Accurate, clinically relevant results depend upon specimen integrity. Antech’s multifaceted approach to improve the standards for clinical testing began last year: we offered two newsletters about specimen integrity, updated several assay techniques, introduced new tests, and provided specimen materials to facilitate transport to the lab (aprotinin tubes for endogenous ACTH samples, urine and viral culture transport vials, cassette holders for smaller tissues). Now we inform you of the shortcomings in the routine analysis of total carbon dioxide (CO₂), caused by problems related to specimen collection and transport.

Over the past year, we have had numerous con plaints from veterinarians about the lack of correlation between total CO₂ levels and clinical findings. After considerable investigative efforts, we determined that these sporadic problems were directly related to the following conditions:

1. Amount of dead air space left in vacuum collection tubes;
   
2. Separation of serum and cells before transport;
   
3. Use of enzymatic assay rather than direct assay;
   
4. Refrigeration of specimen during transport.

Although Antech uses quality reagents and appropriate standards, controls, and equipment, over 50% of the specimen tubes we receive are less than optimally filled. This provides an accurately measured value but one which is often clinically erroneous.

Faced with this problem, we are removing CO₂ from our routine panels effective February 1, 1998.

If there is a specific need for a CO₂ assay on an animal, add it to the chemistry screen by writing CO₂ in the requisition add-on column. The assay will be performed at no charge. If you wish to continue to receive CO₂ levels on all chemistry samples, please notify your customer service representative or laboratory manager by February 1, 1998. Please assure that specimens for CO₂ analysis are drawn into completely filled gel separator tubes, centrifuged and cooled during transport. The CO₂ analysis are drawn into completely filled gel separator tubes, centrifuged and cooled during transport. The CO₂ value will be accurately measured, but it may not correlate with your clinical findings.

• Effects of sample handling on total CO₂ concentrations in canine and feline serum and blood games, K.M. et al, Am J Vet Res 58: 343-347, 1997).
  The author drew 10, 3 or 1 mL of blood into 10 mL tubes. Mean total CO₂ values were 2.0 and 3.7 mEq/L greater for the full 10 mL tube than for the 3 mL and 1 mL filled tubes.
  
• Pseudometabolic acidosis caused by underfilling of Vacutainer tubes (Herr, R.D. Ann Emory Medical 21 (C2): 177-180, 1992).
  The author also drew 10, 3 or 1 mL of blood into 10 mL tubes, Total CO2 levels were 21.7 (10 mL), 19.4 (3 mL) and 16.3 (1 mL) mEq/L, a finding confirmed later by the James et al study.
  
• The magnitude of metabolic acidosis is dependent on differences in bicarbonate assays (Bray, S.G. Am J Kidney Dis 28 (5): 700-703, 1996).
  Mean total CO₂ levels using an enzymatic method (18.7 mEq/L) were substantially lower than a direct measurement with an electrode (22.2 mEq/L).
  
• NCCKS Guideline C × 27- A blood gas (arteria1, venous, capillary). Pre-analytic considerations. Specimen collection, calibration and controls; approved guideline – samples must be separated to ensure that accurate CO₂ levels are reported. This prob1em is enhanced by extended transport times.

The bicarbonate level of blood plays an essential role in acid-base balance and is reflected by the serum total CO₂ level. Acid-base balance is maintained through renal and pulmonary mechanisms. Oxidative metabolism produces large amounts of CO2 each day. On combining with water in the blood stream, CO₂ Forms carbonic acid. This reaction is facilitated by the enzyme carbonic anhydrase from red blood cells. The excess hydrogen formed is buffered by intracellular constituents such as hemoglobin. The bicarbonate leaves the erythrocyte and enters the extra- cellular fluid in exchange for extracellular chloride. The net effect of these reactions is to carry CO2 in the bloodstream as bicarbonate with little change in extrace11ular pH. These processes are reversed in pulmonary alveoli, as CO₂ is excreted by ventilation.

ACID-BASE IMBALANCES AND COMPENSATORY MECHANISMS
DISORDER	pH [H+]	PRIMARY IMBALANCE	COMPENSATORY RESPONSE
METABOLIC ACIDOSIS		[HCO3-]	pCO2
METABOLlC ALKALOSIS		[HCO3-]	pCO2
RESPIRATORY ACIDOSIS		pCO2	[HCO3-]

The body’s buffering capacity includes the extra- and intracellular buffers and bone. Extracellular buffers are the bicarbonate (HCO₃-/H₂CO₃) and phosphate (HPO₄ ²-/H₂PO₄) buffer pairs, and the plasma proteins, organic and inorganic phosphates, and hemoglobin. Bone carbonate provides a large and generally overlooked buffer store which is estimated to contribute up to 40% of the buffering capacity of an acute acid load. A change in hydrogen ion concentration affects all buffer pairs in the body and hence measurement of one buffer pair reflects changes in the others .

Serum/plasma pH is determined by the ratio between bicarbonate and carbonic acid concentration. Respiratory acidosis or alkalosis depends upon CO₂ levels, while metabolic acidosis or alkalosis is determined by HCO₃–. Primary respiratory imbalances in acid-base metabolism are counter-balanced by compensatory changes mediated through the kidneys which alter the excretion or retention of hydrogen ions or bicarbonate. The most commonly used clinical assay measures total CO₂, the combination of CO₂ and HCO₃ . Elevated CO₂ can arise from either respiratory acidosis or metabolic alkalosis. Therefore, accurate assessment of acid- base balance requires information about pH and pCO₂ as well. Unfortunately, the accuracy of these assays depends upon their rapid assessment, so that transport and time delays often render them invalid.