Abstract:
The invention relates to a centrifuge apparatus with a built-in sample tube reader and methods for rapidly obtaining measurements of creamatocrit, fat content and/or energy (caloric) content from low-volume fresh and frozen milk specimens.

Description:
FIELD OF THE INVENTION 
     The invention relates to a centrifuge apparatus with a built-in reader and methods for rapidly obtaining measurements of creamatocrit, fat content and/or energy (caloric) content of fresh and frozen milk specimens. 
     BACKGROUND OF THE INVENTION 
     Continuing efforts have been made to increase human neonatal survival rates with great progress towards improving the chances of survival for full-term and premature babies. Nutrition plays a key role in determining how well a newborn will thrive and develop. Nutritional information is, of course, provided with artificial formulas for infants. However, human breast milk is generally regarded as superior to formulas, giving infants a significant advantage with regards to growth and development, yet human breast milk is rarely analyzed with regards to fat content, which is directly related to energy content. Such information is critical to the care of premature infants and babies that fail to gain weight at a normal rate. 
     Many methods, as well as instruments employing such methods, have been developed for determining the fat content of milk. While some of the methods have enjoyed popularity in the dairy industry, none has gained widespread use or acceptance in clinical or health care settings. The Babcock method developed in the late 1800&#39;s became a standard procedure for determining milkfat content in the dairy industry. The procedure is somewhat hazardous, involving mixing a raw milk sample with concentrated sulfuric acid followed by heating and centrifugation of the mixture. Further, the Babcock method is not suitable for very small test sample volumes, for example, less than 100 microliters. 
     U.S. Pat. No. 3,161,768 to Goulden teaches a method of using infrared absorption, measured at the wavelength of absorption at the ester linkages (approximately 5.72 microns), for determining fat content of the disperse phase in an emulsion or suspension, such as milk. Such measurements are significantly affected by differences in diet and even genetic differences in cattle, but more reliable measurements are obtained when infrared absorption is measured at the carbon-hydrogen stretching wavelength (approximately 3.48 microns) as taught in U.S. Pat. No. 4,247,773 to Nexo et al. A disadvantage is that milk must be homogenized to reduce fat particle size in order to obtain meaningful data by such absorptiometric methods. 
     The International Dairy Federation published a standard procedure (IDF Standard 9C, 1987) for determining fat content of dried milk known as the Rose Gottlieb method. This gravimetric method is complicated, lengthy, and involves the use of solvents. Moreover, the milk sample must first be dried and the method is not suitable for small samples of milk less than 100 ml. 
     Other methods described for determining fat content include colorimetric methods, based upon a color reaction between milk fat and hydroxamic acid, and analysis of fat content by nuclear magnetic resonance (NMR). These procedures are complicated and require relatively expensive, specialized equipment. 
     In an attempt to develop a procedure with clinical applications, Lucas et al. described a simplified method for determining the fat and energy content of human milk based upon centrifugation of a small sample collected in a standard hematocrit capillary tube for fifteen minutes. See Lucas et al., Br. Med. J. 1:1018, 1978. The length of the cream layer is measured and calculated as a percentage of the total length of the milk column, for example, using a standard hematocrit-measuring card to determine the volume percentage of fat, referred to as a creamatocrit. Use of a hematocrit reader card requires the user to visually align several interfaces at once and then use the determined fat content to calculate estimated caloric content. Alternatively, calipers can be used to measure the cream column and the total length of the centrifuged milk specimen in the capillary tube, and the measurements obtained can be used to calculate the percentage of milkfat, with further calculations needed to determine estimated caloric content. In yet another method, micro-capillary readers can be used to mechanically determine fat content by manually aligning the capillary tube after centrifugation with an index mark, then aligning two rotating disks with the total length and the cream column; when a reading is determined, the user then must subtract the number from one hundred to obtain the percentage of milkfat and then perform further calculations to estimate caloric content. 
     Although the creamtocrit method represents a significant simplification in relation to other methods for determining milkfat content, it, too, has failed to secure broad acceptance in clinical settings. Further improvements directed towards shortening centrifugation time and to simplifying measurements and calculations of fat and energy content are needed in order for the creamatocrit technique to enjoy widespread use, particularly in clinical and public health care settings where the determination of fat and energy content of human milk is critical to neonatal and infant nutrition, as well as veterinary and research applications for non-human mammals. 
     SUMMARY OF THE INVENTION 
     The invention is an improvement to the existing creamatocrit technique comprising a centrifuge apparatus with a semi-automatic or fully automatic reader for entering data points determined from the centrifuged sample, as well as its use for rapid calculations of creamatocrit, estimated fat content, energy and/or caloric content. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a perspective view of one embodiment of the present invention, namely a centrifuge containing a sample tube reader. 
