Title of Invention	SYNERGISTIC RED BLOOD CORPUSCLE (RBC) AGGREGATING TONIC SOLUTIONS WITH A LONG SHELF FOR DETERMINING OSMOTIC FRAGILITY OF RED CELLS IN MICROTITRE PLATE WELLS.
Abstract	This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising: homogenized gelatin solutions in the concentration range of 0.1g to 6.0 g gelatin/L of distilled water, RBC haemolyzing cum aggregating solutions of iso-osmotic compound / compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0.1g-2.0 g/L,

Title of Invention

SYNERGISTIC RED BLOOD CORPUSCLE (RBC) AGGREGATING TONIC SOLUTIONS WITH A LONG SHELF FOR DETERMINING OSMOTIC FRAGILITY OF RED CELLS IN MICROTITRE PLATE WELLS.

Abstract

This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising: homogenized gelatin solutions in the concentration range of 0.1g to 6.0 g gelatin/L of distilled water, RBC haemolyzing cum aggregating solutions of iso-osmotic compound / compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0.1g-2.0 g/L,

Full Text	This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells. BACKGROUND It is well known that normal human red blood corpuscles (RBC) when suspended in solutions of reducing tonicity at some stage undergo osmotic haemolysis. However, at isotonic concentration they survive for a considerable length of time. The change in the ratio of red cell volume to surface area produced under pathological conditions changes the ability of these RBCs to resist osmotic haemolysis in the above mentioned solutions as compared to normal RBCs. The available tonic solutions for determining osmotic fragility of red cells consist of simple salt solutions to buffered salt solutions of strength 0.1 % to 1.0 % are for immediate use. These solutions when producing haemolysis of RBCs change turbidity of the solution, thereby causing changes in the transparency of the salt solutions. This optical change in the solution has been utilized for determining osmotic fragility of the RBCs. The said optical change in the solutions can either be measured by determining optical absorption of the suspension of the solution or by visually recording the change in the transmission of light through the solution. Alternatively, a red cell count of the surviving cells under the microscope gives an indication of the degree of osmotic haemolysis and hence the osmotic fragility of the RBCs under examination. All the above mentioned methods are cumbersome; generate voluminous test material and those which are based on visual recording of change in transparency of the solution depend upon a very subjective criteria for assessment of the results. Furthermore, the process requires skilled technicians and doctors for the conduct of the tests and review of the results. The other method for diagnosing a change in red cell volume directly measures the volume of each red cell and gives the mean corpuscular volume of the RBCs. This entails the use of Hi Tech. electronic counters which are capital intensive and require very rigid laboratory conditions for their satisfactory working and as such are neither suitable for field work environment nor affordable for routine use in all but specialized laboratories. Furthermore, they entail transportation of perishable blood samples expeditiously and under controlled conditions of temperature to the medical centers where further processing of the samples must take place immediately. The objective of this invention is to prepare a synergistic red blood corpuscle (RBC) aggregating tonic solution with a long shelf life for determination of the osmotic fragility of RBCs which can be prepared as a standardized solution for transportation and storage. The said solution will simplify the process of determination of osmotic fragility of red cells; make it manageable and use more definitive criteria for interpretation of results which can be followed by the most junior of the technical staff either at field or at laboratory. To achieve the said objective this invention provides synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising: homogenized gelatin solutions in the concentration range of 0.1 g to 6.0 g gelatin/L of distilled water, RBC haemolyzing cum aggregating solutions of iso-osmotic compound / compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0. lg-2.0 g/L, A listing of concentrations of compound / compounds iso-osmotic with normal saline solution reflects the extent of ionic disassociation and means that aqueous solution at the concentration shown can be mixed with normal saline or balanced salt solutions without altering the iso-osmotic relations as shown in table given below: The said solution after the initial osmotic haemolysis of the test red cells causes the remaining surviving cells to aggregate and form clearly defined buttons in the centre of the microtitre plate wells which can be easily visualised by the naked eye. The invention will now be described with reference to the accompanying drawings and following examples: Fig 1 shows micro titre plate wells. Fig 2 shows MOFTI results in Micro titre plate wells The synergistic red blood corpuscle (RBC) aggregating tonic solution with a long shelf life was prepared as follows: EXAMPLE 1: Homogenised gel solution of concentration 0.4 g gelatine/L of distilled water was prepared and lO.Og/L sodium chloride was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.4 g/L of the homogenised RBC aggregating solution was added as a preservative. EXAMPLE 2: Homogenised gel solution of concentration 0.3 g gelatine/L of distilled water was prepared 15.0 g/L sodium bicarbonate was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.3 g/L of the homogenised RBC aggregating solution was added as a preservative. EXAMPLE 3: Homogenised gel solution of concentration 0.3 g gelatine / L of distilled water was prepared and 30.0 g/L NaH2PO4 was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.3 g/L