- 07 Jun 2017
- Anurfeate: Nucleus is absent Ex: RBC of mammal
- Mononucleate: It is very common. Usually the cell contains a single nucleus
- Binucleate: Two nuclei are present. Ex: cells of cartilage.
- Poly nucleate: Three to many nuclei are present. In animals poly nucleate cell is called "Syncytial cell". Ex: Syncytial cell of osteoblast.
- Cylindrical: Ex: Columunar epithelium.
- Horse shoe shape: Ex: Paramecium meganucleus.
- Tri lobed nucleus: Ex: WBC of mammals.
- Branched nucleus: Ex: Silk spining cell of insect larvae.
- Irregular nucleus: Ex: Glandular cell of insect.
- Nuclear membrane.
- Nucleo-plasm or karyolymph.
- Chromatin network.
- It gives protection to the genome.
- Helps in the reflection of DNA.
- During synaptic pairing in meiosis it attracts the homologous chromosomes.
- Granular zone
- Fibrillar zone
- Protenaceious part,
- Nucleolar associated chromatid.
- It is helpful in biogenesis of ribosomes.
- It plays a major role in mitosis.
- Nucleus plays a major role in the general metabolism of the cell
- It is helpful in the synthesis of ribosomes.
- It is helpful in the synthesis of RNA.
- It controls the synthesis of proteins.
- It is the seat of heredity.
- Mechanical support: E.R. will give mechanical support to cytoplasm. Hence it is called cytoskeleton.
- Intracellular transport: E.R. functions as circulatory system of the cell. It transport substance from one place to another place, in the cell it is called intracellular transport.
- Protein synthesis: ER. will provide surface for the attachment of ribosomes. These ribosomes will synthesise the proteins.
- Synthesis of lipoproteins: Smooth E.R. will synthesise lipids. In the Golgi complex the glycerides get associated with proteins produced by the E.R. Thus complex lipoproteins are formed.
- Detoxification: Smooth E.R. will detoxify internal, or external toxins. If toxins are more in the body more smooth E.R. will be produced.
- A.T.P. Synthesis: E.R. membranes are the sites of A.T.P, synthesis in the cell.
- Formation of other membranes: In the cell E.R. gives rise to.
a) Cisternae of Golgi apparatus.
b) Outer membrane of nucleus.
- Impulse conduction: In the muscle sarcoplasmic eticulum will release calcium which is responsible for the muscle action In 1926 "Porter" stated that E.R. membrane shows ionic grandients, and electric potential.
- The outer membrane of the nucleus will give buds. They unite to form annulate lamellae.
- Annulate lamellae will give rough E.R.
- From smooth E.R. golgi will form.
- Vesicles will give plasma lemma.
- According to GERL system Golgi will give rise to primary lysosomes. Thus there is an interrelationship among the membrane systems.
- Phospholipids: These substances have a composition which is in between the structure of phospholipids of endoplasmic reticulum and plasma membrane.
- Enzymes: ATP-ase, CPT-ase, transphorases, etc. enzymes are. present.
- Carbohydrates: Glucose, manose, galactose carbohydrates are seen.
- From E R: Beams & Kessel' in 1968 proposed that Golgi system cisternae arise from Endoplasmic reticulum.
- Rough E.R. produces protein which will be transfer to smooth ER.
- Vesicles are developed from smooth E.R.
- By the fusion of vesicles cisternae are formed. From the cisternae secretory vesicles are formed on the maturing face. (These sides are called "G.E.R.L" - Golgi Endoplasmic Reticulum Lysosome)
2) From nuclear membrane: In 1965 "Bouch" described the origin of golgi from the outer membrane of the nucleus. Vesicles are pinched off from the outer nuclear membrane and they are united to form cistemae.
- Proteins are produced by ribosome.
- They will be transmitted to smooth E.R.
- From there they are concentrated at Golgi complex.
- From the Golgi complex the secretory vesicles are formed.
- 07 Jun 2017
Chemical Composition: Every lysosome will show hydrolytic enzyme. They are important in digestion of food such as 40 enzymes arc recognized in lysosome.
