Method and apparatus for the detection of microfilariae
United States Patent [191 Wilkins et al.  
 [ METHOD AND APPARATUS FOR THE DETECTION OF MICROFILARIAE [76] Inventors: Robert John Wilkins, 339 E. 58th St., New York, NY. 10022; Arthur Hurvitz, 4370 Kissena Blvd., Flushing, NY. 11355 [22] Filed: Nov. 15, 1973 [21] Appl. No.: 416,320  
 [52] US. Cl. 23/230 B, 23/259, 23/292, 128/2 F, 73/4256, l95/103.5 R, 195/127 [51] Int. Cl. G0ln 33/16, GOln H30 [58] Field of Search 23/230 B, 253 R, 259, 292; 195/103.5 R, 127; 128/2 F, 2 G; 424/11; 73/425.6  
 Primary Ii.\&#39;aminerR. E. Serwin Attorney, Agent, or Firm-Burgess, Dinklage &amp; Sprung 1 1 Apr. 1,1975  
 [57] ABSTRACT In the detection of microfilariae wherein a blood sample is drawn&#39;into a syringe, the blood mixed with an aqueous lysing solution, filtered through a pad, the filter pad stained and examined on a glass slide under a microscope, the improvement which comprises mixing said lysing solution with said blood by drawing said lysing solution into said blood-containing syringe, and discharging the mixture through said filter pad, the blood sample thereby being minimally exposed for contamination. Desirably the lysing solution is employed in about 5 to 15 times the volume of the blood sample and comprises about 1% of a polyoxyalkylene phenol ether, about 1.75% of sodium citratecitric acid wherein the sodium citrate is present in about 3 times the amount of the citric acid and about 0.1% of methyl p-hydroxy-benzoate, the pH being about 6. Advantageously the filter pad comprises a polycarbonate film having pores of about 8 microns in diameter and it is stained on the glass slide with a gentian violet solution containing safranin O and propylene glycol. The apparatus includes a syringe having an openended projection which is of such size as snugly to fit in the outlet of the lysing solution dispenser and the inlet of a holder for said filter pad, so as to minimize any possible contamination.  
 10 Claims, 10 Drawing Figures DRAWING BLOOD SAMPLE INTO SYR INGE DRAWING LVSING SOLUTION INTO SYRINGE EXPELLING LIQUID FROM SYRINGE THROUGH FILTER WASHING I TRANSFERRING FILTER PAPER TO MICROSCOPE SLIDE I STAINING l COVERING I EXAMINING MICROSCOPICALLY F I G. I.  
 DRAWING BLOOD SAMPLE INTO SYRINGE DRAWING LYSING SOLUTION INTO SYRINGE FROM SYRINGE THROUGH FILTER EXPELLING LIQUID WASHING TRANSFERRING FILTER PAPER TO MICROSCOPE SLIDE STAINING COVERING EXAMINING MICROSCOPICALLY METHOD AND APPARATUS FOR THE DETECTION OF MICROFILARIAE The present invention relates to the detection of mierofilariae in blood.  
  Mammals. particularly small animals such dogs. have been suffering from hcartworm disease, i.e. worms which find their way into the animals heart with frequently fatal results if not promptly treated. The most satisfactory way of detecting such disease is through examination of a blood sample to detect the presence of [)irofilaria immitis microfilariae.  
  Different procedures have been proposed for such examination. A drop of blood may be placed on a glass slide and directly examined under a microscope. Because the sample is so small, however, microfilariae might not be observed even though present. Use of stains to detect the normally transparent microfilariae helps but other substances present in the blood sample may obscure the microfilariae. ln Journal of the American Veterinary Medical Association, Vol. 156, No. 1970). pages I403 if, there is described an improved technique wherein a larger blood sample is lysed to solubilize unwanted cellular materials and the mixture is filtered. stained on the filter and examined. This resulted in a marked increase in reliability but the procedure was complicated, required considerable apparatus. and was prone to error due to the possibility of contamination and complication if carried out under less than ideal conditions.  
  it is accordingly an object of the present invention to provide a simple, reliable process for detecting the presence of mierofilariae.  
  It is a further object of the invention to provide a simple apparatus suited for carrying out such process.  
