**4. Discussions**

Diagnosis in all these patients has been established in collaboration with clinician doctors from department of hospitals in the system of evidence based medicine, on data encompassed in observation daily sheet of patients.

Anemia of chronic kidney (ACKD) diseases associated with the iron deficiency (IDA) was microcytic and hypochromic, especially once the HGB level fall below 100g/L and HCT are somewhat lower that seen in normochromic, normocytic anemia from chronic diseases(ACKD).

Proportion of hypochromic red cells is a time average marker, was similar in anemic patients like glucose, HbA1c in diabetes patients. The marker for IDA, hypochromic erythrocytes, has been investigated for every patient, on blood film slide, May Gunwald stain. The hypochromic cells >10% were considered functional ID, in correlation low with iron. Various cut off values for functional ID is reported in literature ranging from 2% to 10% of hypocromic cells [17]

Measuring of TIBC was made as an indirect method of assessing transferrin and provided comparable information [18]. TSI indicates the percent of iron binding sites on transferring that is carrying iron. TSI is derived from a calculation using the formula: [(SI/TIBC) x100] and TSI is generally considered to be the most sensitive laboratory test for detecting altered iron metabolism in hereditary hemochromatosis (HH). It may be elevated prior to

**Parameters in Chronic Renal Failure after undergoing the schedules of Dialysis (mean value)** 

Glucose: 105 mg%; (5.76 mmol/L) SD=1.04; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Urea nitrogen: 65 mg%; (23.2 mmol/L); SD = 1.60; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Creatinine; 1.8 mg/%; (1.58mmol/)L; (SD=0.20; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Sodium: 145 mmol/L;SD=0.70 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Potassium7.1 mmol/l;SD=2.90 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, E CO2: 19 mmol/L;SD=2.29; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Hb= 10.5- 11.2mg/dl SD= 2.45;

Table 3.Values of biochemical and hematological parameters in blood samples from patients

Diagnosis in all these patients has been established in collaboration with clinician doctors from department of hospitals in the system of evidence based medicine, on data

Anemia of chronic kidney (ACKD) diseases associated with the iron deficiency (IDA) was microcytic and hypochromic, especially once the HGB level fall below 100g/L and HCT are somewhat lower that seen in normochromic, normocytic anemia from chronic

Proportion of hypochromic red cells is a time average marker, was similar in anemic patients like glucose, HbA1c in diabetes patients. The marker for IDA, hypochromic erythrocytes, has been investigated for every patient, on blood film slide, May Gunwald stain. The hypochromic cells >10% were considered functional ID, in correlation low with iron. Various cut off values for functional ID is reported in literature ranging from 2% to

Measuring of TIBC was made as an indirect method of assessing transferrin and provided comparable information [18]. TSI indicates the percent of iron binding sites on transferring that is carrying iron. TSI is derived from a calculation using the formula: [(SI/TIBC) x100] and TSI is generally considered to be the most sensitive laboratory test for detecting altered iron metabolism in hereditary hemochromatosis (HH). It may be elevated prior to

with Chronic Renal Failure, undergoing the schedules of dialysis

encompassed in observation daily sheet of patients.

**Normal Range of Blood Tests used in Diagnosis of CRF (laboratory reference)** 

Glucose: 65- 115 mg%; (3.9-6.1 mmol/L) ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Urea: 17-45 mg%; (1.7-8.3 mmol/ L) ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Creatinine: 0.2-1.25; (0.07-0.12 mmol/L) ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Sodium: 137-145 mmol/L; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Potassium: 3,6—5mmol/L ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, E CO2: 22-30mmol/L ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Hb=11.4-13.6mg/dl; SD=2.7

**Parameters in Chronic Renal Failure before schedules of Dialysis ( mean value)** 

Glucose: 98mg%; (5.44mmol/L); SD=2.80; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Urea nitrogen: 112 mg%; (40 mmol/L); SD = 2.40; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Creatinine; 5.5 mg/%; ( 4.85 mmol/L); SD=0.15; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Sodium: 170 mmol/L;SD=0.14 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Potassium14.5 mmol/l;SD=2.88 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, E CO2: 11 mmol/L;SD=0.26; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Hb= 8.5g/- 9.2mg/dl; SD=2.20

**4. Discussions** 

diseases(ACKD).

