Activated Partial Thromboplastin Time (aPTT) is a tested used to evaluate the intrinsic coagulation system. It aids the screen for congenital deficiencies of factors II, V, VIII, IX, X, XI and XII of the coagulation pathway. aPTT is also useful in the monitoring of heparin therapy. In addition, aPTT is used in the detection of Hemophilia A, Christmas disease, liver failure and Vitamin K deficiency. Furthermore, the test proveds screening for the presence of dysfibrinogenemia, disseminated intravascular coagulation, congenital deficiency of Fitzgerald factor and prekallikrein. aPTT is prolonged in the deficiency or inhition of any of the coagulation factors except factor VII. Coumadin therapy, heparin, aprotinin and lupus anticoagulants also have the same effect on aPTT. Prolonged aPTT is often evaluated with that of Prothrombin Time (PT) to assess coagulation system.
Coagulation Method
Coagulation Reaction Detection Method (Scattered Light Detection Method)
Irridates red light (660nm) onto a mixture of blood plasma and reagent and detects the change in turbidity (when fibrin clots are formed) as the change in scattered light. It measures the coagulation time.
Coagulation Point Detection Method (Percentage Detection Method)
Calculates the coagulation time as the time required to achieve the amount of scattered light that is set for the coagulation detection point, using the amount of scattered light that is present just after the start of detection as 0% and the amount of scattered light that is present at the completion of coagulation as 100%
Sunday, November 8, 2009
Urine FEME
Urine FEME, also known as Urine Formed Elements with Microscopic Examination, consists of mainly two components. One component involves using reagent strips on patient's urine sample, while the other component involves microscopic examination of the urine.
The reagent strips contains nine separate reagent areas that contain different chemicals for the determination of a particular analyate. It also contains a non-reactive reagent pad which is used to determine the colour of the urine specimen. The automated machine used in the clinical laboratory is called the Clinitek Atlas analyzer. The machines contains the reagent strips mentioned above, and would be able to test several components in a urine specimen. The 12 components to be tested are pH, colour of urine, clarity, protein, occult blood, leukocytes, nitrite, glucose, ketone, bilirubin, urobilinogen and specific gravity. When a patient's urine sample reacts with the reagent pad, there would be a colour change on the respective reagent pad. A manual test can be carried out a spearate manual test reagent strip. The results can be read using a colour chart supplied by the manufacturer which indicate the presence or absence of some analytes, pH of urine or the level of analytes present in the urine specimen. These results may provide information regarding the patient's kidney function, urinary tract infections, carbohydrate metabolism and liver function
Firstly, 5ml of a patient's urine sample is poured into a clear plastic tube and labelled with the patient's particulars and barcode label. The tuve is then loaded onto a tube rack and loaded onto the analyzer. The machine would take up a small volume of the urine specimen and aliquot it onto the 10 reagent pads on the reagent strips. After which, the machine would detect the colour change on the test strip and the test results would be recorded.
The test results might give us some information of a patient's health status. For example, a presence of leukocytes could indicate a possible urinary tract infection. Presence of ketones could indicate a possible carbohydrate metabolic disorder or due to fasting. Presence of casts or blood in the urine could indicate a possible loss of kidney function.
The microscopic examination of the urine specimen involves charging a small volume of urine to a Kova microscopic slide. The slide is then examined using a light microscope and observed for the presence of RBC, WBC, epithelial cells, casts or crystals present in the urine. The results of microscopic examination should be cross-referenced to the results from the reagent strips, such as the presence of RBCs and WBCs. The results of the two different components should tally.
The reagent strips contains nine separate reagent areas that contain different chemicals for the determination of a particular analyate. It also contains a non-reactive reagent pad which is used to determine the colour of the urine specimen. The automated machine used in the clinical laboratory is called the Clinitek Atlas analyzer. The machines contains the reagent strips mentioned above, and would be able to test several components in a urine specimen. The 12 components to be tested are pH, colour of urine, clarity, protein, occult blood, leukocytes, nitrite, glucose, ketone, bilirubin, urobilinogen and specific gravity. When a patient's urine sample reacts with the reagent pad, there would be a colour change on the respective reagent pad. A manual test can be carried out a spearate manual test reagent strip. The results can be read using a colour chart supplied by the manufacturer which indicate the presence or absence of some analytes, pH of urine or the level of analytes present in the urine specimen. These results may provide information regarding the patient's kidney function, urinary tract infections, carbohydrate metabolism and liver function
Firstly, 5ml of a patient's urine sample is poured into a clear plastic tube and labelled with the patient's particulars and barcode label. The tuve is then loaded onto a tube rack and loaded onto the analyzer. The machine would take up a small volume of the urine specimen and aliquot it onto the 10 reagent pads on the reagent strips. After which, the machine would detect the colour change on the test strip and the test results would be recorded.