- It is a newly formed lysosome.
- It is formed from golgi.
- It forms from G.E.R.L, which means 'Golgi associated with Endoplasmic Reticulum will give Lysosome". This was stated by Dyson 1978.
- This is called original lysosome.
- A original lysosome units with a phagocytic or pinocync vesicle and forms a phagosome.
- In this phagosome the food is digested.
List of some hydrolytic enzymes seen in Lysosome:
Functions of Lysosomes:
- 07 Jun 2017
- In 1857 Kolliker observed mitochondria and called them as sarco-somes.
- Flemming called them as Fila.
- Altmann in 1890 called them as Bioplasts.
- Benda gave the name mito-chondria.
- Porter & Palade described their electron microscopic structure.
- Mitochondria are present in all eukaryotic cells.
- Outer chamber
- Inner chamber.
- Luck stated free existing mitochondria will elongate and divide and new mitochondria are formed.
- Morrison stated mitochondria arise from either plasma membrane or endoplasmic reticulum.
- In the cytoplasm small particles may-be present they are called promitochondria they may give rise to mitochondria.
- In Bacteria & Mitochondria electron transport system is present in plasma membrane and in inner membrane respectively.
- Bacterial plasma membrane shows mesosomes, mitochondrial crystae can be compared with them.
- Both bacteria & Mitochondria will show circular D.N.A.
- In both bacteria and mitochondria ribosomes are reported.
- Both bacteria and mitochondria will produce AT.P. and R.N.A. Hence we consider the mitochondria might have originated from bacterial cell. In the cell mitochondria will function as semi-autonomous body.
- A.T.P. Synthesis: It is the power house of the cell. It brings oxidation of food. Hence Kreb's cycle reactions, electron transport system enzymes are located in mitochondria. By the oxidation of food energy is liberated in the form of A.T.P. (Oxidative phosphorelation takes place.)
- Yolk formation: Mitochondria are responsible for the fcri soiydk in the developing ovum Granules are formed in the matrix They oeconie large masses Mitochondrion is converted into yolk storing body.
- Mitochondrian sperm formation: When spermatid become? <pe'm mitochondria will form a spiral around the axial filament. This is called Neben-kem. It forms the middle piece of the sperm.
- Origin of new system: It is believed that some of the ceil organelles may originate from mitochondria.
- Heat production: In the oxidation of food ATP is released. Only 45% of the total energy is trapped in the form of ATP. The remaining 35% of ATP will come out as heat. (In birds and mammals this heat is useful for the maintenance of body temperature.).
- ATP released during respiration (because of mitochondria ) will take part in many biosynthetic paths of the cell.
- Nageli and Cramer called this membrane as cell membrane in 1855.
- In 1931 "Plowe" named this as plasmalemma.
- Modem cell biologists prefer to call it plasma membrane (According to De Robertis).
- Danielli & Davson in 1952 proposed molecular model of plasma membrane.
- Robertson proposed unit membrane concept in 1960.
- Human R.B.C. are kept in dilute solution (Hypotonic). Then it swells. It breakes and protoplasm will go out (Haemolysis). The remaining membrane is called Ghost. It is used for plasma membrane studies.
- Liver cell or the membrane surrounding the nerve fibre are used as a material for the study of plasma membrane.
- Outer layer is made by proteins....20 A°
- Middle layer is bimolecular lipid layer....35A°
- Inner layer is made by proteins ...20A°
- Danielli Davsons model: It shows thin lipid layer with protein absorbed on both the sides. The lipid contains polar heads pointing out sides and ion poiar parts run transversly. In 1954 Danielli modified this model and gave a new model.
- Robertson unit membrane concept: Robertson in 1959 described trilaminar structure of plasma membrane. It contains an outer and inner protein layers in between them a bimolecular lipid layer is present.
1) The unit membrane is 75A° thick.
2) The Outer and inner protein layers are 20A° thick.
3) In between them the lipid layer will show 35A° units.
