Qbc test

It is a test used to diagnose malaria.

1. Qbc test (Diagnostic test for MALARIA) By – MONEY KALASH PharmD

2. Malaria is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes. In the human body, the parasites multiply in the liver, and then infect red blood cells. Symptoms of malaria include fever, headache, and vomiting, and usually appear between 10 and 15 days after the mosquito bite. If not treated, malaria can quickly become life-threatening by disrupting the blood supply to vital organs. Malaria

3. LIFE CYCLE OF MALARIA PARASITE

4. Introduction According to the latest estimates, released in December 2013, there were about 207 million cases of malaria in 2012 (with an uncertainty range of 135 million to 287 million) and an estimated 627 000 deaths (with an uncertainty range of 473 000 to 789 000). Malaria mortality rates have fallen by 42% globally since 2000, and by 49% in the WHO African Region. As Malaria is a life threatning disease. So, the demonstration of malarial parasite is a requisite for definitive diagnosis of malaria for effective treatment & reducing mortality and morbidity of malaria.

5. Cont…. There are many laboratory methods of diagnosing malaria such as- Blood Smear Test Polymerase Chain Reaction Rapid Diagnostic Tests etc. These tests are briefly discussed as follows….

6. a) Microscopic examination of thick and thin film smears

7. Cont…. Conventional light microscopy is the established “gold standard” for confirmation of malaria. These blood smear test should be repeated every 12-24 hours for a total of 3 sets. It also requires trained and skilled technicians ,good supervisory personnel and good equipments (microscopes , reagents) . Although It provides permanent record for the test but it is time consuming(at least 60 minutes from time of sample collection).

8. b) Polymerase Chain Reaction Analysis of blood samples of parasite-specific nucleic acids by nested and real time PCR is reported to be highly sensitive and specific. The technique requires highly trained personnel and is both time and resource intensive. Nested PCR is valuable as a confirmatory test. c) Rapid Diagnostic Test Immuno-chromatographic Method is used to detect antigens derived from malaria parasite in lysed blood. Some substances w/h are detected in RDTs are- Histidine-rich protein II (HRP II) Parasite lactate dehydrogenase (pLDH) These tests have higher sensitivity upto 90% in detection of P.falciparum at densities at or above 100 parasites/ l of blood. Below this level, senstivity decreases markedly.

9. d)QBC  Q uantitative B uffy C oat (Diagnostic test for Malaria)

10. What is QBC Test? The QBC Malaria Test is a fluorescence microscopy-based malaria diagnostic test. It speeds and simplifies malaria detection, with a combination of features and benefits.

11. Principle of QBC Test 1. Fluorescent Stain – The QBC Malaria Test is internally coated with fluorescent A cridine Orange (AO) stain to improve the visibility of malaria parasites. When excited by blue light (~460 nm), parasites will fluoresce brightly against a dark background. 2. Centrifugation - Centrifugation makes parasites easier to locate within the tube . When the tube is centrifuged in the QBC Capillary Centrifuge, blood components and malaria parasites separate based on density, and concentrate in distinct layers.

12. Requirements QBC malaria tubes Blood sample QBC Centrifuge Fluorescent Microscope

13. 1. QBC Malaria Tubes QBC blood tubes are 75 mm glass tubes, internally coated with potassium oxalate , acridine orange, anticoagulants . The tubes are sealed with a plastic closure or rubber stopper and contain a precision- molded float within the blood-filled tube.

14. 2. QBC Centrifuge The key feature of QBC test is Centrifugation.

15. Procedure It consists of 3 parts- Collection of specimen & Loading in capillary tubes Centrifugation Study under Fluorescent Microscope

16. 1. Collection of specimen & loading of QBC tubes  Venous blood for QBC haematology test analysis can be collected in Vacutainer brand tubes containing EDTA anticoagulant. QBC tubes for venous blood are filled by means of a semi-automatic pipetter supplied with the QBC System.QBC capillary-blood tubes are pre-coated with anticoagulants and are filled with blood directly from a finger puncture.

17. QBC Blood tube is filled with 55-65 microlitres of blood. Specimen can be collected from finger, ear or heel puncture(generally finger). A precisely made cylindrical float, designed to be suspended in the packed RBCs , is inserted before loading of sample in capillary tube. After filling with blood, the tubes are sealed with a rubber stopper or plastic closure.

18. 2. Centrifugation The tube is centrifuged at 11,000-12,000 rpm for 5 minutes. The components of the buffy coat separate according to their densities, forming discrete bands. Because the float occupies 90% of the internal lumen of the tube, the leukocyte and the thrombocyte cell band widths and the top-most area of red cells are enlarged to 10 times normal.

19. 3. Study under Microscope The QBC tube is placed on the tube holder and examined using a standard white light microscope equipped with the UV microscope adapter, an epi -illuminated microscope objective.   Parasites can be viewed through the capillary tube using a special long-focal-length objective (paralens) with a fluorescence microscope . Since the parasites contain DNA which takes up the acridine orange stain, they appear as bright specks of light among the non-fluorescing red cells.