  These and other objects and advantages are realized in accordance with the present invention which relates to an improvement in the process of detecting microfilariae wherein a blood sample is drawn into a syringe. the blood mixed with an aqueous lysing solution, filtered through a pad, the filter pad stained and examined on a glass slide under a microscope. Specifically, the improvement comprises mixing said lysing solution into said blood-containing syringe, and discharging the mixture through said filter pad, the blood sample thereby being minimally exposed for contamination.  
  The invention also provides an apparatus for carrying out the novel process, comprising a syringe having an open-ended projection, an annular needle removably disposed in said syringe for drawing a blood sample therein. a lysing solution dispenser having an outlet for said solution. said outlet being of such size as snugly to receive the open-ended projection of said syringe without said needle. whereby said syringe can be operated to draw said lysing solution into the blood sample within said syringe, a filter holder having an inlet and outlet and means for removably supporting a filter pad therebetwcen, said inlet being of such size as snugly to receive the open-ended projection of said syringe so as to expel the lysed blood sample onto said filter pad, and a glass slide being of such size to be received on a microscope viewing stage.  
  1n practicing the invention a blood sample which may be as little as 1 cc or even less is drawn into a syringe through a conventional needle. The sample may be drawn directly from the animal although usually it is drawn from a vial which contains a previously drawn,  
 optionally anti-coagulated, sample of which aliquot portions are to be subjected to a variety of tests.  
  The syringe needle is removed and the tip of the syringe as a male member is snugly mated with the outlet of a dispenser containing lysing solution, the lysing solution being drawn into the syringe by operating its piston. In this fashion the blood sample does not contaminate the lysing solution within the dispenser which is also kept from normal atmospheric contamination. The syringe barrel thus serves as the only mixing vessel.  
  Advantageously the dispenser outlet includes a oneway flapor ball-valve to prevent blood from flowing into the dispenser in the event the syringe piston is moved in the wrong direction.  
  The lysing solution may be any usually employed but especially satisfactory results are realized when it is an aqueous solution comprising by weight about 0.25 to 2.5% of a non-ionic surfactant, about 1 to 2.5% of an alkali metal citrate-citric acid buffer system wherein the citrate is present in about 2 to 4 times the amount of the citric acid, and about 0.01 to 1% of an antifungal agent, the pH being about 5 to 7. It is employed in about 5 to 15 times the volume of the blood sample, e.g. about 9; smaller amounts may not produce rapid and/or sufficient lysis and larger amounts are unneces sary.  
  The preferred non-ionic surfactants are polyoxyalkylated phenol ethers such as polyoxyethylene octylated phenol ether. Suitable materials are sold by Rohm and Haas under the tradenames Triton X- and Triton X-405. lt solubilizes the blood cell components more quickly and reliably than other surface active agents. Preferably it is employed in about 1% by weight of the solution although, as noted, it can be employed in about 0.25 to 2.5%. Lower amounts produce insufficient or too slow lysis; larger amounts result in foaming which interferes with the subsequent filtration.  
  The citrate may be potassium or other soluble salt, although preferably it is sodium citrate. The citratecitric acid system serves to maintain the pH at the desired level of about 5 to 7, advantageously about 5.5 to 6.5 and preferably about 6. More acid systems may result in precipitation of the iron of the hemoglobin which would interfere with the microscopic observation. Less acid systems result in more viscous solutions with much cellular debris which will effect less selective filtration. The citrate-citric acid also serves the beneficial function of an anticoagulant.  
  The overall amount of citrate-citric acid may range from about 1 to 2.5% although preferably it is more than about 1 and especially about 1.75%. Lesser amounts result in too little anticoagulation and too low a pH. Higher amounts are wasteful and may exceed the solubility of both components. The citrate is advantgeously present in about 2 to 4 times, preferably about 3 times, the weight of the citric acid for the desired effect.  
  Coloring agents, stabilizers, and the like may also be present. A particularly suitable component is an antifungal agent to prevent contamination of the lysing solution itself. Particularly suitable for this purpose is methyl p-hydroxybenzoate (methylparaben) employed in about 0.01 to 1%, advantageously about 0.05 to 0.2% and preferably about 0.1% by weight. The balance of the solution is preferably deionized or distilled water.  
  After allowing the blood sample and lysing solution to mix fora few seconds in the barrel of the syringe, the tip of the syringe is inserted in mating relationship into the inlet of a filter holder having an inlet and outlet and means for rcmovably supporting a filter pad therebetween. The syringe plunger is actuated to force the lysed blood sample through the filter pad. retaining thereon insoluble material which will include the microfilariae. if present. The syringe may be used to force wash water once or twice through the filter pad to ensure removal of any debris.  