4) The polar hydrophyllic ends of the lipid layer will face the proteins. Where as the hydrophobic ends of the lipids are away from the proteins.
- Fluid Mosaic model: In 1972 "Singer" & "Nicholson" proposed this model.
- Cell membrane is a mosaic of lipids and proteins.
- Lipids are arranged in a bilayer way. It forms the structural frame work of plasma membrane.
- Protein molecules are arranged in 2 ways.
1) Extrinsic proteins: These proteins are located adjacent to the outer and inner surfaces of the lipid layer.
- Hydrophobic end: It is water hating, non polar end.
- Hydrophilic end: It is water loving part. It is called polar end. It is present near the proteins.
- Structural pore: These are permanent openings.
- Dyanamic pores: They form during the intake of material.
- Protein channel pores: These are small channels of specific proteins through which some ions can diffuse.
- Modification of plasma membrane: To perform specialised functions plasma membrane will show some modifications.
- They are (a) Microvilli, (b) Inter digitations, (c) Desomosomes, tight junctions etc.
a) Microvilli: In certain parts the plasma membrane will show minute infloldings they are called microvilli. They show -1 mili micron diameter and 6 milli micron length. In some cases the villi are connected with endoplasmic reticulum. These microvilli are more in intestinal mucosa cells. The microvilli show cytoplasm with micro filaments. The microvilli will increase the absorptive surface of the intestinal epithelium.
b) Inter digitations: At some places the plasma membrane of adjacent cells will develop into finger like projections they are called inter digitations.
c) Desmosomes: The plasma membranes of adjacent cells become thicker in certain regions. On these thick areas fine, filaments are present. They are called tonbfilaments or tonofibrils. Such parts are called desmosomes. Desmosome is concerned with cell adhesion and maintenance of cell shape.
d) Terminal bar: It is a desmosome without tonofibrils. It is called terminal bar.
e) Zonula acculdens: These are special area of adjacent cells where the 2 plasma membranes fuse. These tight junctions are present below the apical boarder. It gives mechanical attachment between cells.
- Endocytosis: It is the process of by which large particles of food are engulfed. 1) Pinocytosis or cell drinking : Ingestion of liquid through plasma membrane is called pinocytosis.
- Micro pinocytosis is observed at submicroscopic level. When a liquid come nearer to plasma membrane, it forms a vesicles. It is called pinoc vesicle. After some time it is pinched off from the plasma membrane and becomes pinocytic vesicle in the cytoplasm
- Phagocytosis: The process of taking solid food or solid material through the cell membrane into the cell is called phagocytosis. Phagocytosis means to eat. It is first discovered by "Metknikoff
- Exocytosis: It is also called cell vomiting . The process of sending out products from the cell to the out side is called exocytosis or emeiocytosis.
- Active transport: If molecules or ions move against the concentrate gradient through plasma membrane, it is called active transport. For this energy is required. Now a days it is proved that carrier system is seen in the plasma membrane and it is responsible for active transport.
- It is believed that it develop de novo.
- It is formed by the assembly of lipids & proteins.
- It is believed that it develops from other membrane systems.
- 06 Jun 2017
- It gives strength to the cell and forms cytoskeleton.
- Granular endoplasmic reticulum will produce proteins.
- Agranular endoplasmic reticulum will produce lipids.
- It forms the work bench for many biochemical reactions in the cell.
- In the central matrix of mitochondria respiratory enzymes are present. The take up Krebs cycle reactions.
- In the inner membrane of mitochondria electron transport enzymes are present.
- Mitochondria helps in the oxidation of the food material and liberates energy , Hence they are called power houses of cell.
- In the mitochondria a circular DNA is present. Hence mitochondria is also c. led semi autonomous body.
- Lysosome is helpful in the digestion of the food.
- At starvation lysosome will digest cell organelles.
