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bright field and dark field imaging

bright field and dark field imaging Excludes Scleroderma in Raynaud’s

 

he absence of a systemic sclerosis (SSc) nailfold pattern in patients with Raynaud’s phenomenon or suspected connective tissue disease is of high clinical value as a biomarker to rule out SSc, according to a large cohort study from the U.K.

For identifying patients who met the 2013 American College of Rheumatology/European League Against Rheumatism or the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) criteria for SSc, the study found that a nailfold capillaroscopy pattern had a negative predictive value of 90% (95% CI 86 to 93), according to Maya H. Buch, MBChB, PhD, and colleagues from the University of Leeds, writing in BMC Musculoskeletal Disorders.

That pattern also had a sensitivity of 71% (95% CI 61 to 80), a specificity of 95% (95% CI 91 to 97), and a positive predictive value of 84% (95% CI 74 to 91).

“We were very impressed with nailfold capillaroscopy’s utility in negative prediction,” Buch said in an interview with MedPage Today. “The most valuable result here is the low likelihood of scleroderma in patients with Raynaud’s phenomenon who do not have any scleroderma-specific features on nailfold capillaroscopy. In practice, this means we can more confidently reassure such a patient and discharge care back to the patient’s general practitioner.”

The researchers noted that to the best of their knowledge, this is the first study to demonstrate that the absence of any SSc pattern on nailfold capillaroscopy maintains its known negative predictive value, including for patients with secondary Raynaud’s phenomenon, who are considered at increased risk of SSc. “This study is only one of two to include a large unselected cohort of patients with Raynaud’s phenomenon — mirroring clinical practice in which rheumatology departments frequently receive referrals of patients with Raynaud’s from GPs,” Buch said.

Primary Raynaud’s phenomenon is associated with normal microcirculation architecture, whereas microangiopathies are associated with secondary Raynaud’s, she explained. The SSc nailfold capillaroscopy pattern correlates with disease duration and severity, and also predicts future vascular and visceral organ damage. Nailfold capillaroscopy also detects vascular problems in glaucoma.

Although an SSc nailfold capillaroscopy pattern is sometimes present in other connective tissue diseases, “nailfold capillaroscopy could be performed to provide reassurance to the rheumatologist in the assessment of both [primary and secondary] Raynaud’s phenomenon,” the researchers wrote.

Buch and colleagues studied 347 patients referred for nailfold capillaroscopy to a tertiary-care center from January 2009 to October 2013. The mean age of the cohort was 47 years and 83% were female. Clinical review showed that 54 patients (16%) did not have true Raynaud’s phenomenon, 69 (20%) had primary Raynaud’s, 172 (50%) had secondary Raynaud’s, and 52 (15%) had SSc.

At referral, 46 patients (89%) met either VEDOSS or the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for SSc. Of the patients with secondary Raynaud’s, 71 (41%) were being managed for connective tissue disease or inflammatory arthritis, while 101 (59%) had an antibody and/or a red-flag feature for SSc.

A nailfold capillaroscopy pattern for SSc was detected in 80 patients (23%) — 43 with early, 31 with active, and six with late-pattern vasculopathy. This pattern was observed in 37 patients (71%) diagnosed with SSc, 30 (17%) with secondary Raynaud’s, nine (13%) with primary Raynaud’s, and four (7%) without Raynaud’s.

Considering only those patients with non-SSc connective tissue disease or inflammatory arthritis, 16 of 71 patients (23%) had an SSc pattern. This was detected in two of five patients with SLE, eight of 42 with undifferentiated connective tissue disease, four of six with mixed connective tissue disease, one of three with Sjogren’s syndrome, and one of 14 with inflammatory arthritis.

Interestingly, the team said, participants meeting ACR/EULAR criteria were more likely to have an SSc nailfold capillaroscopy pattern than those meeting the VEDOSS criteria were: 84% versus 42%, respectively. “This may be related to the earlier stage of disease in those meeting VEDOSS with less time for detectable vasculopathic changes at the nailfold to develop,” the researchers wrote. “These findings are important as the earlier detection and management of SSc may lead to reduced morbidity and earlier detection of its complications.”

Among the study limitations were the lack of formal measurements to determine enlarged capillaries and the use of two different nailfold capillaroscopy methods, which might have introduced bias. As in clinical practice, the examiners were not blinded to the clinical diagnosis, possibly introducing investigator bias. In addition, the retrospective analysis may have missed important data, particularly the presence of telangiectasia.

The authors cited the need for larger, more defined prospective studies of a heterogeneous group of Raynaud’s patients. Buch noted that the current study is part of a larger Leeds program to identify biomarkers for accurately identifying patients at risk for scleroderma or those with scleroderma at risk for poorer outcomes.

bright field and dark field imaging

What is Blood and What Does it Do?

 

Two types of blood vessels carry blood throughout our bodies: The arteries carry oxygenated blood (blood that has received oxygen from the lungs) from the heart to the rest of the body.

The blood then travels through the veins back to the heart and lungs, where it receives more oxygen. As the heart beats, you can feel blood traveling through the body at your pulse points – like the neck and the wrist – where large, blood-filled arteries run close to the surface of the skin.