  The filter pad is preferably a membrane or film of carefully controlled pore size. c.g. about 2.5 to l microns and advantageously about to 8 microns. Smaller pores result in slow filtration and may remove some of the cellular debris which is undesirable. Too large a pore size will obviously pass the microfilariae. A General Electric Nucleopore polycarbonate film having pores of about 8 microns is especially suitable.  
  After filtration the membrane is removed and placed on a glass microscope slide. Conventional stains may be used to facilitate detection of the normally transparent microfilariae but it has been found that methylene blue is far less satisfactory than gentian violet which gives sharper and deeper coloration. The gentian violet is preferably employed in combination with safranin (I and a non-volatile humectant such as a polyhydroxyalkane, e.g. a lower alkylene glycol or polyglyeol, glycerine or the like. Propylene glycol is particularly satisfactory. This serves to prevent drying out of the filter pad when placed between two glass plates and viewed under the microscope. The two glass plates may be squeezed to expel any excess liquid.  
  By staining the filter pad or the glass slide rather than in the filter holder. less stain is used and the excess stain remains on the pad rather than being washed therethrough. In addition there is less mess and no coloration of the filter holder.  
  Viewing is thereafter conducted in conventional fashion. It is obvious that detection of microfilariae will be simplified because of concentration from the larger volume of blood and the freedom from contamination.  
  The invention will now be described with reference to the accompanying drawing wherein:  
  FIG. I is a flowsheet of the steps involved in the process in accordance with the present invention;  
  FIG. 2 is a side view of the syringe at the outset of the process;  
  FIG. 3 is a view of the syringe and hypodermic needle after drawing a blood sample.  
  FIG. 4 is a view of the syringe coupled to a lysing solution dispenser:  
  FIG. 5 is an enlarged sectional view of the outlet of the dispenser of FIG. 4;  
  FIG. 6 is a view of the syringe discharging its contents through a filter holder;  
  FIG. 7 is a longitudinal sectional view through a portion of FIG. 6;  
  FIG. 8 is a perspective view of a glass slide carrying a filter pad during the act of staining;  
  FIG. 9 is a view of the slide of FIG. 8 surmounted by a glass plate: and  
  FIG. 10 is an enlarged view of a portion of what is seen when viewing the slide of FIG. 9 on a microscope.  
  Referring now more particularly to the drawings, in FIG. 2 there is shown a syringe 12 comprising a piston or plunger 14 fitting within a barrel 16. A cap 18 is removably fitted over the tip or projection 20 of the syringe 12. The cap 18 is removed and replaced by a hypodermic needle 22 (FIG. 3) which is inserted into a vial (not shown) containing a blood sample or into an animal. The plunger 14 is retracted. drawing a blood sample into the barrel 16.  
  The needle 22 is then removed and the tip 20 is mated with the outlet 24 of a plastic wash bottle 26 (FIG. 4). As seen in FIG. 5 the outlet 24 in inoperative position has a cap 28 which is removed when the syringe is to be attached. A one-way valve, e.g. a flap 30 prevents blood from the syringe 12 being expelled into the dispenser 26. Instead. the syringe plunger 14 is further retracted to draw lysing solution into the barrel 16 to mix with the blood sample.  
  The syringe projection 20 is removed from the dispenser outlet 24, the cap 28 replaced on the dispenser, and the projection 20 is mated with the inlet of a filter assembly 32 as shown in FIG. 6. As shown in FIG. 7, the assembly 32 comprises threadedly engaging inlet and outlet members 34 and 36, respectively. A wire screen support 38 is mounted in one of the members, e.g. outlet 36 and it supports a filter membrane 40. The syringe contents are expelled through the membrane 40, any solids therein being retained on the membrane. Desirably the syringe is uncoupled, water drawn therein and expelled into the assembly 32 as a wash.  
  The membrane 40 is placed on a glass slide 42 (FIG. 8) and a stain is applied through a dropper 44, whereafter a second glass plate 46 is superposed (FIG. 9) and the prepared slide is viewed under a microscope (not shown). As seen in FIG. 10 there can be seen holes 48 of fairly regular size. which are the openings in the membrane 40. There are also visible stained elongated microfilariae 50.  
  The invention will now be described in the following illustrative example which was carried out with the apparatus of the drawing.  