of the homogenised RBC aggregating solution was added as a preservative. EXAMPLE 4: Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 50.0 g/L Calcium lactate - 5H2O was added to form the aggregating tonic solution. Thereafter 0.4g/L Sodium Azide of the homogenised RBC aggregating solution was added as a preservative. EXAMPLE 5: Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 60.0 g/L glucose anhydride was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.5 g/L of the homogenised RBC aggregating solution was added as a preservative. EXAMPLE 6: Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 10.0 g/L NaH2PO4 + 15.0 g/L Na2HPO4.7H2O was added to form the aggregating tonic solution. ^Thereafter Sodium Azide 0,4 g/L of the homogenised RBC aggregating solution was added as a preservative. The above examples illustrate that synergistic isotonic RBC aggregating solutions can thus be prepared from a variety of iso-osmotic compounds singly or in combination when they are dissolved in the above mentioned homogenised gelatine solution and preserved with sodium Azide. The choice of the tonic strength of the actual synergistic tonic red cell aggregating solution will depend upon the requirement of the pathological RBC condition under investigation i.e. whether increase or decrease in osmotic fragility of RBCs occurs in that particular pathological condition. For the purpose, a range of tonic solutions : hypotonic and isotonic solutions can be obtained by simply changing the amounts and proportions of iso-osmotic compounds : sodium azide : gelatine. Within the range specified for the compounds, the said homogenised RBC aggregating tonic solution will aggregate the intact surviving RBCs to form button in microtitre plate wells for easy display. The most important use of the above solutions in hypotonic concentrations is in the diagnosis of microcytemia which is an integral feature of the carrier state of the genetic disorder called thalassemia. Although originally brought to India by the Alexander, thalassemic gene has persisted due to the protection it affords against Malaria. In our population the average incidence is 3.4%. However, in the high risk communities in entire Indogangetic belt, incidence as high as 14 % has been reported. WHO recommends mass screening for any genetic disorder if it exceeds 2.5%. Thalassemic gene carrier has no overt disability or clinical manifestation. The procedure for detecting microcytemia in a thalassemia carrier individual is: PROCEDURE The test process is very SIMPLE but requires METICULOUS care in regard to the quantity of blood added to the MOFTI (Micro Osmotic Fragility Test India) reagent bottles. a. label two bottles containing MOFTI reagent for each person to be tested. b. Open the bottle by removing the top cover and the dropper. Add to each of the bottles one drop of blood from either a finger prick or veinepuncture. In case of finger prick sample ensure complete drying of the cleansing agent i.e. alcohol / spirit used for cleaning the finger so as to prevent chemically induced haemolysis of RBC"s. Mix the contents of the bottle gently and close it with the dropper and the cover. c. Leave it undisturbed for 30 minutes. d. Mix the sample contained in the bottle gently to cause uniform suspension of the sample. e. Pour 2 drops of sample from eachbottle into the respective microtitre plate well, (shown in fig 1 of the accompanying drawings f. After all the samples have been poured; cover the microtitre plate and leave it undisturbed for 2 hours at room temperature at 25°C±5°C. g. Place the microtitre plate on a horizontal view box and observe HAEMOLYSIS or BUTTON formation in the wells, in duplicate sample of each patient. In case of dissimilarity the test will need to be repeated, (shown in fig. 2 of the accompanying drawings) RESULTS The reagent for MOFTI screening is so calibrated that only a BUTTON is reported as POSITIVE. Complete haemolysis or partial haemolysis with varying degree of ring formation is reported as NEGATIVE. CLINICAL SIGNIFICANCES 1. Decreased osmotic fragility has been primarily reported in two common clinical conditions e.g. Beta Thalassemia Trait (BTT) and iron deficiency Anaemia. The severity of Microcytosis in BTT however is far more pronounced than the fall in the haemoglobin leVel which may actually be within the normal range. 2. Other conditions where decreased osmotic fragility has been recorded are: Thalassemia Major, Sickle Thalassemia, Sickle Cell Trait, Haemoglobinopathy SS, E Thalassemia, D Thalassemia, HbH Disease, 3. Positive MOFTI has also been observed with elevated sedimentation rate. I claim: 1. Synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising: homogenized gelatin solutions in the concentration range of 0.1 g\|to 6.0 g gelatin/L of distilled water, RBC haemolyzing cum aggregating solutions of iso-osmojtic compound /- compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of O.lg-2.0 g/L, 2. Synergistic red blood corpuscle (RBC) aggregating tonic solutions as claimed in claim 1 wherein the preferable iso-osmotic compounds used in the concentration are given below: • Sodium Chloride - 1.0 g-20.0 g/L • Sodium bicarbonate - 1.5 g - 30.0 g/L • NaH2PO4 - 2.3 g-46.6g/L • Calcium Lactate - 5 H2O - 5.0 g - 100 g / L • Glucose, anhydrous - 5.6 g - 112 g / L • NaH2PO4 - 1.65 g-23.3 g/L + • + Na2HPO4 7H2O 1.84-36.8g/L 3. Synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells substantially as herein described with reference to the forego ing examples and accompanying drawings. This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising: homogenized gelatin solutions in the concentration range of 0.1 g to 6.0 g gelatin/L of distilled water, RBC haemolyzing cum aggregating solutions of iso-osmotic compound / compounds as herein described which are isoosmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0.1 g-2.0 g/L,

