Principle

This test, presented for the first time in1986, in an essentially hematological and veterinary application (count with approach of the formula), involves the acridine orange staining of the figurative elements of a fresh blood sample, which then separate according to their density through gravimetry in a precision-made micro-capillary tube.

The principle of its use in parasitological diagnosis is based on 3 elements :

• Centrifugation, which separates the elements according to their density, then concentrates and stacks the identical elements at certain predictable levels of the tube. 
• During centrifugation, a float which has been previously inserted in the tube is suspended among the buffy coat and the erythrocytic layer and provokes a mechanical expansion of the surrounding cells into the residual 40 µm space, in contact with the wall of the tube where they can be easily observed. 
• Acridine orange, a specific agent of nucleic acids, after being excited by an appropriate light source (UV or dichroïsm filtered), creates in the cells possessing nucleic matter a metachromatic staining differential between the DNA (apple green to yellow-green) and the RNA (green to orange-red). It will reveal the presence of the parasites on the dark background of the red blood cells (RBCs) a priori deprived of systematized nucleic structures.     

Equipment

1. The kit comprises different elements :   

• a 75 mm long micro-hematocrit glass capillary tube. The internal walls are coated with various reagents: acridine orange at one end; an anticoagulant (sodium heparinate, EDTA) at the opposite end ; potassium oxalate intended to increase the density of the red blood cells by osmotic action, thus facilitating their separation from the white blood cells (particularly between reticulocytes and granulocytes). 
• A cylindrical plastic float, of the same relative gravity as that of the leukocytes, measuring 20 mm long. 
• A plastic stopper.     

2. Implementation requires the following equipment : 

• a micro-hematocrit centrifuge fitted with 20 slots adapted to the dimensions of the tubes, 
  a plastified support, the "Paraviewer", comprising a gutter intended to receive the tube and enabling microscopic examination,  
• a microscope fitted with a UV light source and FITC filters, an immersible lens with X60 or X50 magnification and a ³ 0,34 mm focal distance so as to pass the thickness of the wall of the tube and be able to examine several cell thickness. A field model exists based on an original principle, mains mode or battery operated: it is adaptable to any standard microscope; it comprises a cold light source linked by fiber optics to an immersible X60 lens, modified by the addition of dichroic filters placed on the light trajectory, which excites the acridine orange and provides a fluorescent image     

Method

1. The sample : 

The required 55 to 65 µl blood sample is obtained by capillary action from venous blood taken on a anticoagulant or capillary blood from a finger puncture. The tube is filled from the end coated with anticoagulants, the filling level indicated by two marks inscribed on the tube. 

2. The preparation : 

The tube, held between two gloved fingers, is alternatively inclined to one side then the other so as to enable contact between the figurative elements of the blood with the acridine orange. A stopper is then placed at the opposite end of the tube to the filling end and the float is introduced into the tube with the aid of forceps to avoid contact with the fingers. According to experiments, the sample may be centrifuged up to 5 hours later without any alteration to the legibility. The tube prepared in this way, is placed in the centrifuge where it is subjected to a 12,000 g acceleration for 5 minutes. 

3. The reading: 

The result can be read immediately. It shows below the plasma, the buffy coat of platelets, the lympho-monocytic ring, the layer of granulocytes and finally the erythrocytic sediment. 

Practice shows that the classic protocol (examination in UV light of the erythrocytic layer at the red cell/white cell interface, on several lower fields) may be taken by default in certain situations, in particular among patients who are carriers of gametocytes of Plasmodium falciparum and/or asexual forms of Plasmodium vivax,  Plasmodium ovale or Plasmodium malariae.

Practical conduct:

It is preferable to take a slightly larger sample of capillary or venous blood than the manufacturer recommends. The tube is half filled which corresponds to approximately 100 microliters. In these conditions, there are no negative repercussions on the quality of the acridine staining nor on the separation of the different layers during centrifugation. The tube is then examined under fluorescence, at a X50 magnification, with the initial focus centered on the erythrocytic layer at the level of the interface with the leukocytes. A positive diagnosis often appears instantaneously. In the event of negativity, the complete height of the cellular sediment is examined by exploring, field by field, the four main discrete bands by adjusting the micrometric screw. 

It is indispensable to add to this classic protocol a fundamental stage, which consists of the examination of the "buffy coat" under white light, i.e. without fluorescence. This stage facilitates the examination, thanks to their clearly discernable pigment, of the sexual stages of the different species which migrate preferentially in the leukocytic layers and are represented in the erythrocytic sediment, especially in the event of low parasitemia. This procedure also assists in the location of eventual melaniferous leukocytes, sometimes the only remaining indications of a recent contact with the parasite.  

Positive diagnosis:

The morphology of the parasites remains perfectly comparable to that observed on thin blood film smear tests, even during treatment. Against a dark background, where the erythrocytic membranes create a lattice effect, the trophozoites may be observed in an intraerythrocytic position. The nucleus gives off an intense green homogeneous fluorescence, from 1 to 2 µm in diameter. The cytoplasm, variable in size and shaped like a crescent, emits a green-orange fluorescence, less intense, with well-defined margins, and variable width and thickness. It encapsulates the nucleus within its extremities and delimits a non-fluorescent area corresponding to the food vacuole.

