Discloser. Objectives LETTING GO OF THE RULES OF 3. I am receiving an honorarium from Sysmex for this presentation

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1 LETTING GO OF THE RULES OF 3 and how not to let the MCHC rule your life Jeri Walters, SH(ASCP) Technical Supervisor Esoteric Testing, WI Core Lab, ACL Laboratories Discloser I am receiving an honorarium from Sysmex for this presentation Objectives Discuss the history of the Rules of Three and their original purpose in the hematology testing. Explain the evolution of RBC Indices and how they are used as indicators of abnormal or problem samples. Apply your knowledge of the RBC Indices to troubleshoot real life case examples. 1

2 THE RULES OF 3 WHAT THEY ARE WHERE THEY CAME FROM WHY THEY NEED TO GO AWAY THE RULES OF 3 HGB X 3 = HCT +/- 3% RBC X 3.3 = HGB +/- 1.5 gm/dl RBC X 9 = HCT +/- 3% ACTUALLY.THERE IS A 4 TH RULE..and it is 2

3 4 TH RULE THE RULES OF 3 ONLY WORK WHEN THE RED CELLS ARE NORMAL ( LIKE NORMAL SIZE AND NORMAL HGB CONTENT) SO IF THE RED CELLS ARE NORMAL WHY ARE WE DOING RULES? A BRIEF HISTORY LESSON 3

4 A LONG TIME AGO.. Methods were primitive VERY PRIMITIVE AND THEN LABORATORY SCIENCE ADVANCED TO THESE INSTRUMENTS OF TORTURE 4

5 A YOUNG MAN NAMED WINTROBE TULANE.WAS ANNOYED AND FRUSTRATED BECAUSE: - THERE WERE NO ESTABLISHED HEMATOLOGY NORMALS - MANY RBC RESULTS WERE ERRONEOUS ( duh!...i wonder why ) SO WINTROBE INVENTED A RELIABLE AND REPRODUCIBLE HCT AND DERIVED THE RELATIONSHIP OF RESULTS (RBC, HCT, HGB) AND HE CALLED THIS RELATIONSHIP THE RBC INDICES 5

6 WHAT WINTROBE S RBC INDICES DID Provided information about RBC size, HGB concentration, and HGB weight used primarily to categorize anemias. WINTROBE INDICES MCV = (HCT/RBC) x 10 MCH = (HGB/RBC) x 10 MCHC = (HGB/HCT) x 100 THE ORIGINAL WINTROBE REFERENCE RANGES 6

7 AND THUS. A RELATIONSHIP BETWEEN RESULTS WAS BORN WHAT THE INDICES TOLD US RBCs WERE NORMAL, BIG, OR SMALL RED CELL HGB CONTENT WAS NORMAL, INCREASED OR DECREASED WE MESSED UP BEFORE WE HAD INSTRUMENTS THAT DID INDICES YOU COULD SEE IF YOUR RESULTS MADE SENSE 7

8 BUT WHO COULD DO THESE IN THEIR HEAD? MCV = (HCT/RBC) x 10 MCH = (HGB/RBC) x 10 MCHC = (HGB/HCT) x 100 SO WE MADE THE MATH EASIER WINTROBE INDICES AND THE RULES OF 3 MCV = (HCT/RBC) x 10 RBC X 9 = HCT +/- 3% MCH = (HGB/RBC) x 10 RBC X 3.3 = HGB +/- 1.5 gm/dl MCHC = (HGB/HCT) x 100 HGB X 3 = HCT +/- 3% 8

9 OR TA DA! THE RULES OF 3 RBC x 3 = HGB HGB x 3 = HCT The rules of 3 told you if your results (HGB, HCT, RBC) made sense.but you still had to manually calculate the indices, unless you had one of these WHY WE SHOULD HAVE RETIRED THE RULES OF 3 ALONG WITH THESE THINGS 9

10 WE HAVE THESE INDICES ON EVERY SAMPLE REGARDLESS OF WHAT INSTRUMENT YOU USE.YOU KNOW WHEN YOU HAVE A PROBELM WHY THEY DIDN T GO AWAY EARLY CLASSIC IMPEDANCE HAD LIMITATIONS AND IT WAS BECAUSE OF THE WAY IT MEASURED RED CELLS 10