Full Text

This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells.
BACKGROUND
It is well known that normal human red blood corpuscles (RBC) when suspended in solutions of reducing tonicity at some stage undergo osmotic haemolysis.
However, at isotonic concentration they survive for a considerable length of time. The change in the ratio of red cell volume to surface area produced under pathological conditions changes the ability of these RBCs to resist osmotic haemolysis in the above mentioned solutions as compared to normal RBCs.
The available tonic solutions for determining osmotic fragility of red cells consist of simple salt solutions to buffered salt solutions of strength 0.1 % to 1.0 % are for immediate use. These solutions when producing haemolysis of RBCs change turbidity of the solution, thereby causing changes in the transparency of the salt solutions. This optical change in the solution has been utilized for determining osmotic fragility of the RBCs. The said optical change in the solutions can either be measured by determining optical absorption of the suspension of the solution or by visually recording the change in the transmission of light through the solution. Alternatively, a red cell count of the surviving cells under the microscope gives an indication of the degree of osmotic haemolysis and hence the osmotic fragility of the RBCs under examination. All the above mentioned methods are cumbersome; generate voluminous test material and those which are based on visual recording of change in transparency of the solution depend upon a very subjective criteria for assessment of the results. Furthermore, the process requires skilled technicians and doctors for the conduct of the tests and review of the results.
The other method for diagnosing a change in red cell volume directly measures the volume of each red cell and gives the mean corpuscular volume of the RBCs. This entails the use of Hi Tech. electronic counters which are capital intensive and require very rigid laboratory conditions for their
satisfactory working and as such are neither suitable for field work environment nor affordable for routine use in all but specialized laboratories. Furthermore, they entail transportation of perishable blood samples expeditiously and under controlled conditions of temperature to the medical centers where further processing of the samples must take place immediately.
The objective of this invention is to prepare a synergistic red blood corpuscle (RBC) aggregating tonic solution with a long shelf life for determination of the osmotic fragility of RBCs which can be prepared as a standardized solution for transportation and storage. The said solution will simplify the process of determination of osmotic fragility of red cells; make it manageable and use more definitive criteria for interpretation of results which can be followed by the most junior of the technical staff either at field or at laboratory.
To achieve the said objective this invention provides synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising:
homogenized gelatin solutions in the concentration range of 0.1 g to 6.0 g gelatin/L of distilled water,
RBC haemolyzing cum aggregating solutions of iso-osmotic compound / compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and
sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0. lg-2.0 g/L,
A listing of concentrations of compound / compounds iso-osmotic with normal saline solution reflects the extent of ionic disassociation and means that aqueous solution at the concentration shown can be mixed with normal saline or balanced salt solutions without altering the iso-osmotic relations as shown in table given below:
The said solution after the initial osmotic haemolysis of the test red cells causes the remaining surviving cells to aggregate and form clearly defined buttons in the centre of the microtitre plate wells which can be easily visualised by the naked eye.
