Lactate Dehydrogenase

Lactate dehydrogenase (LD, LDH) is an enzyme that is found in all cells in all tissues of the body. It catalyzes the reversible conversion of lactate to pyruvic acid in glycolysis and gluconeogenesis. It is released from various anatomical sites of the body in response to cellular injury and damage. It is used as a common marker for tissue damage and disease such as heart failure. Its down-fall is that it isn’t very specific to which tissue is damaged, but there are subtle hints that can clue a physician in particular directions.

Lactate dehydrogenase is structurally composed of four subunits, but the two common subunits are LDHA known as LDH-M, and LDHB, known as LDH-H. The only difference between the two subunits is that their is an amino acid substitution of alanine with glutamine within the H subunit. This amino acid change slightly changes the biochemical properties of the two subunits slightly in that the H subunit can bind faster, but the catalytic activity of the M subunit does not deteriorate at the same rate as the H subunit, it holds well.

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The two subunit of LDH can form five isomers which are found in various sites within the body;

LDH-1 (4H)- Found in the heart, RBCs, and the brain.

LDH-2 (3H1M)- Found in the RES.

LDH-3 (2H2M)- Found in the lungs.

LDH-4 (1H3M)- Found in the kidneys, placenta, and the pancreas.

LDH-5 (4M)- Found in the liver and striated muscle.

LDH is a protein that is found in small amounts normally in the body and there are various conditions that can cause an elevation. Cancer can raise the LDH levels within the body. Cancer cells rely on increased glycolysis due to their high energy demand. LDH elevation in cancer is often times referred to the Warburg effect which allows malignant cells to convert glucose stores into lactate even in the presence of aerobic respiration. This shifts glucose metabolism from simple energy production to accelerate cell growth and proliferation.

Hemolysis can be measured as LDH is abundant in RBCs and can be measured. Although measures should be taken to correctly receive the sample as incorrect procedures an cause hemolysis and a false-positive elevation in LDH levels among other substrates and electrolytes.

It can also be used as a marker for myocardial infarction. Normally LDH-2 is at a higher level than LDH-1. When someone experiences a myocardial infarction, levels of LDH-1 will be significantly elevated to a level higher than LDH-2. This is known as the LDH flip and is diagnostic in patients who have experienced myocardial infarction. Elevation of LDH peaks 3-4 days after MI, and can remain elevated for up to 10 days. LDH has since been replaced by the troponin test, which is a much more specific and sensitive test in diagnosing MI.

High levels of LDH in the cerebrospinal fluid (CSF) can indicate bacterial meningitis. Elevated LDH levels in viral meningitis is indicative of a poor prognosis.

LDH is an important tool that physicians don’t always utilize to its lack of specificity, but it can still be helpful in a diagnosis. Its important not to ignore any test and any result as it still contributes to the whole picture.

-Caleb

 

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Hemoglobin A1C and Diabetes

Diabetes mellitus is a metabolic disorder which is characterized by an elevated blood sugar level over a prolonged period of time. Type 1 diabetes is often referred to as juvenile diabetes or insulin-dependent diabetes mellitus. There is loss of the insulin producing cells in the pancreatic islets called the beta cells. This leads to insulin deficiency. The cause is most often idiopathic or sometimes is immune-mediated where the T-cells lead an autoimmune attack on the beta cells. Type 1 diabetes is often times inherited and occurs mostly in children which is why it is sometimes referred to juvenile diabetes.

Type 2 diabetes mellitus is characterized by insulin resistance, which sometimes can be coupled by decreased synthesis of insulin. There is a defective response to insulin by the bodies different cells. Often a problem with the insulin receptors of these cells. Type 2 diabetes is a life-style problem and some genetic variability. Physical stress, poor diet, stress and excessive BMI are correlated with type 2 DM. In the early stages of type 2 DM it is manageable and the high blood sugar can be reversed by medications and be controlled. As it progresses it may come with complications similar to type 1 DM and treatment may need to be changed and monitored.