The positive diagnosis of trophozoites is ALWAYS based on an image associating a nucleus and a cytoplasm, in an intraerythrocytic situation that can be verified by varying the focusing levels. 

In the event of the hemolysis of the RBCs, the intraerythrocytic situation disappears, but the morphology of the parasite is conserved.

Differential diagnosis:

• The distinction between parasites and leukocytes is very easy. The size of the elements, compared with that of red blood cells, is discriminant: the nucleus of the parasite is around 2 µm, that of the leukocytes 5 times bigger; its shape is compact in the trophozoite, lobe-shaped in the polynuclear. 
• These criteria enable the easy elimination of the cellular debris which will eventually contaminate the layer of red blood cells for they do not form a systematized image compatible with the diagnosis and their reddish fluorescence disappears in a few seconds under luminous excitation, unlike that of the parasites. 
• In the same way, the Howell-Jolly bodies are presented in the form of a very brilliant, apple green dot, perfectly circular, often centered with a small dark non fluorescent point. Sometimes, they are encircled by reddish fragments which could evoke a cytoplasm, but this fluorescence also fades very quickly. 
• The platelets could pose a difficult problem for they frequently contaminate the layer of RBCs. In fact, their size is close to that of a trophozoite and, as on the smear tests, they tend to superimpose themselves on the RBCs. However, there is no morphology associating nucleus and cytoplasm, but a rather heterogeneous and micro-granulous fluorescence, with pale green tones and yellowish punctuations, with uncertain limits. The examination takes place at different levels of focus. 
• The only real problem with differential diagnosis, which could arise exceptionally, is constituted by the distinction between Plasmodium and Babesia due to the similarity in appearance, size and intracellular situation. However in the case of babesiosis, it has been possible to find, by adjusting the micrometric screw, a daisy-like appearance never previously seen in cases of malaria.     

The reader rapidly becomes familiar, but in the event of any doubt, AN ATYPICAL APPEARANCE SHOULD BE CONSIDERED NEGATIVE. The theoretical limit case is represented by an extremely weak parasitemia, a morphology that has been altered considerably by treatment prior to the examination and the presence of platelets with abnormal morphology, as is encountered at times in malaria.

Orientation of the species diagnosis:

The species diagnosis is based on a whole range of arguments. Dispersion and monomorphism of the image constitute a very good argument in favor of an infection by Plasmodium falciparum. Polymorphism and concentration enable another species to be suggested. However, these are ideal conditions, rarely encountered (weak parasitemia, morphology altered by taking medicine, presence of two species). The examination of the MGG stained thin blood film remains indispensable for species diagnosis. It is also necessary for the evaluation of parasitic density.

Reading in fluorescence: study of the leukocytic layers and red blood cells  

 Reading in white light:study of the leukocytic layers  

The performances of the test appear closely dependent on the training of the reader, the use of adequate equipment especially the lens and a complete reading protocol.  

Sensitivity, specificity:

The results show the test to be superior to classical methods. The test detection threshold, evaluated on a cascade of dilutions of a culture of Plasmodium falciparum blocked at the stage of young trophozoites, is estimated at 0.1 to 1 infected RBC per micro-liter (HPM). 

Studies have shown that the test identifies a parasitemia more precociously than the thick blood film method. In the same way, the parasitic clearance is higher with this test (84 ± 13 hours) than with the smear test (66 ± 11 hours). Compared with the tests of molecular biology (nucleic catheters) and flow cytometry, the QBC provides the best performance. It is thus currently the most sensitive diagnostic method.

By applying the positive diagnosis criteria of the genus Plasmodium, the results show a specificity of 100 %.

Feasibility:

The most remarkable aspect of the test is its response speed, even in the event of very week parasitemia: reading is possible 6 minutes after the sample and if, on average, a positive diagnosis takes less than one minute, three minutes suffice to make a negative test within the limits of the detection threshold. 

The safety and the simplicity of the implementation are proven and the qualities approved by all users. The quality of the acridine orange marking of the parasites is constant. Thanks to the very discriminant character of the image of the parasite in a fresh state, the reading apprenticeship with formal recognition of the parasites requires less than 2 hours to a technician already trained in parasitological diagnosis. 

Kept in obscurity at + 4 °C, a centrifuged tube remains legible for 10 to15 days. This time period drops to 48 hours in the event of conservation at + 37 °C. A smear test performed simultaneously enables positive samples to be stored in file. 

Despite its limits, the QBC-malaria test® constitutes the only real alternative to the reference methods which are the smear blood test and the thick blood film test. It provides clear progress in terms of sensitivity, speed and reliability. It is perfectly adapted to diagnosis and in particular to the new clinical forms of malaria with low parasitemia.

Its interest has been proven in epidemiological surveys and it is now difficult to do without it in this precise context.

It is extremely regrettable that, on purely economic grounds, the manufacturer should have chosen not to commercialize the equipment any longer. The reagents are still available.