombocyte-bound P-selectin is analysed with help from flow cytometry and sP-selectin is determined with the ELISA technique. Micha’s research uses these analyses for illnesses such as acute strokes (12) and Alzheimer’s (13).

Inflammation parameters

C-reactive protein (CRP) is an acute-phase protein. The analysis is used as a bio-marker to show inflammation. Interleukin-6 (IL-6) is secreted by monocytes during inflammation. IL-6 stimulates the production of CRP. Myeloperoxidase (MPO) can be found in neutrophil granulocytes. Upon activation, MPO is secreted and the enzyme is used as an activity marker for these cells. Micha has first and foremost applied these analyses to rheumatoid arthritis and acute strokes (3, 16).