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  • 1. Clin Lab Lecture 10/24/05 Week Five Class Bulletin: For the in class exam you should know: • Stats • Retic Ct. • Indices • Terms The in class exam is open note; but not open book— you can bring one thing, a binder or whatever. You can cram it full of whatever you want— my handouts, anything. I would allow this to be open book but books take up too much space and they make a lot of noise during an exam. Tipson taking this exam successfully: • Prep as if it were a closed-book exam • Tab your notes so you can find stuff • You do not have to memorize normals right now. I’ ll tell you that the reference values for WBC, for example, are 5000-11,000 • If you are prepped, you’ ll be able to leave early. There are two parts to your exam: Part One: The in-class exam on Nov 7 Part Two has two sections, both are due Nov 14 A Flow diagram (please add IDA, it’ s missing from handout) B Case studies The key to part A is to be able to utilize it: you must include every one of these anemias— these are common in a primary care practice. Please do not pull stuff off the internet or from a book. It’ s okay to start from a book or website to get your feet wet; but you want to be thinking, “ How does a PRIMARY CARE doc approach this. NOT a pathologist or lab tech (who might list bone marrow tap and retic ct. as second test to run).” So don’ t list every test you can do, list the tests YOU would do. In addition, you cannot prove a negative. You must have two confirmed tests for each diagnosis. If you call this “ anemia due to renal disease” , you must say confirm with renal function tests (you don’ t need to know the names of
  • 2. tests we haven’ t covered, just “ renal function test” is fine). If there are two functions of the same anemia, you must follow both. For example, anemia due to liver disease (dis) can be macro or microcytic, you must follow both. I will come up with practice problems for you for next week. You may not discuss your exam with others. You may talk to me about it. You can talk about Clin Lab Diag general topics with others; but not exam. If you want to know that you are heading down the right track, email me and I will point you in the right direction. Use your best judgment— have integrity. MEGALOBLASTIC ANEMIAS This is excerpted from the patient handout on anemia in MD consult. For me, it helps to have a “big picture” overview for this lecture. Megaloblastic anemia is the end product of deficiencies in the B vitamins: folate, and vitamin B12 (also called cobalamin), or both. Such deficiencies produce: abnormally large red blood cells (megaloblastic) that have a shortened life span. Neurologic problems are also associated with these deficiencies. There are a number of conditions that can cause these deficiencies. Causes of Vitamin B12 Deficiency: • Pernicious anemia. Pernicious anemia is an autoimmune disease in which antibodies are tricked into attacking stomach’s parietal cells. This results in impaired production of intrinsic factor (IF), a compound that is critical for absorption of vitamin B12; and HCl. Pernicious anemia is diagnosed in about 1% of people over 60, with women having a higher risk than men. • H. pylori and atrophic gastritis. A 2000 study suggested that the H. pylori bacterium is a player in many cases of vitamin B12 deficiency. The bacteria are not only major culprits in peptic ulcers, but also are strongly associated with atrophic gastritis. This condition is a gradual loss of the stomach lining and is a known cause of vitamin B12 deficiency. (Some researchers theorize that H. pylori-induced injuries in the stomach lining may actually be the first step in the destructive process that leads
  • 3. to pernicious anemia.) • Complications of gastrointestinal surgery. Surgeries such as stomach bypass or stapling, which remove part or all of the stomach, pose a 15% to 30% chance of causing vitamin B12 deficiencies. • Overgrowth of intestinal bacteria. Tropical sprue (an acquired malabsorption disease occurring in tropical climates). • Overexposure to nitrous oxide. • Low intake Vitamin B12 deficiency from diet is very rare, since the liver stores over a three-year supply. It usually does not occur even in alcoholism, vegetarianism, or in malnourished people with kidney failure or cancer. Since animal products are the chief source, however, true vegan vegetarians may need a supplement, fortified food, or appropriate food selection known to contain adequate amounts of this vitamin. Causes of Folate Deficiency. • Intake and Intake +Alcoholism The body stores only about 100 times its daily requirements for folate and can exhaust this supply within about three months if the diet is deficient in folate. Poor diet coupled with alcoholism is the most common cause of folate deficiency. Alcohol abuse not only contributes to malnutrition, but alcohol causes chemical changes that can result in lower folate levels. • Malabsorption Any condition that disturbs the small intestine and impairs its absorption ability can cause a deficiency. Such disorders include the following: Inflammatory bowel disease Celiac sprue (a sensitivity reaction to gluten) Parasitic diseases such as giardiasis Short bowel syndrome (due to bowel resection) • Demand