11 CLASSIC IMPEDANCE MCHC LOCKED INTO A NARROW, NORMAL RANGE MCHC BECAME AN INSTRUMENT FUNCTION INDICATOR RATHER THAN A CLINICAL PARAMETER (ADDING SHEATH FLOW TO CLASSIC IMPEDANCE FIXED THIS) CLASSIC IMPEDANCE vs IMPEDANCE WITH SHEATH FLOW IT S ALL ABOUT DIFFERENCES.. FLUIDICS NATURAL DEFORMABILITY OF RBC HYPERTONIC vs ISOTONIC DILUENT CLASSIC IMPEDANCE SHEATH FLOW IMPEDANCE 11

12 NATURAL DEFORMABILITY OF RED BLOOD CELLS NORMAL CELLS DO THIS BUT WHEN THE RED CELLS ARE NOT NORMAL HYPOCHROMIC CELLS OVER DEFORM IN CLASSIC IMPEDANCE SHEATH FLOW CONTROLS THIS CELLS ZIP THROUGH TOO FAST SIZE IS UNDERESTIMATED HCT ARTIFICIALLY DECREASED MCHC ARTIFICIALLY INCREASED CONTROLED BY SHEATH FLOW ACCURATELY SIZED ACCURATE HCT AND MCHC WHEN THE RED CELLS ARE NOT NORMAL HYPERCHROMIC CELLS DON T DEFORM ENOUGH IN CLASSIC IMPEDANCE SHEATH FLOW CONTROLS THIS CELLS MOVE THROUGH TOO SLOW SHEATH FLOW SIZE IS OVER ESTIMATED HCT ARTIFICIALLY INCREASED MCHC ARTIFICIALLY DECREASED CONTROLED BY ACCURATELY SIZED ACCURATE HCT AND MCHC 12

13 THE RESULT IS A Clamped MCHC WITH CLASSIC IMPEDANCE Classic Conductance MCHC High MCHC s normalized Clamped MCHC Range Low MCHC s normalized Reference MCHC Result Reference: B.Bull, The Validity of the MCHC as Measured By Various Analyzers, presented at AACC, July SO WITH CLASSIC IMPEDANCE IF YOUR MCHC GOES OVER 35.5 OR SO SOMETHING IS MORE LIKELY WRONG WITH YOUR INSTRUMENT AND NOT THE PATIENT CLASSIC IMPEDANCE WITH HYDRODYNAMIC FOCUSING GIVES A WIDER DYNAMIC RANGE 37.1 Hydrodynamic MCHC Reference MCHC

14 WE HAVE BETTER TECHNOLOGY BUT. WE ARE STILL STUCK ON THE IDEA THAT AN MCHC CANNOT BE HIGHER THAN 36 AND THAT THE HCT HAS TO BE 3 X THE HGB MCHC REFERENCE RANGES AND 95 % CONFIDENCE LIMITS IF THE REFERENCE RANGE ENCOMPASSES 95 % OF THE NORMAL PEOPLE...WHERE ARE THE OTHER 5 %? 14

15 MCHC WHAT WE NEED TO DO STOP DOING THE RULES OF 3 IN OUR HEAD THEY DON T WORK IF THE RED CELLS ARE ABNORMAL WE HAVE THE RED CELL INDICES- USE THEM IF THE INDICES ARE OUT OF WHACK, INVESTIGATE ACCEPT THE FACT THAT AN MCHC OF 37 IS A POSSIBILITY WHAT S OK LOW MCV, LOW MCHC -THERE ARE MICROCYTIC HYPOCHROMIC ANEMIAS LOW MCV, NORMAL MCHC IS OK THERE ARE HEMOGLOBINOPATHIES 15

16 IRON DEFICIENCY THALASSEMIA MINOR WHAT S NOT OK MCHC > 37..AND WHAT CAN CAUSE IT RBC AGGLUTINATION LIPEMIA HEMOLYSIS LOW SODIUM ABNORMAL OSMOLALITY (OFTEN CHEMOTHERAPY) 16

17 FASTEST WAY TO INVESTIGATE AND I KNOW YOU DON T WANT TO HEAR THIS SPIN A HEMATOCRIT LIPEMIA HEMOLYSIS AGGLUTINATION ABNORMAL SODIUM OR OSMOLALITY and if you can t spin a HCT you could centrifuge the tube WHAT IF IT LOOKS LIKE THIS? 17

18 WHAT TO DO ABOUT IT HEMOLYSIS If it s the tube re-draw If it s the patient that s a problem WHAT TO DO ABOUT IT HEMOLYSIS If it s the patient that s a problem Free HGB isn t as functional as HGB in a cell RBC and HCT are reliable indicators The MCV will be correct Nobody cares about the MCH, MCHC at this point WHAT IF IT LOOKS LIKE THIS? 18