         FIG. 2  shows a cross-sectional view of an embodiment of a rotor for a centrifuge of the present invention. 
         FIG. 2A  shows an enlarged cross-sectional view through the capillary tube and tube holder within a cavity in the rotor. 
         FIG. 3  shows a perspective view of an encoder and encoder strip with etched slots. 
         FIG. 4  shows a perspective view of an exemplary capillary tube containing a milk sample after centrifugation. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In one embodiment, the invention comprises a centrifuge for separation of milkfat (cream) from the aqueous phase in small samples (≦100 microliters) of milk, including, but not limited to, human breast milk. Prior to the present invention, creamatocrit procedures have been conducted using standard centrifuge devices with cavities for containing samples oriented either horizontally or at a fixed angle of about 45° with respect to the rotational axis. U.S. Pat. No. 4,738,655 to Brimhall et al. and U.S. Pat. No. 5,354,254 to Zabriskie et al., each incorporated herein in entirety, teach the use of a steeper angle, for example 70° to the horizontal plane (about 20° to the rotor axis), in order to significantly shorten the time required to sediment blood cells. The same principle is applied in the present invention with regards to centrifugation of milk samples with beneficial results in terms of decreasing centrifugation time needed for separation of the fat and aqueous phases. The time required for separating the cream and milk phases in the present invention is approximately no more than about three minutes. 
       FIG. 1  shows a perspective view of one embodiment of a centrifuge  10  with a fixed angle rotor  12  according to the present invention. The rotor  12  contains a plurality of circumferentially and evenly spaced cavities  14  for receiving standard non-heparinized hematocrit capillary tubes  16  containing milk samples. Preferably, the capillary tubes are placed within tube holders  17 , as shown in  FIGS. 2 and 2A , to contain milk samples in the event a capillary tube breaks during centrifugation. Such tube holders  17  prevent contamination of the centrifuge device  10  and operator by containing a potentially biohazardous specimen. A particularly useful type of tube holder  17  is disposable, made of transparent plastic and accepts capillary tubes with a maximum outer diameter of about 0.08 inches. Such tube holders are available from Separation Technology, Inc. (Altamonte Springs, Fla.). The cavities  14  are formed at a fixed angle to the rotor axis  15 , indicated by the broken line in  FIG. 2 . A preferred angle is approximately 20° to the rotor axis  15 , but can be within a range from about 15° to about 25° to the rotor axis  15 . 
     The rotor  12  can be made from any number of materials known in the art that provide adequate corrosion resistance and strength, for example, aluminum, titanium, carbon fibers, or plastic polymers, such as acetal. Depending upon the material used, the rotor  12  can be machined, molded, stamped or otherwise manufactured by methods known in the art. 
     As further shown in  FIG. 2 , the rotor  12  connects to a shaft  18  driven by a motor (not shown), preferably one that operates on DC voltage, capable of spinning the rotor  12  at a maximum speed of about 5,500 to 7,000 r.p.m. and about 1,500 to 2,500 RCF. At a speed of about 6,000 r.p.m (about 1,750 RCF), a milk sample approximately 75 microliters in volume contained within a standard non-heparinized hematocrit capillary tube  16 , is completely separated into cream and aqueous milk phases within approximately three minutes. 
     Preferably, a microprocessor is used to control the speed of the centrifuge  10 , as is conventional in the art. For example, a Hall effect sensor can provide rotational speed input such that motor speed is continuously monitored and adjusted by the microprocessor. A particularly useful type of speed control is pulse width modulation (PWM) with a frequency of modulation at about 2,000 Hz. In a preferred embodiment, the motor is turned off if motor speed drops below a certain minimum speed, for example, about 5,670 r.p.m., and an optional error message indicating low speed is displayed. Additionally, the centrifuge can be designed so that the motor shuts down if the Hall effect sensor fails. Further, in a preferred embodiment, the motor is connected to a timer that fixes the spin time to a particular time span, for example 180 seconds, permitting the user to merely push a button or use some other form of initiation switch. 
     Additionally, the centrifuge  10  can include a battery pack (not shown), as known in the art, for operation as an alternative to other power sources. Rechargeable batteries, such as nickel metal hydride, are particularly suited for such use since they can charge while the centrifuge  10  is connected to an external power supply. If rechargeable batteries are used, the battery voltage is preferably monitored by the microprocessor to prevent overcharging. 