  • 4. Deficiencies can also be caused by high demand for folic acid caused by conditions such as: Cancer Pregnancy Severe psoriasis severe hyperthyroidism hemolytic anemia. • Some drugs may also hinder folate absorption Dilantin for seizures methotrexate for immunosuppression in Crohn’s and cancer trimethoprim for bacterial UTI triamterene for high BP and fluid retention MEGALOBLASTIC ANEMIAS (MA) Presentation for this type of anemia is subtle. Unless the pat tells you “I’ve been a vegan 20 y and not taken any vitamin supplements. My chief complaint (CC) is weakness and fatigue”; you are not going to know off the bat that the pat has MA. Until you do the labs you may not see a MA pic right away…. The other thing is, folate and B12 deficiency, no matter what they are due to: demand, intake, pernicious anemia (PA), will look the same. The only difference in presentation will be that B12 neurological symptoms. Personality changes, loss of cognition, neuropathy….The anemia is reversible, but the neurological symptoms may not be. So if you suspect folate deficiency (def), pls also ck B12 levels. Folate def can be due to: • Increased util • Lo intakenutrient def B12 def can be due to: • Lo intake • Malabsorption • Parasites • PA Clinical Note: In the US and Canada #1 Reason for B12 def is PA PA is an autoimmune disease (usu in pats with a history of AI dis). Ab formed ag. stomach parietal cells that make intrinsic factor and HCL or Ab ag. intrinsic factor (IF) itself. Many people have both. Intrinsic factor is necessary for absorption of B12 in the terminal ileum.
  • 5. Presentation Regardless of cause, B12 def presents as: Clin symptoms that are evolving over weeks to months Nutritional def not common you have a big store, takes a while to deplete PA manifest in middle adulthood 40s and 50s Parasites suck B12 right out of terminal ileum Signs and Symptoms include: 1. Weakness 2. Shortness of breath 3. Lemon yellow pallor (is usully [usu] mentioned in textbks. but this depends on skin color) 4. Tongue changes 5. Glossitis Inflammation (Infl) of the tongue. B12 is required (req) for maturation of epithelium (epit) of tongue and rest of GI tract. Tongue will appear raw and red; or sore, pale and smooth. 6. GI symptoms usu with PA: abdominal pain, diarrhea, vomiting, nausea. 7. 40-45% will have neurological symptoms at diagnosis. Fingers tingling and numb, can’t feel where floor is…. Key is getting to Dx of B12 def before neurological symptoms happen. If the cause of vit B12 def is pernicious anemia, the Ab to the parietal will show up in 75 % and Ab to IF 90%. Labs When we look at hematology changes—and these occur usu before neurological symptoms you have an opportunity to catch B12 def. Hematology changes include: 1. Pancytopenia: all the cels are dec in number. 2. WBC : 4-5K, (normal 5-11K/microliter (uL ) 3. HCT LO 4. Hb low/normal Women: mild anemia 10-12 Men: mild anemia 11.5-13 5. Indices MCV HI MCH HI
  • 6. MCHC HI Despite these values, we would still call this macrocytic/normachromic. Why? Because the cels are bigger, so more Hb can load onto each cel. This gives us a misleading HI MCH and MCHC. When you actually look at the cels on a slide they don’t look full of Hb the way a HI MCH normally looks 6. Blood smear: We would see Ovalocytes oval shaped cels Macrocytes larger than normal cels Dacrocytes tear drop shaped cels This is because, is MA there is an asynchrony bet the development of the nucleus and the cytoplasm RBC morphology Anisocytosisvariation in size Poikilocytosisvariation in shape Basophilic stipplingaggregate of ribosomes Nucleated RBCs Howell-Jolly bodiesfragments of DNA See p.66-86 in text: Practical Hematology for pics of RBC abnormalities and above terms WBC morphology NP and lymphocytes bigger than usu Hypersegmented NP: usu 3-4lobesnow 6-10 lobes nuclei Thrombocytopenia (low platelet ct.) + giant platelets This is b/c with megakaryocytes, as we remember from last week polyploidy and thencytoplasm breaks off to form platelets. But if we have asynchrony as we do with MA, this development is off and cytoplasm doesn’t pinch off in time resulting in LO #’s of platelets and plucked off in big chunks, monster platelets. B12 def is interfering with DNA. Look at biochemistry See the following website: http://www.biology.arizona.edu/biochemistry/problem_sets/b12/01t.html For a tutorial on B12/folate biochemistry Biochemistry of B12 and its relationship to folate: Cobalamin is found in the liver of animals bound to protein as methycobalamin or 5'- deoxyadenosylcobalamin. The vitamin must be hydrolyzed from protein in order to be active.