- Lysosome can dissolve the cell. It is called suicide. Henc lysosomes are called suicidal bags of cells.
|Feature||Prokaryotic Cell||Eukaryotic Cell|
|2.||DNA||single, circular and naked||more than one and combined with proteins|
|5.||Division||amrtosis||mitosis or meiosis|
|6.||Ribosomes||70S(50S+30S scattered in cytoplasm||80S(60S+40S) Found attached to ER or free in cytoplasm.|
|9.||Chloroplasts||absent||present in plant cells, absent in animal cell|
|13.||Cell wall||non-cellulose||cellulose only in plants cells|
|14.||Respiratory enzymes||located in plasma membrane||enclosed in mitochondria.|
- 06 Jun 2017
- In blue green algae cell shows a cell wall, enclosing protoplasm. In the protoplasm the peripheral coloured part is the chromateplasm and the central colorless part is the centroplasm. In the centroplasm there is DNA as the genetic material.
- Bacterial cell consists of a rigid cell enclosing protoplasm. The rigid cell is surrounded by slime layer or capsule. The protoplasm shows a peripheral plasma membrane which is often producing coiled mesosomes to carry respiration. The central part of the cell shows a long coiled thread like DNA. Protoplasm contains many ribosomes, fat bodies, volutin granules etc. The cells also show pili and flagella.
|Year||Names of contributor||Cytological contribution|
|1824||R.J.H. Dutrochet||Showed that all animals and plants composed of cells.|
|1826||Turpin||Reported the occurrence of cell division.|
|1831||R. Brown||Described the nucleus|
|1835||Felix Dujardin||Described protoplasm as ("Sarcode")|
|1838||M.J.Schleiden||Proposed "Cell theory"|
|1839||T.Schwann||Applied "Cell theory" to animals.|
|1840||J.E. Purkinje||Named the cell contents as Protoplasm.|
|1855||R.Virchow||Stated that all cells arise from pre-existing cell.|
|1863||Waldeyer||Chromosomes of cell|
|1871||F. Miescher||discovered nucleo-protein and nucleic acid.|
|1882||Strasburger||described mitosis in plant cells|
|1887||E.Van Benden||discovered centrioles.|
|1888||T.Boweri||described the centrioles.|
|1888||Waldeyer||Introduced the term chromosome.|
|1902||W.S.Sutton||Proposed "the chromosome theory" heredity.|
|1905||J.B.Farmer along with J.E.Moore.||Coined the term meiosis|
|1943||A.Claude||Isolated cell components like ribosomes, mitochondria and nuclei|
|1952||C. Du Duve||Identified hysosomes.|
|1953||J.D.Watson and F.H.C. Crick||Proposed the double helixmodel for the DNA molecule.|
|1959||S.Ochoa||Synthesis of polyribonuclotide in vitro.|
|1959||A.Kornberg||Synthesis of polydeoxiri-|
|1968||M.W.Nirenberg and H.G.Khorana.||Triplet genetic code.|
|1968||R.H.HoIley||discovery of base sequence of RNA|
This method is optional for estimation of hemoglobin and this method is recommended by International Committee for Standardization in hemotology. This is because in this method all type of hemoglobin are transformed to cyanmethemoglobin (except sulfhemoglobin), and a firm and trustworthy standard is available.
When Blood is mixed with a solution of potassium cynide, potassium ferricyanide and Drabkin’s solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. Potassium ferricyanide transforms hemoglobin to methemoglobin, and methemoglobin combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). This way all types of hemoglobin present in blood are entirely transformed to a single compound cyanmethemoglobin. When the reaction is entire, absorbance of the solution is deliberate in a spectrophotometer at 540 nanometer. Hemoglobincyanide has a wide absorbance peak at this wavelength. The absorbance is compared with that of the standard hemiglobincyanide solution by using a formula to obtain the amount of hemoglobin.
- Spectrophotometer or photoelectric colorimeter
- Pipette 5 ml
- Sahli’s pipette
- Drabkin’s Solution
- Cyanmethemoglobin standard solution with known hemoglobin value
Blood obtained from skin puncture or EDTA-anticoagulated venous blood.