Hemoglobin is a protein found in the red blood cells in the body. For an in depth look at what hemoglobin is exactly take a look at the previous article written, titled “Hemoglobin”. Hemoglobin A1C, which is going to be talked about today refers to the glycolated hemoglobin within the body. Glycolated hemoglobin refers to glucose that is bound to the hemoglobin in the red cells.

Hemoglobin A1C is used to monitor diabetes and can tell doctors whether the dose of insulin is sufficient enough to bring the patients glucose to a healthy level and it can also diagnose diabetes. It gives a snapshot of glucose control over a period of 3-4 months. What happens with diabetes is that  glucose builds up in the blood. When too much glucose is present it begins binding to the hemoglobin within the red cells. This is called glycolated hemoglobin. It gives a fairly accurate analysis of glucose control over a period of 3-4 months because red cells live for 120 days.

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The test is measured as a percentage. The percentage is pertaining to how much hemoglobin is saturated with glucose. The reference range is 4-5.6% although levels between 5.7% and 6.4% raise the risk of developing diabetes. A acceptable range for everyone is different when they have diabetes, but typically the individuals physician will work to keep the A1C level below 7%.  Its standard practice to order the hemoglobin A1C test every 3 months as a good way to monitor therapy and change doses, brands, etc. if needed. As the A1C levels rise or remain high it means that either the patient is non-compliant to their treatment regimen or that the treatment itself is not working and puts the patient at a higher risk of diabetic complications. Such complications include eye disease, heart disease, kidney disease, nerve damage and strokes.

Its important as a physician to recognize that certain conditions can alter the results of the test. Chronic anemias, kidney disease or specific blood disorders such as thalassemia can affect the results of the test and must be taken into consideration when using the A1C test to guide treatment.

-Caleb

Urinalysis

A urinalysis is exactly what the name entails. An analysis of a patients urine. A urinalysis is a fairly common test that may be ordered as part of an annual physical or part of diagnostic testing. It can be used as an evaluation of UTIs, Diabetes Mellitus, kidney disease, kidney stones, proteinuria, rhabdomyolysis, liver disease, or if a patient presents with particular symptoms such as abdominal pain, flank pain, painful urination, blood upon urination, and fever. Pregnancy testing is also part of a routine urinalysis if ordered.

The first step in an urinalysis is collection of the specimen from the patient. An optimal sample is an early morning sample, as it is the most concentrated produced during the day. There are no fasting requirements or medication schedule dosage changes unless otherwise directed by the patients ordering physician. 30-60 mL of urine is collected in a clean urine specimen cup through a clean catch method that should be explained to the patient upon request of sample.

There are different variants of urinalysis. One is the macroscopic observation of the urine. This is the direct visual observation which includes noting its quantity, clarity, and color. Urine is normally yellow and clear without any cloudiness. Abnormalities can mean different things and further analysis is needed.

Cloudy: Infection

Dark Yellow: Dehydration

Brown: Liver disease (caused by an accumulation of bilirubin)

Red: Blood (Indicates UTI, stones, tumors, renal trauma)

Orange/Tea Colored: Rhabdomyelitis (Breakdown of muscle)

Foamy: May suggest excess protein

It is important to note that certain medications taken for UTIs can change the color of the urine, Phenazopyridine in particular.

Macroscopic -Urinalysis

Dipstick chemical analysis is performed on a narrow plastic strip which has individual tests denoted with different colored squares. The entire strip is dipped into the urine sample and color changes of the squares are noted either by the technologist or by inserting it into an instrument to read it. Each square takes a specific amount of time to react so its important to allow the reaction to come to fruition before resulting. The color change of particular squares are compared to a reference guide and can point out abnormalities.

In no particular order the squares on the dipstick indicate;

Specific gravity (concentration of the urine)

pH

Protein concentration

Glucose concentration

Presence of Ketones

Hemoglobin

Leukocyte esterase (Suggestive of WBC in urine)

Nitrite (Suggestive of bacteria in urine)

Bilirubin

Urobilinogen

Multistix-Urinalysis-Color-Key

Dipsticks are convenient and are easy to interpret and cost-effective. Its important to keep in mind that dipsticks are qualitative and not quantitative in that they only suggest that there is an abnormality, they don’t quantify that abnormality. Further analysis is required for such results.