20. Observations

21. (Cont….) When the  QBC Malaria Tube  is centrifuged, the blood becomes concentrated & the blood components and malaria parasites separate based on density and concentrate in distinct layers. Parasites and leucocytes nuclei fluoresce a yellowish-green. It is in the Buffy Coat layer where the malaria parasites will be visible as shown above.

22. (Cont….) Red cells containing Plasmodia are less dense than normal ones and concentrate just below the leukocytes, at the top of the erythrocyte column. It appears so because the blood components and malaria parasites present in blood sample separate on centrifugation based on density and concentrate in distinct layers.

23. Malarial Parasite Identification The float occupies the area midpoint between red cells and plasma. The amber layer surrounding the float is the buffy coat. Seen enlarged above, the three separate layers of the buffy coat are comprised of orange-yellow fluorescing platelets  (top) , green lympocytes / monocytes   (middle)  and yellow granulocytes  (bottom) . Below the granulocytes is the red blood cell layer (some overlap of RBC's and granulocytes occurs). Red cells containing  Plasmodium  parasites concentrate at the top of this layer and appear as multiple "pinpoints" of fluorescence. A negative specimen shows no fluorescing points in this area.

24. Falciparum rings in RBC layers WBC layer & fluorescent malaria (buffy coat)  

25.  Blood cells & parasites have DNA in them and certain fluorescent dyes have an affinity for the nucleic acid in the parasite nucleus and will attach to the nuclei. When excited by UV light at an appropriate wavelength(470-490 nm), the nucleus will fluoresce strongly. Reason for flourescence of blood components

26. Things to remember Red blood cells (RBC’s) containing malaria parasites are less dense than non-infected RBC’s. AO is a very intense fluorescent stain, it is nonspecific and stains nucleic acids from all cell types.(The sensitivity of AO staining for detection of malaria parasites in infections with parasite levels of <100 parasites/ μl (0.002% parasitemia ) has been reported to range from 41 to 93%.)

27. Other stains for QBC test An alternative fluorochrome procedure uses a solution of BCP( Benzothio-carboxy-purine ).  In contrast, RBC inclusions and the nuclei of leukocytes are poorly stained. This method overcomes some of the problems inherent in some fluorescence systems, such as the need for rapid examination to prevent fading or precipitation of the dye.

28. Advantages of QBC test 1. Sensitivity The most sensitive test available for the diagnosis of malaria due to: The high volume of blood The concentration of the parasites by centrifugation The fluorescent staining of parasites The  QBC Malaria Test  is 5.5 to 7 percent more sensitive than Giemsa thick films. It can detect as little of one parasite per µL of blood and establish diagnosis earlier than thick film in 47% of low parasitemia (<10 parasites per µL) cases. Up to 18% more positives are reported by QBC than the conventional thick film method.

29. 2. Diagnosis All Plasmodium species are identified and quantified in the QBC Malaria Tube using the QBC Paralens Advance system (or traditional microscope); Species differentiation is possible in the QBC Malaria Test.  A QBC test is highly specific and a negative result will enable the patient to avoid a multi-drug therapy. 3. Time saving A negative test can be reported within one minute and positive result within minutes. 4. Proven Technology The QBC Malaria Test was the first malaria diagnostic test to receive FDA clearance, and its technology has been proven in dozens of scientific studies. (Cont….)

30. QBC malaria test kit

31. Comparative study of QBC & Smear test Comparative Analysis of QBC and Thick Film Microscopy: Study done at Kasturba Medical College Diagnostic Centre, Mangalore ( Urmila Shenoi et al) Total number of samples examined 18,845 Total number positive by QBC 4,824 Total number positive by thick film 3,490 Positivity rate of QBC 25% Positivity rate of thick film 18%  

32. PERIPHERAL SMEAR QBC TEST Method Cumbersome Easy Time Longer, 60 - 120 minutes Faster, 15 - 30 minutes Sensitivity 5 parasites/µl in thick film and 200 / µl in thin film Claimed to be more sensitive, at least as good as a thick film Specificity Gold standard ? False positive , artifacts may be reported as positive by not so well trained technicians Species identification Accurate, gold standard Difficult to impossible Cost Inexpensive Costly equipment and Consumables Acceptability 100% Not so Availability Everywhere Limited Other -- Accidentally can detect filarial worms

33. R eferences The National Centre for Biotechnology Information http://www.ncbi.nlm.nih.gov/pmc/articles/PMC118060 / 2. http://www.malariasite.com http://www.malariasite.com/malaria/QBC.htm# 3. http://www.woodleyequipment.com/laboratory-diagnostics/qbc-europe-products/qbc-malaria-kits-329-94-.php 4.. http:// newmodernlabservices.com / userfiles /file/The%20QBC%20Test.pdf

34. Thank you