The invention will now be described with reference to the accompanying drawings and following examples:
Fig 1 shows micro titre plate wells.
Fig 2 shows MOFTI results in Micro titre plate wells
The synergistic red blood corpuscle (RBC) aggregating tonic solution with a long shelf life was prepared as follows:
EXAMPLE 1:
Homogenised gel solution of concentration 0.4 g gelatine/L of distilled water was prepared and lO.Og/L sodium chloride was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.4 g/L of the homogenised RBC aggregating solution was added as a preservative.
EXAMPLE 2:
Homogenised gel solution of concentration 0.3 g gelatine/L of distilled water was prepared 15.0 g/L sodium bicarbonate was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.3 g/L of the homogenised RBC aggregating solution was added as a preservative.
EXAMPLE 3:
Homogenised gel solution of concentration 0.3 g gelatine / L of distilled water was prepared and 30.0 g/L NaH2PO4 was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.3 g/L of the homogenised RBC aggregating solution was added as a preservative.
EXAMPLE 4:
Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 50.0 g/L Calcium lactate - 5H2O was added to form the aggregating tonic solution. Thereafter 0.4g/L Sodium Azide of the homogenised RBC aggregating solution was added as a preservative.
EXAMPLE 5:
Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 60.0 g/L glucose anhydride was added to form the aggregating tonic solution. Thereafter Sodium Azide 0.5 g/L of the homogenised RBC aggregating solution was added as a preservative.
EXAMPLE 6:
Homogenised gel solution of concentration 3 g gelatine / L of distilled water was prepared 10.0 g/L NaH2PO4 + 15.0 g/L Na2HPO4.7H2O was added to form the aggregating tonic solution. ^Thereafter Sodium Azide 0,4 g/L of the homogenised RBC aggregating solution was added as a preservative.
The above examples illustrate that synergistic isotonic RBC aggregating solutions can thus be prepared from a variety of iso-osmotic compounds singly or in combination when they are dissolved in the above mentioned homogenised gelatine solution and preserved with sodium Azide. The choice of the tonic strength of the actual synergistic tonic red cell aggregating solution will depend upon the requirement of the pathological RBC condition under investigation i.e. whether increase or decrease in osmotic fragility of RBCs occurs in that particular pathological condition. For the purpose, a range of tonic solutions : hypotonic and isotonic solutions can be obtained by simply changing the amounts and proportions of iso-osmotic compounds : sodium azide : gelatine. Within the range specified for the compounds, the said homogenised RBC aggregating tonic solution will aggregate the intact surviving RBCs to form button in microtitre plate wells for easy display.
The most important use of the above solutions in hypotonic concentrations is in the diagnosis of microcytemia which is an integral feature of the carrier state of the genetic disorder called thalassemia. Although originally brought to India by the Alexander, thalassemic gene has persisted due to the protection it affords against Malaria. In our population the average incidence is 3.4%. However, in the high risk communities in entire Indogangetic belt, incidence as high as 14 % has been reported. WHO recommends mass screening for any genetic disorder if it exceeds 2.5%. Thalassemic gene carrier has no overt disability or clinical manifestation. The procedure for detecting microcytemia in a thalassemia carrier individual is:
PROCEDURE
The test process is very SIMPLE but requires METICULOUS care in regard to the quantity of blood added to the MOFTI (Micro Osmotic Fragility Test India) reagent bottles.
a. label two bottles containing MOFTI reagent for each person to be tested.