The test results might give us some information of a patient's health status. For example, a presence of leukocytes could indicate a possible urinary tract infection. Presence of ketones could indicate a possible carbohydrate metabolic disorder or due to fasting. Presence of casts or blood in the urine could indicate a possible loss of kidney function.
The microscopic examination of the urine specimen involves charging a small volume of urine to a Kova microscopic slide. The slide is then examined using a light microscope and observed for the presence of RBC, WBC, epithelial cells, casts or crystals present in the urine. The results of microscopic examination should be cross-referenced to the results from the reagent strips, such as the presence of RBCs and WBCs. The results of the two different components should tally.
Saturday, November 7, 2009
Streptococcus pneumoniae Antigen Test
Hi everyone. This is Jeremy (0702919B) from TG01 posting. This post will be about a test kit used in the lab to detect Streptococcus pneumoniae. Any questions, please feel free to ask.
Streptococcus pneumoniae Antigen Test
The BinaxNOW® Streptococcus pneumoniae Test is a rapid, in vitro immunochromatographic assay. It detects S. pneumoniae antigen in the urine of patients with pneumonia and also in the cerebral spinal fluid (CSF) of patients with meningitis. Both pneumonia and bacterial meningitis pose many complications to patients, and are almost always fatal if left untreated. Thus, it is necessary for an early detection and appropriate treatment to be administered. The Streptococcus pneumoniae Antigen Test, used together with culture and other methods, can also aid in the diagnosis of both pneumococcal pneumonia and pneumococcal meningitis.
Principle
The test kit involves the use of anti-S. pneumoniae antibody and control antibody. Both antibodies are adsorbed onto a nitrocellulose membrane (the anti-S. pneumoniae antibody on the sample line, while control antibody on the control line). The antibodies are conjugated to visualizing particles that are dried onto an inert fibrous support.
The presence of pneumococcal antigen in the sample will reacts to bind anti-S. pneumoniae conjugated antibody. The resulting antigen-conjugate complexes are captured by immobilized anti-S. pneumoniae antibody, forming the Sample Line. Immobilized control antibody captures anti-species conjugate, forming the Control Line.
Results Interpretation
Positive results:
Two pink-to-purple coloured lines will be formed. This means that an antigen was detected. Specimens with low levels of antigen may give a faint sample line. Any visible line is positive.
Negative results:
Only one pink-to-purple coloured control line in the top half of the window is formed. Formation of only the control line means that detection part of the test was done correctly, however no S. pneumoniae antigen was detected.
Invalid results:
No lines are seen, or only the sample line is seen. An invalid test must be repeated.
*Read results after 15 minutes.
That is all for the test kit. Thank you for reading.
Sunday, October 25, 2009
G6PD screening & Quantitative assays
hello hello hello!
stella here for the final blog posting!
and i apologize for the late entry.
G6PD (Glucose-6-Phosphate Dehydrogenase) test is performed mainly on patients who develop symptoms of hemolytic anemia and neonatal jaundice. Hemolytic anemia takes place when the bone marrow is not able to compensate for the destruction by elevating the RBC production. G6PD is necessary for the maintenance of an intact red cell membrane because G6PD deficiency may lead to hemolysis of red cells. During the abnormal state, the RBCs will not be able to regenerate NADPH, a reaction that will be catalysed by G6PD in normal situations.
Both G6PD Quantitative and Qualitative (screening) assays are performed to enable differential diagnosis in G6PD deficiency to be made.
The principle of both G6PD assays is based on this reaction:
Glucose-6-Phosphate + NADP (no fluorescence) ---(G6PD)---> Gluconate-6-Phosphate + NADPH (fluorescence)
The NADPH produced will fluoresce under long-wave UV light during the reaction.
There is maerked deficiency or lack of enzyme G6PD when no fluorescnece is observed.
If Glucose-6-Phosphate is oxidised to Gluconate-6-Phosphate, the coenzyme NADP is reduced to NADPH with a corresponding elevation in fluorescence.
The procedures for both assays are different, with the screening assay much simpler as compared to the quantitative assay.