- Take 5 ml of Drabkin’s solution in a test tube and add 20 μl of blood. This way, we will get the dilution of 1:25. Now mix the mixture and allow to stand for atleast 5 minutes. This time is adequate for transformation of hemoglobin to hemiglobincyanide.
- Pour the test sample to a cuvette and read the absorbance of the test sample in a spectrophotometer at 540 nanometer or in a photoelectric colorimeter using a yellow-green filter. Also read the absorbance of the standard solution. Absorbance must be read against Drabkin’s solution.
- From the formula given below, the hemoglobin value is derived.
Hemoglobin in gm/dl = [Absorbance of test sample ÷ Absorbance of standard] x concentration of standard x [Dilution factor ÷ 100]
Preparation of table and graph
Result can be obtained quickly, if the table of graph is prepared which correspond absorbance with hemoglobin concentration. This is markedly acceptable when huge number of samples are daily processed on the same instrument.
For the preparation of a calibration graph, adulterate cyanmethemoglobin standards are commercially available. As another option, standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution. Concentration of hemoglobin (horizontal axis) in each dilution is arranged against the absorbance (vertical axis) on a linear graph paper. A straight line connecting the points and passing through the origin is obtained. A table can be prepared relating absorbance to concentration of hemoglobin from the help of this graph.
- The hemiglobincyanide solution is stable so that delay in getting the reading of absorbance does not influence the result.
- High TLC (total leukocyte cunt) (> 25,000/μl), abnormal plasma proteins (e.g. in Waldenström’s macroglobulinemia, multiple myeloma) or lipemic blood (hypertriglyceridemia), can cause the error in results.
When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is estimated by reading the value directly from the scale.
- Sahli’s hemoglobinometer: This equipment consist of a comparator with a brown glass standard and Sahli’s graduated hemoglobin tube which is marked in percent and gram.
- Hemoglobin pipette or Sahli’s pipette (marked at 0.02 ml or 20 μl).
- Stirrer (a small glass rod).
- Dropping pipette (dropper).
- N/10 hydrochloric acid
- Distilled water
Blood is obtained directly by skin puncture or EDTA-anticoagulated venous blood.
- The N/10 hydrochloric acid is place into Sahli’s graduated tube up to mark 2 grams.
- With the help of Sahli’s pipette, take blood sample exactly up to 20 μl mark. Blood adhering to the outer part of the pipette is wiped away with the help of tissue paper (absorbent paper) or cotton (gauze piece).
- Add blood sample to the N/10 hydrochloric acid solution which is placed into Sahli’s graduated tube, mix the mixture with the help of a glass stirrer, and allow the tube to stand for 10 minutes.
- Add distilled water drop by drop into the mixture placed in Sahli’s graduated tube, till the color of the solution matches that of the brown glass standard.
- Take the reading of the lower meniscus from the Sahli’s graduated tube in grams.
- All the Sahli’s graduated tube is marked in both percent and grams figures, this is because (a) different manufacturers of hemoglobinometers have different values as 100%, so that blood sample will yield different results on different instruments and (b) no single hemoglobin is can be evaluate as 100% since it is different according to the sex and age of the individual and altitude.
- Disadvantages of Sahli’s method:
• It is impossible to match the color perfectly of the mixture into the Sahli’s graduated tube with the brown glass standard.
• Minimum 1 hour is required for the maximum color development of acid hematin because 95% color of acid hematin is attained at the end of 10 minutes.
• Sulfhemoglobin, methemoglobin and carboxyhemoglobin cannot be converted into acid hematin. Fetal hemoglobin is also not converted to acid hematin and therefore this technique is not appropriate for in small infants.
• The acid hematin solution is not firm and stable, and the color development is slow.
• Lights may affect the visual comparison of color.
• Color of the brown glass standard dims with time.
• Personal error in matching the color of the mixture in Sahli’s graduated tube with the brown glass standard is 10%.
Hemoglobin is composed of heme (iron + protoporphyrin) and globin polypeptide chains. It is present in the red blood cells of all vertebrates except Channichthyidae (the family of fish: white-blooded fish also called crocodile fish found in southern South America and the Southern Ocean around Antarctica). It carries oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs.