19 WHAT TO DO ABOUT IT LIPEMIA PLASMA REPLACEMENT (YUCK!) PLASMA BLANK (UGH!) SIMPLE MATH.Hmmm SIMPLE MATH BASED ON CONSTANT RATIO (2.98) BETWEEN THE MCV AND MCH FORMULA CAN BE USED WHEN MCV IS IN THE NORMAL RANGE CORRECTED HGB = MCV x RBC 2.98 x 10 ALSO CAN BE USED FOR ICTERIC AND HIGH WBC INTERFERENCE Pathology, 1991 (23) SIMPLE MATH BASED ON CONSTANT RATIO (2.98) BETWEEN THE MCV AND MCH THIS IS IMPORTANT FORMULA CAN BE USED WHEN MCV IS IN THE NORMAL RANGE CORRECTED HGB = MCV x RBC 2.98 x 10 ALSO CAN BE USED FOR ICTERIC AND HIGH WBC INTERFERENCE Pathology, 1991 (23) 19

20 WHAT TO DO IF THE MCV IS NOT NORMAL PLASMA REPLACEMENT Or try DILUTE 1:3 or 1:5 WHAT IF IT LOOKS LIKE THIS? COLD AGGLUTININ 20

21 HEAT IT Probably best to heat just an aliquot LOW SODIUM WHY DOES THAT HAPPEN? HYPO OR HYPERNATREMIA (low or high sodium for us non-chemists) HIGH NA IN VIVO IN ISOTONIC DILUENT RESULT HIGH HCT HIGH MCV H 2 O OUT H 2 O IN LOW MCHC LOW NA LOW HCT LOW MCV HIGH MCHC H 2 O IN H 2 O OUT 21

22 SODIUM 132 HOW DO I FIX IT? RECALCULATE INDICES WITH SPUN HCT DILUTE WITH INSTRUMENT DILUENT AND LET EQUILIBRATE NORMAL NA NO LIPEMIA NO AGGLUTINATION NO HEMOLYSIS NOW WHAT? 22

23 WHAT IT MIGHT BE AND WHAT TO DO look at a slide HGB SS, C, OR SC DISEASE LET IT GO (UNLESS THE MCHC IS > 38) or SPHEROCYTOSIS DEHYDRATION CAN CAUSE A HIGH MCHC IF THE PATIENT IS DEHYDRATED SO ARE THE RED CELLS 23

24 WHAT S NOT OK NORMAL OR HIGH MCV, LOW MCHC HIGH SODIUM AGED SAMPLE VERY HIGH GLUCOSE VERY HIGH WBC SODIUM 176 HIGH GLUCOSE VERY HIGH 1494 mg/dl 24

25 HOW DO YOU FIX THESE? SPIN A HEMATOCRIT OR. DILUTE THE SAMPLE AND LET IT EQUILIBRATE FOR 5 OR 10 MINUTES Original RBC histogram VERY HIGH WBC CLL 25

26 CHOICES.. DO THE MATH 1. Perform a manual hematocrit - carefully read below the buffy layer (this is going to be difficult) 2. Subtract the WBC from the RBC 3. Recalculate the indices OR.see this?. that dimorphic thing on the RBC RBC WBC 26

27 MORE TYPICAL CLL INTERFERENCE WHEN ALL ELSE FAILS COLD AGGLUTININ 27

28 COLD AGGLUTININ Heated to 37 o C for 20 minutes that didn t work cold agglutinin Heated to 37 o C and diluted 1:5 with warm diluent LAST RESORT OPTION Replace plasma with warm saline or diluent May have to do this twice (this is very time consuming and annoying and hemolysis may be a problem) 28

29 AM I GOING TO REPORT THESE RESULTS? IF HGB IS 9.1 AND HCT 25.0.MCHC IS 36.4 THAT IS CLINICALLY THE SAME ANSWER THE REALLY LAST RESORT OPTION SEVERE COLD AGGLUTININ AFTER HEATING, DILUTING, REPLACING (and cussing) 29

30 NO ONE EVER DIED BECAUSE THEY DID NOT HAVE AN MCHC (THAT I KNOW OF) RESULTS REPORTED WBC 6.4 K/mcL RBC UNABLE TO REPORT DUE TO SEVERE COLD AGGLUTININ HEMOGLOBIN 9.2 [g/dl HEMATOCRIT 27.5 % (SPUN) MCV MCH MCHC UNABLE TO REPORT DUE TO SEVERE COLD AGGLUTININ UNABLE TO REPORT DUE TO SEVERE COLD AGGLUTININ UNABLE TO REPORT DUE TO SEVERE COLD AGGLUTININ THANK YOU QUESTIONS??? 30