10% of hypocromic cells [17]

significant deposition of tissue iron. TS levels increase as additional iron is accumulated. A drawback to using the TS is that it is dependent on performing both the SI and TIBC.

Current guidelines from the American College of Physicians include a normal level of TSI encompassed between 20-40%, a cut off level of TSI >55% identifying iron overload and TSI < 15% meaning IDA. Red distribution width (RDW) is a mathematical expression of size variation used to quantify anisocytosis. The higher the RDW means the greater the anisocytosis. Increased RDW may be an early indication of iron deficiency, where it may precede the onset of microcytosis.

These measurements, known as erythrocyte or red blood cell indices, provide an important information about various types of anemia. If the MCV is low, the cells are microcytic or smaller than normal. Microcytic red blood cells have been seen in iron deficiency anemia and thalassemia minor. If the MCV is high, the cells are macrocytic, or larger than normal. Macrocytic red blood cells were associated with pernicious anemia or folic acid deficiencies. If the MCV is within the normal range, the cells are referred to as normocytic and nomocytic anemia was met with more frequency in chronic diseases/inflammation, small MCH under 27% show hypocromic erythrocytes, frequently encountered in IDA. In the same correlation with MCHC less than 32% indicates that the red blood cells are deficient in hemoglobin concentration.

This situation is most often seen with iron deficiency anemia. RDW is a measurement of anisocytosis. IDA and thalassemia are both microcytic-hypochromic anemia. As screening tests for discovery of anemia to elderly we used, beside additional tests, erythrocytes indexes such as MCV, MCH, and RBC number to distinguish this anemia types. MCH is just the equivalent of Retyculocites –Hemoglobin (Ret-He) that indicates the long term of life span of erythrocytes.

Both serum transferrin receptor and erythrocyte zinc protoporphyrin have been demonstrated to be useful in a variety of clinical situations. Serum transferrin receptor can be best used in diagnosing iron disorders, especially for patients with pathologies that may affect iron metabolism. Erythrocyte zinc protoporphyrin can be best used as a primary screening test for assessing iron status, especially in patients likely to have uncomplicated iron deficiency hemoglobin status and life span of erythrocytes [18].

Other anemia, most notably thalassemia, are also characterized by low MCV, MCH, MCHC and additional tests are needed for confirmation of thalassemia Patient with a ratio target cells/normal cells > 1% in low power field and with >20% microcytic red cells on blood film ( magnification x 400), were suspicious for beta-thalssemia. RBC count result higher in thalassemia minor group in comparison with IDA. Microcytic, hypochromic and polyglobulia are more evident in thalassemia minor compared with IDA and hemoglobin and hematocrit can be normally but only MCV and MCH decreased in thalassemia silent carrier. (Graphic 3)

The bone morrow hemosiderrin and microscopic bone marrow examination have been recommended in clinical management in most elderly patients with anemia in Mielodysplastic Syndrome (MDS) The problems in diagnostic anemia occurs when the iron reserves are depleted and not.

The peptide hormone Hepcidin appears to play a central role in the pathogenesis of the anemia of chronic disease, but is extremely difficult to measure in the serum. Thus the "anemia of chronic disease" may include patients with a variety of patho-physiological mechanisms. The peptide hormone Hepcidin, secreted by the liver, controls plasma iron concentration by inhibiting iron export from macrophages cells(cut off, 15 ng/d L, Elisa

Variability of Biological Parameters in Blood Samples

is present.

proportional to cell density [21].

Between Two Consecutive Schedules of Hemodialysis 117

Fig. 2. Qualitative platelets disorders: platelets with abnormality bizarre in shape and size The optical conventional techniques used for platelet counting have limits that are influenced by the human eye, especially for detection of objects <5 microns. Thus, the modern trend is to replace optical systems and introduce some electronic optical systems. Electronic microscopy with Beta rays and wave lengths thousands of times smaller than the white light gives a higher power of resolution and thus, analyzers well-suited for platelet count in biological fluids are ones that use either of two methods: WCS technology of impedance (Volume, Conductivity and Scatter Light), {Graphic 4} and WOC analysis by laser ray (White Cell Optical Count) [20], Graphic 5. In WCS, the fat within the cell membrane behaves as an object that facilitates generation of an electronic impulse with an amplitude proportionate as the cell volume and helps create a potential difference next to the count cleft Since VCS technology includes a highly accurate measure of cell volume, we can use this information to correct the conductivity and scatter signals. The result of this volumetric compensation is a pair of measurements that are very powerful, and unique to Beckman Coulter. The HMX Coulter Analyzer utilizes the Coulter principle to provide cellular information for the complete WBC differential. The system measures the amount of light "lost" due to diffraction and absorbance as compared to full transmission when no cell