In humans, hemoglobin is not homogeneous and normally different variants and derivatives exist. Normal hemoglobin variants are fetal hemoglobin (Hb F), adult hemoglobin (Hb A), Hb A2 and embryonic hemoglobins (Gower I, Gower II and Portland). They differ from each other on the basis of the structure and the type of polypeptide chains.
INDICATION FOR HEMOGLOBIN ESTIMATION
- Screening for polycythemia: Polycythemia is a disease state in which the hemoglobin level and hematocrit (HCT) or packed cell volume (PCV) value is elevated. It may be primary, secondary or relative.
- To determine presence and severity of anemia: Anemia is a disease state in which the hemoglobin concentration or oxygen-carrying capacity of blood is low. Clinical signs and symptoms (conjunctival vessels, polar of skin, mucosal membranes) are unreliable for the diagnosis of anemia. Anemia is best determined by estimation of hemoglobin and hematocrit (HCT) or packed cell volume (PCV).
- To assess response to specific therapy in anemia.
- Estimation of red cell indices (along with hematocrit (HCT) or packed cell volume (PCV) and red cell count) i.e. mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC).
- Selection of blood donors in the blood bank.
METHOD FOR ESTIMATION OF HEMOGLOBIN
There are different methods for estimation of hemoglobin. These are:
(1) Colorimetric methods: In these methods, the color comparison is made between the standard and the test sample, either visually or by colorimetric methods.
- Visual methods: Sahli’s acid hematin method, WHO hemoglobin color scale and Tallqvist chart.
- Photoelectric or colorimetric methods: Cyanmethemoglobin (hemoglobin-cyanide) method, oxyhemoglobin method, and alkaline hematin method.
(2) Gasometric method: In this method, oxygen-carrying capacity of red blood cells (RBCs) is measured in a Van Slyke apparatus. The amount of hemoglobin is then derived from the formula that 1 gram of hemoglobin carries 1.34 ml of oxygen. However, this method measures only physiologically active hemoglobin, which can carry oxygen. It does not measure methemoglobin, sulfhemoglobin, and carboxyhemoglobin. Also, this method is expensive and time-consuming, and the result is about 2% less than other methods.
(3) Chemical method: In this method, iron-content of hemoglobin is first evaluated. The value of hemoglobin is then derived indirectly from the formula that 100 grams of hemoglobin contain 374 mg of iron. This method is tiresome and time-consuming.
(4) Specific gravity method: In this method, an approximate value of hemoglobin is estimated from the specific gravity of blood as determined from copper sulfate technique. This method is simple and rapid. This method is useful and most common in mass screening like the selection of blood donors. See procedure.
Tallqvist Hemoglobin Chart
Tallqvist hemoglobin chart consists of a series of lithographed colors said to correspond to hemoglobin values ranging from 10% to 100%. In this method, a drop of blood obtained by finger puncture is placed on a piece of absorbent paper. The color produced is matched against the color on the chart and the corresponding reading is taken. The room of error is 20-50%. Although this method is very cheap and simple.
- 19 Jul 2016
USES OF RED CELL INDICES
(2) Differentiation of iron deficiency anemia from thalassemia trait: In iron deficiency, MCV, MCH, and MCHC are low, while in thalassemia trait, MCV and MCH are low and MCHC is normal.
RBC count in million/cmm
• Non-megaloblastic macrocytosis: Chronic alcoholism, liver disease, hypothyroidism, normal pregnancy, reticulocytosis
In the presence of large number of abnormal red cells like sickle cells, and in dimorphic anemia (e.g. mixed normocytic and microcytic), MCV may be normal (since it is an average value) and thus unreliable for morphological classification.
Mentzer index is derived by dividing MCV with red cell count. Ratio of less than 13 is seen in thalassemia while ratio is more than 13 in iron deficiency anemia.
RBC count in millions/cmm
MCH is decreased in microcytic hypochromic anemia, and increased in macrocytic anemia and in newborns.
PCV in %
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentra-tion: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
1. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company, 2001.