  • 7. Hydrolysis occurs in the stomach by gastric acids; or the intestines by trypsin following consumption of animal meat. The vitamin is then bound by intrinsic factor, a protein secreted by parietal cells of the stomach, and carried to the ileum where it is absorbed. Following absorption the vitamin is transported to the liver in the blood bound to transcobalamin II. There are only two clinically significant reactions in the body that require vitamin B12 as a cofactor. During the catabolism of fatty acids, the amino acids valine, isoleucine and threonine and the resultant propionyl-CoA is converted to succinyl-CoA for oxidation in the TCA cycle. One of the enzymes in this pathway, methylmalonyl-CoA mutase, requires vitamin B12 as a cofactor in the conversion of methylmalonyl-CoA to succinyl- CoA. The second reaction requiring vitamin B12 catalyzes the conversion of homocysteine to methionine and is catalyzed by methionine synthase. This reaction results in the transfer of the methyl group from N5-methyltetrahydrofolate to hydroxycobalamin generating tetrahydrofolate (THF) and methylcobalamin during the process of the conversion. Several forms of folate must pass through THF. The form trapped by a B12 deficiency is N5-methyl THF. Without B12, this form of folate cannot donate it’s methyl group to make the necessary folate precursor. So with a B12 def, a significant portion of the body’s folate winds up in a dead end form and becomes useless. Diagnostic Tests Gastric pH Test With vit B12 def, the epit in the GI tract is not developing normally. In addition, if this def is due to PA there is the inhibition of HCl: hypochloridic. To test for low levels of HCl, you would do a gastric pH test. In this test, you swallow a capsule with a string attached, it goes into the stomach, the color changes depending on the pH. After a certain time, you pull it up and read the color. The Heidelberg capsule is a version of this test that uses a capsule with a radio transmitter on ittransmit pH. The Heidelberg capsule is little more specific. Another way to test gastric pH is to administer sodium bicarbarbonate or food prior to giving capsule and see reaction. Low moderate sensitivity with both tests. Gastroscopy Scope has a higher sensitivity but is better for lesions Clinical notes:Some NDs will skip the whole thing and administer HCl and enzymes orally
  • 8. Stool sample: O& P Test B12 def could be due to intake, PA, malabsorption and parasites: Let’s talk about parasites. Parasites are common in developing countries and Scandavian countries ( b/c raw fish consumption) So what is common in these cases is a fish tapeworm. Worldwide, this is the leading cause of vitamin B12 def : diphyllobothrium latum http://www.emedicine.com/ped/topic597.htm http://www.thelifetree.com/fishtapeworms.htm If person has just returned from overseas, you can do an O & P Test (Ova and Parasites): and you should be doing this with three consecutive stool samples on a symptomatic person. 1st Stool sample60% sens 3 stool samples consecutive on a symptomatic pat 80-85% sens If per is asymptomatic 40% sens Tests/Labs/Hx to Isolate etiology of MA as malabsorption: Three conditions to look out for: 7. Crohn’s/IBD 8. Celiac Sprue 9. Long term effective gastric bypass Clinical Symptoms In these cases, you are not going to see only Vitamin B12 def. Vit D, E, A, K may also be def. The result would be reduced night vision, increased bruising and ptikiai (sp?). There may also be poor fat absorption resulting in stool that floats or is malodorous. There may be blood in the stool resulting in black, tarry, sticky stool. There may also be bloody diarrhea and abdominal cramping/ pain CBC CBC won’t just be macrocytic or microcytic due to iron def, there will be coagulation problems Dx of IBD: Intestinal Biopsy Scope: sigmoid, colon If intake: do HX Following the Yellow Brick Road, the labs to differentiate between vit B12/folate def and their etiology