The signals collected are converted into voltage pulses and are processed. The size and shape of the voltage pulses are equivalent to the unique nuclear and morphologic structure of the cells being analyzed conductivity offers information about opacity, which is directly

In WOC technology the laser light measures cellular elements in 4 specific angles and every angle of light scatter from 0° through 90° is influenced by cellular size. The low angles are the most affected, and are often used as an indirect estimation of cellular size. The zero angle measures the dimension of cells and impedance is used to count RBC corpuscles > 36

fL and platelets, corpuscles with the dimensions between 2-20 fL [22].

method). The effect of Hepcidin is to increase intracellular iron stores in ACD, decreased dietary iron absorption and decrease circulating iron concentration in chronic anemia from inflammations and infections [19].


Graphic 3. The RBC count is increased for the amout of hemoglobin present. The concentration of hemoglobin in the RBCs is slighlty decreased (hypochromic) and the cells are small (microcytic). The varioation in RBC size (RDW) is whitin normal limits.

In chronic renal failure (CRF), the peripheral blood smear can reveal activated thrombocytes with fingers( burr cells) as isolated cells or organized in groups. By contrast, with diabetic ketoacidosis, one can see the reverse phenomenon, thrombocytes that are isolated, with round shape form and without activated fringes. Figure 2 It is interesting that platelet activation markers were associated with the severity of DIC and erroneous platelet counts, suggesting that platelet activation is a potential source for the inter-method variation in platelet counts. More attention needs to be given to improve the accuracy of platelet counts, especially in clinical conditions with high levels of platelet activation.

It is well known that white light is comprised of luminous waves with different wave lengths of 750-250 nm. Optical microscopy uses light diffraction but can have light reflection, refraction, diffusion and dispersion phenomena, especially through media with non-homogenous densities.

Thus, it has been recommended that one conduct platelet counts using phase-contrast microscopy, which helps eliminate such light interference phenomena because it the image is formed by a diffraction process in two stages: incident light diffraction and diffraction of the light refracted in the objective.

method). The effect of Hepcidin is to increase intracellular iron stores in ACD, decreased dietary iron absorption and decrease circulating iron concentration in chronic anemia from

Graphic 3. The RBC count is increased for the amout of hemoglobin present. The

are small (microcytic). The varioation in RBC size (RDW) is whitin normal limits.

especially in clinical conditions with high levels of platelet activation.

non-homogenous densities.

the light refracted in the objective.

concentration of hemoglobin in the RBCs is slighlty decreased (hypochromic) and the cells

In chronic renal failure (CRF), the peripheral blood smear can reveal activated thrombocytes with fingers( burr cells) as isolated cells or organized in groups. By contrast, with diabetic ketoacidosis, one can see the reverse phenomenon, thrombocytes that are isolated, with round shape form and without activated fringes. Figure 2 It is interesting that platelet activation markers were associated with the severity of DIC and erroneous platelet counts, suggesting that platelet activation is a potential source for the inter-method variation in platelet counts. More attention needs to be given to improve the accuracy of platelet counts,

It is well known that white light is comprised of luminous waves with different wave lengths of 750-250 nm. Optical microscopy uses light diffraction but can have light reflection, refraction, diffusion and dispersion phenomena, especially through media with

Thus, it has been recommended that one conduct platelet counts using phase-contrast microscopy, which helps eliminate such light interference phenomena because it the image is formed by a diffraction process in two stages: incident light diffraction and diffraction of

inflammations and infections [19].

The optical conventional techniques used for platelet counting have limits that are influenced by the human eye, especially for detection of objects <5 microns. Thus, the modern trend is to replace optical systems and introduce some electronic optical systems.