     Referring again to the particular embodiment shown  FIG. 1 , the centrifuge housing  19  has an external platform  20  with a channel  22  for receiving a sample tube  16  after centrifugation, serving to facilitate the sample “reading” capabilities of the invention. A similar reader with semiautomatic capabilities is described in U.S. Pat. No. 4,887,458 to Baker et al., incorporated herein in entirety. The platform  20  and channel  22  are shown in a horizontal position in  FIG. 1 , but a vertical position is likewise suitable. In one embodiment, the user repositions a movable marker  24  with an alignment mark  26  to enter data points via a data entry button or switch  28 . Referring to the embodiment shown in  FIG. 3 , the marker  24  is mechanically coupled to an encoder  25 , for example an LED encoder, straddling an encoder strip  27 . In a preferred embodiment, the encoder strip  27  is fabricated from a thin strip of electroformed nickel with etched vertical slots  29  approximately 0.003 inches wide. A spacing of approximately 0.0037 inches between the etched slots  29  provides a microprocessor with a resolution input of about 150 slots per inch. When the marker  24  is moved, for example by using a sliding motion as indicated by the arrow in  FIG. 3 , the signal is interrupted by the slots  29  providing data input to a microprocessor, enabling the microprocessor to monitor the exact position of the marker  24  during the tube reading process. Alternatively, the marker  24  can be mechanically linked to a rotary disk with slots designed to interrupt an LED or other emitter and sensor device. Likewise, instead of slots, the disk can have one or more magnets that rotate past a reed switch or Hall effect sensor. 
     The positional information produced by the movable marker  24  is in turn used by a microprocessor to determine the length of the cream column and the total length of the sample within the centrifuged capillary tube  16 . A preferred microprocessor is a Motorola type 68HC11 microprocessor, but other suitable microprocessors are commercially available. A display window  30  located on the housing  19  displays results of calculations, as well as showing optional error messages and/or user prompts when appropriate. Several different types of displays are suitable including, but not limited to, diode array, ferroelectrics, plasma display panel, LED or preferably, a commercially available liquid crystal display (LCD). 
     In one embodiment, after a sample is centrifuged for approximately three minutes, two phases are observable, an aqueous phase  31  and a cream phase  33 , and three interfaces are present as shown in  FIG. 4 : a first interface  32  between the sealant  37  and aqueous milk phase  31 , a second interface  34  between the aqueous milk phase  31  and cream phase  33 , and a third interface  36  between the cream phase  33  and air space  38  in the capillary tube  16 . In the event a clear yellow fluid layer is present on top of the spun specimen (not shown), the yellow fluid layer is included as part of the cream layer  33 . As an example of one method of using the device, the user moves the marker  24  so that the alignment mark  26  is collimated with the sealant/milk interface  32  and presses or otherwise activates the data entry switch  28  to input the first data point. The marker  24  is then moved until the alignment mark  26  is positioned in the approximate center of the diagonal milk/cream interface  34 , and the data entry switch  28  is again activated to input the second data point. The marker  24  is then moved until the alignment mark  26  is approximately centered on the cream/air interface  36 . The third data point is entered via the data entry switch  28 . 
     The electronics in the reader are conventional and are programmed so that after entry of all three data points, the creamatocrit reading is automatically displayed. The creamatocrit value is calculated automatically by comparing electronically the distance between the milk/cream and cream/air interfaces (length of cream phase) with the distance between the sealant/milk and cream/air interfaces (total length of sample), wherein the ratio of the measurements is multiplied by 100. Fat content can optionally be calculated, electronically as known in the art, according to the formula provided in Lucas et al. wherein estimated fat grams/liter=3.968+(5.917×creamatocrit percentage). The result is displayed in the display window  30 . Estimated energy content in kilocalories (kcal) per liter can be calculated as 385.422+(55.656×creamatocrit percentage). Alternatively, other formulas may be programmed as desired, such as Calories per ounce determined by dividing kcal/L by 33.8141. 
     In addition to the semi-automatic format described above, another embodiment is a centrifuge with a fully automatic reader wherein a sensor comprised of a radiation emitter, for example, infrared or visible light, or other measurable emitter, and a sensor for detecting the emissions is used to scan the length of the sample tube after centrifugation. The sensor and electronic interface to a micro-controller (not shown), and software automatically determines the interfaces  32 ,  34 ,  36  and displays the creamatocrit and/or other calculations. 
     While several embodiments have been described, the present invention may be embodied in other specific forms, as apparent to those of ordinary skill in the art, without departing from the spirit of the invention.