Alkaline Information and Courses from MediaLab, Inc.

After considering the results of the brilliant cresyl blue stain, the clinical laboratory scientist decided to repeat the hemoglobin electrophoresis on this patient. This time, she shortened the electrophoresis time by fifteen minutes.The results of the electrophoresis, represented in the image below, show a band in the area of Hb H. Hemoglobin H travels quickly during alkaline electrophoresis, and a shorter electrophoresis time was needed to ensure that HbH remained on the acetate paper. HbF is still present as it was on the original electrophoresis, but it is blended into the Hb A band.

Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A2 moves only slightly from the point of origin near the cathode.Abnormal hemoglobins show the following migration patterns: Hb C migrates with Hb A2 near the cathode. Hb S lies between Hb A2 and Hb F. Hb H and Bart's hemoglobin are unstable and very fast moving placing them past Hb A and near the anode with Hb H being the fastest of the two.Relative migrations of hemoglobin variants on alkaline electrophoresis can be seen below.

Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry.

The following list corresponds to this image of an alkaline hemoglobin electrophoresis.Lanes 1 and 2: normal patient specimenHb A is over 98% with a small amount of Hb A2 visibleLanes 3 and 4: Beta thalassemia minorHb A is decreased to 94%, Hb A2 is increased at 5%, and Hb F is 1%Lanes 5 and 6: Delta-beta thalassemia majorNo Hb A or A2 is present, Hb F is 100%Lanes 7 & 8: Delta-beta thalassemia intermediaHb A is 8.5%, Hb A2 is 3.5% and Hb F is 88%Lane 9: AF control Lane 10: ASC control(Remember, AF and ASC are labels and do not indicate the order of migration.)

Reasons for acidic urine pH include: a high-meat diet, respiratory/metabolic acidosis, and hypochloridemia. A urine with a high concentration of glucose may also have a lower pH. An alkaline pH may be the result of a vegetarian diet, respiratory/metabolic alkalosis, or a bacterial infection caused by urease-producing bacteria. Urine that contains bacteria can become more alkaline if the specimen remains at room temperature for an extended period of time. A pH can be falsely interpreted as more acidic than it actually is if improper technique is used and excess urine is allowed to pool on the reagent strip. The reagents from the protein pad, that includes an acid buffer, can run over into the pH pad if the strip has these two tests located next to each other.

A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, or quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

A routine reagent strip protein method, based on the principle of "protein error of indicators," produces a visible colorimetric reaction that is capable of detecting most instances of proteinuria.Traditionally, laboratories have used sulfosalicylic acid (SSA) to confirm all positive protein reagent strip results, but this practice is not as common in today's laboratories. SSA is a precipitation method that reacts with all forms of protein. However, any substance that is precipitated by acid will produce false-positive SSA results. This includes radiographic dyes, cephalosporins, penicillins, and sulfonamides. SSA may be used as a secondary protein detection method if the urine is highly alkaline (pH of 9.0 or greater) which would overwhelm the buffering capacity of the reagent on the protein reagent stick. SSA may also be used as an alternative protein detection method if the urine is highly colored so that the colorimetric reaction is masked on the reagent strip.

These methodologies use principles that amplify or multiply the target of interest, usually incorporating an enzyme to produce millions or billions of copies in a relatively short time.Some enzymes used in amplification include: DNA ligase DNA polymerase RNA polymerase Reverse transcriptase Alkaline phosphatase Cleavase Note: the steps in amplified testing will vary depending on the target amplified, requirement for thermal cycling and detection techniques.

Amplification Method Amplifies Use of Thermal Cycling (Thermocycling) Polymerase Chain Reaction (PCR) Target amplification using DNA polymerase Yes Ligase Chain Reaction (LCR) Target amplification using DNA ligase Yes Transcription- based or Transcription-mediated amplification(TMA) Target amplification using reverse transcriptase and RNA polymerase No Strand Displacement (SDA) Target amplification using DNA polymerase that continuously displaces strands of DNA containing the target sequence No Branched DNA (bDNA) Signal amplification using alkaline phosphatase No Loop Mediated (LAMP) Target amplification of multiple DNA sequences in a loop pattern using DNA polymerase No Nucleic acid sequence based (NASBA) Target amplification using 3 enzymes No Q-beta Replicase Probe amplification- The concentration of an RNA probe increases if the target is present No

Most drugs are bound to proteins when they circulate in the body. Albumin is a major drug-binding protein in serum. Albumin is an alkaline protein, so acidic and neutral drugs primarily bind to it. If albumin binding sites become saturated, acidic and neutral drugs can bind to lipoproteins. Alkaline drugs tend to bind to globulins, particularly to the globulin, alpha-1 acid glycoprotein. Only free, unbound drugs are able to bind drug receptors and have therapeutic effects. An equilibrium exists in the systemic circulation between a free and protein-bound drug and between a free and receptor-bound drug. This is illustrated in the image to the right.

Albumin (Alb) Bilirubin (Bili) Alkaline phosphatase (Alk Phos) Total protein (TP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

In contrast, RBCs appear swollen in dilute or alkaline urine, having taken on water from their surroundings.

Crystals found in normal alkaline urine include triple phosphate, ammonium biurate, calcium carbonate, and amorphous phosphates. This slide shows an example of triple phosphate crystals. These may appear as four to six sided prisms resembling coffin lids. They indicate either stasis of the bladder or a stale sample.