Microscopic analysis can detect cells, cellular debris, bacteria, crystals, and certain casts to confirm the dipstick results and further quantify analysis. Once the samples are received they must be centrifuged and discarding the supernatant. Epithelial cells may suggest inflammation or damage to the gallbladder and casts and cellular debris suggest inflammation of the kidneys and upper urinary tract. On very rare occasions tumor cells can be seen which are diagnostic for certain renal carcinomas and other urinary tract cancers.

If red cells are noted it could indicate either an infection, trauma, or stones. They can also indicate glomerulonephritis which is inflammation of the kidneys. Sometimes small amounts of red cells will be seen in healthy individuals.

Urine is considered a sterile body fluid therefore there should be no white blood cells or bacteria. Any amount of WBC or bacteria within the urine is considered abnormal and is suggestive of an UTI, cystitis, or pyelonephritis.

Identifying crystals if any is important and lend diagnostic information as to what is pathologically going on within the body.

Uric acid crystals can vary in size and shape, but usually resemble a rhomboid shape. These crystals are common in individuals with urate nephrolithiasis or acute urate nephropathy.

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Cystine crystals are usually colorless hexagonally shaped and look similar to benzene rings (bringing it back to organic chemistry days). These occur in patients with cystinuria which is a genetic defect in renal cystine transport and in acidic urine (pH <6.0).

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Struvite crystals are often described as having a coffin-lid appearance. These crystals are typically magnesium ammonium phosphate and are seen in alkaline urine (pH >7.0). Seen in patients with UTIs caused by urea-splitting bacteria (Proteus mirabilis) or in patients with infected calculi (struvite stones).

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Calcium oxalate crystals are typically found in acidic urine and can take on multiple shapes. Some may look like colorless ovoids, biconcave discs, or even rods. Usually seen in patients with high dietary oxalate ingestion, patients with nephrolithiasis or those in ethylene glycol toxicity with renal failure.

Triple-phosphate-crystal

These are some of the more common crystals that will be seen. Urinalysis isn’t flashy and isn’t always fun as it is someones urine, but it is an important part of routine testing to better deliver care to the patient.

-Caleb

 

 

Blood Draw Tube Colors and Order

The tube order may not seem like a big deal and may seem unnecessary to some, but it is very important to pay attention too. It also matters as to what type of needle is being used for the draw. If a butterfly needle is being used it is important to have a spit tube because with a butterfly there is air within the hose that connects the needle to the vacutainer. Its important to get this air out before filling any tubes used for patient testing. If a standard needle is being used, you typically don’t need the spit tube, but its good practice. The order still remains the same for each.

Light Blue: The typical tube for routine coagulation studies. The additive is sodium citrate (3.2% or 3.8%). Citrate is a anticoagulant which binds to calcium within the blood so the blood can’t clot. Calcium plays an important role in primary and secondary homeostasis. See my post on DIC for that information, in short it is used in the coagulation cascade. An important aspect of coagulation studies is that the light blue tube must, must be filled completely. There is a ratio of sodium citrate to whole blood and that must remain constant. The tube must be rejected if it is not filled completely.

Green or Mint Tubes: These tubes are used for chemistry studies. Often referred to as PST or plasma separator tubes. The additive in these tubes are sodium heparin, lithium heparin or ammonia heparin. The heparin, being an anticoagulant activates antithrombin, which blocks the coagulation cascade and produces a whole blood with plasma sample instead of a clotted blood and serum sample. When these tubes are centrifuged, the gel barrier moves upwards creating a barrier that separates the plasma from the red cells allowing the plasma to be aspirated directly for testing.

Gray Tube: The gray tube tops are typically used for glucose testing, ethanol levels or lactate level testing. The additive is potassium oxalate and sodium fluoride. Potassium oxalate is an anticoagulant which prevents clotting and the sodium fluoride is an anti-glycolytic which prevent the cells from using the glucose in the sample.