The blood that flows through this network of veins and arteries is called whole blood. Whole blood contains three types of blood cells:

Red Blood Cells
White Blood Cells
Platelets

These blood cells are mostly manufactured in the bone marrow (the soft tissue inside our bones), especially in the bone marrow of the vertebrae (the bones that make up the spine), ribs, pelvis, skull, and sternum (breastbone). These cells travel through the circulatory system suspended in a yellowish fluid called plasma (pronounced: plaz-muh). Plasma is 90% water and contains nutrients, proteins, hormones, and waste products. Whole blood is a mixture of blood cells and plasma.
Red Blood Cells

Red blood cells (RBCs, and also called erythrocytes, pronounced: ih-rith-ruh-sytes) are shaped like slightly indented, flattened disks. Red blood cells contain an iron-rich protein called hemoglobin (pronounced: hee-muh-glow-bun). Blood gets its bright red color when the hemoglobin in RBCs picks up oxygen in the lungs. As the blood travels through the body, the hemoglobin releases oxygen to the tissues. The body contains more RBCs than any other type of cell, and each has a life span of about 4 months. Each day, the body produces new RBCs to replace those that die or are lost from the body.

White Blood Cells

White blood cells (WBCs, and also called leukocytes, pronounced: loo-kuh-sytes) are a key part of the body’s system for defending itself against infection. They can move in and out of the bloodstream to reach affected tissues. The blood contains far fewer white blood cells than red cells, although the body can increase production of WBCs to fight infection. There are several types of white blood cells, and their life spans vary from a few days to months. New cells are constantly being formed in the bone marrow.

Several different parts of blood are involved in fighting infection. White blood cells called granulocytes (pronounced: gran-yuh-low-sytes) and lymphocytes (pronounced: lim-fuh-sytes) travel along the walls of blood vessels. They fight germs such as bacteria and viruses and may also attempt to destroy cells that have become infected or have changed into cancer cells.

Certain types of WBCs produce antibodies, special proteins that recognize foreign materials and help the body destroy or neutralize them. Someone with an infection will often have a higher white cell count than when he or she is well because more WBCs are being produced or are entering the bloodstream to battle the infection. After the body has been challenged by some infections, lymphocytes “remember” how to make the specific antibodies that will quickly attack the same germ if it enters the body again.
Platelets

Platelets (also called thrombocytes, pronounced: throm-buh-sytes) are tiny oval-shaped cells made in the bone marrow. They help in the clotting process. When a blood vessel breaks, platelets gather in the area and help seal off the leak. Platelets survive only about 9 days in the bloodstream and are constantly being replaced by new cells.

Drop of Blood

Blood also contains important proteins called clotting factors, which are critical to the clotting process. Although platelets alone can plug small blood vessel leaks and temporarily stop or slow bleeding, the action of clotting factors is needed to produce a strong, stable clot.

Platelets and clotting factors work together to form solid lumps to seal leaks, wounds, cuts, and scratches and to prevent bleeding inside and on the surfaces of our bodies. The process of clotting is like a puzzle with interlocking parts. When the last part is in place, the clot happens – but if only one piece is missing, the final pieces can’t come together.

When large blood vessels are severed (or cut), the body may not be able to repair itself through clotting alone. In these cases, dressings or stitches are used to help control bleeding.

In addition to the cells and clotting factors, blood contains other important substances, such as nutrients from the food that has been processed by the digestive system. Blood also carries hormones released by the endocrine glands and carries them to the body parts that need them.

bright field and dark field imaging

How to made the bright field and dark field imaging?

How to made the bright field and dark field imaging ?

It is very easy to make bright field and dark field imaging yourself. What you have to do is place an opaque round stop in the condenser. An easy way is to cut a piece of black paper and put it on a filter in your filterholder. You can put the stop on a piece of clear acetate sheet. You can even try to draw the stop on it with black paint. The most important thing is to have it big enough to stop all light going directly into the objective. Only the light that is reflected by the objects in the sample reaches the objective then. Stronger objectives are more difficult because their NA is often too high. The NA of your condenser should always be higher then the NA of the objective. If patch-stops of 8, 10, 12 and 15mm are made you can’t go wrong really. For objectives of around x10 the middle sizes prove best.If you like to make the patchstop as precise as possible: The best way is to set up as normal (brightfield), remove the eyepiece and close/open the substage iris until it is *just* visible. Then, either bending your neck over double, or carefully removing the condenser, measure the diameter of the iris diaphragm as it is now set. A pair of calipers is useful here. This diameter is that for the patch stop. Very often, to be on the safe side it is best to add about 10% to this figure, this avoids leakage, especially if you have no means of centering the stop in the filter holder. If you have a phase contrast condenser, the largest phase contrast annuli often make excellent patch stops for darkfield!The real connoisseurs must have recognized the skills of Klaus Kemp in the arranged (cleaned) diatom slide photographed by Mike Samworth.

bright field and dark field imaging

What is bright field and dark field imaging?

Dark Field microscopy is a microscope illumination technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

When light hits an object, rays are scattered in all directions. The design of the dark field microscope is such that it removes the dispersed light so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light.

These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques.

Dark field can be used to study marine organisms such as algae and plankton, diatoms, insects, fibres, hairs, yeast, live bacterium, protozoa as well as cells and tissues and is ideal for live blood analysis enabling the practitioner to see much more than is possible with other lighting methods.

bright field and dark field imaging

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