Steps for screening assay:
1) The EDTA consisting of patient's blood sample is inverted gently for several times for thorough mixing
2) 5ul of whole blood is added, 100ul of working reagent and 100ul of reagent blank into the wells of the titration plates
3) Working reagent is added and start timing
4) After 10 minutes, a small amount of reaction is taken and i spotted onto filter paper
5) The spots are dried for 5 mins using a hair-dryer
6) The spots are then viewed under long-wave UV light
Steps for quantitative assay:
1) The EDTA blood is washed 1 time with 0.9% saline
2) It is then sent for centrifugation to pack the cells at 3000rpm for 10 mins
3) The saline and the buffer coat are removed completely
4) 200ul of washed packed cells are pipetted into 200ul normal saline to obtain 1:1 rbc suspension
5) 50ul of this 1:1 suspension is pipetted into 250ul of !% saponin for lysis
6) It is then mixed well and left to stand for 10 mins at 2-8 degree celsius
7) The hemolysate is ready for testing
Reporting of results for Qualitative (screening) assay:
When fluorescence is observed, enzyme activity is indicated. This explains the presence of G6PD.
Reporting of results for Quantitative assay:
Reference ranges for adults/children = 7.2-17.4 ug/Hb
newborns/cord blood = 13.4-25.4 ug/Hb
stella here for the final blog posting!
and i apologize for the late entry.
G6PD (Glucose-6-Phosphate Dehydrogenase) test is performed mainly on patients who develop symptoms of hemolytic anemia and neonatal jaundice. Hemolytic anemia takes place when the bone marrow is not able to compensate for the destruction by elevating the RBC production. G6PD is necessary for the maintenance of an intact red cell membrane because G6PD deficiency may lead to hemolysis of red cells. During the abnormal state, the RBCs will not be able to regenerate NADPH, a reaction that will be catalysed by G6PD in normal situations.
Both G6PD Quantitative and Qualitative (screening) assays are performed to enable differential diagnosis in G6PD deficiency to be made.
The principle of both G6PD assays is based on this reaction:
Glucose-6-Phosphate + NADP (no fluorescence) ---(G6PD)---> Gluconate-6-Phosphate + NADPH (fluorescence)
The NADPH produced will fluoresce under long-wave UV light during the reaction.
There is maerked deficiency or lack of enzyme G6PD when no fluorescnece is observed.
If Glucose-6-Phosphate is oxidised to Gluconate-6-Phosphate, the coenzyme NADP is reduced to NADPH with a corresponding elevation in fluorescence.
The procedures for both assays are different, with the screening assay much simpler as compared to the quantitative assay.
Steps for screening assay:
1) The EDTA consisting of patient's blood sample is inverted gently for several times for thorough mixing
2) 5ul of whole blood is added, 100ul of working reagent and 100ul of reagent blank into the wells of the titration plates
3) Working reagent is added and start timing
4) After 10 minutes, a small amount of reaction is taken and i spotted onto filter paper
5) The spots are dried for 5 mins using a hair-dryer
6) The spots are then viewed under long-wave UV light
Steps for quantitative assay:
1) The EDTA blood is washed 1 time with 0.9% saline
2) It is then sent for centrifugation to pack the cells at 3000rpm for 10 mins
3) The saline and the buffer coat are removed completely
4) 200ul of washed packed cells are pipetted into 200ul normal saline to obtain 1:1 rbc suspension
5) 50ul of this 1:1 suspension is pipetted into 250ul of !% saponin for lysis
6) It is then mixed well and left to stand for 10 mins at 2-8 degree celsius
7) The hemolysate is ready for testing
Reporting of results for Qualitative (screening) assay:
When fluorescence is observed, enzyme activity is indicated. This explains the presence of G6PD.
Reporting of results for Quantitative assay:
Reference ranges for adults/children = 7.2-17.4 ug/Hb
newborns/cord blood = 13.4-25.4 ug/Hb
Thursday, October 15, 2009
Culturing Of Cord Blood
Hey!
This is Hakim (0703555C) doing my 4th and final blog posting.
Culturing of cord blood is essential for those who want to store their baby's cord blood.
Cord blood is the blood that is in the umbilical cord and placenta after the birth of the baby. The blood can be used to treat haematopoietic diseases such as leukemia, as the cord blood contains stem cells. The cord blood can be stored for up to 20 years for transfusion for future use to the baby or their family members. Therefore, it is essential that the cord blood is sterile and is free of any microorganisms. In addition, storage and maintenance of cord blood is very expensive.
The cord blood that is received for culturing is just a small amount of blood (2 eppendorf tubes).