Lavender/Pink Tube: The lavender tube is typically used for hematological testing or for Hemoglobin A1C testing. The pink tube is used primarily for blood bank testing such as type and screen and cross-matching. The additives in the lavender and pink tubes are EDTA K2 or EDTA K3. The EDTA binds to calcium which blocks the coagulation cascade in the same way that citrate in the light blue tube does. Red cells, leukocytes, and platelets are in EDTA anticoagulated blood for 24 hours. Blood smears should be done within 3 hours of receiving the sample.

SST/Mustard Tube: Serum separator and clot activator that will separate the blood from the serum upon centrifugation. This tube is usually used to test for aldosterone, B12, ferritin and folate levels.

There are not all the different tubes that are used, but these are the most common tubes that I listed and the ones that a laboratory professional will most likely come across. Its important to understand the additive in each one to make sure that they are appropriate for the testing that needs to be done on the patient sample itself within the tube.

phb_Order of Blood Draw with labels72dpi

-Caleb

Case Study Mini-Series; Diagnostic Process and Treatment

Diagnostic workup of a suspected patient with APL should include a case history and physical examination with focus on bleeding tendencies, recurrent infections and anemic symptoms such as fatigue or pallor. A complete blood count with a differential should be performed. During a peripheral blood smear the technologist should be looking for abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods. A bone marrow aspirate with cytology, cytochemistry, immunophenotyping, FISH, RT-PCR, and cytogenetics should be included. Diagnostic coagulation tests such as PT, aPTT, fibrinogen, and a D-dimer should be performed. During the immunophenotyping the characteristic phenotype of APL is CD33, CD13, CD45, CD64, and CD117 positive. Also APL is HLA-Dr negative which differentiates it from other AMLs which are HLA-Dr positive.

Early initiation of induction therapy ATRA before confirmation of diagnosis has changed the management of APL. APL is curable due to the initiation of ATRA. APL is considered a severe hematologic emergency due to its rapidly progressing bleeding diathesis and risk of intracerebral hemorrhage. Making a presumptive diagnosis based on the peripheral blood smear and bone marrow aspirate along with the patient history is important because the earlier that the patient begins therapy the better the outcome. ATRA and blood product support should be started as early as possible. APL blasts are highly sensitive to anthracyclines. Anthracycline chemotherapy with combination ATRA boasts remission rates of more than 90%. ATRA otherwise known as all-trans retinoic acid is a derivative of retinoic acid which reverses the differentiation block of APL blasts. Arsenic therapy with arsenic trioxide is approved in Europe and the United States for relapsed and refractory APL.

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Aggressive supportive therapy involves FFP, cryoprecipitate and platelets to maintain platelet levels greater than 30,000-50,000/uL and fibrinogen levels above 150 mg/dL. This regimen typically lasts during the first week of induction therapy while the coagulation disorder resolves.

There are significant adverse effects with therapy for APL. A common complication during induction therapy with ATRA or ATO (arsenic) is the development of hyperleukocytosis. APL differentiation syndrome is a life-threatening complication that develops a fever, edema/weight gain, respiratory distress, lung infiltrates, and pleural or pericardial effusions. Differentiation syndrome typically occurs within the first two weeks of the onset of therapy. Intravenous Dexamethasone is recommended immediately in the suspicion of APL differentiation syndrome. In mild cases of differentiation syndrome, ATRA or ATO therapy can just be interrupted and continued after symptoms regressed and when leukocyte counts decrease. Arsenic trioxide toxicity causes electrolyte shifts, particularly involving potassium and magnesium which to no surprise can alter ECG readings causing most commonly a QT interval prolongation. ATO therapy must be discontinued in severe prolongations due to the increased risk of cardiac arrhythmias. Documented chemotherapy adverse effects include the typical nausea and vomiting, increased infections, anemia, thrombocytopenia, increased bleeding tendencies which is exacerbated due to the coagulopathy associated with APL, and cardiac effects. With long-term chemotherapy there is an increased risk of drug-induced secondary malignancies.

Choice of treatment and timing of treatment is extremely important. As mentioned earlier it is very important to start induction therapy upon the first suspicion of APL, even before molecular confirmation occurs.

-Caleb