  • 9. If based on the CBC and Hx/presentation, you suspect folate and B12 def, you may choose to do the following: Folate Def 1. Serum folate a. low sensitivity b. not a good test i. snapshot is dependent on what you just consumed 2. RBC folate a. moderately sens b/c RBC live for 120 days. Clinical note: I would not recommend serum. I would recommend RBC and VERY GOOD Hx including very good diet history. Vit B12 def 1. Serum B12 2. Methyl Malonic (MM) acid measured in urine a. methyl malonyl CoA succinyl CoA in the presence of Vit B12. If no B12 go directly to MM acid. Higher amts of MM acid are correlated to def of B12. 3. Homocysteine levels measured in the serum a. HS Methionine in the presence of B12 and folate. So if you have a def in either, you’ll end up with higher amts of HS. This is a serum test. So serum HS levels will be higher with decreasing levels of B12 and folate. Clinical Note: Not one good test for vitB12 def so run 2/3 Etiology But we still haven’t discussed a way to differentiate the cause of B12: demand, intake etc. For this, we have the highly moderate sensitivity test, the Schilling test, which will tell you the etiology of your def. Step One: We begin with a suspicion that pat is B12 def, even if we are running low/normal levels of other values. We give radioactive B12 (colbalt) in excess orally. Assuming normal renal function, this is absorbed circulationexcreted in kidney. We are looking for radioactive B12 in the urine. Result of Schilling Etiology If we excrete appropriate amt LO intake of B12/folate If we don’t excrete it Malabsorption and/or PA Step Two: To differentiate between malabsorption and PA, take radioactive B12 + IF in excess orally. Again measure urine output.
  • 10. Result of Schilling Etiology If we excrete normal or greater amts LO IF PA If we excrete low levels Malabsorption Why the 1st result? Because you dump in so much IF, you have outnumbered the antibodies available to block IF. A parallel is with diabetics. Over the years, b/c of insulin intake you develop antibodies to insulin and have to take larger doses to overcome the inhibitory effect of those antibodies. Your Schilling results should be reconfirmed. Parasites will not be not detected with Schilling. Recap of Megaloblastic Anemia Pat walks in with CC of weakness and fatigue. Your reponse: Hx Physical Exam CBC Serum Vit B12 RBC folate Or MM acid in urine Or HS levels Other things to look at might be: Parasitic exams Tests for malabsorption like fatty stool, intestinal biopsy, scope, vitamin A, or iron, or coagulation factors. I don’t expect you to use all these tests—think logically. QUESTION: How do you treat PA? ANSWER: Allopathic: Immunosuppressives OR Naturopathic: HCl + Digestive enzymes; regular B12 injections and nutritional stuff to treat inflammation QUESTION: Would it help to administer long term IF? ANSWER:
  • 11. I don’t know, the vit B12 injection may aggravate AI dis so it’s something worth researching. Take Home Message MA implies an asynchrony in development between the cytoplasm and nucleus. Anecdote: B12 is needed for all developing cels in body: hematopoietic and GI epit. I don’t remember mechanism of neurological damage, it has something to do with FA synthesis. Anemias associated with interference in Synthesis Iron Deficiency Anemia and Anemia of Chronic Disease Number One Anemia is ACD usu found in in-patient clinics Number two Anemia is IDA, usu found in out-patient Number One nutrition def is IDA IDA Iron depleted, no longer sufficient store. Total amt of Iron in body 4g In circ 60% Stored 40% Two forms it’s stored