The cord blood is cultured on 4 different agar plates:
1. TSA (Trypticase Soy Agar) with sheep blood stored in aerobic conditions - an enriched media to support a wide variety of microorganisms
2. TSA (Trypticase Soy Agar) with sheep blood stored in anaerobic conditions - an enriched media to support a wide variety of anaerobic microorganisms
3. MacConkey Agar- selective media for gram negative bacteria and also to differentiate between lactose fermenters and non-lactose fermenters
4. Sabouraud Dextrose Agar - selective media for yeast cells
The barcode number of the baby must be written on the underside of the media plate. A sterile swab is inserted into the cord blood and is innoculated onto the media plates. Then, a streaker is used to streak the plate.
The anaerobic TSA is inserted into an anaerobic jar. An anaerobic pack which absorbs oxygen and releases carbon dioxide is added into the jar before incubation. In addition, a control plate of a Mueller Hinton Agar innoculated with pseudomonas aeruginosa is put inside the anaerobic jar. The reason for this is because pseudomonas aeruginosa is a strict aerobe and it is the only organism that grows green colonies on Mueller Hinton. Therefore, if there isn't any green colonies on the agar after incubation, we can conclude that the conditions is an anaerobic condition.
After 24 hours of incubation, the agar plates are checked for any signs of bacterial growth. If there is no bacterial growth, the plates are incubated further for another 24 hours. If there is presence of bacterial growth, we must report the findings so that the cord blood will not be stored.
Please feel free to ask any questions!
Thanks!
This is Hakim (0703555C) doing my 4th and final blog posting.
Culturing of cord blood is essential for those who want to store their baby's cord blood.
Cord blood is the blood that is in the umbilical cord and placenta after the birth of the baby. The blood can be used to treat haematopoietic diseases such as leukemia, as the cord blood contains stem cells. The cord blood can be stored for up to 20 years for transfusion for future use to the baby or their family members. Therefore, it is essential that the cord blood is sterile and is free of any microorganisms. In addition, storage and maintenance of cord blood is very expensive.
The cord blood that is received for culturing is just a small amount of blood (2 eppendorf tubes).
The cord blood is cultured on 4 different agar plates:
1. TSA (Trypticase Soy Agar) with sheep blood stored in aerobic conditions - an enriched media to support a wide variety of microorganisms
2. TSA (Trypticase Soy Agar) with sheep blood stored in anaerobic conditions - an enriched media to support a wide variety of anaerobic microorganisms
3. MacConkey Agar- selective media for gram negative bacteria and also to differentiate between lactose fermenters and non-lactose fermenters
4. Sabouraud Dextrose Agar - selective media for yeast cells
The barcode number of the baby must be written on the underside of the media plate. A sterile swab is inserted into the cord blood and is innoculated onto the media plates. Then, a streaker is used to streak the plate.
The anaerobic TSA is inserted into an anaerobic jar. An anaerobic pack which absorbs oxygen and releases carbon dioxide is added into the jar before incubation. In addition, a control plate of a Mueller Hinton Agar innoculated with pseudomonas aeruginosa is put inside the anaerobic jar. The reason for this is because pseudomonas aeruginosa is a strict aerobe and it is the only organism that grows green colonies on Mueller Hinton. Therefore, if there isn't any green colonies on the agar after incubation, we can conclude that the conditions is an anaerobic condition.
After 24 hours of incubation, the agar plates are checked for any signs of bacterial growth. If there is no bacterial growth, the plates are incubated further for another 24 hours. If there is presence of bacterial growth, we must report the findings so that the cord blood will not be stored.
Please feel free to ask any questions!
Thanks!
Monday, September 28, 2009
Biochemical Tests
Good evening everyone. This is Jeremy (0702919B) from TG01. Today, I will be elaborating on several biochemical tests performed in the lab to aid in identifying bacteria.
Jeremy.
Kliger’s Iron Agar (KIA)
KIA is a differential medium. It is similar to the Triple Sugar Iron (TSI) agar that we used in the school's lab. It tests for organisms’ abilities to ferment glucose and lactose to acid and acidic gas products. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose. These lactose non-fermenting enterics are generally more pathogenic in the gastrointestinal tract.
Glucose fermentation creates acidic byproducts that turn the phenol red indicator in the media yellow. Upon depletion of glucose, organisms that can ferment lactose will continue to produce acidic byproducts and the media will remain yellow. Gaseous products produce might lift the agar off the bottom of the tube.