2. Wallach J. Interpretation of Diagnostic Tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000⁻¹⁵
- 24 May 2016
Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.
A few drops of blood (collected in EDTA) are incubated with new methylene blue solution which stains granules of RNA in red cells. A thin smear is prepared on a glass slide from the mixture and reticulocytes are counted under the microscope. Number of reticulocytes is expressed as a percentage of red cells.
New methylene blue solution is prepared as follows:
- New methylene blue: 1.0 gm
- Sodium citrate: 0.6 gm
- Sodium chloride: 0.7 gm
- Distilled water: 100 ml
Reagent should be kept stored in a refrigerator at 2-6°C and filtered before use.
Suitable alternatives to new methylene blue are brilliant cresyl blue and azure B.
Capillary blood or EDTA anticoagulated venous blood can be used.
(1) Take 2-3 drops of filtered new methylene blue solution in a 12 × 75 mm test tube.
(2) Add equal amount of blood and mix well.
(3) Keep the mixture at room temperature or at 37°C for 15 minutes.
(4) After gentle mixing, place a small drop from the mixture on a glass slide, prepare a thin smear, and allow to dry in the air.
(5) Examine under the microscope using oil-immersion objective. Mature red cells stain pale green blue. Reticulocytes show deep blue precipitates of fine granules and filaments in the form of a network (reticulum). Most immature reticulocytes show a large amount of precipitated material in the form of a mass, while the most mature reticulocytes show only a few granules or strands. Any nonnucleated red cell is considered as a reticulocyte if it contains 2 or more blue-stained particles of ribosomal RNA.
(6) Count 1000 red cells and note the number of red cells that are reticulocytes. Counting error is minimized if size of the microscopic field is reduced. This is achieved by using a Miller ocular disk inserted in the eyepiece; it divides the field into two squares (one nine times larger in size than the other). Reticulocytes are counted in both the squares and the red cells are counted in the smaller square.
REPORTING THE RESULT
(1) Reticulocyte percentage: The most common method of reporting is reticulocyte percentage which is calculated from the following formula:
Where NR is the Number of reticulocyte counted and NRBC is number of red blood cell counted.
Reference range is 0.5%-2.5% in adults and children. Reticulocyte count is higher in newborns.
(2) Absolute reticulocyte count = Reticulocyte percentage × Red cell count
Normal: 50,000 to 85,000/cmm
(3) Corrected reticulocyte count (Reticulocyte index)
Corrected reticulocyte count > 2% indicates reticulocyte release appropriate for the degree of anemia. If < 2%, reticulocyte release is inappropriate.
(4) Reticulocyte maturation production index
Estimated maturation time in days
- Reticulocyte percentage: 0.5 2.5%
- Absolute reticulocyte count: 50,000-85,000/cmm
- 24 May 2016
- As one of the baseline studies in anemia with no obvious cause
- To diagnose anemia due to ineffective erythropoiesis (premature destruction of red cell precursors in bone marrow seen in megaloblastic anemia and thalassemia) or due to decreased production of red cells: In hypoplastic anemia or in ineffective erythropoiesis, reticulocyte count is low as compared to the degree of anemia. Increased erythropoiesis (e.g. in hemolytic anemia, blood loss, or specific treatment of nutritional anemia) is associated with increased reticulocyte count. Thus reticulocyte count is used to differentiate hypoproliferative anemia from hyperproliferative anemia.
- To assess response to specific therapy in iron deficiency and megaloblastic anemias.
- To assess response to erythropoietin therapy in anemia of chronic renal failure.
- To follow the course of bone marrow transplantation for engraftment
- To assess recovery from myelosuppressive therapy
- To assess anemia in neonate
Reticulocyte Count Test
Reticulocyte Values In Sickle Cell Anemia
Reticulocyte Count Reference Range
Supravital Stain Reticulocytes
Reticulocyte Count Method
Reticulocyte Staining Procedure
Reticulocytes With Supravital Stain
Miller Disc Retic Count
Reticulocyte Count Procedure Miller Disc