in Ferritin and hemosiderin Where is it stored Liver and Bone marrow (bm) What stores it Macrophages Iron/Blood 1mg/1ml Amt of blood we lose a day 25 ml Amt of blood recycled/day 24 ml Net loss of iron/day 1 mg Avg intake of daily iron in our diets 3-5 mg So if you were a man, short of a blood loss like a GI bleed or cancer, you wouldn’t really lose iron. Good multis for men don’t have iron because it leads to iron loading other problems. Women lose iron every month and in pregnancy will have increased util. Presentation with IDA So things you want to think about when you do your history: • is this a child • is this a pregnant woman
  • 12. • Nutrit def and loss • menstrual in women • Other losses in women and men. Again for malabsorption, you want to ask questions about • night vision • easy bruising • ptikyi (sp?) • quality of stool Clinical note: When you ask, “Are your periods heavy?” Most women will say ‘yes’. You need to ask, “how long is your period, and how often do you go through tampons etc” Ask about the GI tract: • Blood in stool origin is lower down • Tarry, sticky, black stool origin is high up possible stomach/duod ulcers • Ulcers prior Hx • Meds • Lots of aspirin ? • Drug history • Nausea • Diet • Supplements Labs for IDA CBC: Iron def is viewed in three stages: Stage One Proactive heading that way, showing predisposition to signs and symptoms 1st stage: iron stores decreased. Test Result CBC normal Ferritin serum normals have become wider and wider. Low/normal iron stores No real signs and symptoms at this point and hematology symptoms are normal. But as a primary care doc you are trying to prevent stuff sot hink about how to get more dietary iron in. Stage Two We have progressed used our iron in circ and started deep into the stores.
  • 13. Some signs and symptoms will include: • Post running, hard to catch breath • Tachycardia • Tires easily Hematology picture: Pretty normal; but slight changes Test Result HCT 38- 39 Serum IRON Normal slightly Low (when you are down, stores dump so this won’t read very low) Hb Slightly LO Indices Might start to have a dimorphic pop. RBCs live 120 days, and less iron to play with Microcytic/NChrom to NCytic/NChrom Serum ferritin LO Your HCT usu runs about 42, but now 38, 39 trending down. Slight, slight abnormal; but if you don’t know individual reference values.. you may not catch this. Stage 3: Classic iron def. Anemia considered moderate to severe Clinical Guideline for Hb: Women Normals: 12-14 slight anemia 10-12 moderate 8-10 severe: below 8 Men Normals: 13-15 Mild: 11-13 Moderate: 9-11 Severe : below 9 Test Result HCT/Hb LO Indices Microcytic/hypochromic RDW HI Morphology Anisocytosis Serum iron LO
  • 14. Serum ferritin LO TIBC HI Transferrin LO Note: Can differentiate bet IDA and ACD with lab tests for: 1. TIBC 2. Ferritin 3. transferrin Transferrin (TF) is a transport protien made by liver. TF levels increase in iron def. TF is a bus, intended to take iron from one place to another. Normally this bus is half full. If we have more buses made and less iron riding the buses, TBIC total iron binding capacity will be increased. I think this the best test, b/c it’s the most sens. Transferrin will usu be the opposite of TBIC because it is calculated as: Serum Fe x 100/ TIBC. And transferring will be low in IDA. Note: You can use all three readings or just one. And by the way, liver dis will f$ck up the picture. Anemia of Chronic Dis IDA and ACD mimic each other. Big picture in ACD: Acute and chronic inflammationIL-1, IL-6 and TNFsequestration of Fe and increased transport from circ back to stores. In an acute infection, bacteria use Fe, so there may be an evolutionary benefit to this phenom. Initial pic of ACD looks IDA. The big difference is in the iron stores. End.