Non lactose fermenters metabolize amino acids and proteins in the media, creating alkaline byproducts that turn the indicator red at the slant.
Non glucose and lactose fermenters will use solely amino acids and proteins, creating a red slant and unchanged color at the butt.
Organisms that sulfer reducing enteric will produce H2S that creates a black precipitate at the butt.
| | Slant | Butt | Examples |
| Lactose fermenters | Yellow | Yellow | Escherichia coli |
| Non lactose fermenters | Red | Yellow OR Black (H2S production) | Shigella dysenteriae Proteus mirabilis (produce black ppt) |
| Non glucose and lactose fermenters | Red | Unchanged | Pseudomonas aeruginosa |
Simmons’ Citrate Agar
Simmons’ citrate agar tests the ability of an organism to use citrate as a sole carbon source and ammonium ions as a sole nitrogen source. The medium contains citrate, ammonium ions, and also a pH indicator, bromothymol blue. Organism that can grow on Simmons’ citrate agar are capable of metabolizing citrate as the sole carbon source and ammonium ions as the sole nitrogen source. This will create alkaline byproducts which will turn bromothymol blue indicator blue.
Growth on Simmon’s citrate agar or an intense blue color formation is an indication of a positive result.
Urease Test
This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea.
Jeremy.
Immunohaematology (Blood Bank)
Firstly, I would like to discuss on how a patient's blood sample is being processed when it arrives at the blood bank. When a blood sample is first received, the Medical Technologist would check the EDTA blood tubes for any blood clots. This is being done by using 2-3 wooden sticks to "dig" the blood sample and try to "fish" out for any clots. If clots are absent, the sample would be suitable to be processed using the Ortho Autovue Innova System. If clots are present, the form accompanying the patient's sample should have "clotted" indicated on it. The blood sample with blood clots present cannot be processed by the Ortho Autovue Innova System, this the type and screen has to be carried manually.
After the blood samples in the EDTA are checked for presence of clots, the tube is loaded into the Ortho Autovue Innova System for the machine to read and screen the sample. The machine is able to carry out forward, reverse & Rh grouping of the blood samples. In addition, it is also able to detect abnormal antibodies that might be present in the blood. If the machine is malfunctioned, a manual type and screen can be carried out instead.
In type and screen which is done manually, forward group involves placing patient's blood sample into commercial sera. The 3 main groups of anti-sera are anti-A, anti-B & anti-AB. In reverse grouping, patient's serum (or plasma) is added to commercial blood. The 3 types of commericial blood used are blood group A, B & O. Lastly, to determine the presence of antigen D in patient's blood, patient's blood is added to anti-D commercial serum. This concludes the process of type and screen, and from this the patient's blood group and Rh status can be determined.
In an event that a patient requires blood transfusion, a cross-match must be carried out to check for compatiblity. In cross-matching, patient's serum is added into a sample of packet of blood (to be transfused). It is incubated at room temperature for 5 minutes, then centrifuged @ 3000rpm for 15s. The glass tube is inspected visually for any agglutination present. If there is no agglutination present, it can be concluded that the packet of blood can be safely transfused into the patient.
Regards,
Zhu Zhijie Dennis
0700847G
TG01
After the blood samples in the EDTA are checked for presence of clots, the tube is loaded into the Ortho Autovue Innova System for the machine to read and screen the sample. The machine is able to carry out forward, reverse & Rh grouping of the blood samples. In addition, it is also able to detect abnormal antibodies that might be present in the blood. If the machine is malfunctioned, a manual type and screen can be carried out instead.
In type and screen which is done manually, forward group involves placing patient's blood sample into commercial sera. The 3 main groups of anti-sera are anti-A, anti-B & anti-AB. In reverse grouping, patient's serum (or plasma) is added to commercial blood. The 3 types of commericial blood used are blood group A, B & O. Lastly, to determine the presence of antigen D in patient's blood, patient's blood is added to anti-D commercial serum. This concludes the process of type and screen, and from this the patient's blood group and Rh status can be determined.
In an event that a patient requires blood transfusion, a cross-match must be carried out to check for compatiblity. In cross-matching, patient's serum is added into a sample of packet of blood (to be transfused). It is incubated at room temperature for 5 minutes, then centrifuged @ 3000rpm for 15s. The glass tube is inspected visually for any agglutination present. If there is no agglutination present, it can be concluded that the packet of blood can be safely transfused into the patient.
Regards,
Zhu Zhijie Dennis
0700847G
TG01
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