Electronic microscopy with Beta rays and wave lengths thousands of times smaller than the white light gives a higher power of resolution and thus, analyzers well-suited for platelet count in biological fluids are ones that use either of two methods: WCS technology of impedance (Volume, Conductivity and Scatter Light), {Graphic 4} and WOC analysis by laser ray (White Cell Optical Count) [20], Graphic 5. In WCS, the fat within the cell membrane behaves as an object that facilitates generation of an electronic impulse with an amplitude proportionate as the cell volume and helps create a potential difference next to the count cleft Since VCS technology includes a highly accurate measure of cell volume, we can use this information to correct the conductivity and scatter signals. The result of this volumetric compensation is a pair of measurements that are very powerful, and unique to Beckman Coulter. The HMX Coulter Analyzer utilizes the Coulter principle to provide cellular information for the complete WBC differential. The system measures the amount of light "lost" due to diffraction and absorbance as compared to full transmission when no cell is present.

The signals collected are converted into voltage pulses and are processed. The size and shape of the voltage pulses are equivalent to the unique nuclear and morphologic structure of the cells being analyzed conductivity offers information about opacity, which is directly proportional to cell density [21].

In WOC technology the laser light measures cellular elements in 4 specific angles and every angle of light scatter from 0° through 90° is influenced by cellular size. The low angles are the most affected, and are often used as an indirect estimation of cellular size. The zero angle measures the dimension of cells and impedance is used to count RBC corpuscles > 36 fL and platelets, corpuscles with the dimensions between 2-20 fL [22].

Variability of Biological Parameters in Blood Samples

phenomenon.

interference phase.

manual counting.

**5. Conclusions** 

Between Two Consecutive Schedules of Hemodialysis 119

failure), errors in platelet counts occur in optical microscopy due to the double refraction

This phenomenon occurs because particles <5μ create reflection, refraction, diffusion and diffraction of light through environments with different properties (ε) and in solutions with higher osmolality. The diffraction of rays by objects < 5μ are not sufficiently dispersed and

The angle comprised between the rays which delimit the light cone represents the numerical aperture(A) and the resolution power or the spectral separation power, dependent of light

Optical instruments contain light separation media that are non-homogenous, including glass (ocular, objectives, prisms, air) and thus yield losses in the intensity of the incidence, reflection, refraction and diffraction rays through the media crossed by them. After the expression: S = [n1 - n2/n1 x n2]², where "n" represent the refraction index from the environment, the losses of the incidental ray, because of interference, is 4% from the intensity of incidental fascicle [25]. Platelets with dimensions <2μ and are met by light rays, with a very high speed of propagation through liquid environments may not be seen in optical microscopy if increased osmolarity concentrations are present. In accordance with Huygens interference principle, clefts S1 and S2 become secondary oscillation sources. The sources of secondary vibration of the light generated waves can overlap between the interference areas and

Thus, the average of the intensity values of the object light image in the ocular may has the range between 0 value and 4 'e" (e= ¹/4nS) in the minimal, respectively, maximum

The minimal intensity state of the light reflected on the object in order to create its reversed

The normal thrombocytes having the diameter of 2-4 microns, create reflection, refraction, diffusion and diffraction of light through microscopy and become more less visible to

There has been some debate over which counting principle, between the impedance and optical methods, measures platelet counts more accurately. Some studies suggested that the accuracy of the optical methods was superior for thrombocytopenic specimens, while recent studies demonstrated the impedance method to be more accurate for samples from patients

The anemia of hospitalized patients with chronic or acute renal diseases undergoing hemodialysis exists in our study in 60% from studied cases and must be managed of laboratory medicine in collaborative with the clinician. A routine anemia screening should be recommended using HGB, HCT and erythrocytes indexes MCV, MCH, MCHC and must

An iron panel (serum iron, TIBC, IST% and RPI) is useful in differentiating anemia of chronic disease from iron deficiency. By this study the anemia can be defined as a decrease of HGB and or hematological indexes with 10%from initial normal values, with cut of

The methods used to assess platelet counts of hemodialysis patients, optical microscopy, peripheral blood smear and use of the cytometry principle with impedance principle (VIC),

only a part of the issued light falls on the object from the objective of microscope.

diffraction (D), light wave length (L) and numerical aperture (A) , (D = L / A ), [24].

fringes, thus yielding what is termed the interference domain.

image in the ocular leads image loss for the human eye.

be redefined the anemia by these common parameters.

