Erythrocyte Morphology, Phase Contrast Microscopy

Phase contrast microscopy is a diagnostic procedure used in urology and nephrology (study of the kidneys) for the examination and evaluation of cytomorphological vital specimens (specimen with living cells) in hematuria and urinary sediment diagnostics. The outstanding importance of this method is based in particular on the possibility, relevant to diagnostics, of assessing the erythrocyte morphology (shape of the red blood cells), which can provide valuable information about the origin of a possible hematuria (blood in the urine) or erythrocyturia (red blood cells in the urine). Phase contrast microscopy of urine sediment is ideal for evaluating various forms of erythrocytes.The introduction of phase contrast microscopy into routine diagnostics can be seen as a major advance, as it makes it easier to distinguish pathognomonic urine components from non-pathological artifacts. Phase contrast microscopy is superior to brightfield microscopy, which was used until the introduction of phase contrast microscopy, with regard to the differentiation of artifacts. If an unclear or inconclusive finding is present, resorting to this procedure may save less useful measures such as radiological or cystoscopic diagnosis (cystoscopy) for glomerular hematuria (see below), thus reducing costs and saving treatment time.

Indications (areas of application)

Phase-contrast microscopy is used in urology and nephrology to study erythrocyte morphology:

• Assessment of cytomorphologic vital specimens in hematuria and urinary sediment diagnosis.Indication: > 6-8 erythrocytes/l detectable in urine or positive streak test.In the context of erythrocyte morphology, the differentiation of glomerular hematuria from non-renal erythrocyturia is performed by the detection of dysmorphic (glomerular) and eumorphic erythrocytes.
• Dysmorphic hematuria of glomerular origin is found in patients with membranous and membranoproliferative glomerulonephritis, as well as IgA nephropathy, focal and segmental nephrosclerosis, and lupus nephritis. In glomerular hematuria, erythrocytes are forced through damaged basement membranes of glomerular capillaries, causing damage. Microscopically, one recognizes dysmorphic erythrocytes, which are also called acanthocytes (= erythrocytes with “Mickey Mouse ears”).
• Eumorphic hematuria (postglomerular hematuria) is found in patients with renal cysts in rupture into the hollow system, bladder bilharzia, blood admixture from the vagina (vagina), injury after placement of a bladder catheter, blood clotting disorders, etc.

The procedure

The principle of phase contrast microscopy is based on the interference of light rays in the beam path of the microscope, which can achieve a high-contrast image of unstained cells. Structure and function of the phase contrast microscope:

• The annular aperture, condenser, annular phase plate and objective are of particular importance to the function of the phase contrast microscope. By inserting the annular aperture into the condenser and the phase plate into the objective, a separation of non-diffracted and diffracted light rays is caused. The resulting delay in the wavelength of the diffracted light rays evokes a phase difference that optically makes the structural details of the microscopic object under examination appear darker than the surrounding medium. Physically, the phase platelets convert the phase or path differences into amplitude differences. As a result of these amplitude differences, the image of the object appears in the microscope by mutual cancellation or amplification of the direct light rays passing through the object and diffracted at the object.
• Thus, a disadvantage of this principle is the fact that ideal phase preparations are not the rule, since amplitude effects are usually superimposed on the phase effects.
• Based on the principle of imaging the structures by amplitude differences, the existing cell structures are represented as graded shades of gray depending on their own optical density. To improve the reproduction of the structures, the use of thin preparations and phase platelets is indicated.Depending on the nature of the various phase platelets, both positive and negative phase contrasts can be achieved, resulting in the objects of interest being displayed either darker or brighter than their surroundings.
• In phase contrast microscopy, it is further important to note that necrosis (death of cells) can occur relatively quickly due to exposure to light and additional damage to the vital cells. As a result of this rapid necrosis development, the assessment of the slides must be performed within one to two hours in order to maintain the validity of the diagnostic measure. Thus, this time limitation on the evaluation of vital specimens usually requires documentation by photomicrography or, if necessary, by performing microkinematography. A photomicrograph is a photograph of a microscopic image taken with a camera attached to a microscope. Through microkinematography, there is the possibility to capture objects under microscopic observation in moving images.