 EXAMPLE 1 cc of blood was drawn from a stoppered vial containing blood drawn from a heartworm-infected dog, using a 25 cc syringe equipped with a standard needle. The needle was removed and the syringe tip inserted into the uncovered outlet of a lysing solution dispenser containing lysing solution at a pH of 6 and of the following composition;  
 oetyl phenoxy polycthoxy cc ethanol (Triton X- l ()0) sodium citrate 13.2 grams citric acid 4.4 grams methyl p-hydroxy-henzoate l gram Yellow No. S (FDSC) coloring 1 gram agent deionized water to 1 liter 9cc of lysing solution were withdrawn and after 5 seconds the syringe tip was placed in the inlet of a Gelman membrane filter holder whose screen had been provided with a General Electric Nucleopore polycarbonate filter having an 8 micron pore size. The lysed solution was expelled through the filter membrane and the syringe was used to effect two water washes of 10 ee each time. The filter membrane was removed, placed on a glass slide and a few drops of staining solution were applied. the staining solution having been formulated as set out hereinbelow. A second glass slide was placed over the filter membrane, the glass slides squeezed together and expelled liquid wiped away. The slides were then placed under a microscope and microfilariae were readily visible.  
 Preparation of Staining Solution A. gentian violet 3.0 grams ammonium oxalate 0.8 gram ethyl alcohol. 95% 20 cc distilled water to I00 ce B. sat&#39;ranin 0 (Certified 0.25 gram Biological Stain CI. 50240) ethyl alcohol. 95% cc distilled water to I00 cc 5 parts by volume of A were mixed with 95 parts of B, the mixture in turn mixed with one-half its volume of propylene glycol and filtered.  
  It will be appreciated that the instant specification and example are set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from the spirit and scope of the present invention.  
 What is claimed is:  
  1. In the detection of microfilariae wherein a blood sample is drawn into a syringe, the blood mixed with an aqueous lysing solution, filtered through a pad, the fil-. ter pad stained and examined on a glass slide under a microscope, the improvement which comprises mixing said lysing solution with said blood by drawing said lysing solution into said blood-containing syringe, and discharging the mixture through said filter pad, the blood sample thereby being minimally exposed for contamination.  
  2. The process of claim 1, wherein said aqueous lysing solution comprises by weight about 0.25 to 2.5% of a non-ionic surfactant, about l to 2.5% of an alkali metal citrate-citric acid buffer system wherein the citrate is present in about 2 to 4 times the amount of the citric acid. and about 0.01 to 1% of an antifungal agent, the pH being about 5 to 7.  
  3. The process of claim 2, wherein said aqueous lysing solution is employed in about 5 to times the volume of the blood sample and comprises about 1% of a polyoxyalkylene phenol ether, about 1.75% of sodium citrate-citric acid wherein the sodium citrate is present in about 3 times the amount of the citric acid, and about 0.1% of methyl p-hydroxy-benzoate, the pH being about 6.  
  4. The process of claim I, wherein said filter pad comprises an inert permeable membrane having pores of about 2.5 to 10 microns in diameter.  
  5. The process of claim 4, wherein said filter pad comprises a polycarbonate film having pores of about 8 microns in diameter.  
  6. The process of claim 1, wherein said filter pad is stained on said glass slide with a gentian violet solution.  
  7. The process of claim 6, wherein said gentian violet solution further contains safranin 0 and a nonvolatile humectant.  
  8. The process of claim 3, wherein said filter pad comprises a polycarbonate film having pores of about 8 microns in diameter, and said film is stained on said glass slide with a gentian violet solution containing safranin 0 and propylene glycol.  
  9. An apparatus for carrying out the detection of microfilariae in a blood sample comprising a syringe having an open-ended projection, an annular needle removably disposed in said syringe for drawing a blood sample therein, a lysing solution dispenser having an outlet for said solution, said outlet being of such size as snugly to receive the open-ended projection of said syringe without said needle. whereby said syringe can be operated to draw said lysing solution into the blood sample within said syringe, a filter holder having an inlet and outlet and means for removably supporting a filter pad therebetween, said inlet being of such size as snugly to receive the open-ended projection of said syringe so as to expel the lysed blood sample onto said filter pad, and a glass slide for receiving said filter pad, said glass slide being of such size as to be received on a microscope viewing stage.  
  10. The apparatus according to claim 9 including a one-way valve in said outlet of said lysing solution dispenser to prevent accidental expulsion of blood from said syringe into said dispenser.