117g/L HGB for men and 108g/L HGB for women.

undergoing cytotoxic chemotherapy [26].

Graphic 4. VCS Technology includes a highly accurate measure of cell volume and this information is added to the Conductivity and Scatter signals. Every angle of light scatter from 0° through 90° is influenced by cellular size. The low angles are most affected, and are often used as an indirect estimation of cellular size.

Graphic 5. WOC channel (White Cell Optical Count) is used for counting blood cells and differential count by laser technokigy. The result of this volumetric compensation is a pair od measurements that are very powerful, and unique to Beckman Coulter.

The main elements that maintain the plasma osmolality in normal values (310 Osm/l) are; Na, K, urea and glucose. Serum osmolality is normal whenever the osmotic pressure set by urea and glucose is negligible and the Na+ concentration can largely define osmolality [Osm = 2.1 x conc Na mEq/L).Whenever the level of plasma urea or glucose is high, the osmolality becomes: 2.1 (Na + K) mmol/L + urea mg% / 2.8 + glucose mg% /18.02), result expressed in Osm /L[23]. In metabolic states with high osmolality (e.g. from chronic renal

Graphic 4. VCS Technology includes a highly accurate measure of cell volume and this information is added to the Conductivity and Scatter signals. Every angle of light scatter from 0° through 90° is influenced by cellular size. The low angles are most affected, and are

Graphic 5. WOC channel (White Cell Optical Count) is used for counting blood cells and differential count by laser technokigy. The result of this volumetric compensation is a pair

The main elements that maintain the plasma osmolality in normal values (310 Osm/l) are; Na, K, urea and glucose. Serum osmolality is normal whenever the osmotic pressure set by urea and glucose is negligible and the Na+ concentration can largely define osmolality [Osm = 2.1 x conc Na mEq/L).Whenever the level of plasma urea or glucose is high, the osmolality becomes: 2.1 (Na + K) mmol/L + urea mg% / 2.8 + glucose mg% /18.02), result expressed in Osm /L[23]. In metabolic states with high osmolality (e.g. from chronic renal

od measurements that are very powerful, and unique to Beckman Coulter.

often used as an indirect estimation of cellular size.

failure), errors in platelet counts occur in optical microscopy due to the double refraction phenomenon.

This phenomenon occurs because particles <5μ create reflection, refraction, diffusion and diffraction of light through environments with different properties (ε) and in solutions with higher osmolality. The diffraction of rays by objects < 5μ are not sufficiently dispersed and only a part of the issued light falls on the object from the objective of microscope.

The angle comprised between the rays which delimit the light cone represents the numerical aperture(A) and the resolution power or the spectral separation power, dependent of light diffraction (D), light wave length (L) and numerical aperture (A) , (D = L / A ), [24].

Optical instruments contain light separation media that are non-homogenous, including glass (ocular, objectives, prisms, air) and thus yield losses in the intensity of the incidence, reflection, refraction and diffraction rays through the media crossed by them. After the expression: S = [n1 - n2/n1 x n2]², where "n" represent the refraction index from the environment, the losses of the incidental ray, because of interference, is 4% from the intensity of incidental fascicle [25].

Platelets with dimensions <2μ and are met by light rays, with a very high speed of propagation through liquid environments may not be seen in optical microscopy if increased osmolarity concentrations are present. In accordance with Huygens interference principle, clefts S1 and S2 become secondary oscillation sources. The sources of secondary vibration of the light generated waves can overlap between the interference areas and fringes, thus yielding what is termed the interference domain.

Thus, the average of the intensity values of the object light image in the ocular may has the range between 0 value and 4 'e" (e= ¹/4nS) in the minimal, respectively, maximum interference phase.

The minimal intensity state of the light reflected on the object in order to create its reversed image in the ocular leads image loss for the human eye.

The normal thrombocytes having the diameter of 2-4 microns, create reflection, refraction, diffusion and diffraction of light through microscopy and become more less visible to manual counting.

There has been some debate over which counting principle, between the impedance and optical methods, measures platelet counts more accurately. Some studies suggested that the accuracy of the optical methods was superior for thrombocytopenic specimens, while recent studies demonstrated the impedance method to be more accurate for samples from patients undergoing cytotoxic chemotherapy [26].