b. Open the bottle by removing the top cover and the dropper. Add to each of the bottles one drop of blood from either a finger prick or veinepuncture. In case of finger prick sample ensure complete drying of the cleansing agent i.e. alcohol / spirit used for cleaning the finger so as to prevent chemically induced haemolysis of RBC"s. Mix the contents of the bottle gently and close it with the dropper and the cover.
c. Leave it undisturbed for 30 minutes.
d. Mix the sample contained in the bottle gently to cause uniform suspension of the sample.
e. Pour 2 drops of sample from eachbottle into the respective microtitre plate well, (shown in fig 1 of the accompanying drawings
f. After all the samples have been poured; cover the microtitre plate and leave it undisturbed for 2 hours at room temperature at 25°C±5°C.
g. Place the microtitre plate on a horizontal view box and observe HAEMOLYSIS or BUTTON formation in the wells, in duplicate sample of each patient. In case of dissimilarity the test will need to be repeated, (shown in fig. 2 of the accompanying drawings)
RESULTS
The reagent for MOFTI screening is so calibrated that only a BUTTON is reported as POSITIVE. Complete haemolysis or partial haemolysis with varying degree of ring formation is reported as NEGATIVE.
CLINICAL SIGNIFICANCES
1. Decreased osmotic fragility has been primarily reported in two common clinical conditions e.g. Beta Thalassemia Trait (BTT) and iron deficiency Anaemia. The severity of Microcytosis in BTT however is far more pronounced than the fall in the haemoglobin leVel which may actually be within the normal range.
2. Other conditions where decreased osmotic fragility has been recorded are: Thalassemia Major, Sickle Thalassemia, Sickle Cell Trait, Haemoglobinopathy SS, E Thalassemia, D Thalassemia, HbH Disease,
3. Positive MOFTI has also been observed with elevated sedimentation rate.
I claim:
1. Synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising:
homogenized gelatin solutions in the concentration range of 0.1 g|to 6.0 g gelatin/L of distilled water,
RBC haemolyzing cum aggregating solutions of iso-osmojtic compound /- compounds as herein described which are iso-osmotically equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl) solution, and
sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of O.lg-2.0 g/L,
2. Synergistic red blood corpuscle (RBC) aggregating tonic solutions as claimed in claim 1 wherein the preferable iso-osmotic compounds used in the concentration are given below:
• Sodium Chloride - 1.0 g-20.0 g/L
• Sodium bicarbonate - 1.5 g - 30.0 g/L
• NaH2PO4 - 2.3 g-46.6g/L
• Calcium Lactate - 5 H2O - 5.0 g - 100 g / L
• Glucose, anhydrous - 5.6 g - 112 g / L
• NaH2PO4 - 1.65 g-23.3 g/L
+ • +
Na2HPO4 7H2O 1.84-36.8g/L
3. Synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells substantially as herein described with reference to the forego ing examples and accompanying drawings.
This invention relates to synergistic red blood corpuscle (RBC) aggregating tonic solutions with a long shelf life for determining osmotic fragility of red cells in microtitre plate wells comprising:
homogenized gelatin solutions in the concentration range of 0.1 g to 6.0 g gelatin/L of distilled water,
RBC haemolyzing cum aggregating solutions of iso-osmotic
compound / compounds as herein described which are isoosmotically
equivalent to 0.1-2.0 % of iso-tonic normal saline (NaCl)
solution, and
sodium azide preserved RBC haemolyzing cum aggregating solutions in the range of 0.1 g-2.0 g/L,

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Patent Number

216317

Indian Patent Application Number

1408/CAL/1998

PG Journal Number

11/2008

Publication Date

14-Mar-2008

Grant Date

12-Mar-2008

Date of Filing

06-Aug-1998

Name of Patentee

DR. MANJIT KAUR SHARMA.

Applicant Address

18/74/2,DOVER LANE, CALCUTTA-29

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. MANJIT KAUR SHARMA.	18/74/2, DOVER LANE, CALCUTTA-29

PCT International Classification Number

A 61 K 47/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA